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                            STANDARD METHODS
                                FOR THE
                              EXAMINATION
                                   OF
                            WATER AND SEWAGE



                            _FOURTH EDITION_

    Revised by committees of the American Public Health Association,
 American Chemical Society, and referees of the Association of Official
                         Agricultural Chemists


                   AMERICAN PUBLIC HEALTH ASSOCIATION
                        169 MASSACHUSETTS AVENUE
                                 BOSTON
                                  1920




                       _Copyright, 1917 and 1920_

              _By the American Public Health Association_

------------------------------------------------------------------------




                               CONTENTS.


                                                                    PAGE
 PREFACE TO THE FOURTH EDITION                                       vii

 COLLECTION OF SAMPLES                                                 1
     QUANTITY OF WATER REQUIRED FOR ANALYSIS                           1
     BOTTLES                                                           1
     TIME INTERVAL BETWEEN COLLECTION AND ANALYSIS                     2
     REPRESENTATIVE SAMPLES                                            3

 PHYSICAL EXAMINATION                                                  4
     TEMPERATURE                                                       4
     TURBIDITY                                                         4
         TURBIDITY STANDARD                                            4
         PLATINUM WIRE METHOD                                          5
         TURBIDIMETRIC METHOD                                          7
         COEFFICIENT OF FINENESS                                       8
     COLOR                                                             9
         COMPARISON WITH PLATINUM-COBALT STANDARDS                     9
         COMPARISON WITH GLASS DISKS                                  10
         COMPARISON WITH NESSLER STANDARDS                            10
         LOVIBOND TINTOMETER                                          11
     ODOR                                                             12
         COLD ODOR                                                    12
         HOT ODOR                                                     12
         EXPRESSION OF RESULTS                                        12

 CHEMICAL EXAMINATION                                                 14
     EXPRESSION OF RESULTS                                            14
     FORMS OF NITROGEN                                                15
     AMMONIA NITROGEN                                                 15
         DETERMINATION BY DISTILLATION                                15
             MEASUREMENT OF AMMONIA NITROGEN                          16
                 COMPARISON WITH AMMONIA STANDARDS                    16
                 COMPARISON WITH PERMANENT STANDARDS                  17
             MODIFICATION FOR SEWAGE                                  18
         DETERMINATION BY DIRECT NESSLERIZATION                       19
     ALBUMINOID NITROGEN                                              20
     ORGANIC NITROGEN                                                 21
     NITRITE NITROGEN                                                 22
     NITRATE NITROGEN                                                 23
         PHENOLDISULFONIC ACID METHOD                                 23
         REDUCTION METHOD                                             24
     TOTAL NITROGEN                                                   25
     OXYGEN CONSUMED                                                  25
         RECOMMENDED METHOD                                           26
         OTHER METHODS                                                27
     RESIDUE ON EVAPORATION                                           29
         TOTAL RESIDUE                                                29
         FIXED RESIDUE AND LOSS ON IGNITION                           29
     SUSPENDED MATTER                                                 30
         DETERMINATION WITH GOOCH CRUCIBLE                            30
         DETERMINATION BY FILTRATION                                  30
         DETERMINATION OF VOLUME                                      30
         FIXED RESIDUE AND LOSS ON IGNITION                           30
     HARDNESS                                                         30
         TOTAL HARDNESS BY CALCULATION                                31
         TOTAL HARDNESS BY SOAP METHOD                                31
         TOTAL HARDNESS BY SODA REAGENT METHOD                        34
         TEMPORARY HARDNESS BY TITRATION WITH ACID                    34
         NON-CARBONATE HARDNESS BY SODA REAGENT METHOD                34
         NON-CARBONATE HARDNESS BY SOAP METHOD                        35
     ALKALINITY                                                       35
         PROCEDURE WITH PHENOLPHTHALEIN                               36
         PROCEDURE WITH METHYL ORANGE                                 37
         PROCEDURE WITH LACMOID                                       37
         PROCEDURE WITH ERYTHROSINE                                   37
         BICARBONATE                                                  37
         NORMAL CARBONATE                                             38
         HYDROXIDE                                                    38
         ALKALI CARBONATES                                            39
     ACIDITY                                                          39
         TOTAL ACIDITY                                                40
         FREE CARBON DIOXIDE                                          40
         FREE MINERAL ACIDS                                           41
         MINERAL ACIDS AND SULFATES OF IRON AND ALUMINIUM             41
     CHLORIDE                                                         41
     IRON                                                             43
         TOTAL IRON                                                   44
             COLORIMETRIC METHOD                                      44
                 COMPARISON WITH IRON STANDARDS                       45
                 COMPARISON WITH PERMANENT STANDARDS                  46
             VOLUMETRIC METHOD                                        46
         DISSOLVED IRON                                               47
         SUSPENDED IRON                                               47
         FERROUS IRON                                                 47
         FERRIC IRON                                                  48
     MANGANESE                                                        48
         PERSULFATE METHOD                                            48
         BISMUTHATE METHOD                                            49
     LEAD, ZINC, COPPER, AND TIN                                      50
         LEAD                                                         51
         ZINC                                                         52
         COPPER                                                       53
         TIN                                                          54
     MINERAL ANALYSIS                                                 56
         RESIDUE ON EVAPORATION                                       56
         ALKALINITY AND ACIDITY                                       56
         CHLORIDE                                                     56
         NITRATE NITROGEN                                             56
         SEPARATION OF SILICA, IRON, ALUMINIUM, CALCIUM, AND
           MAGNESIUM                                                  56
             SILICA                                                   56
             IRON AND ALUMINIUM                                       57
             CALCIUM                                                  57
             MAGNESIUM                                                57
         SEPARATION OF SULFATE, SODIUM, AND POTASSIUM                 58
             SULFATE                                                  58
             SODIUM, POTASSIUM AND LITHIUM                            58
             POTASSIUM                                                59
         LITHIUM                                                      60
         BROMINE, IODINE, ARSENIC, AND BORIC ACID                     61
             BROMINE AND IODINE                                       61
             ARSENIC                                                  63
             BORIC ACID                                               63
     HYDROGEN SULFIDE                                                 63
     CHLORINE                                                         64
     DISSOLVED OXYGEN                                                 65
     ETHER-SOLUBLE MATTER                                             69
     RELATIVE STABILITY OF EFFLUENTS                                  69
     BIOCHEMICAL OXYGEN DEMAND OF SEWAGES AND EFFLUENTS               71
         RELATIVE STABILITY METHOD                                    71
         SODIUM NITRATE METHOD                                        72

 ANALYSIS OF SEWAGE SLUDGE AND MUD DEPOSITS                           73
     COLLECTION OF SAMPLE                                             73
     REACTION                                                         73
     SPECIFIC GRAVITY                                                 74
     MOISTURE                                                         74
     VOLATILE AND FIXED MATTER                                        74
     TOTAL ORGANIC NITROGEN                                           74
     ETHER-SOLUBLE MATTER                                             75
     FERROUS SULFIDE                                                  76
     BIOCHEMICAL OXYGEN DEMAND                                        76

 ANALYSIS OF CHEMICALS                                                77
     REAGENTS                                                         77
     SULFATE OF ALUMINIUM                                             78
         INSOLUBLE MATTER                                             78
         OXIDES OF ALUMINIUM AND IRON                                 78
         TOTAL IRON                                                   79
         FERRIC IRON                                                  79
         FERROUS IRON                                                 80
         BASICITY RATIO                                               80
     LIME                                                             80
     SULFATE OF IRON                                                  81
         INSOLUBLE MATTER                                             81
         IRON AS FERROUS SULFATE                                      81
         ACIDITY                                                      81
     SODA ASH                                                         82
         INSOLUBLE MATTER                                             82
         AVAILABLE ALKALI                                             82

 CHEMICAL BIBLIOGRAPHY                                                82

 MICROSCOPICAL EXAMINATION                                            89
     MICROSCOPICAL BIBLIOGRAPHY                                       91

 BACTERIOLOGICAL EXAMINATION                                          92
     APPARATUS                                                        92
         SAMPLE BOTTLES                                               92
         PIPETTES                                                     92
         DILUTION BOTTLES                                             92
         PETRI DISHES                                                 92
         FERMENTATION TUBES                                           92
     MATERIALS                                                        93
         WATER                                                        93
         MEAT EXTRACT                                                 93
         PEPTONE                                                      93
         SUGARS                                                       93
         AGAR                                                         93
         GELATIN                                                      93
         LITMUS                                                       93
         GENERAL CHEMICALS                                            93
     METHODS                                                          93
         PREPARATION OF CULTURE MEDIA                                 93
             TITRATION                                                93
             STERILIZATION                                            94
             NUTRIENT BROTH                                           95
             SUGAR BROTHS                                             95
             NUTRIENT GELATIN                                         95
             NUTRIENT AGAR                                            96
             LITMUS OR AZOLITMIN SOLUTION                             96
             LITMUS-LACTOSE-AGAR                                      97
             ENDO’S MEDIUM                                            97
         COLLECTION OF SAMPLE                                         98
         STORAGE AND TRANSPORTATION OF SAMPLE                         98
         DILUTIONS                                                    98
         PLATING                                                      99
         INCUBATION                                                   99
         COUNTING                                                     99
         THE TEST FOR THE PRESENCE OF MEMBERS OF THE B. COLI GROUP   100
             PRESUMPTIVE TEST                                        100
             PARTIALLY CONFIRMED TEST                                101
             COMPLETED TEST                                          102
             APPLICATION OF THESE TESTS                              102
         EXPRESSION OF RESULTS                                       103
             SUMMARY OF THESE TESTS                                  104
         INTERPRETATION OF RESULTS                                   106
         DIFFERENTIATION OF FECAL FROM NON-FECAL MEMBERS OF THE B.
           COLI GROUP                                                106
             METHYL RED TEST                                         107
             VOGES-PROSKAUER TEST                                    108
         ROUTINE PROCEDURE FOR BACTERIOLOGICAL EXAMINATION           108
     BACTERIOLOGICAL BIBLIOGRAPHY                                    110

 INDEX                                                               113




                       PREFACE TO FOURTH EDITION.


The Committee on Standard Methods of Bacteriological Water Analysis was
reorganized in 1918 with the following membership: F. P. Gorham,
chairman, L. A. Rogers, W. G. Bissell, H. E. Hasseltine, H. W. Redfield,
with M. Levine as adjunct member. This committee made a report in 1918
which was not acted on by the Laboratory Section, and in 1919 made a
revised report, recommending certain changes in Standard Methods, which
were adopted by the section and which are now incorporated in this
present fourth edition.

Following are the more important changes:

New brands of peptone authorized.

Phenol Red Method of Hydrogen-ion Concentration.

Five-tenths per cent of sugar specified for broths instead of 1 per
cent.

Sterilization of sugar is media specified in greater detail.

Preparation of Endo Medium.

Synthetic Medium for the Methyl Red Test.

There are no changes in the chemical methods in this edition.




                  AMERICAN PUBLIC HEALTH ASSOCIATION.
                         _LABORATORY SECTION._
       STANDARD METHODS FOR THE EXAMINATION OF WATER AND SEWAGE.

Compiled and revised by committees of the American Public Health
Association and the American Chemical Society and referees of the
Association of Official Agricultural Chemists.




                         COLLECTION OF SAMPLES.


                    QUANTITY REQUIRED FOR ANALYSIS.

The minimum quantity necessary for making the ordinary physical,
chemical, and microscopical analyses of water or sewage is 2 liters; for
the bacteriological examination, 100 cc. In special analyses larger
quantities may be required.


                                BOTTLES.

The bottles for the collection of samples shall have glass stoppers,
except when physical, mineral, or microscopical examinations only are to
be made. Jugs or metal containers shall not be used.

Sample bottles shall be carefully cleansed each time before using. This
may be done by treating with sulfuric acid and potassium bichromate, or
with alkaline permanganate, followed by a mixture of oxalic and sulfuric
acids, and by thoroughly rinsing with water and draining. The stoppers
and necks of the bottles shall be protected from dirt by tying cloth,
thick paper or tin foil over them.

For shipment bottles shall be packed in cases with a separate
compartment for each bottle. Wooden boxes may be lined with corrugated
fibre paper, felt, or similar substance, or provided with spring corner
strips, to prevent breakage. Lined wicker baskets also may be used.

Bottles for bacteriological samples shall be sterilized as directed on
page 98.


                       INTERVAL BEFORE ANALYSIS.

In general, the shorter the time elapsing between the collection and the
analysis of a sample the more reliable will be the analytical results.
Under many conditions analyses made in the field are to be commended, as
data so obtained are frequently preferable to data obtained in a distant
laboratory after the composition of the water has changed.

The time that may be allowed to elapse between the collection of a
sample and the beginning of its analysis cannot be stated definitely. It
depends on the character of the sample, the examinations to be made, and
other conditions. The following are suggested as fairly reasonable
maximum limits.

                   _Physical and chemical analysis._

       Ground waters                       72 hours
       Fairly pure surface waters          48   "
       Polluted surface waters             12   "
       Sewage effluents                     6   "
       Raw sewages                          6   "

       _Microscopical examination._

       Ground waters                       72 hours
       Fairly pure surface waters          24   "
       Waters containing fragile organisms Immediate  examination

       _Bacteriological examination._

       Samples kept at less than 10°C      24 hours

If a longer period elapses between collection and examination the time
should be noted. If sterilized by the addition of chloroform,
formaldehyde, mercuric chloride, or some other germicide samples for
sanitary chemical examination may be allowed to stand for longer periods
than those indicated, but as this is a matter which will vary according
to circumstances, no definite procedure is recommended. If unsterilized
samples of sewage, sewage effluents, and highly polluted surface waters
are analyzed after greater intervals than those suggested caution must
be used in interpreting analyses of the organic content, which
frequently changes materially upon standing.

Determinations of dissolved gases, especially oxygen, hydrogen sulfide,
and carbon dioxide, should be made at the time of collection in order to
be reasonably accurate, in accordance with the directions given
hereafter in connection with each determination.


                        REPRESENTATIVE SAMPLES.

Care should be taken to obtain a sample that is truly representative of
the liquid to be analyzed. With sewages this is especially important
because marked variations in composition occur from hour to hour.
Satisfactory samples of some liquids can be obtained only by mixing
together several portions collected at different times or at different
places—the details as to collection and mixing depending upon local
conditions.




                         PHYSICAL EXAMINATION.


                              TEMPERATURE.

The temperature of the sample, if taken, shall be taken at the time of
collection, and shall be expressed preferably in degrees Centigrade, to
the nearest degree, or closer if more precise data are required. The
thermophone[109] is recommended for obtaining the temperature of water
at various depths below the surface.


                               TURBIDITY.

The turbidity of water is due to suspended matter, such as clay, silt,
finely divided organic matter, microscopic organisms, and similar
material.


                        TURBIDITY STANDARD.[110]

The standard of turbidity shall be that adopted by the United States
Geological Survey, namely, a water which contains 100 parts per million
of silica in such a state of fineness that a bright platinum wire 1
millimeter in diameter can just be seen when the center of the wire is
100 millimeters below the surface of the water and the eye of the
observer is 1.2 meters above the wire, the observation being made in the
middle of the day, in the open air, but not in sunlight, and in a vessel
so large that the sides do not shut out the light so as to influence the
results. The turbidity of such water is arbitrarily fixed at 100 parts
per million.

For preparation of the silica standard dry Pear’s “precipitated fuller’s
earth” and sift it through a 200–mesh sieve. One gram of this
preparation in 1 liter of distilled water makes a stock suspension which
contains 1,000 parts per million of silica and which should have a
turbidity of 1,000. Test this suspension, after diluting a portion of it
with nine times its volume of distilled water, by the platinum wire
method to ascertain if the silica has the necessary degree of fineness
and if the suspension has the necessary degree of turbidity. If not,
correct by adding more silica or more water as the case demands.[A]

Footnote A:

  This method of correction very slightly alters the coefficient of
  fineness of the standard, but does not noticeably affect its use.

Standards for comparison shall be prepared from this stock suspension by
dilution with distilled water. For turbidity readings below 20,
standards of 0, 5, 10, 15, and 20 shall be kept in clear glass bottles
of the same size as that containing the sample; for readings above 20,
standards of 20, 30, 40, 50, 60, 70, 80, 90, and 100 shall be kept in
100 cc. Nessler tubes approximately 20 millimeters in diameter.

Comparison with the standards shall be made by viewing both standard and
sample sidewise toward the light by looking at some object and noting
the distinctness with which the margins of the object can be seen.

The standards shall be kept stoppered, and both sample and standards
shall be thoroughly shaken before making the comparison.

In order to prevent any bacterial or algal growths from developing in
the standards a small amount of mercury bichloride may be added to them.


                       PLATINUM WIRE METHOD.[42]

This method requires a rod with a platinum wire 1 mm. in diameter
inserted in it about 1 inch from one end of the rod and projecting from
it at a right angle at least 25 mm. Near the other end of the rod, at a
distance of 1.2 meters from the platinum wire, a small ring shall be
placed directly above the wire through which, with his eye directly
above the ring, the observer shall look when making the examination.

The rod shall be graduated as follows: The graduation mark of 100 shall
be placed on the rod at a distance of 100 mm. from the center of the
wire. Other graduations shall be made according to Table 1, which is
based on the best obtainable data. The distances recorded in Table 1 are
intended to be such that when the water is diluted the turbidity
readings will decrease in the same proportion as the percentage of the
original water in the mixture. These graduations are those on what is
known as the U. S. Geological Survey Turbidity Rod of 1902.[105]

                  Table 1.—GRADUATION OF TURBIDITY ROD.

 ───────────────────────────────────┬───────────────────────────────────
              Turbidity             │  Vanishing depth of wire (mm.).
        (parts per million).        │
                                    │
 ───────────────────────────────────┼───────────────────────────────────
                                   7│                             1095
                                   8│                              971
                                   9│                              873
                                  10│                              794
                                  11│                              729
                                  12│                              674
                                  13│                              627
                                  14│                              587
                                  15│                              551
                                  16│                              520
                                  17│                              493
                                  18│                              468
                                  19│                              446
                                  20│                              426
                                  22│                              391
                                  24│                              361
                                  26│                              336
                                  28│                              314
                                  30│                              296
                                  35│                              257
                                  40│                              228
                                  45│                              205
                                  50│                              187
                                  55│                              171
                                  60│                              158
                                  65│                              147
                                  70│                              138
                                  75│                              130
                                  80│                              122
                                  85│                              116
                                  90│                              110
                                  95│                              105
                                 100│                              100
                                 110│                               93
                                 120│                               86
                                 130│                               81
                                 140│                               76
                                 150│                               72
                                 160│                               68.7
                                 180│                               62.4
                                 200│                               57.4
                                 250│                               49.1
                                 300│                               43.2
                                 350│                               38.8
                                 400│                               35.4
                                 500│                               30.9
                                 600│                               27.7
                                 800│                               23.4
                                1000│                               20.9
                                1500│                               17.1
                                2000│                               14.8
                                3000│                               12.1
 ───────────────────────────────────┴───────────────────────────────────

_Procedure._—Lower the rod vertically into the water as far as the wire
can be seen and read the level of the surface of the water on the
graduated scale. This will indicate the turbidity.

The following precautions shall be taken to insure correct results:

Observations shall be made in the open air, preferably in the middle of
the day and not in direct sunlight. The wire shall be kept bright and
clean. If for any reason observations cannot be made directly under
natural conditions a pail or tank may be filled with water and the
observation taken in that, but if this is done care shall be taken that
the water is thoroughly stirred before the observation is made, and no
vessel shall be used for this purpose unless its diameter is at least
twice as great as the depth to which the wire is immersed. Waters which
have a turbidity greater than 500 shall be diluted with clear water
before the observations are made, but if this is done the degree of
dilution shall be reported.


                         TURBIDIMETRIC METHOD.

Several forms of turbidimeter or diaphanometer[73] have been suggested
for use. The simplest and most satisfactory form is the candle
turbidimeter.[116] This consists of a graduated glass tube with a flat
polished bottom, enclosed in a metal case. This is supported over an
English standard candle and so arranged that one may look vertically
down through the tube at the flame of the candle. The observation is
made by pouring the sample of water into the tube until the image of the
flame of the candle just disappears from view. Care shall be taken not
to allow soot or moisture to accumulate on the lower side of the glass
bottom of the tube so as to interfere with the accuracy of the
observations. The graduations on the tube correspond to turbidities
produced in distilled water by certain numbers of parts per million of
silica standard. In order to insure uniform results it is necessary to
have the distance between the top rim of the candle and the bottom of
the tube constant, and this distance shall be 7.6 cm. or 3 inches. The
observations shall be made in a darkened room or with a black cloth over
the head.

It is allowable to substitute for the candle an electric light.
Calibrate the apparatus to correspond with the United States Geological
Survey scale. The figures in Table 2 on page 8 are believed to be
approximately correct for the candle turbidimeter but should be checked
by the experimenter. It is allowable to calibrate the tube of the
instrument with waters of known turbidity prepared by making a series of
dilutions of the silica standard with distilled water. From the figures
obtained in calibrating plot a curve from which the turbidity of a
sample may be read when the depth of water in the tube has been
obtained.

               Table 2.—GRADUATION OF CANDLE TURBIDIMETER.

 ───────────────────────────────────┬───────────────────────────────────
           Depth of liquid          │             Turbidity
               (cm.).               │  (parts per million of silica).
 ───────────────────────────────────┼───────────────────────────────────
                                 2.3│                               1000
                                 2.6│                                900
                                 2.9│                                800
                                 3.2│                                700
                                 3.5│                                650
                                 3.8│                                600
                                 4.1│                                550
                                 4.5│                                500
                                 4.9│                                450
                                 5.5│                                400
                                 5.6│                                390
                                 5.8│                                380
                                 5.9│                                370
                                 6.1│                                360
                                 6.3│                                350
                                 6.4│                                340
                                 6.6│                                330
                                 6.8│                                320
                                 7.0│                                310
                                 7.3│                                300
                                 7.5│                                290
                                 7.8│                                280
                                 8.1│                                270
                                 8.4│                                260
                                 8.7│                                250
                                 9.1│                                240
                                 9.5│                                230
                                 9.9│                                220
                                10.3│                                210
                                10.9│                                200
                                11.4│                                190
                                12.0│                                180
                                12.7│                                170
                                13.5│                                160
                                14.4│                                150
                                15.4│                                140
                                16.6│                                130
                                18.0│                                120
                                19.6│                                110
                                21.5│                                100
 ───────────────────────────────────┴───────────────────────────────────

The results of turbidity observations shall be expressed in whole
numbers which correspond to parts per million of silica and recorded as
follows:

      Turbidity between │   1│and│     50│recorded to nearest│unit
          "        "    │  51│ " │    100│   "     "     "   │   5
          "        "    │ 101│ " │    500│   "     "     "   │  10
          "        "    │ 501│ " │   1000│   "     "     "   │  50
          "        "    │1001│ " │greater│   "     "     "   │ 100


                      COEFFICIENT OF FINENESS[80]

The quotient obtained by dividing the weight of suspended matter in the
sample by the turbidity, both expressed in the same unit, shall be
called the coefficient of fineness. If the quotient is greater than
unity the matter in suspension is coarser and if it is less than unity
it is finer than the standard.


                                 COLOR.

The “color,” or the “true color,” of water shall be considered the color
that is due only to substances in solution; that is, it is the color of
the water after the suspended matter has been removed. In stating
results the word “color” shall mean the “true color” unless otherwise
designated.

The “apparent color” shall be considered as including not only the true
color but also any color produced by substances in suspension. It is the
color of the original unfiltered sample.

The platinum-cobalt method of measuring color shall be considered as the
standard, and the unit of color shall be that produced by 1 part per
million of platinum.


             COMPARISON WITH PLATINUM-COBALT STANDARDS.[43]

_Reagents._—Dissolve 1.246 grams of potassium platinic chloride
(PtCl_{4}2KCl), containing 0.5 gram platinum, and 1.00 gram crystallized
cobalt chloride (CoCl_{2}.6H_{2}O), containing 0.25 gram of cobalt, in
water with 100 cc. concentrated hydrochloric acid, and dilute to 1 liter
with distilled water. This solution has a color of 500. Dilute this
solution with distilled water in 50 cc. Nessler tubes to prepare
standards having colors of 0, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, and
70. Keep these standards in Nessler tubes of such diameter that the
graduation mark is between 20 and 25 cm. above the bottom and of such
uniformity that they match within such limit that the distance from the
bottom to the graduation mark of the longest tube shall not exceed that
of the shortest tube by more than 6 mm. Protect the tubes from dust and
light when not in use.

_Procedure._—The color of a sample shall be observed by filling a
standard Nessler tube to the height equal to that in the standard tubes
with the sample and by comparing it with the standards. The observation
shall be made by looking vertically downward through the tubes upon a
white or mirrored surface placed at such angle that light is reflected
upward through the column of liquid.

Water that has a color greater than 70 shall be diluted before making
the comparison, in order that no difficulties may be encountered in
matching the hues.

Water containing matter in suspension shall be filtered, before the
color observation is made, until no visible turbidity remains. If the
suspended matter is coarse, filter paper may be used for this purpose;
if the suspended matter is fine, the use of a Berkefeld filter is
recommended. The Pasteur filter shall not be used as it exerts a marked
decolorizing action.

The apparent color, if determined, shall be determined on the original
sample without filtration. The true and the apparent color of clear
waters or waters with low turbidities are substantially the same.

The results of color determinations shall be expressed in whole numbers
and recorded as follows:

           Color between   1 and  50 recorded to nearest unit
             "       "    51  "  100    "    "     "        5
             "       "   101  "  250    "    "     "       10
             "       "   251  "  500    "    "     "      20.


                   COMPARISON WITH GLASS DISKS.[105]

As the platinum-cobalt standard method is not well adapted for field
work, the color of the water to be tested may be compared with that of
glass disks held at the end of metallic tubes through which they are
viewed by looking toward a white surface. The glass disks are
individually calibrated to correspond with colors on the platinum scale.
Experience has shown that the glass disks used by the U. S. Geological
Survey give results in substantial agreement with those obtained by the
platinum determinations, and their use is recognized as a standard
procedure.


                   COMPARISON WITH NESSLER STANDARDS.

Inasmuch as the Nessler scale[62] and the natural water scale[22][49]
which agrees with it except for colors less than 20, have been largely
used in the past, the old results may be converted[117] into terms of
the platinum standard by means of the ratios in Table 3, but they must
not be considered as universally applicable as the variable
sensitiveness of the Nessler solution introduces an uncertain factor.

  Table 3.—VALUES FOR CONVERTING COLORS BY THE NATURAL WATER SCALE INTO
        COLORS BY THE PLATINUM STANDARD IN PARTS PER MILLION.[B]

 ───────────┬─────┬─────┬─────┬─────┬─────┬─────┬─────┬─────┬─────┬─────
  Modified  │     │     │     │     │     │     │     │     │     │
 Nessler or │     │     │     │     │     │     │     │     │     │
   natural  │0.00.│0.01.│0.02.│0.03.│0.04.│0.05.│0.06.│0.07.│0.08.│0.09.
    water   │     │     │     │     │     │     │     │     │     │
  standard. │     │     │     │     │     │     │     │     │     │
 ───────────┴─────┴─────┴─────┴─────┴─────┴─────┴─────┴─────┴─────┴─────
                     Platinum-cobalt standard color.
        0.00│    0│    2│    4│    6│    8│    9│   11│   13│   15│   17
         .10│   18│   19│   20│   20│   21│   22│   23│   24│   24│   26
         .20│   26│   27│   27│   28│   29│   29│   30│   31│   32│   32
         .30│   33│   34│   34│   35│   35│   36│   37│   37│   38│   38
         .40│   39│   40│   40│   41│   42│   42│   43│   44│   45│   45
         .50│   46│   47│   47│   48│   48│   49│   50│   50│   51│   51
         .60│   52│   53│   53│   54│   54│   55│   56│   56│   57│   57
         .70│   58│   58│   59│   59│   60│   60│   61│   61│   62│   62
         .80│   63│   64│   64│   65│   66│   66│   67│   68│   69│   69
         .90│   70│   71│   72│   73│   74│   75│   77│   78│   79│   80
        1.00│   81│   82│   82│   83│   84│   84│   85│   86│   87│   87
        1.10│   88│   89│   89│   90│   91│   91│   92│   93│   94│   94
        1.20│   95│   96│   96│   97│   98│   98│   99│  100│  101│  101
        1.30│  102│  103│  103│  104│  105│  105│  106│  107│  108│  108
        1.40│  109│  110│  110│  111│  112│  112│  113│  114│  115│  115
        1.50│  116│  117│  117│  118│  118│  119│  120│  120│  121│  121
        1.60│  122│  123│  123│  124│  125│  125│  126│  127│  128│  128
        1.70│  129│  130│  130│  131│  132│  132│  133│  134│  135│  136
        1.80│  136│  137│  137│  138│  139│  139│  140│  141│  142│  142
        1.90│  143│  144│  144│  145│  146│  146│  147│  148│  149│  149
        2.00│  150│     │     │     │     │     │     │     │     │
 ───────────┴─────┴─────┴─────┴─────┴─────┴─────┴─────┴─────┴─────┴─────

Footnote B:

  Zero on the true Nessler scale is about 15 on the platinum scale.


                          LOVIBOND TINTOMETER.

The value of the readings of tint and shade by the Lovibond
tintometer[66][82][83] has not been commensurate with the labor
involved, but it is necessary to make a record of the reflected tint and
shade[50] of some waters. The standard color disks used in teaching
optics may be used for the purpose.

_Procedure._—The white disk supports three movable standard color
sectors, red, yellow, and blue, and one movable black sector. All are
mounted on a device which can be revolved rapidly, blending the colors
into a uniform tint or shade. A scale around the circumference of the
disk is used to indicate the percentage of each color or white or black
in the blend.

Place the sample in a battery jar on a white ground; adjust the sectors
so that when blended the tint or shade will match the reflected tint or
shade of the sample. Report the percentages of red, yellow blue, white,
and black in the blended tint or shade.


                ODOR.[4][14][53][72][92][114][115][121c]

The observation of the odor, cold and hot, of samples of surface water
is important as the odors are usually indicative of organic growths or
sewage contamination or both. The odor of some ground waters is caused
by the earthy constituents of the water-bearing strata. The odor of a
contaminated well water is often contributory evidence of its pollution.
A study of the organisms as directed under Microscopical Examination (p.
90) is a valuable adjunct to physical and chemical examination of water.
Certain odors distinguish or identify certain organisms, as, for
example, the “fishy” odor of _Uroglena_, the “aromatic” or “rose
geranium” odor of _Asterionella_ and the “pig pen” odor of _Anabaena_.
Observe and record the odor, both at room temperature and at just below
the boiling point, as follows:


                               COLD ODOR.

Shake the sample violently in one of the collecting bottles, when it is
half to two-thirds full and when the sample is at room temperature
(about 20° C.). Remove the stopper and smell the odor at the mouth of
the bottle.


                               HOT ODOR.

Pour about 150 cc. of the sample into a 500 cc. Erlenmeyer flask. Cover
the flask with a well-fitting watch glass. Heat the water almost to
boiling on a hot plate. Remove the flask from the plate and allow it to
cool not more than five minutes. Then agitate it with a rotary movement,
slip the watch glass to one side, and smell the odor.


                         EXPRESSION OF RESULTS.

Express the quality of the odor by a descriptive epithet like the
following, which may be abbreviated in the record:

                           a—aromatic
                           C—free chlorine
                           d—disagreeable
                           e—earthy
                           f—fishy
                           g—grassy
                           m—moldy
                           M—musty
                           P—peaty
                           s—sweetish
                           S—hydrogen sulfide
                           v—vegetable.

Express the intensity of the odor by a numeral prefixed to the term
expressing quality, which may be defined as follows:

 Numerical   Term.                       Definition.
  value.

     0     None.     No odor perceptible.

     1     Very      An odor that would not be detected ordinarily by
           faint.      the average consumer, but that could be detected
                       in the laboratory by an experienced observer.

     2     Faint.    An odor that the consumer might detect if his
                       attention were called to it, but that would not
                       attract attention otherwise.

     3     Distinct. An odor that would be detected readily and that
                       might cause the water to be regarded with
                       disfavor.

     4     Decided.  An odor that would force itself upon the attention
                       and that might make the water unpalatable.

     5     Very      An odor of such intensity that the water would be
           strong.     absolutely unfit to drink. (A term to be used
                       only in extreme cases.)




                         CHEMICAL EXAMINATION.


                         EXPRESSION OF RESULTS.

The results of chemical analyses shall be expressed in parts per
million, which in most analyses is practically equivalent to milligrams
per liter. In some laboratories other forms of expression have been
used. Results expressed in parts per 100,000 or in grains per gallon may
be transformed to parts per million, or conversely, by the use of the
following table:

         Table 4.—FACTORS FOR TRANSFORMING RESULTS OF ANALYSES.

 ───────────────────────────────┬───────────────────────────────────────
              Unit.             │              Equivalent.
 ───────────────────────────────┼─────────┬─────────┬─────────┬─────────
                                │ Grains  │ Grains  │         │
                                │per U.S. │   per   │Parts per│Parts per
                                │ gallon. │Imperial │100,000. │million.
                                │         │ gallon. │         │
 ───────────────────────────────┼─────────┼─────────┼─────────┼─────────
 1 grain per U. S. gallon       │    1.000│     1.20│     1.71│     17.1
 1 grain per Imperial gallon    │     .835│     1.00│     1.43│     14.3
 1 part per 100,000             │     .585│      .70│     1.00│     10.0
 1 part per million             │     .058│      .07│      .10│      1.0
 ───────────────────────────────┴─────────┴─────────┴─────────┴─────────

The following general rules shall govern the use of significant figures
in the expression of results:

1. If the results show quantities greater than 10 parts per million use
no decimals; record only whole numbers. If the quantities reach hundreds
and thousands of parts record only two significant figures.

2. If the results are between 1 and 10 parts do not retain more than one
decimal place.

3. If the results are between 0.1 and 1 part do not retain more than two
decimal places.

4. Estimates of ammonia, albuminoid, and nitrite nitrogen alone justify
the use of three decimals.

5. If the results of analyses are tabulated ciphers should not be added
at the right of the decimal point to make the column uniform.


                           FORMS OF NITROGEN.

Nitrogenous organic matter passes through several intermediate compounds
during its natural decomposition, and that which does not gasify
ultimately forms nitrate. Nitrogen in organic matter is determined by
the Kjeldahl process.[13][14][58] An indication of the amount present is
obtained by the albuminoid nitrogen determination.[14][15][67][106][107]
It has not been found possible to differentiate the nitrogen in the
organic matter that readily decomposes from that in stable or
non-putrescible compounds. Decomposition of organic matter produces
nitrogen combined in ammonia, which is the first step between
nitrogenous organic matter and the completely mineralized nitrate.
Ammonia nitrogen may be determined by distillation and Nesslerization or
by direct Nesslerization of the clarified sample. The next step is
oxidation to nitrite, and the final step, oxidation to nitrate. It is
recommended that all forms of nitrogen be reported as the element
nitrogen (N).


                           AMMONIA NITROGEN.

There are two methods for estimating ammonia nitrogen—distillation and
direct Nesslerization. Distillation is recommended for most waters and
direct Nesslerization is recommended for sewages, sewage effluents, and
highly polluted surface waters.


           DETERMINATION BY DISTILLATION.[38][68b][111][121]

_Procedure._—Use a metal or a glass flask connected with a condenser so
that the distillate may drop from the condenser tube directly into a
Nessler tube or a flask. Free the apparatus from ammonia by boiling
distilled water in it until the distillate shows no trace of ammonia.
After this has been done empty the distilling flask and measure into it
500 cc. of the sample, or a smaller portion diluted to 500 cc. with
ammonia-free water. If the sample is acid or if the presence of urea is
suspected add about 0.5 gram of sodium carbonate before distillation.
Omit this if possible as it tends to increase “bumping.” Apply heat so
that the distillation may proceed at the rate of not more than 10 cc.
nor less than 6 cc. per minute. Collect the distillate in four Nessler
tubes, 50 cc. to each tube, or if the nitrogen is high in a 200 cc.
graduated flask. These receptacles contain the ammonia nitrogen to be
measured as hereafter described.

Use Nessler tubes of such diameter that the graduation mark is between
20 and 25 cm. above the bottom and of such uniformity of diameter that
the distance from the bottom to the graduation mark of the longest tube
shall not exceed that of the shortest tube by more than 6 mm. The tubes
must be of clear white glass with polished bottoms.


                    MEASUREMENT OF AMMONIA NITROGEN.

The amount of ammonia in the distillates may be measured either by (1)
comparison of the Nesslerized distillates with Nesslerized solutions
containing known quantities of nitrogen as ammonium chloride, or by (2)
comparison of the Nesslerized distillates with permanent standard
solutions in which the colors of Nesslerized standard ammonia solutions
are duplicated by solutions of platinum and cobalt chlorides.


                   COMPARISON WITH AMMONIA STANDARDS.

_Reagents._—1. Ammonia-free water.

2. Standard ammonium chloride solution. Dissolve 3.82 grams of ammonium
chloride in ammonia-free water and dilute to 1 liter; dilute 10 cc. of
this to 1 liter with ammonia-free water. One cc. equals 0.00001 gram of
nitrogen.

3. Nessler reagent.[8] Dissolve 50 grams of potassium iodide in a
minimum quantity of cold water. Add a saturated solution of mercuric
chloride until a slight precipitate persists permanently. Add 400 cc. of
50 per cent solution of potassium hydroxide, made by dissolving the
potassium hydroxide and allowing it to clarify by sedimentation before
using. Dilute to 1 liter, allow to settle, and decant. This solution
should give the required color with ammonia within five minutes after
addition and should not produce a precipitate with small amounts of
ammonia within two hours.

_Procedure._—Prepare a series of 16 Nessler tubes containing the
following amounts of the standard ammonium chloride solution, diluted to
50 cc. with ammonia-free water, namely: 0.0, 0.1, 0.3, 0.5, 0.7, 1.0,
1.4, 1.7, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 6.0 cc. These solutions
will contain 0.00001 gram of nitrogen for each cubic centimeter of the
standard solution.

Nesslerize the standards and the distillates by adding approximately 1
cc. of Nessler reagent to each tube. Do not stir the contents of the
tubes. The temperature of the tubes should be practically the same as
that of the standards; otherwise the colors will not be directly
comparable.[45] Allow the tubes to stand at least 10 minutes after
Nesslerizing. Compare the color produced in the tubes with that in the
standards by looking vertically downward through them at a white or
mirrored surface placed at an angle in front of a window so as to
reflect the light upward.

If the color obtained by Nesslerizing the distillates is greater than
that of the darkest tube of the standards, mix the contents of the tube
thoroughly, pour out half of the liquid, and dilute the remainder to the
original volume with ammonia-free water; then make the color comparison
and multiply the result by two. If the color is still too dark after
pouring out half the liquid, repeat this process of division until a
reading can be made. The process of dilution may be shortened by mixing
together the distillates from one sample before making the comparison
and comparing an aliquot portion with the standards.

After the readings have been recorded add the results obtained by
Nesslerizing each portion of the entire distillate. If 500 cc. of the
sample is distilled this sum, expressed in cubic centimeters and
multiplied by 0.02, will give the number of parts per million of ammonia
nitrogen in the sample. If x cc. of sample is used multiply the sum of
the readings by 10/x.

If the ammonia is known to be high the distillate may be collected in
200 cc. flasks and an aliquot part Nesslerized.


              COMPARISON WITH PERMANENT STANDARDS.[62][65]

_Reagents._—Platinum solution. Dissolve 2.00 grams of potassium platinic
chloride (PtCl_{4}.2KCl) in a small amount of distilled water, add 100
cc. of strong hydrochloric acid, and dilute to 1 liter.

Cobalt solution. Dissolve 12 grams of cobaltous chloride
(CoCl_{2}.6H_{2}O) in distilled water, add 100 cc. of strong
hydrochloric acid, and dilute to 1 liter.

Prepare standards by putting various amounts of these two solutions into
Nessler tubes and diluting to the 50 cc. mark with distilled water as
indicated in Table 5. These standards may be kept for several months if
protected from dust.

  Table 5.—PREPARATION OF PERMANENT STANDARDS FOR THE DETERMINATION OF
                                AMMONIA.

 ───────────────────────┬───────────────────────┬───────────────────────
    Value in standard   │ Solution of platinum. │  Solution of cobalt.
   ammonium chloride.   │                       │
 ───────────────────────┼───────────────────────┼───────────────────────
                   _cc._│                  _cc._│                  _cc._
                     0.0│                    1.2│                    0.0
                      .1│                    1.8│                     .0
                      .2│                    2.8│                     .0
                      .4│                    4.7│                     .1
                      .7│                    5.9│                     .2
                        │                       │
                     1.0│                    7.7│                     .5
                     1.4│                    9.9│                    1.1
                     1.7│                   11.4│                    1.7
                     2.0│                   12.7│                    2.2
                     2.5│                   15.0│                    3.3
                        │                       │
                     3.0│                   17.3│                    4.5
                     3.5│                   19.0│                    5.7
                     4.0│                   19.7│                    7.1
                     4.5│                   19.9│                    8.7
                     5.0│                   20.0│                   10.4
                        │                       │
                     6.0│                   20.0│                   15.0
                     7.0│                   20.0│                   22.0
 ───────────────────────┴───────────────────────┴───────────────────────

The amounts in Table 5 are approximate, and the actual amount necessary
will differ with the character of the Nessler solution, the color
sensitiveness of the analyst’s eye, and other conditions. The final test
of the standard is best obtained by comparing it with Nesslerized
standards and modifying the tint accordingly. Such comparison should be
made for each new batch of Nessler solution and should be checked by
each analyst.

_Procedure._—In comparison with permanent standards, Nesslerize the
distillates in the manner above described and compare the resulting
colors at the end of about 10 minutes with the permanent standards. The
method of calculating results is precisely the same as with the ammonia
standards.


                        MODIFICATION FOR SEWAGE.

Ammonia nitrogen and albuminoid nitrogen in sewages, soils, and other
materials of high nitrogen content may be satisfactorily determined by
diluting the sample with ammonia-free distilled water and proceeding as
described in the preceding sections, but it is permissible to distill
with steam.[40]

_Procedure._—Use a 200 cc. long-necked Kjeldahl flask connected with a
condenser so that the distillate may drop from the condenser tube
directly into a Nessler tube or a flask. Connect the Kjeldahl flask with
a steam generator by a tube reaching almost to the bottom of the flask.

After the apparatus is freed from ammonia put the sample to be tested
into the flask. Use 10 to 100 cc. of the sample according to its ammonia
content. Pass ammonia-free steam through the liquid in the Kjeldahl
flask and collect the distillate in the usual way. It is usually
convenient to collect the distillate in a 200 cc. flask and to take an
aliquot part of it for Nesslerization. Compare with standards and
calculate the nitrogen content in the usual manner.

This method has the advantage, when the sample is treated with an
alkaline solution of potassium permanganate, of avoiding bumping,
permitting the assay of solid matter, and yielding the ammonia more
rapidly than by the ordinary process of distillation.


            DETERMINATION BY DIRECT NESSLERIZATION.[21][75]

 _Reagents._— 1. Ten per cent solution of copper sulfate
                (CuSO_{4}.5H_{2}O).

              2. Ten per cent solution of lead acetate
                (Pb(C_{2}H_{3}O_{2})_{2}.3H_{2}O).

              3. Fifty per cent solution of sodium hydroxide (NaOH) or
                potassium hydroxide (KOH).

_Procedure._—To 50 cc. of the sample to be tested, diluted if necessary
with an equal volume of ammonia-free water, in a short tube, add a few
drops of the copper sulfate solution. After thoroughly mixing, add 1 cc.
of the alkali hydroxide solution and again thoroughly mix. Allow the
tube to stand for a few minutes, when a heavy precipitate should fall to
the bottom, leaving a colorless supernatant liquid. Nesslerize an
aliquot part. Compare with standards and compute the ammonia nitrogen in
the same manner as in the distillation procedure.

Samples containing hydrogen sulfide may require the use of lead acetate
in addition to the copper sulfate. Some samples may require a few trials
before the right combination of the three solutions to bring about the
best results can be found.

Instead of adding copper sulfate to sewages of high magnesium content
satisfactory clarification of the sample can be obtained by mixing it
with the alkali hydroxide alone.[54]


                          ALBUMINOID NITROGEN.

The addition of an alkaline permanganate solution to liquids containing
nitrogenous organic matter causes the formation of ammonia, which can be
distilled and determined by Nesslerization of the distillate. The
nitrogen of the ammonia, thus obtained, is called albuminoid nitrogen.
As the ratio of nitrogenous organic matter to the ammonia obtained by
distillation is decidedly variable[6][30][75] in sewages and other
substances containing much nitrogenous organic matter albuminoid
nitrogen results on such substances are less accurate[29] than organic
(Kjeldahl) nitrogen. Therefore in sewage work, including analysis of
influents and effluents of purification plants and the water of highly
polluted streams, it is recommended that determinations of organic
nitrogen be substituted for determinations of albuminoid nitrogen. For
ground waters and surface waters containing but little pollution, the
albuminoid nitrogen is approximately one-half the organic nitrogen;
accordingly the continuance of albuminoid nitrogen determinations for
this class of work is approved.

_Reagents._—Alkaline potassium permanganate. Pour 1,200 cc. of distilled
water into a porcelain dish holding 2,500 cc., boil 10 minutes, and turn
off the gas. Add 16 grams of C. P. potassium permanganate and stir until
solution is complete. Then add 800 cc. of 50 per cent clarified solution
of potassium hydroxide or an equivalent amount of sodium hydroxide and
enough distilled water to fill the dish. Boil down to 2,000 cc. Test
this solution for ammonia by making a blank determination. Correct
determinations by the amount of this blank.

_Procedure._—After the collection of the distillate for ammonia nitrogen
described on page 15 add 50 cc. (or more if necessary to insure the
complete oxidation of the organic matter) of alkaline potassium
permanganate and continue the distillation until at least four portions,
and preferably five portions, of 50 cc. each, of distillate have been
collected in separate tubes. Determine the albuminoid nitrogen in the
distillate by Nesslerization. If the albuminoid nitrogen is known to be
high it is convenient to collect the distillate in a 200 cc. flask and
to Nesslerize an aliquot part of it.

Dissolved albuminoid nitrogen may be determined in a sample from which
suspended matter has been removed by filtration either through filter
paper or through a Berkefeld filter. Suspended albuminoid nitrogen is
the difference between the total and the dissolved albuminoid nitrogen.


                 ORGANIC NITROGEN.[24b][69][71][76][84]

_Procedure for water._—Boil 500 cc. of the sample in a round-bottomed
flask to remove ammonia nitrogen. This usually causes the loss of 200
cc. of the sample, which may be collected for the determination of
ammonia nitrogen. Add 5 cc. of nitrogen-free concentrated sulfuric acid
and a small piece of ignited pumice. Mix by shaking and place over a
flame under a hood. Digest until copious fumes of sulfuric acid are
given off and the liquid finally becomes colorless or pale straw color.
Remove from the flame, and add potassium permanganate crystals in small
portions until a heavy green precipitate persists in the liquid. Cool.
Dilute to about 300 cc. with ammonia-free water. Make alkaline with 10
per cent ammonia-free sodium hydroxide. Distill the ammonia, collect the
distillate in Nessler tubes, Nesslerize, and compare with standards as
described (pp. 16–18).

_First procedure for sewage_[76].—Distill the ammonia nitrogen directly
from 100 cc. or less of the sample, diluted to 500 cc. with
nitrogen-free water. Collect the distillate and determine the ammonia
nitrogen in it. Add 5 cc. of nitrogen-free sulfuric acid and 1 cc. of 10
per cent nitrogen-free copper sulfate, and digest the liquid for half an
hour after it has become colorless or pale straw color. Add 0.5 gram of
potassium permanganate crystals to the hot acid solution, and dilute to
500 cc. with ammonia-free water. Dilute 10 cc. or more of this liquid,
in a Kjeldahl distilling flask, to about 300 cc. with ammonia-free
water. Make alkaline with 10 per cent sodium hydroxide, distill, and
Nesslerize. With some samples direct Nesslerization may be used. (See p.
19.)

In this determination care must be taken to digest thoroughly, to add
potassium permanganate to the point of precipitation, to sample
carefully after dilution, and to add enough sodium hydroxide to insure
the separation of the ammonia from the precipitated manganese hydroxide.
Potassium permanganate should not be added during digestion because it
causes loss of nitrogen.

_Second procedure for sewage._—Omit the separation of ammonia nitrogen
and determine the ammonia nitrogen and organic nitrogen together.
Determine the ammonia nitrogen in a separate sample by direct
Nesslerization as described on page 19. The organic nitrogen is equal to
the difference.


                NITRITE NITROGEN.[51][63a][64][94c][108]

_Reagents._—1. Sulfanilic acid solution. Dissolve 8.00 grams of the
purest sulfanilic acid in 1,000 cc. of 5 N acetic acid (sp. gr. 1.041)
or in 1,000 cc. of water containing 50 cc. of concentrated hydrochloric
acid. This is practically a saturated solution.

2. α-naphthylamine acetate or chloride solution. Dissolve 5.00 grams
solid α-naphthylamine in 1,000 cc. of 5 N acetic acid or in 1,000 cc. of
water containing 8 cc. of concentrated hydrochloric acid. Filter the
solution through washed absorbent cotton or an alundum filter.

3. Sodium nitrite stock solution. Dissolve 1.1 gram silver nitrite in
nitrite-free water; precipitate the silver with sodium chloride solution
and dilute the whole to 1 liter.

4. Standard sodium nitrite solution. Dilute 100 cc. of solution 3 to 1
liter, then dilute 50 cc. of this solution to 1 liter with sterilized
nitrite-free water, add 1 cc. of chloroform, and preserve in a
sterilized bottle. One cc. = 0.0005 mg. nitrogen.

5. Fuchsine solution. 0.1 gram per liter.

_Procedure._—Place in a standard Nessler tube 50 cc. of the sample,
decolorized if necessary with nitrite-free aluminium hydroxide (see p.
42) or a smaller amount diluted to 50 cc. At the same time prepare in
Nessler tubes a set of standards, by diluting to 50 cc. with
nitrite-free water, various amounts of the standard nitrite solution.
The following amounts of standard solution are suggested: 0.0, 0.1, 0.2,
0.4, 0.7, 1.0, 1.4, 1.7, 2.0, and 2.5 cc. Add 1 cc. of the sulfanilic
acid solution and 1 cc. of the α-naphthylamine acetate or hydrochloride
solution to the sample and to each standard. Mix thoroughly and allow to
stand 10 minutes; then compare the sample with the standards. Do not
allow the sample to stand more than one-half hour before making the
comparison. If the color of the sample is deeper than that of the
highest standard repeat the test on a diluted sample. If 50 cc. of the
sample is used 0.01 times the number of cc. of the standard matched
equals parts per million of nitrite nitrogen. Satisfactory results can
be obtained by using either hydrochloric or acetic acid in preparing the
test solutions, but the speed of the reaction is more rapid if acetic
acid is used.[112]

Permanent standards may be prepared by matching the nitrite standards
with dilutions of the fuchsine solution. Fuchsine standards have been
found to be sufficiently accurate for waters high in nitrite and for
sewage. The standards should be checked once a month and kept out of
bright sunlight.


                   NITRATE NITROGEN.[16][36][90][100]

Two methods are recommended for the determination of nitrate nitrogen in
water, sewage, and sewage effluents.


                PHENOLDISULFONIC ACID METHOD.[1][5][32]

_Reagents._—1. Phenoldisulfonic acid. Dissolve 25 grams of pure white
phenol in 150 cc. of pure concentrated sulfuric acid. Add 75 cc. of
fuming sulfuric acid (15 per cent SO_{3}), stir well, and heat for 2
hours at about 100°C.

2. Potassium hydroxide solution. Prepare an approximately 12 N solution,
10 cc. of which will neutralize about 4 cc. of the phenoldisulfonic
acid.

3. Standard nitrate solution. Dissolve 0.72 gram of pure recrystallized
potassium nitrate in 1 liter of distilled water. Evaporate cautiously to
dryness 10 cc. of the solution on the water bath. Moisten residue
quickly and thoroughly with 2 cc. of phenoldisulfonic acid and dilute to
1 liter. This is the standard solution, 1 cc. of which equals 0.001 mg.
of nitrate nitrogen.

4. Standard silver sulfate solution. Dissolve 4.4 grams of silver
sulfate free from nitrate in 1 liter of water. One cc. of this solution
is equal to 1 mg. of chloride.

_Procedure._—The alkalinity, chloride, and nitrite content, and color of
the sample must first be determined. If the sample is highly colored
decolorize it with freshly precipitated aluminium hydroxide. Measure
into an evaporating dish 100 cc. of the sample, or if nitrate is very
high such volume as will contain about 0.01 mg. of nitrate nitrogen. Add
sufficient N/50 sulfuric acid nearly to neutralize the alkalinity. Then
add sufficient standard silver sulfate to precipitate all but about 0.1
mg. of chloride. The removal of chloride may be omitted if the sample
contains less than 30 parts per million of chloride. Heat the mixture to
boiling, add a little aluminium hydroxide, stir, filter, and wash with
small amounts of hot water. Evaporate the filtrate to dryness, and add 2
cc. of the phenoldisulfonic acid, rubbing with a glass rod to insure
intimate contact. If the residue becomes packed or appears vitreous
because of the presence of much iron, heat the dish on the water bath
for a few minutes. Dilute the mixture with distilled water, and add
slowly a strong solution of potassium hydroxide or ammonium hydroxide
until the maximum color is developed. Transfer the solution to a Nessler
tube, filtering if necessary. If nitrate is present a yellow color will
be formed. Compare the color with that of standards[52][55] made by
adding 2 cc. of strong potassium hydroxide or ammonium hydroxide to
various amounts of standard nitrate solution and diluting them to 50 cc.
in Nessler tubes. The following amounts of standard nitrate solution are
suggested: 0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, 20.0, and
40.0 cc. These standards may be kept several weeks without
deterioration. If 100 cc. of water is used the number of cubic
centimeters of the standard multiplied by 0.01 is equal to parts per
million of nitrate nitrogen.

Standards that will remain permanent for several years if stored in the
dark may be prepared from tripotassium nitrophenoldisulfonate.[5]

If nitrite nitrogen is present in excess of 1 part per million it should
be oxidized by heating the samples a few minutes with a few drops of
hydrogen peroxide free from nitrate repeatedly added[95] or by adding
dilute potassium permanganate in the cold until a faint pink coloration
appears; the nitrogen equivalent of the nitrite thus oxidized to nitrate
is then subtracted from the final nitrate nitrogen reading.


                        REDUCTION METHOD.[2][46]

_Reagents._—1. Sodium or potassium hydroxide solution. Dissolve 250
grams of the hydroxide in 1.25 liters of distilled water. Add several
strips of aluminium foil and allow the evolution of hydrogen to continue
over night. Concentrate the solution to 1 liter by boiling.

2. Aluminium foil. Use strips of pure aluminium about 10 cm. long, 6 mm.
wide, and 0.33 mm. thick and weighing about 0.5 gram.

_Procedure._—To 100 cc. of the sample in a 300 cc. casserole add 2 cc.
of the hydroxide solution and concentrate by boiling to about 20 cc.
Pour the contents of the casserole into a test tube about 16 cm. long
and 3 cm. in diameter, or of approximately 100 cc. capacity. Rinse the
casserole several times with nitrogen-free water and add the rinse water
to the liquid already in the tube, thus making the contents of the tube
approximately 75 cc. Add a strip of aluminium foil. Close the tube by
means of a rubber stopper through which passes a bent glass tube about 5
mm. in diameter. Put the shorter arm of the tube flush with the lower
side of the rubber stopper and let the longer arm extend below the
surface of distilled water in another test tube. This apparatus serves
as a trap through which the evolved hydrogen escapes freely. The small
amount of ammonia escaping into the trap may be neglected. Allow the
action to proceed for a minimum period of four hours or over night. Pour
the contents of the tube into a distilling flask, dilute with 250 cc. of
ammonia-free water, distill, collect the distillate in Nessler tubes,
and Nesslerize. If the nitrate content is high collect the distillate in
a 200 cc. flask and Nesslerize an aliquot part. If the supernatant
liquid in the reduction tube is clear and colorless the solution may be
diluted to a definite volume and an aliquot part Nesslerized without
distillation.


                          TOTAL NITROGEN.[93]

In sewage work it is frequently of assistance to know the total nitrogen
content. This is ordinarily computed by adding together the organic,
ammonia, nitrite, and nitrate nitrogen, each of which is determined as
already described.


            OXYGEN CONSUMED.[24][67][84a][85][94f][101][102]

Oxygen consumed means the oxygen that the oxidizable compounds of sewage
and water consume when treated in an acid solution with potassium
permanganate. The expression is synonymous with oxygen required, oxygen
absorbed, and oxygen-consuming capacity. It should not be confused with
biochemical oxygen demand.

As the carbon, not the nitrogen, in organic matter is oxidized by
potassium permanganate, oxygen consumed is considered by some an
indication of the amount of carbonaceous organic matter present. The
determination indicates, however, only part of the carbon, the
proportion varying in different samples because the carbon in
nitrogenous matter is not so readily oxidized as that in carbonaceous
organic matter. Furthermore, it does not directly differentiate the
carbon present in unstable organic matter from that in fairly stable
organic matter, such as is sometimes referred to as residual humus
matter. As nitrite nitrogen, ferrous iron, sulfide, and other oxidizable
mineral substances reduce potassium permanganate, corrections for them
should be made in the determination.


                          RECOMMENDED METHOD.

_Reagents._—1. Dilute sulfuric acid. Dilute 1 part of concentrated
sulfuric acid with 3 parts of distilled water and free the solution from
oxidizable matter by adding potassium permanganate until a faint pink
color persists after the solution has stood several hours.

2. Standard ammonium oxalate. Dissolve 0.888 gram of the pure salt in 1
liter of distilled water. One cc. is equivalent to 0.1 mg. of oxygen. An
equivalent quantity of oxalic acid or sodium oxalate may be used.

3. Standard potassium permanganate. Dissolve 0.4 gram of the
crystallized salt in 1 liter of distilled water. Add 10 cc. of the
dilute sulfuric acid and 10 cc. of this solution of potassium
permanganate to 100 cc. of distilled water, and digest 30 minutes. Add
10 cc. of the ammonium oxalate solution, and then add potassium
permanganate till a pink coloration appears. This destroys the
oxygen-consuming capacity of the water used. Now add another 10 cc. of
ammonium oxalate solution and titrate with potassium permanganate.
Adjust the potassium permanganate solution so that 1 cc. is equivalent
to 1 cc. of ammonium oxalate solution or 0.1 mg. of available oxygen.

_Acid digestion._—Place in a flask 100 cc. of the water, or, if the
water is of high organic content, a smaller portion diluted to 100 cc.
Add 10 cc. of sulfuric acid solution and 10 cc. of standard potassium
permanganate and digest the liquid exactly 30 minutes in a bath of
boiling water the level of which is kept above the level of the contents
of the flask.[70][71a] If the quantity of permanganate is insufficient
for complete oxidation repeat the digestion with a larger quantity; at
least 5 cc. excess of the standard permanganate should be present when
the ammonium oxalate solution is added. Remove the flask, add 10 cc. of
the ammonium oxalate solution, and titrate with the standard
permanganate until a faint but distinct color is obtained. If 100 cc. of
water is used the number of cubic centimeters of potassium permanganate
solution in excess of the number of cubic centimeters of ammonium
oxalate solution is equal to parts per million of oxygen consumed.

If oxidizable mineral substances, such as ferrous iron, sulfide, or
nitrite, are present in the sample corrections should be applied as
accurately as possible by suitable procedures. Direct titration of the
acidified sample in the cold, using a three-minute period of digestion,
serves this purpose quite well for polluted surface waters and fairly
well for purified sewage effluents. Few raw sewages containing no trade
wastes need such a correction, but raw sewages containing “pickling”
liquors do need it. If the sample contains both oxidizable mineral
compounds and gaseous organic substances the latter should be driven off
by heat and the sample allowed to cool before applying this test for the
correction factor. If such corrections are made the fact should be
stated with the amount of correction.

_Period and temperature of digestion._—As the practice in regard to the
period and temperature of digestion has varied widely it is difficult to
compare the results obtained at one laboratory with those obtained at
another. None of the methods gives absolute results. They are all
relative[26][29][57] at best. Digesting 30 minutes at the boiling
temperature is herein designated the recommended method. If samples are
analyzed by any other method the method should be noted, and,
representative results by the standard method should be placed on record
for purposes of comparison.


                             OTHER METHODS.

_Additional reagents._—1. Potassium iodide solution. Ten per cent
solution, free from iodate.

2. Standard sodium thiosulfate. Dissolve 1.0 gram of the pure
crystallized salt in 1 liter of distilled water. Standardize this
solution against the standard potassium permanganate. As the thiosulfate
solution does not keep well determine its actual strength at frequent
intervals.

3. Starch indicator. Prepare as directed in the section on dissolved
oxygen (pp. 65–66).

4. Sodium hydroxide solution. Dissolve 1 part of pure sodium hydroxide
in 2 parts of distilled water.

Certain widely practiced deviations from the standard procedure just
described are noted in the following paragraphs.

1. Heat the acidified sample to boiling, add the permanganate solution,
and digest for two minutes[16] at boiling temperature. This procedure is
facilitated by agitating the liquid constantly with a small current of
air to guard against bumping.

2. Same method as No. 1 except that the period of digestion is five
minutes.[121a]

3. Same method as No. 2 except that the permanganate solution is added
to the acidified sample when cold, and digestion is continued five
minutes after the sample reaches the boiling point. The advantage of
this method is that there is included the oxygen-consuming power of the
volatile matter present in some sewages and sewage effluents, which is
driven off by heat and thus escapes when the test is made in accordance
with procedures 1 and 2.

4. Same method as No. 3 except that the period of digestion is 10
minutes.[63][68c]

5. Digestion of the sample after the acid and permanganate solutions are
added is carried out abroad, especially in England, at approximately the
room temperature,[24a][69a][94f][100a] apparently to guard against
decomposition[17] of permanganate in the presence of high chloride, for
periods of three minutes, fifteen minutes, and four hours; many
observers record the oxygen consumed after all three periods, while some
record the result only for the four-hour period. At the end of the
period of digestion, add 0.5 cc. of potassium iodide solution to
discharge the pink color; mix; titrate the liberated iodine with
thiosulfate until the yellow color is nearly destroyed, then add a few
drops of starch solution and continue titration until the blue color is
just discharged. The number of cubic centimeters of potassium
permanganate solution in excess of the number of cubic centimeters of
sodium thiosulfate solution is equal to parts per million of oxygen
consumed.

6. Digestion in alkaline solution[104] is preferable to digestion in
acid solution for brines or waters high in chlorine. Place in a flask
100 cc. of the sample, or if it is of high organic content a smaller
portion diluted to 100 cc. Add 0.5 cc. of sodium hydroxide solution and
10 cc. of standard potassium permanganate and digest exactly 30 minutes.
Remove the flask, add 5 cc. of sulfuric acid and 10 cc. of the standard
ammonium oxalate, and titrate with the standard potassium permanganate
as in the acid digestion.


                        RESIDUE ON EVAPORATION.


                           TOTAL RESIDUE.[16]

Ignite and weigh a clean platinum dish, and measure into it 100 cc. of
the thoroughly shaken sample. Evaporate to dryness on a water bath. Then
heat the dish in an oven at 103° C. or 180° C. for one hour. Cool in a
desiccator and weigh. The temperature of drying should be mentioned in
the report. The increase in weight gives the total solids or residue on
evaporation. If 100 cc. of the sample was taken this weight expressed in
milligrams and multiplied by 10 is equal to parts per million of residue
on evaporation. The residue from waters low in organic matter but
relatively high in iron may be used, as a matter of convenience, for the
determination of iron.


              FIXED RESIDUE AND LOSS ON IGNITION.[13][96]

The residue from sewages and waters high in organic matter may be
ignited to burn off the organic matter, which, with some volatile
inorganic matter, constitutes the loss on ignition.

_Procedure._—Ignite the residue in the platinum dish at a low red heat.
If great accuracy is desired this should be done in an electric muffle
furnace or in a radiator, which consists of a platinum or a nickel dish
large enough to allow an air space of about half an inch between it and
the dish within it, the inner dish being supported by a triangle of
platinum wire laid on the bottom of the outer dish. A disc of platinum
or nickel foil large enough to cover the outer dish is suspended over
the inner dish to radiate the heat into it. The larger dish is heated to
bright redness until the residue is white or nearly so. Allow the dish
to cool, and moisten the residue with a few drops of distilled water.
Dry the residue in the oven, cool in a desiccator, and weigh. The fixed
residue on evaporation is the difference between this weight and the
weight of the dish.

The loss on ignition is the difference between the total residue on
evaporation and the fixed residue on evaporation.

If the odor and color on ignition of some residues give helpful clues to
the character of the organic matter record them.


                       SUSPENDED MATTER.[56][110]


                   DETERMINATION WITH GOOCH CRUCIBLE.

_Reagent._—Prepare a dilute cream of asbestos fibre which has been
finely shredded, thoroughly ignited, treated with strong hydrochloric
acid for at least 12 hours, and washed with distilled water till free
from acid.

_Procedure._—1. Prepare a mat of the asbestos fibre 1/16 inch thick in a
Gooch crucible. Dry it in an oven at 103 or 180° C., cool and weigh.
Filter 1,000 cc. of samples having a turbidity of 50 parts per million
or less. If the turbidity is higher use sufficient water to obtain 50 to
100 mg. of suspended matter. Dry for one hour at 103 or 180° C., cool
and weigh. Report the temperature at which the residue was dried. If
1,000 cc. is filtered the increase in weight expressed in milligrams is
equal to parts per million of suspended matter.


                      DETERMINATION BY FILTRATION.

The difference between the total solids in filtered and unfiltered
portions of a sample may be used as a basis for calculating suspended
matter.


                        DETERMINATION OF VOLUME.

The determination of the volume[9][69b] of suspended matter in sewages
has received considerable attention abroad. Imhoff recommends the use of
conical glass vessels holding 1 liter with the lower portions graduated
in cubic centimeters. Others recommend centrifuges with sediment tubes.


                  FIXED RESIDUE AND LOSS ON IGNITION.

Treat the total residue from a filtered sample in the same manner as
described for the total residue, and obtain the loss on ignition due to
dissolved matter, and by difference the loss on ignition due to
suspended matter.


                             HARDNESS.[94e]

A water containing certain mineral constituents in solution, chiefly
calcium and magnesium, which form insoluble compounds with soap, is said
to be hard. Carbon dioxide in water increases the solubility of calcium
and magnesium carbonates, forming bicarbonate. If carbon dioxide is
removed from the water by boiling the bicarbonate is decomposed and
calcium and magnesium are partly precipitated. The proportion of calcium
or magnesium carbonate that a water can hold in solution depends on the
concentration of carbon dioxide, which in turn depends on the
temperature of the water and the proportion of carbon dioxide in the
atmosphere with which the water has been in contact. Consequently, when
the carbon dioxide is removed from the water by boiling or otherwise the
carbonates of calcium and magnesium are partly, but not completely,
precipitated, and the hardness of the water is thus diminished and the
water is softened to the extent to which these substances are
precipitated. The hardness thus removed is called temporary hardness.
The hardness which still remains after boiling is due mainly to calcium
and magnesium in equilibrium with sulfate, chloride, and nitrate, and
residual carbonate, and it is called permanent hardness. Non-carbonate
hardness is the hardness caused by sulfates, chlorides, and nitrates of
calcium, magnesium, iron, and other metals that form insoluble soaps.


                     TOTAL HARDNESS BY CALCULATION.

The most accurate method of ascertaining total hardness is to compute it
from the results of determinations of calcium and magnesium in the
sample. (See methods, pp. 57–58.) Iron and other metals must be included
in the calculation if they are present in significant amounts. Total
hardness as CaCO_{3} equals 2.5 Ca plus 4.1 Mg.


                  TOTAL HARDNESS BY SOAP METHOD.[121b]

The determination of hardness by the soap method roughly approximates
the amount of calcium and magnesium in a water, though it actually
measures the soap-consuming power of the water.

_Reagents._—1. Standard calcium chloride solution. Dissolve 0.2 gram of
pure calcite (calcium carbonate) in a little dilute hydrochloric acid,
being careful to avoid loss of solution by spattering. Evaporate the
solution to dryness several times with distilled water to expel excess
of acid. Dissolve the residue in distilled water and dilute the solution
to 1 liter. One cc. of this dilution is equivalent to 0.2 mg. of calcium
carbonate.

2. Standard soap solution. Dissolve 100 grams of dry white Castile soap
in 1 liter of 80 per cent alcohol, and allow this solution to stand
several days before standardizing. Pure potassium oleate made from lead
plaster and potassium carbonate may be used in place of Castile soap.

_First method of standardization._—Dilute 20 cc. of the calcium chloride
solution in a 250 cc. glass-stoppered bottle to 50 cc. with distilled
water which has been recently boiled and cooled. Add soap solution from
a burette, 0.2 or 0.3 cc. at a time, shaking the bottle vigorously after
each addition until a lather remains unbroken for five minutes over the
entire surface of the water while the bottle lies on its side. Then
adjust the strength of the stock solution with 70 per cent alcohol so
that the resulting diluted soap solution will give a permanent lather
when 6.40 cc. of it is properly added to 20 cc. of standard calcium
chloride solution diluted to 50 cc. Usually 75 to 100 cc. of the stock
soap solution is required to make 1 liter of the standard soap solution.
The quantity of calcium carbonate equivalent to each cubic centimeter of
the standard soap solution consumed in the titration is indicated in
Table 6.

 Table 6.—TOTAL HARDNESS IN PARTS PER MILLION OF CaCO_{3} FOR EACH TENTH
   OF A CUBIC CENTIMETER OF SOAP SOLUTION WHEN 50 CC. OF THE SAMPLE IS
                                TITRATED.

 ───────────┬─────┬─────┬─────┬─────┬─────┬─────┬─────┬─────┬─────┬─────
    Cubic   │     │     │     │     │     │     │     │     │     │
 centimeters│ 0.0.│ 0.1.│ 0.2.│ 0.3.│ 0.4.│ 0.5.│ 0.6.│ 0.7.│ 0.8.│ 0.9.
   of soap  │     │     │     │     │     │     │     │     │     │
  solution. │     │     │     │     │     │     │     │     │     │
 ───────────┼─────┼─────┼─────┼─────┼─────┼─────┼─────┼─────┼─────┼─────
         0.0│     │     │     │     │     │     │     │  0.0│  1.6│  3.2
         1.0│  4.8│  6.3│  7.9│  9.5│ 11.1│ 12.7│ 14.3│ 15.6│ 16.9│ 18.2
         2.0│ 19.5│ 20.8│ 22.1│ 23.4│ 24.7│ 26.0│ 27.3│ 28.6│ 29.9│ 31.2
            │     │     │     │     │     │     │     │     │     │
         3.0│ 32.5│ 33.8│ 35.1│ 36.4│ 37.7│ 38.0│ 40.3│ 41.6│ 42.9│ 44.3
         4.0│ 45.7│ 47.1│ 48.6│ 50.0│ 51.4│ 52.9│ 54.3│ 55.7│ 57.1│ 58.6
         5.0│ 60.0│ 61.4│ 62.9│ 64.3│ 65.7│ 67.1│ 68.6│ 70.0│ 71.4│ 72.9
            │     │     │     │     │     │     │     │     │     │
         6.0│ 74.3│ 75.7│ 77.1│ 78.6│ 80.0│ 81.4│ 82.9│ 84.3│ 85.7│ 87.1
         7.0│ 88.6│ 90.0│ 91.4│ 92.9│ 94.3│ 95.7│ 97.1│ 98.6│100.0│101.5
 ───────────┴─────┴─────┴─────┴─────┴─────┴─────┴─────┴─────┴─────┴─────

This table does not provide for the use of so large volume of soap
solution for a single determination as former ones because the end-point
becomes somewhat obscured in the presence of magnesium if more than 7
cc. is used.

_Second method of standardization._—Dilute 100 cc. of the stock soap
solution to 1 liter with 70 per cent alcohol. This dilute solution
should be of such strength that approximately 6.4 cc. of it will give a
permanent lather when 20 cc. of standard calcium chloride solution
diluted to 50 cc. with distilled water is titrated with it. Determine
the amount of soap solution required to give a permanent lather with 50
cc. of distilled water and with 5, 10, 15, and 20 cc. of standard
calcium chloride solution diluted to 50 cc. with distilled water.
Finally plot on cross-section paper a curve showing the relation of
various quantities of soap solution to corresponding quantities of
standard calcium carbonate solution and therefore to parts per million
of hardness.

_Procedure._—Measure 50 cc. of the water into a 250 cc. bottle and add
to it soap solution in small quantities in precisely the same manner as
described under the standardization of the soap solution. From the
number of cubic centimeters of soap solution used obtain from Table 6 or
from the plotted curve the total hardness of the water in parts per
million of calcium carbonate.

To avoid mistaking the false or magnesium end-point for the true one[35]
when adding the soap solution to waters containing magnesium salts, read
the burette after the titration is apparently finished, and add about
0.5 cc. more of soap solution. If the end-point was due to magnesium the
lather will disappear. Soap solution must then be added until the true
end-point is reached. Usually the false lather persists for less than
five minutes.

If more than 7 cc. of soap solution is required for 50 cc. of the water
take less of the sample and dilute it to 50 cc. with distilled water
which has been recently boiled and cooled. This step reduces somewhat
the disturbing influence of magnesium,[107a] which consumes more soap
than an equivalent weight of calcium.

At best the soap method is not a precise test on account of the
different relative amounts of calcium and magnesium in different waters.
For hard waters, especially in connection with processes for
purification and softening, it is advised that this method be not
exclusively used. If the same water is frequently analyzed it may be of
assistance to standardize the soap solution against a mixture of calcium
and magnesium salts, the relative proportions of which approximate those
found in the water.

The strength of the soap solution should be determined from time to
time, to make sure that it has not materially changed. Record all
results in parts per million of calcium carbonate.

One English degree of hardness, Clark’s scale, is equivalent to 1 grain
per Imperial gallon of calcium carbonate. One French degree of hardness
is equivalent to 1 part per 100,000 of calcium carbonate. One German
degree of hardness is equivalent to 1 part per 100,000 of calcium oxide,
and multiplied by 17.9 gives parts per million of calcium carbonate. The
relations of these various scales are indicated in Table 7.

                 Table 7.—CONVERSION TABLE FOR HARDNESS.

 ───────────────────────────┬───────────────────────────────────────────
            Unit.           │                Equivalent.
 ───────────────────────────┼──────────┬──────────┬──────────┬──────────
                            │Parts per │  Clark   │  French  │  German
                            │ million. │ degrees. │ degrees. │ degrees.
 ───────────────────────────┼──────────┼──────────┼──────────┼──────────
 One part per million       │      1.00│      0.07│      0.10│     0.056
 One Clark degree           │      14.3│      1.00│      1.43│       .80
 One French degree          │      10.0│       .70│      1.00│       .56
 One German degree          │      17.9│      1.24│      1.78│      1.00
 ───────────────────────────┴──────────┴──────────┴──────────┴──────────


        TOTAL HARDNESS BY SODA REAGENT METHOD.[47][74][81][94d]

Add standard sulfuric acid to 200 cc. of the sample until the alkalinity
is neutralized. (See Procedure with methyl orange, p. 37.) Then apply
the non-carbonate hardness method (pp. 34–35). This method gives fairly
satisfactory estimates of total hardness of hard waters.


               TEMPORARY HARDNESS BY TITRATION WITH ACID.

Determine the alkalinity in presence of methyl orange (see p. 37) in the
original sample and also in the sample after boiling, cooling, restoring
to the original volume with boiled distilled water, and filtering. The
difference between the two, if any, is the temporary hardness. This is
the most accurate method of determining the temporary hardness of
ordinary waters. Iron bicarbonate is included as a part of the temporary
hardness.


    NON-CARBONATE HARDNESS BY SODA REAGENT METHOD.[47][74][81][94d]

The use of soda reagent does not avoid entirely the error due to
solubility of the salts of calcium and magnesium; consequently, if much
depends on the results, as in water softening, gravimetric
determinations of the calcium and magnesium that remain in solution
should be made and a correction should be applied for those amounts.

_Reagent._—Prepare soda reagent from equal parts of sodium hydroxide and
sodium carbonate. It should be approximately tenth normal.

_Procedure._—Measure 200 cc. of the sample and 200 cc. of distilled
water into 500 cc. Jena or similar glass Erlenmeyer flasks. Treat the
contents of each flask in the following manner. Boil 15 minutes to expel
free carbon dioxide. Add 25 cc. of soda reagent. Boil 10 minutes, cool,
rinse into 200 cc. graduated flasks, and dilute to 200 cc. with boiled
distilled water. Filter, rejecting the first 50 cc., and titrate 50 cc.
of each filtrate with N/50 sulfuric acid in the presence of methyl
orange or erythrosine indicator. The non-carbonate hardness in parts per
million of calcium carbonate is equal to 20 times the difference between
the number of cubic centimeters of sulfuric acid required for the soda
reagent in distilled water and the number of cubic centimeters of N/50
sulfuric acid required for the soda reagent in the sample.

Water naturally containing bicarbonate and carbonate in excess of
calcium and magnesium requires a larger amount of acid to neutralize the
sample after it has been treated than is required to neutralize the
volume of soda reagent originally added. (See p. 39.)


                 NON-CARBONATE HARDNESS BY SOAP METHOD.

Non-carbonate hardness may be calculated for waters which are soft or
moderately hard in a fairly satisfactory manner by deducting the total
alkalinity from the total hardness by the soap method (pp. 31–34). For
waters that are very hard, and particularly those that contain much
magnesium, this method is not advised.


                      ALKALINITY.[11][18][47][97]

The alkalinity of a natural water represents its content of carbonate,
bicarbonate, borate, silicate, phosphate, and hydroxide. Alkalinity is
determined by neutralization with standard sulfuric acid or potassium
bisulfate in the presence of phenolphthalein and either methyl orange,
erythrosine, or lacmoid as indicators. Methyl orange may be used except
in waters containing aluminium sulfate or iron sulfate. The relations
between estimates in presence of these indicators and the carbonate,
bicarbonate, and hydroxide radicles are indicated in Table 8. The
alkalinity of carbonates in the presence of phenolphthalein is different
from that in the presence of methyl orange, partly because of loss of
carbon dioxide and partly because of defects in phenolphthalein as an
indicator in such conditions.

  Table 8.—RELATIONS BETWEEN ALKALINITY TO PHENOLPHTHALEIN AND THAT TO
   METHYL ORANGE, ERYTHROSINE, OR LACMOID, IN PRESENCE OF BICARBONATE,
                        CARBONATE, AND HYDROXIDE.

 ─────────────────┬─────────────────────────────────────────────────────
     Result of    │   Value of radicle expressed in terms of calcium
   titration.[C]  │                     carbonate.
 ─────────────────┼─────────────────┬─────────────────┬─────────────────
                  │  Bicarbonate.   │   Carbonate.    │   Hydroxide.
 ─────────────────┼─────────────────┼─────────────────┼─────────────────
 P = 0            │        T        │        0        │        0
 P < 1/2T         │     T − 2P      │       2P        │        0
 P = 1/2T         │        0        │       2P        │        0
 P > 1/2T         │        0        │    2(T − P)     │     2P − T
 P = T            │        0        │        0        │        T
 ─────────────────┴─────────────────┴─────────────────┴─────────────────

Footnote C:

  T = Total alkalinity in presence of methyl orange, erythrosine, or
  lacmoid. P = Alkalinity in presence of phenolphthalein.

_Reagents._—1. Sulfuric acid or potassium bisulfate. A N/50 solution.

2. Phenolphthalein indicator. Dissolve 5 grams of a good quality of
phenolphthalein in 1 liter of 50 per cent alcohol. Neutralize with N/10
potassium hydroxide. The alcohol should be diluted with boiled distilled
water.

3. Methyl orange indicator. Dissolve 0.5 gram of a good grade of methyl
orange in 1 liter of distilled water. Keep the solution in the dark.

4. Lacmoid indicator. Dissolve 2.0 grams of lacmoid in 1 liter of 50 per
cent alcohol. Dilute the alcohol with freshly boiled distilled water.

5. Erythrosine indicator. Dissolve 0.5 gram of erythrosine (the sodium
salt) in 1 liter of freshly boiled distilled water.


                    PROCEDURE WITH PHENOLPHTHALEIN.

Add 4 drops of phenolphthalein indicator to 50 or 100 cc. of the sample
in a white porcelain casserole or an Erlenmeyer flask over a white
surface. If the solution becomes colored, hydroxide or normal carbonate
is present. Add N/50 sulfuric acid from a burette until the coloration
disappears.

The phenolphthalein alkalinity in parts per million of calcium carbonate
is equal to the number of cubic centimeters of N/50 sulfuric acid used
multiplied by 20 if 50 cc. of the sample was used, or by 10 if 100 cc.
was used.


                     PROCEDURE WITH METHYL ORANGE.

Add 2 drops of methyl orange indicator to 50 or 100 cc. of the sample,
or to the solution to which phenolphthalein has been added, in a white
porcelain casserole or an Erlenmeyer flask over a white surface. If the
solution becomes yellow, hydroxide, normal carbonate, or bicarbonate is
present. Add N/50 sulfuric acid from a burette until the faintest pink
coloration appears. The methyl orange alkalinity in parts per million of
calcium carbonate is equal to the total number of cubic centimeters of
N/50 sulfuric acid used multiplied by 20 if 50 cc. of the sample was
used, or by 10 if 100 cc. was used.


                        PROCEDURE WITH LACMOID.

Add 4 drops of lacmoid indicator to 50 or 100 cc. of the sample in a
porcelain casserole or an Erlenmeyer flask. Add N/50 sulfuric acid from
a burette until within 1 or 2 cc. of the amount necessary for
neutralization has been added. Heat the solution until bubbles of steam
begin to break at the surface. Remove the dish from the source of heat
and continue the titration until a drop of the acid striking the surface
of the liquid and sinking to the bottom of the vessel produces no change
in the uniform reddish or purple color of the solution. The calculation
is the same as for phenolphthalein alkalinity.


                      PROCEDURE WITH ERYTHROSINE.

Add 5 cc. of neutral chloroform and 1 cc. of erythrosine indicator to 50
or 100 cc. of the sample in a 250 cc. clear glass-stoppered bottle. If
the chloroform becomes rose colored on shaking, hydroxide, bicarbonate,
or normal carbonate is present. Add N/50 sulfuric acid from a burette
until the chloroform becomes colorless. A white surface behind the
bottle facilitates detection of a trace of color as the end-point is
approached. The calculation is the same as with phenolphthalein
alkalinity.


                              BICARBONATE.

Bicarbonate is present if the alkalinity to phenolphthalein is less than
one-half the alkalinity to methyl orange, erythrosine, or lacmoid. The
alkalinity to methyl orange, erythrosine, or lacmoid is due entirely to
bicarbonate if there is no phenolphthalein alkalinity. If there is
phenolphthalein alkalinity the bicarbonate, in terms of calcium
carbonate, is equal to the methyl orange, erythrosine, or lacmoid
alkalinity minus twice the phenolphthalein alkalinity. Bicarbonate,
carbon dioxide as bicarbonate, and half-bound carbon dioxide can be
calculated as follows:

Bicarbonate (HCO_{3}) = 1.22 times the bicarbonate expressed in terms of
calcium carbonate.

Carbon dioxide (CO_{2}) as bicarbonate = 0.88 times the bicarbonate
expressed in terms of calcium carbonate.

Half-bound carbon dioxide (CO_{2}) = 0.44 times the bicarbonate
expressed in terms of calcium carbonate.


                       NORMAL CARBONATE.[20][94]

Normal carbonate is present if the alkalinity to phenolphthalein is
greater than zero but less than the alkalinity to methyl orange,
erythrosine, or lacmoid. If the phenolphthalein alkalinity is exactly
equal to one-half the methyl orange, erythrosine, or lacmoid alkalinity
the alkalinity is due entirely to normal carbonate. If the
phenolphthalein alkalinity is less than one-half the methyl orange,
erythrosine, or lacmoid alkalinity normal carbonate expressed in terms
of calcium carbonate is equal to twice the phenolphthalein alkalinity.
If the phenolphthalein alkalinity is greater than one-half the methyl
orange, erythrosine, or lacmoid alkalinity the normal carbonate is equal
to twice the difference between the methyl orange, erythrosine, or
lacmoid alkalinity and the phenolphthalein alkalinity. The carbonate,
carbon dioxide as carbonate, and bound carbon dioxide can be calculated
as follows:

Carbonate (CO_{3}) = 0.6 times the normal carbonate expressed in terms
of calcium carbonate.

Carbon dioxide as carbonate (CO_{2}) = 0.44 times the normal carbonate
expressed in terms of calcium carbonate.

Bound carbon dioxide (CO_{2}) is the sum of the carbon dioxide as
carbonate and one-half that as bicarbonate.


                           HYDROXIDE.[20][94]

If hydroxide, or caustic alkalinity, is present the alkalinity to
phenolphthalein is greater than one-half the alkalinity to methyl
orange, erythrosine, or lacmoid; the alkalinity is due entirely to
hydroxide if the phenolphthalein alkalinity is equal to the methyl
orange, erythrosine, or lacmoid alkalinity. If the phenolphthalein
alkalinity is more than half and less than all the methyl orange,
erythrosine, or lacmoid alkalinity, hydroxide, expressed in terms of
calcium carbonate, is equal to twice the phenolphthalein alkalinity
minus the methyl orange, erythrosine, or lacmoid alkalinity.


                           ALKALI CARBONATES.

Waters which contain sodium or potassium carbonates or bicarbonates
contain all of their calcium and magnesium as carbonates or
bicarbonates. That is, they possess no non-carbonate hardness (sulfates,
nitrates or chlorides of calcium and magnesium).

The most accurate method is to determine the total alkalinity by
titration with N/50 sulfuric acid, using methyl orange, erythrosine, or
lacmoid as an indicator; then determine the calcium and magnesium
content; and subtract from the total alkalinity the computed alkalinity
due to the calcium and magnesium expressed in terms of calcium
carbonate. The remainder is the alkalinity due to carbonates and
bicarbonates of sodium and potassium.

This determination may also be made by applying the method, for
non-carbonate hardness with soda reagent (see p. 35), and by noting the
excess of acid required to neutralize the alkaline carbonates originally
present.

With present information as to solubilities of the normal carbonates of
calcium and magnesium, it is difficult in their presence to measure
slight quantities of carbonates of sodium or potassium.


                           ACIDITY.[24d][37]

Waters may have an acid reaction because of the presence of free carbon
dioxide, mineral acids, or some of their salts, especially those of iron
and aluminium.

_Reagents._—1. N/50 sodium carbonate. Dissolve 1.06 grams of anhydrous
sodium carbonate in 1 liter of boiled distilled water that has been
cooled in an atmosphere free from carbon dioxide. Preserve this solution
in bottles of resistant glass protected from the air by tubes filled
with soda-lime. One cc. is equivalent to 1 mg. of CaCO_{3}.

2. N/22 sodium carbonate. Dissolve 2.41 grams of anhydrous sodium
carbonate in 1 liter of boiled distilled water that has been cooled in
an atmosphere free from carbon dioxide. Preserve this solution in
bottles of resistant glass protected from the air by tubes filled with
soda-lime. One cc. is equivalent to 1 mg. of CO_{2}.

3. Phenolphthalein indicator (see p. 36).

4. Methyl orange indicator (see p. 36).


                             TOTAL ACIDITY.

_Procedure._—Add 4 drops of phenolphthalein indicator to 50 or 100 cc.
of the sample in a white porcelain casserole or an Erlenmeyer flask over
a white surface. Add N/50 sodium carbonate until the solution turns
pink. The total acidity in parts per million of calcium carbonate is
equal to the number of cubic centimeters of N/50 sodium carbonate used
multiplied by 20 if 50 cc. of the sample was used, or by 10 if 100 cc.
was used.


           FREE CARBON DIOXIDE.[20][23][61][87][88][94a][118]

Carbon dioxide may exist in water in three forms—free carbon dioxide,
bicarbonate (pp. 37–38), and carbonate (p. 38). One-half the carbon
dioxide as bicarbonate is known as the half-bound carbon dioxide. The
carbon dioxide as carbonate plus one-half that as bicarbonate is known
as the bound carbon dioxide.

_Procedure._—Pour 100 cc. of the sample into a tall narrow vessel,
preferably a 100 cc. Nessler tube. Add 10 drops of phenolphthalein
indicator, and titrate rapidly with N/22 sodium carbonate, stirring
gently, until a faint but permanent pink color is produced. The free
carbon dioxide (CO_{2}) in parts per million is equal to 10 times the
number of cubic centimeters of N/22 sodium carbonate used.

Because of the ease with which free carbon dioxide escapes from water,
particularly when the gas is present in large amount, a special sample
should be collected for this determination, which should preferably be
made at the time of collection. If the analysis cannot be made at the
time of collection approximate results with water not too high in free
carbon dioxide may be obtained on samples collected in bottles
completely filled so as to leave no air space under the stopper. Bottled
samples should be kept, until tested, at a temperature lower than that
of the water when collected. If mineral acids or certain salts are
present correction must be made. At best, the results of the titration
are uncertain because the proper end-point for correct results differs
in color with different types of water.


                          FREE MINERAL ACIDS.

_Procedure._—Add 2 drops of methyl orange indicator to 50 or 100 cc. of
the sample in a white porcelain casserole or an Erlenmeyer flask over a
white surface. Add N/50 sodium carbonate from a burette until the pink
coloration of the solution disappears. The acidity due to free mineral
acids, expressed in terms of calcium carbonate, is equal to the number
of cubic centimeters of N/50 sodium carbonate used multiplied by 20 if
50 cc. of the sample was used, or by 10 if 100 cc. was used.


       MINERAL ACIDS AND SULFATES OF IRON AND ALUMINIUM.[24d][37]

_Procedure._—Modify the method for free mineral acids by titrating the
water at boiling temperature in the presence of phenolphthalein
indicator. The acidity due to free mineral acids and sulfates of iron
and aluminium, expressed in terms of calcium carbonate, is equal to the
number of cubic centimeters of N/50 sodium carbonate used multiplied by
20 if 50 cc. of the sample was used, or by 10 if 100 cc. was used.

The acidity due to sulfates of iron and aluminium is equal to the
acidity due to mineral acids and sulfates minus the acidity due to
mineral acids. The acidity due to ferrous and ferric sulfate can be
calculated from the determined amount of these salts (pp. 43–48). The
acidity due to aluminium sulfate is equal to the acidity due to total
acid sulfates minus that due to iron sulfates.

Acidity shall be reported in parts per million of calcium carbonate
(CaCO_{3}). Sulfate (SO_{4}) equals parts per million of calcium
carbonate multiplied by 0.96.

Carbon dioxide (CO_{2}) equals parts per million of calcium carbonate
multiplied by 0.44.


                             CHLORIDE.[16]

Chloride in water and sewage has its origin in common salt, from mineral
deposits in the earth, from ocean vapors carried inland by the wind, or
from polluting materials like sewage and trade wastes, which contain the
salt used in the household and in manufacturing. Comparison of the
chloride content of a water with that of other waters in the vicinity
known to be unpolluted frequently affords useful information as to its
sanitary quality. If, however, the chloride normally exceeds 20 parts
per million because of chloride-bearing mineral deposits the chloride
content of a water has little sanitary significance.

_Reagents._—1. Standard sodium chloride solution. Dissolve 16.48 grams
of pure fused sodium chloride in 1 liter of distilled water. Dilute 100
cc. of this stock solution to 1 liter in order to obtain a standard
solution each cubic centimeter of which contains 0.001 gram of chloride.

2. Standard silver nitrate solution. Dissolve about 2.40 grams of silver
nitrate crystals in 1 liter of distilled water. Standardize this with
the standard salt solution, and adjust, correcting for volume (see p.
43), so that 1 cc. will be exactly equivalent to 0.0005 gram of
chloride.

3. Potassium chromate indicator. Dissolve 50 grams of neutral potassium
chromate in a little distilled water. Add enough silver nitrate to
produce a slight red precipitate. Filter and dilute the filtrate to 1
liter with distilled water.

4. Aluminium hydroxide. Electrolyze ammonia-free water, using aluminium
electrodes. Wash the precipitate until it is free from chloride,
ammonia, and nitrite. Or dissolve 125 grams of potassium or ammonium
alum in 1 liter of distilled water. Precipitate the aluminium by adding
cautiously ammonium hydroxide. Wash the precipitate in a large jar by
successive additions and decantations of distilled water until free from
chloride, nitrite, and ammonia.

_Procedure._—Add 1 cc. of potassium chromate indicator to 50 cc. of the
sample in a 6–inch white porcelain evaporating dish or a 150 cc.
Erlenmeyer flask over a white surface. Titrate with the silver nitrate
solution under similar conditions of volume, light, and temperature as
were used in standardizing the silver nitrate until a faint reddish
coloration is perceptible. The detection of the end-point is facilitated
by comparison of the contents of the porcelain dish with those of
another dish containing the same quantity of potassium chromate
indicator in 50 cc. of distilled water. Some analysts prefer to make the
titration in a dark-room provided with a yellow light. The end-point is
very sharp by electric light and also by daylight with photographic
yellow glass. The titration may be made in Nessler tubes[68a] if the
solutions are standardized under similar conditions.

If the amount of chloride is very high use 25 cc., or even a smaller
quantity, dilute the volume taken to 50 cc. with distilled water. If the
amount of chloride is very low concentrate 250 cc. of the sample to 50
cc. by evaporation. Rotate the liquid to make sure that no residue
remains undissolved on the walls of the dish, and, if necessary, use a
rubber-tipped glass rod to assist in this operation.

Chloride is determined by some observers by extracting with hot
distilled water the residue in the platinum dish in the determination of
the residue on evaporation and proceeding as just described. This is
permissible if a little sodium carbonate is added before evaporation to
prevent loss of chloride through decomposition of magnesium chloride in
the residue.

If the sample has a color greater than 30 it should be decolorized by
shaking it thoroughly with washed aluminium hydroxide (3 cc. to 500 cc.
of the sample) and allowing the precipitate to settle. Make the
determination on a portion of the clarified sample, filtered if
necessary. If the sample is acid, neutralize it with sodium carbonate;
if hydroxide is present, add dilute sulfuric acid until the cold liquid
will just discharge the color of phenolphthalein. If the presence of
sulfide and sulfocyanate renders it necessary, make proper
corrections[24c][100b] or modifications in treatment.

Make correction for the error due to variations in the volume of the
liquid and precipitate by means of the formula[39] X = 0.003V + 0.02, in
which X = the correction in cubic centimeters of silver nitrate solution
and V = cubic centimeters of liquid at the end of the titration. If 50
cc. of the sample is titrated chloride (Cl) in parts per million is
equal to the number of cubic centimeters of silver nitrate solution
multiplied by 10. The correction to be applied is 0.2 cc. unless unusual
accuracy is required.


                             IRON.[94b][98]

Iron occurs in natural waters in both ferrous and ferric condition,
depending on the source of the sample. In ground waters the iron is
usually in an unoxidized and soluble condition, sometimes combined with
carbonic or sulfuric acid, and also in combination with organic matter.
Many waters, especially those that have been exposed to the air, contain
the iron in the form of a colloidal hydroxide. Silt-bearing waters often
contain much iron in suspension, usually in an oxidized form. Sewages
and sewage effluents, particularly those receiving manufacturing wastes,
contain various forms of iron of different degrees of solubility,
oxidation, and coagulation.


                          TOTAL IRON.[59][63b]


                          COLORIMETRIC METHOD.

_Reagents._—1. Standard iron solution. Dissolve 0.7 gram of crystallized
ferrous ammonium sulfate in 50 cc. of distilled water to which 20 cc. of
dilute sulfuric acid has been added. Warm the solution slightly and add
potassium permanganate until the iron is completely oxidized. Dilute the
solution to 1 liter. One cc. of the standard solution equals 0.1 mg. Fe.

2. Potassium sulfocyanide solution. Dissolve 20 grams of the salt in 1
liter of distilled water.

3. Dilute hydrochloric acid. One volume of acid (Sp. gr. 1.2) and one
volume of distilled water. This shall be free from nitric acid.

4. N/5 potassium permanganate. Dissolve 6.30 grams of the salt in
distilled water and dilute to 1 liter.

5. Hydrochloric acid. Concentrated, free from iron.

6. Nitric acid. Concentrated, free from iron.

7. Nitric acid. 5N, free from iron.

_First procedure._—Evaporate 100 cc. of the water to dryness, or use the
residue left after the determination of residue on evaporation (p. 29).
Ignite the residue at a low red heat taking care not to heat it hot
enough to make the iron difficultly soluble. Cool the dish and add 5 cc.
of concentrated hydrochloric acid. Moisten the inner surface of the
dish. Warm the solution for two or three minutes, and again moisten the
inner surface of the dish by permitting the hot acid to flow over it.
Wash the hot solution from the dish into a 50 cc. Nessler tube,
filtering if necessary through paper that has been washed with hot
water. Dilute to 50 cc., and add 3 drops of potassium permanganate
solution. Add 5 cc. of potassium sulfocyanide solution, mix, and compare
with standards.

If it is not convenient to use the residue on evaporation and if the
sample is relatively free from organic matter, boil 50 cc. of the sample
with 5 cc. of 5N nitric acid for five minutes. Add a few drops of
permanganate and 5 cc. of potassium sulfocyanide and compare with
standards, using nitric acid in place of hydrochloric acid in the
standards. This method is excellent for ground waters. The permanganate
and acid liberate chlorine in water high in chloride, and produce a
permanent yellow color which interferes with the determination, unless
the sample is first diluted to 50 cc. An excess of permanganate,
reacting with hydrochloric acid, causes similar trouble. The amounts of
hydrochloric acid, 5 cc., and of sulfocyanide, 5 cc., should be
approximately measured because more acid lightens the color whereas more
sulfocyanide deepens it. This is especially important if permanent
standards are used.

_Second procedure._—For surface waters containing small amounts of
organic matter, the method of Klut[59] is recommended. Samples
containing small amounts of iron should be concentrated, if possible,
until at least 0.5 mg. of iron is present in the volume tested. Boil the
sample in a beaker with 2 to 3 cc. of concentrated nitric acid free from
iron, adding permanganate if necessary to destroy the organic matter. To
the hot liquid add ammonia in slight excess and warm until the smell of
ammonia is hardly discernible. Filter and wash with water at 70° to 80°
C. containing a little ammonia. Dissolve the iron in the beaker and on
the filter paper in 5 cc. of concentrated hydrochloric acid, and wash
with hot water until the iron is all dissolved, collecting the filtrate
in a 50 cc. Nessler tube. Dilute to 50 cc. Add potassium sulfocyanide
and determine the iron by comparison with standards.


                    COMPARISON WITH IRON STANDARDS.

_First procedure._—Prepare standards containing amounts of standard iron
solution ranging from 0.05 to 4 cc. according to the quantity of iron in
the sample. Dilute these amounts with water to about 40 cc. Add 5 cc. of
dilute hydrochloric acid and 3 drops of potassium permanganate to each
tube and dilute to 50 cc. Add 5 cc. of the potassium sulfocyanide to
each of the standard solutions at the same time that it is added to the
samples of water under examination, and compare immediately after
mixing. If 100 cc. of the sample is used the iron in parts per million
is equal to the number of cubic centimeters of the standard iron
solution in the standard that the sample matches.

_Second procedure._—For a small number of determinations it is more
convenient to run the standard iron solution into a Nessler tube
containing the acid, distilled water, and potassium sulfocyanide until
the color matches that of the sample tested. When determining iron in
three or four samples the colors may be matched in the order of their
intensity and the volumes of standard iron solution required for each
tube may be read from the burette.


                  COMPARISON WITH PERMANENT STANDARDS.

_Reagents._—1. Platinum solution. Dissolve 2 grams of potassium platinic
chloride (PtCl_{4}.2KCl) in distilled water, add 100 cc. of concentrated
hydrochloric acid, and dilute to 1 liter with distilled water.

2. Cobalt solution. Dissolve 24 grams of dry cobaltous chloride crystals
(CoCl_{2}.6H_{2}O) in a small amount of distilled water, add 100 cc. of
strong hydrochloric acid, and dilute to 1 liter with distilled water.

_Procedure._—Prepare a series of permanent standards by diluting to 50
cc. with distilled water the amounts of platinum and cobalt solutions,
in 50 cc. Nessler tubes, indicated in Table 9. Compare the sample with
these standards, and calculate the parts per million of iron.

  Table 9.—PREPARATION OF PERMANENT STANDARDS FOR THE DETERMINATION OF
                                  IRON.

 ───────────────────────┬───────────────────────┬───────────────────────
 Value in standard iron │  Platinum solution.   │   Cobalt solution.
        solution.       │                       │
 ───────────────────────┼───────────────────────┼───────────────────────
          _cc._         │         _cc._         │         _cc._
                     0.0│                      0│                    0.0
                      .1│                      2│                    1.0
                      .3│                      6│                    3.0
                      .5│                     10│                    5.0
                      .7│                     14│                    7.5
                     1.0│                     20│                   11.0
                     1.5│                     28│                   17.0
                     2.0│                     35│                   24.0
                     2.5│                     39│                   32.0
                     3.0│                     39│                   43.0
                     3.5│                     40│                   55.0
 ───────────────────────┴───────────────────────┴───────────────────────


                        VOLUMETRIC METHOD.[24f]

Some samples of sewage and water mixed with trade wastes and mine
drainage contain so much iron that it is preferable to use the
volumetric method described on page 57 for the determination of both
total and dissolved iron, rather than to work with quantities small
enough to permit application of the colorimetric methods just described.
If iron is present in large quantities in suspension, as in some sewages
and septic tank effluents, it may be filtered off and the residue
washed, ignited, and fused with potassium and sodium carbonate. The
fusion is then extracted with hydrochloric acid and the iron determined
as on page 57.

Samples containing much organic matter should be evaporated to dryness
with 0.5 cc. of concentrated sulfuric acid and the residue then ignited
before estimation of iron.


                            DISSOLVED IRON.

Determine, by the method described for total iron, the iron in the
sample after filtration. Iron may precipitate from some samples during
filtration.


                            SUSPENDED IRON.

The suspended iron is the difference between total iron in the
unfiltered sample and dissolved iron in the filtered sample.


                           FERROUS IRON.[24e]

Determine the total ferrous iron in an unfiltered sample and the
dissolved ferrous iron in a filtered sample.

_Reagents._—1. Standard iron solution. Dissolve 0.7 gram of crystallized
ferrous ammonium sulfate in a large volume of freshly boiled distilled
water to which 10 cc. of dilute sulfuric acid has been added and dilute
to 1 liter. This solution should be freshly prepared when needed. One
cc. of this standard solution contains 0.1 mg. of Fe.

2. Potassium ferricyanide solution. Dissolve 5 grams of the salt in 1
liter of distilled water. Use a freshly prepared solution.

3. Dilute sulfuric acid. Dilute 1 part of sulfuric acid, specific
gravity 1.84, with 5 parts of distilled water.

_Procedure._—Add 10 cc. of dilute sulfuric acid to 50 cc. of the sample,
remove the suspended matter by filtration if necessary, and add 15 cc.
of potassium ferricyanide solution. Dilute the solution to 100 cc. with
distilled water. Compare the color developed in the sample with that in
standards made at the same time from the ferrous iron solution. Place in
100 cc. Nessler tubes, in the following order, 75 cc. of distilled
water, 10 cc. of dilute sulfuric acid, and 15 cc. of potassium
ferricyanide solution, and mix well the contents of each tube. Prepare
as many tubes in this way as are needed. Add various quantities of
standard ferrous iron solution to several tubes, mix well, and compare
the resulting colors with the samples _immediately_.


                              FERRIC IRON.

The amount of ferric iron in solution and suspension is equal to the
difference between the total iron and the ferrous iron obtained by the
methods described.


                               MANGANESE.

If the sample contains less than 10 parts per million of manganese, use
a colorimetric method in which the manganous salt is oxidized to
permanganate and the color produced thereby is compared with that of a
standard solution similarly treated. The persulfate method and the
bismuthate method are suitable. If the sample contains more than 10
parts per million of manganese it is sometimes preferable to use a
volumetric or gravimetric method.


                           PERSULFATE METHOD.

_Reagents._—1. Nitric acid. Dilute concentrated nitric acid with an
equal volume of distilled water. Free the diluted acid from brown oxides
of nitrogen by aeration.

2. Silver nitrate. Dissolve 20 grams of silver nitrate in 1 liter of
distilled water.

3. Standard manganous sulfate. Dissolve 0.288 gram of purest potassium
permanganate in about 100 cc. of distilled water. Acidify the solution
with sulfuric acid and heat to boiling. Add slowly a sufficient quantity
of dilute solution of oxalic acid to discharge the color. Cool and
dilute to 1 liter. One cc. of this solution contains 0.1 mg. of
manganese.

4. Ammonium persulfate. Crystals, free from chloride.

_Procedure._—Use an amount of the sample that contains not more than 0.2
mg. of manganese. Add 2 cc. of nitric acid and boil down to about 50 cc.
Precipitate the chloride with silver nitrate solution, adding at least 1
cc. in excess. Shake and heat to coagulate the precipitate, and filter.
A sample that contains much chloride should be evaporated with a few
drops of sulfuric acid until white fumes appear and then diluted before
the nitric acid and silver nitrate are added as directed above. If the
sample is highly colored by organic matter it should be evaporated with
sulfuric acid, and the residue ignited and dissolved in dilute nitric
acid. Add about 0.5 gram of ammonium persulfate crystals and warm the
solution until the maximum permanganate color is developed. This usually
takes about ten minutes. At the same time prepare standards by diluting
portions of 0.2, 0.4, 0.6 cc., etc. of the standard manganous sulfate
solution to about 50 cc. and treating them exactly as the sample was
treated. Transfer the sample and the standards to 50 cc. Nessler tubes,
and compare the colors immediately. Manganese in parts per million is
equal to the number of cubic centimeters of standard manganous sulfate
solution in the tube that the sample matches multiplied by 100, divided
by the number of cubic centimeters of the sample used.


                      BISMUTHATE METHOD.[2a][113]

_Reagents._—1. Nitric acid. Dilute 1 part of concentrated nitric acid
with 4 parts of distilled water. Free the dilute acid from brown oxides
of nitrogen by aeration.

2. Sulfuric acid. Dilute 1 part of concentrated sulfuric acid with 3
parts of distilled water.

3. Dilute sulfuric acid. Dilute 25 cc. of concentrated acid to 1 liter
with distilled water. Add enough permanganate solution to color faintly
the dilute acid.

4. Standard manganous sulfate. The standard solution of manganous
sulfate prepared as described under persulfate method (p. 48) should be
used and the standards should be prepared by following the same
procedure as is used for the sample. This solution is more permanent
than a solution of potassium permanganate, which may, however, be used.
To prepare it dissolve 0.288 gram of potassium permanganate in distilled
water and dilute the solution to 1 liter.

5. Sodium bismuthate. Purest dry salt.

_Procedure._—Use an amount of the sample that contains not more than 0.2
mg. of manganese. Add 0.5 cc. of sulfuric acid and evaporate to dryness.
Heat until the sulfuric acid is volatilized and ignite the residue.
Dissolve in 40 cc. of nitric acid, add about 0.5 gram of sodium
bismuthate, and heat until the permanganate color disappears. Add a few
drops of a solution of ammonium or sodium bisulfate to clear the
solution and again boil to expel oxides of nitrogen. Remove from the
source of heat, cool to 20° C., again add 0.5 gram of sodium bismuthate,
and stir. When the maximum permanganate color has developed, filter
through an alundum or Gooch crucible containing an asbestos mat ignited
and washed with potassium permanganate. Wash the precipitate with dilute
sulfuric acid until the washings are colorless. Transfer the filtrate to
a 50 cc. Nessler tube and compare the color of it with that of standards
prepared from the potassium permanganate solution. To prepare the
standards, dilute portions of 0.2, 0.4, 0.6 cc., etc. of the
permanganate solution to 50 cc. with dilute sulfuric acid. The content
of manganese is calculated as described under persulfate method (p. 49).


                  LEAD, ZINC, COPPER, AND TIN.[7][60]

Determinations of lead, zinc, copper, and tin are important in certain
mining regions and in places where the water has a solvent action on
pipes and other containers. The use of certain “germicides” also makes
it necessary to test for some of these metals.

Lead, zinc, and copper may be determined colorimetrically or
electrolytically. The colorimetric methods are not so accurate as a
combination of both, and are chiefly of value as qualitative tests.

It is possible to make a rough estimation of the amount of lead in clear
waters by acidifying with acetic acid, saturating with hydrogen sulfide,
and comparing the color produced with that produced by standard lead
solutions in Nessler tubes, treated in similar manner. This method,
however, is not applicable if the water is colored or contains iron.

_Reagents._—1. Standard lead solution. Dissolve 1.60 grams of lead
nitrate (Pb(NO_{3})_{2}) in 1 liter of distilled water. One cc. of this
solution contains 1 mg. of lead (Pb). As a check it is desirable to
determine lead as sulfate in a measured portion of this solution.

2. Standard copper solution. Dissolve about 0.8 gram of copper sulfate
crystals (CuSO_{4}.5H_{2}O) in water and, after the addition of 1 cc. of
concentrated sulfuric acid, dilute the solution to 1 liter. Determine
the copper in 100 cc. of this solution in the usual way by electrolytic
deposition. Dilute the solution so that 1 cc. contains 0.2 milligram
copper (Cu). This solution is permanent.

3. Ammonium chloride. Twenty-five per cent solution.

4. Ammonium acetate. Fifty per cent solution.

5. Ammonium hydroxide. (Sp. gr. 0.96.)

6. Hydrogen sulfide. Saturated solution.

7. Potassium sulfide. An alkaline solution of potassium sulfide made by
mixing equal volumes of 10 per cent potassium hydroxide and a saturated
aqueous solution of hydrogen sulfide.

8. Potassium oxalate. Crystals.

9. Potassium sulfate. Crystals.

10. Alcohol. Ninety-five per cent.

11. Alcohol. Fifty per cent.

12. Acetic acid. Fifty per cent.

13. Nitric acid. Concentrated acid (Sp. gr. 1.42).

14. Nitric acid. Dilute 1 part of the concentrated acid to 10 parts with
distilled water.

15. Hydrochloric acid. (Sp. gr. 1.20.)

16. Sulfuric acid. Concentrated acid (Sp. gr. 1.84).

17. Sulfuric acid. Dilute the concentrated acid with an equal volume of
distilled water.

18. Urea. Crystals.


                                 LEAD.

Concentrate (1)[D] rapidly by boiling in a 7–inch porcelain dish over a
free flame 3 or 4 liters of the sample to be tested, or more if very
small amounts of the metals are present, to a volume of about 30 cc. Add
10 or 15 cc. of ammonium chloride solution to assist in the separation
of the sulfides, then add a few drops of concentrated ammonium
hydroxide, and saturate with hydrogen sulfide. Allow to stand some time,
preferably over night, add a little more ammonium hydroxide and hydrogen
sulfide, boil the contents of the dish a few minutes, and filter. The
precipitate (2) may consist of lead, zinc, copper, and iron sulfides and
the suspended organic matter. The soluble coloring matter is in the
filtrate (3). Wash the precipitate a few times with hot water, place the
precipitate and the filter paper in the original dish and boil with
dilute nitric acid, rubbing down the sides of the dish, if necessary, to
detach any adhering sulfide precipitate. After again filtering and
washing several times with hot water, evaporate the filtrate and
washings in the original dish to a bulk of 10 to 15 cc., cool, add 5 cc.
of concentrated sulfuric acid, and heat until copious fumes of sulfuric
acid are evolved.

Footnote D:

  The numbers in parentheses refer to tables 10–12, pages 55–56.

If lead is present dilute the contents of the dish slightly with water,
and treat them with 150 cc. of 50 per cent alcohol, in which the lead
sulfate is insoluble. Allow to stand some time, preferably over night,
filter off the lead sulfate, and wash it with 50 per cent alcohol. Save
the filtrate for the determination of zinc.

Dissolve the precipitate of lead sulfate by boiling the filter
containing it in ammonium acetate solution in a porcelain dish. (4).
Filter into a 50 cc. Nessler tube and wash the filter with boiling water
containing a little ammonium acetate. Divide this filtrate in halves and
treat one-half with saturated hydrogen sulfide water in order to get an
approximation of the amount of lead present. To the other half, or an
aliquot portion, if a large amount of lead is present, add a few drops
of acetic acid, then an excess of saturated hydrogen sulfide solution,
and compare the color with that of standards made by treating known
amounts of the standard lead solution with a little acetic acid,
ammonium acetate, and hydrogen sulfide.


                                 ZINC.

If zinc is present and copper is absent concentrate the filtrate from
the lead sulfate to expel the alcohol, and remove the iron by adding an
excess of ammonium hydroxide. Filter, wash, and acidify the filtrate
with sulfuric acid. Concentrate the filtrate to about 150 cc. and
transfer to a weighed platinum dish. Add 2 grams of potassium oxalate
and 1.5 grams of potassium sulfate. Deposit the zinc electrolytically by
means of a current of about 0.3 ampere for three hours. After deposition
is complete and while the current is on, siphon off the solution and at
the same time run into the dish a stream of distilled water in order to
expel the free sulfuric acid, which might dissolve some of the zinc if
the circuit were broken. After the acid has been removed break the
circuit, wash the dish with water, then with 95 per cent alcohol, dry at
70° C., cool, and weigh it. The difference between this weight (10) and
the weight of the platinum dish equals the amount of metallic zinc. Some
difficulty has been experienced in this determination in obtaining pure
reagents. It is therefore advisable to make blank determinations with
each new lot of reagents and to correct the results if necessary.

If copper also is present (5) concentrate the filtrate from the lead
sulfate until the alcohol is expelled, and add an excess of ammonium
hydroxide. (6) Remove any iron precipitate by filtration. Neutralize the
filtrate (7) with sulfuric acid, and add 2 cc. of concentrated sulfuric
acid and 1 gram of urea. Electrolyze the solution and determine copper
colorimetrically as described in the procedure for copper (p. 54). After
the copper has been deposited add ammonium hydroxide to the solution
containing the zinc until nearly all the sulfuric acid has been
neutralized, concentrate to slightly less than the capacity of the
platinum dish, add 1.5 grams of potassium sulfate and 2 grams of
potassium oxalate, and electrolyze for zinc. As this solution is usually
saturated with ammonium salts due to neutralizing the large quantity of
sulfuric acid, it is frequently impossible to get the zinc deposited
firmly on the dish before the salts interfere by crystallization. To
avoid this difficulty, dilute half the solution and electrolyze it for
zinc; or, if the amount of zinc is very small, precipitate the zinc as
sulfide in acetic acid solution, wash, ignite to oxide, and weigh the
precipitate. This difficulty will not be encountered if copper is absent
as there will then be no excess of ammonium salts.

If lead and copper are known to be absent and zinc alone is to be
determined (13), after treating with sulfuric acid for separation of
lead, slightly dilute the contents of the dish. Add an excess of
ammonium hydroxide to precipitate iron and filter. Make the filtrate
slightly acid with sulfuric acid, concentrate to about 150 cc., transfer
to a weighed platinum dish, add potassium oxalate and sulfate, and
electrolyze the solution as described for deposition of zinc.


                              COPPER.[77]

Use 1 liter of a sample containing 0.1 to 1.0 part per million of
copper, and proportionate amounts for other concentrations. Evaporate to
about 75 cc., and wash into a 100 cc. platinum dish. Add 2 cc. of dilute
sulfuric acid for clear and soft waters; add more acid to very alkaline
waters to offset the alkalinity; add 5 cc. of acid to waters carrying
much organic matter or clay to insure the formation of a soluble copper
salt. Then place the dish as the anode in a direct current circuit,
suspend a spiral wire cathode in the solution so that it is parallel to
and about half an inch from the bottom of the dish, and close the
circuit.

Electrolyze for about four hours with occasional stirring, or over
night, if convenient. The current may be supplied by two gravity cells
in series, yielding a current through the solution of about 0.02 ampere.
Lift out the cathode without previously opening the circuit, and immerse
the spiral in a small amount of dilute nitric acid previously heated to
boiling. Wash off the wire and evaporate the nitric acid solution to
dryness on the water bath. If the presence of silver is suspected add a
few drops of hydrochloric acid before evaporation. Dissolve the residue
in water and wash it into a 50 cc. Nessler tube. Dilute to 50 cc. and
add 10 cc. of the potassium sulfide solution. The color of the copper
sulfide develops at once and is fairly permanent, lasting at least
several hours. Add 10 cc. of the potassium sulfide solution to a similar
tube containing 50 cc. of distilled water, and then add to it standard
copper solution in 0.2 cc. portions until the colors of the two tubes
match. If 1 liter of the sample is used copper in parts per million is
equal to the number of cubic centimeters of standard copper solution
required to match the color of the sample multiplied by 0.2.


                                  TIN.

Small quantities of tin are occasionally found in waters that have
passed through tin or tin-lined pipes. This metal, if present, is
precipitated with the iron by ammonia in the lead, zinc, and copper
separations. In the method for copper alone, it is removed in the same
way and may be further avoided by dissolving the sulfides in
concentrated nitric acid. Any tin present will then separate as an
insoluble compound, which may be ignited and weighed as the oxide
(SnO_{2}).

The following schematic tables illustrate the procedures given.

     Table 10.—SCHEME FOR THE SEPARATION OF LEAD, ZINC, AND COPPER.

 ───────────────────────────────────────────────────────────────────────
 1. Concentrate sample. Add 10 cc. NH_{4}Cl, a few drops NH_{4}OH and
 saturate with H_{2}S. Allow to stand, add more NH_{4}OH and H_{2}S.
 Boil, filter, and wash.
 ────────────────────────────────────────────────────────┬──────────────
 2. Dissolve the precipitate in dilute HNO_{3}. Filter   │3. Reject the
 and wash. Evaporate to 10 or 15 cc. Cool. Add 5 cc.     │filtrate which
 concentrated H_{2}SO_{4}, and heat until white fumes are│contains the
 given off. Dilute slightly and treat with 150 cc. of 50 │coloring
 per cent alcohol. Allow to stand; filter, and wash with │matter.
 50 per cent alcohol.                                    │
 ──────────────────────────┬─────────────────────────────┤
 4. The precipitate        │5. The filtrate contains the │
 contains the Pb. Dissolve │Zn and Cu. Concentrate to    │
 in NH_{4}C_{2}H_{3}O_{2}  │expel alcohol. Add excess of │
 solution. Filter into a 50│NH_{4}OH, filter and wash    │
 cc. Nessler tube and wash │precipitate.                 │
 with water containing     ├──────────────┬──────────────┴──────────────
 NH_{4}C_{2}H_{3}O_{2}.    │6. Reject the │7. The filtrate contains the
 Divide filtrate in halves.│precipitate   │Zn and Cu. Neutralize with
 Saturate one-half with    │which contains│H_{2}SO_{4}. Add 10 cc.
 H_{2}S. Determine the Pb  │the Fe.       │concentrated H_{2}SO_{4} and
 in the other half by      │              │1 g. urea. Electrolyze for
 adding HC_{2}H_{3}O_{2}   │              │two hours with a current of
 and H_{2}S and comparing  │              │0.5 ampere. Break circuit,
 with standards containing │              │empty dish and wash.
 known amounts of Pb.      │              │
 ──────────────────────────┼──────────────┴─────────────────────────────
 8. The deposit is Cu.     │9. The solution contains the Zn. Nearly
 Immerse the cathode in a  │neutralize with NH_{4}OH. Concentrate to
 small amount of hot,      │less than the capacity of the dish. Add 2 g.
 dilute HNO_{3}; wash off  │K_{2}C_{2}O_{4} and 1.5 g. K_{2}SO_{4}.
 and evaporate to dryness. │Electrolyze for 3 hours with a current of
 Take up in water and wash │0.3 ampere. Siphon off solution, break
 into a Nessler tube. Make │circuit, wash with water, then alcohol, dry
 up to mark, and add 10 cc.│at 70° C., cool and weigh.
 of potassium sulfide      ├────────────────────────────────────────────
 solution. Compare with    │10. The weighed residue is metallic Zn.
 standard. If large amount │
 is present, dry and weigh │
 as Cu.                    │
 ──────────────────────────┴────────────────────────────────────────────

           Table 11.—SCHEME FOR DETERMINATION OF COPPER ONLY.

 ───────────────────────────────────────────────────────────────────────
 11. Concentrate sample to 75 cc. Add 2 cc. conc. H_{2}SO_{4} for clear,
 soft waters and 5 cc. for alkaline or turbid waters. Electrolyze
 following procedure in 7 and 8.
 ───────────────────────────────────────────────────────────────────────

            Table 12.—SCHEME FOR DETERMINATION OF ZINC ONLY.

 ───────────────────────────────────────────────────────────────────────
 13. Follow scheme for all three metals as given in Table 10 through
 section 5. Nearly neutralize the filtrate with H_{2}SO_{4}, concentrate
 to less than the capacity of the dish and electrolyze as directed in
 section 9.
 ───────────────────────────────────────────────────────────────────────


                           MINERAL ANALYSIS.


                        RESIDUE ON EVAPORATION.

See description of method (p. 29). The residue should be dried one hour
at 180° C. Turbid waters should be filtered, and the composition of the
suspended matter should be determined separately or the amount of it
reported as suspended matter.


                        ALKALINITY AND ACIDITY.

                 See description of method (pp. 35–41).


                               CHLORIDE.

                 See description of method (pp. 41–43).


                           NITRATE NITROGEN.

                 See description of method (pp. 23–25).


 SEPARATION OF SILICA, IRON, ALUMINIUM, CALCIUM, AND MAGNESIUM.[10][48]


                                SILICA.

Evaporate in platinum 100 to 1,000 cc. of the sample or sufficient if
possible to form a residue weighing 0.4 to 0.6 gram, and preferably
containing 0.1 to 0.2 gram of calcium. When the residue is nearly dry
add 1 cc. of hydrochloric acid (1 to 1) and, after moistening the sides
of the dish, evaporate to dryness. Dry at 180° C. and if much organic
matter is present char it in a radiator. Moisten the residue with dilute
hydrochloric acid and expel the excess of acid by heating on the water
bath. Add a few drops of hydrochloric acid, dissolve in hot water, and
filter. Wash the residue with hot water. Evaporate the filtrate to
dryness, repeat the filtration, and combine the two residues. If great
accuracy is not required the second evaporation with hydrochloric acid
may be omitted. Ignite and weigh the insoluble residue. Add 2 drops of
concentrated sulfuric acid and a little hydrofluoric acid, volatilize
the acids, ignite, and weigh again. Report the loss in weight as silica
(SiO_{2}). A weight of non-volatile matter exceeding 0.5 mg. should be
analyzed.


                          IRON AND ALUMINIUM.

Heat to boiling the filtrate from the insoluble residue, oxidize with
concentrated nitric acid or bromine, and concentrate to about 25 cc. Add
ammonium hydroxide in slight excess, boil one minute, and filter.
Dissolve the precipitate on the filter in a small amount of hot dilute
hydrochloric acid. Reprecipitate with ammonium hydroxide, filter, and
wash. Unless very accurate results are necessary this solution and
reprecipitation may be omitted. Unite the two filtrates for
determination of calcium. Ignite and weigh the precipitate. It will
comprise oxides of iron and aluminium and phosphate. If much phosphate
is present it should be determined in a separate sample and a correction
for the amount applied; otherwise it may be neglected. Determine the
iron in the ignited precipitate by fusion with sodium or potassium
pyrosulfate, reduction with zinc, and titration with potassium
permanganate. Aluminium (Al) is calculated as follows:

            Al = 0.53[(Fe_{2}O_{3} + Al_{2}O_{3}) − 1.43 Fe]


                                CALCIUM.

Concentrate the filtrate from the separation of iron and aluminium to
about 100 cc., and add an excess of concentrated solution of ammonium
oxalate, little by little, to the hot ammoniacal solution. Keep the
solution warm and stir at intervals till the precipitate settles readily
and leaves a clear supernatant liquid. Filter, dissolve the precipitate
in a little hot dilute hydrochloric acid, and reprecipitate with
ammonium hydroxide and ammonium oxalate. If great accuracy is not
required this solution and reprecipitation may be omitted, and the first
precipitate may be washed clean with hot water[64a]. Save the filtrate
for determination of magnesium. Ignite the precipitate and weigh it as
calcium oxide, 71.5 per cent of which is the equivalent of calcium (Ca);
or dissolve the precipitate in hot 2 per cent sulfuric acid and titrate
with a standard solution of potassium permanganate.


                               MAGNESIUM.

Acidify with hydrochloric acid the filtrate from the separation of
calcium and concentrate it to about 100 cc. Add 20 cc. of a saturated
solution of microcosmic salt, cool, and make slightly but distinctly
alkaline by adding ammonium hydroxide, drop by drop. Allow the solution
to stand four hours, then filter and wash with 3 per cent ammonium
hydroxide. Dissolve the precipitate, especially in the presence of large
amounts of sodium or potassium, in a slight excess of dilute
hydrochloric acid and reprecipitate the magnesium with ammonium
hydroxide and a few drops of microcosmic salt solution. If great
accuracy is not required this solution and reprecipitation may be
omitted. Ignite the precipitate and weigh it as magnesium pyrophosphate
(Mg_{2}P_{2}O_{7}), 21.9 per cent of which is the equivalent of
magnesium (Mg.). If manganese is present[64a] it is precipitated with
the magnesium and a correction for it should be applied after having
determined manganese in a separate sample. The weight of manganese
pyrophosphate (Mn_{2}P_{2}O_{7}) is 2.58 times the weight of manganese.


             SEPARATION OF SULFATE, SODIUM, AND POTASSIUM.


                                SULFATE.

Evaporate to dryness 100 to 1,000 cc. of the sample, or sufficient to
obtain a residue weighing 0.4 to 0.6 gram and containing preferably 0.05
to 0.2 gram of sodium. Acidify the residue with hydrochloric acid and
remove the silica, iron, and aluminium (pp. 56–57). Make acid and add a
hot solution of barium chloride in slight excess to the hot filtrate,
and warm it, stirring at intervals for one-half hour, until the
precipitate settles readily and leaves a clear supernatant liquid. Dry,
ignite, and weigh the precipitate of barium sulfate, 41.1 per cent of
which is equal to the content of sulfate (SO_{4}).


                    SODIUM, POTASSIUM, AND LITHIUM.

Evaporate to dryness the filtrate from the precipitation of barium
sulfate. Add a few cubic centimeters of hot water and then a saturated
solution of barium hydroxide until a slight film collects on the top of
the solution. Filter and wash the precipitate with hot water. Add to the
filtrate an excess of ammonium hydroxide and ammonium carbonate
solution. Filter, evaporate the filtrate to dryness, dry, and ignite at
low red heat to expel ammonium salts. Repeat the operations including
the addition of barium hydroxide until no precipitate is obtained by
barium hydroxide or by ammonium hydroxide and ammonium carbonate.
Evaporate the final filtrate to dryness in a weighed platinum dish, dry,
cool, and weigh the residue. Dissolve the residue in a few cubic
centimeters of water, filter, wash the filter paper twice with hot
water, then ignite the filter paper in the platinum dish. Cool and weigh
the residue. Subtract this weight from the first weight including the
residue. The difference is the weight of the chlorides of sodium and
potassium and lithium. If it is not desired to separate sodium and
potassium the weight of sodium and potassium as sodium may be calculated
from this difference by multiplying it by 0.394.


                               POTASSIUM.

_First procedure._—Add to the solution of the chlorides of sodium and
potassium a few drops of dilute hydrochloric acid (1 to 3) and 1 cc. of
10 per cent platinic chloride (PtCl_{4}) for each 30 mg. of the combined
chlorides. Evaporate to a thick syrup on the water bath, then remove
dish and allow it to come to dryness at laboratory temperature. Treat
the residue cold with 80 per cent alcohol and filter. Wash the
precipitate with 80 per cent alcohol until the filtrate is no longer
colored. Dry the precipitate and dissolve it in hot water. Evaporate the
solution to dryness in a platinum dish and weigh it as potassium
platinic chloride (K_{2}PtCl_{6}). The weight of potassium (K) is 16.1
per cent of this weight and the equivalent of potassium chloride (KCl)
is 30.7 per cent of this weight. Subtract the equivalent weight of
potassium chloride from the weight of the combined chlorides. The weight
of the sodium is 39.4 per cent of the difference.

_Second procedure._[86][103a]—Add to the hot solution of the combined
chlorides 20 per cent perchloric acid (HClO_{4}) slightly in excess of
the amount required to combine with the bases. One cubic centimeter of
20 per cent perchloric acid is equivalent to 90 mg. of potassium.
Evaporate the solution to dryness, dissolve the residue in 10 cc. of hot
water and a small amount of perchloric acid, and again evaporate to
dryness. Repeat the addition of water, perchloric acid, and evaporation
until white fumes appear on evaporating to dryness. Add to the residue
25 cc. of 96 per cent alcohol containing 0.2 per cent of perchloric acid
(1 cc. of 20 per cent perchloric acid in 100 cc. of 98 per cent
alcohol). Break up the residue with a stirring rod. Decant the
supernatant liquid through a weighed Gooch crucible that has been washed
with 0.2 per cent perchloric acid in alcohol. If the precipitate is
unusually large dissolve it in hot water and repeat the evaporation with
perchloric acid. Wash the precipitate once by decantation with the 0.2
per cent perchloric acid in alcohol, transfer the precipitate to the
crucible, and wash it several times with a 0.2 per cent perchloric acid
in alcohol. Dry the crucible at 120–130° C. for one hour, cool, and
weigh it. The increase in weight is potassium perchlorate (KClO_{4}).
The equivalent weight of potassium is 28.2 per cent and the equivalent
weight of potassium chloride is 53.8 per cent of the potassium
perchlorate. Calculate the content of sodium by difference.


                              LITHIUM.[34]

Use a large quantity of the sample. Obtain the combined chlorides of
sodium, potassium, and lithium (see pp. 58–59). Transfer the combined
chlorides to a small Erlenmeyer flask (50 or 100 cc. capacity) and
evaporate the solution nearly, but not quite, to dryness. Add about 30
cc. of redistilled amyl alcohol. Connect the flask, the stopper of which
carries a thermometer, with a condenser[E] and boil until the
temperature rises approximately to the boiling point of amyl alcohol
(130° C.), showing that all the water has been driven off. Cool slightly
and add a drop of hydrochloric acid to convert small amounts of lithium
hydroxide to lithium chloride. Connect with the condenser and continue
the boiling to drive off again all water and until the temperature
reaches the boiling point of amyl alcohol. The content of the flask at
this time is usually 15 to 20 cc. Filter through a small paper or a
Gooch crucible into a graduated cylinder and note exact quantity of
filtrate, which determines the subsequent correction. Wash the
precipitate with small quantities of dehydrated amyl alcohol. Evaporate
the filtrate and washings in a platinum dish to dryness on the steam
bath, dissolve the residue in water, and add a few drops of sulfuric
acid. Evaporate on a steam bath and expel the excess of sulfuric acid by
gentle heat over a flame. Repeat until carbonaceous matter is completely
burned off. Cool and weigh the dish and contents. Dissolve in a small
quantity of hot water, filter through a small filter, wash, and return
filter to dish, ignite, and weigh. The difference between the original
weight of dish and contents and the weight of the dish and small amount
of residue equals the weight of impure lithium sulfate. The purity of
the lithium sulfate should be tested by adding small amounts of ammonium
phosphate and ammonium hydroxide, which will precipitate any magnesium
present with the lithium sulfate. Any precipitate appearing after
standing over night should be collected on a small filter and weighed as
magnesium pyrophosphate, calculated to sulfate, and subtracted from the
weight of impure lithium sulfate. From this weight subtract 0.00113 gram
for every 10 cc. of amyl alcohol filtrate exclusive of the amyl alcohol
used in washing residue because of the slight solubility of solid mixed
chlorides in amyl alcohol. Calculate lithium from the corrected weight
of lithium sulfate. Dissolve the mixed chlorides from flask and filter
with hot water, evaporate to dryness, ignite gently to remove amyl
alcohol, filter and thoroughly wash; concentrate the filtrates and
washings to 25 to 50 cc.

Footnote E:

  The amyl alcohol may be boiled off without the use of a condenser, but
  the vapors are very disagreeable.

To the weight of potassium chloride add 0.00051 gram for every 10 cc. of
amyl alcohol used in the extraction of the lithium chloride, which
corrects for the solubility of the potassium chloride in amyl alcohol.
Calculate to potassium.

The weight of sodium chloride is found by subtracting the combined
weights of lithium chloride and potassium chloride (corrected) from the
total weight of the three chlorides. Calculate sodium chloride to
sodium.


               BROMINE, IODINE, ARSENIC, AND BORIC ACID.

Evaporate to dryness a large quantity of the sample to which a small
amount of sodium carbonate has been added. Boil the residue with
distilled water, transfer it to a filter, and thoroughly wash it with
hot water. Dilute the alkaline filtrate to a definite volume, and
determine bromine and iodine, arsenic, and boric acid in aliquot
portions of it.


                        BROMINE AND IODINE.[10]

_Reagents._—1. Sulfuric acid. 1 to 5.

2. Potassium nitrite or sodium nitrite. Two per cent solution.

3. Carbon bisulfide. Freshly purified by distillation.

4. Iodine standards. Acidify with dilute sulfuric acid measured
quantities of a standard solution of potassium iodide in small tubes.
Add 3 or 4 drops of the potassium nitrite solution and extract with
carbon bisulfide as in the actual determination. Transfer to small
flasks the standards from which the iodine has been removed.

5. Chlorine water. Saturated solution.

6. Bromine standards. Add measured quantities of a standard solution of
a bromide to the liquid in each of the small flasks from which the
iodine has been removed. Add to each 5 cc. of purified carbon bisulfide,
and proceed exactly as with the sample.

_Procedure._—Evaporate to dryness an aliquot portion of the alkaline
filtrate. Dissolve the residue in 2 or 3 cc. of water, and add enough
absolute alcohol to make the percentage of alcohol about 90. Boil and
filter and repeat the extraction of the residue with alcohol once or
twice. Add 2 or 3 drops of sodium hydroxide to the combined alcoholic
filtrates and evaporate to dryness. Dissolve the residue in 2 or 3 cc.
of water and repeat the extraction with alcohol and the filtration. Add
a drop of sodium hydroxide to the filtrate and evaporate it to dryness.
Dissolve the residue in a little water. Acidify this solution with
dilute sulfuric acid, adding 3 or 4 drops excess, and transfer it to a
small flask. Add 4 drops of the solution of potassium nitrite or sodium
nitrite and about 5 cc. of carbon bisulfide. Shake the mixture until all
the iodine is extracted. Separate the acid solution from the carbon
bisulfide by filtration. Wash the flask, filter, and contents with cold
distilled water, and transfer the carbon bisulfide containing the iodine
in solution to Nessler tubes by means of about 5 cc. of pure carbon
bisulfide. In washing the filter, dilute the contents of the tube to a
definite volume, usually 12 or 15 cc., and compare the color with that
of known amounts of iodine dissolved in carbon bisulfide in other tubes.

Transfer to a small flask the sample from which the iodine has been
removed. Add saturated chlorine water, 1 cc. at a time, shaking after
each addition until all the bromine is freed. Care must be taken not to
add much more chlorine than that necessary to free the bromine, since an
excess of reagent may form a bromochloride that spoils the color
reaction. Separate the water solution from the carbon bisulfide by
filtration through a moistened filter, wash the contents of the filter
two or three times with water, and then transfer them to a Nessler tube
by means of about 1 cc. of carbon bisulfide. Repeat this extraction of
the filtrate twice, using 3 cc. of carbon bisulfide each time. The
combined carbon bisulfide extracts usually amount to 11.5 to 12 cc. Add
enough carbon bisulfide to the tubes to bring them to a definite volume,
usually 12 to 15 cc., and compare the sample with the standards. If much
bromine is present it is not always completely extracted by the amounts
of carbon bisulfide recommended. If the extraction is incomplete,
therefore, make one or two extra extractions with carbon bisulfide,
transfer the extracts to another tube, and compare the color with that
of the standards.


                              ARSENIC.[31]

Evaporate to dryness an aliquot portion of the alkaline filtrate (p.
61). Acidify the residue with arsenic-free sulfuric acid, and subject it
to the action of arsenic-free zinc and sulfuric acid in a
Marsh-Berzelius apparatus. Compare the mirror obtained with a mirror
obtained from an arsenious oxide solution of known strength.


                              BORIC ACID.

Evaporate to dryness an aliquot portion of the alkaline filtrate (p.
61), treat the residue with 1 or 2 cc. of water, and slightly acidify
the solution with hydrochloric acid. Add about 25 cc. of absolute
alcohol, boil, filter, and repeat the extraction of the residue. Make
the filtrate slightly alkaline with sodium hydroxide, and evaporate it
to dryness. Add a little water, slightly acidify with hydrochloric acid,
and place a strip of turmeric paper in the liquid. Evaporate to dryness
on the steam bath, and continue the heating until the turmeric paper is
dry. If boric acid is present the turmeric paper becomes cherry red. It
is not usually necessary to determine quantitatively boric acid; the
quantitative method devised by Gooch[33] is recommended.


                         HYDROGEN SULFIDE.[103]

Hydrogen sulfide should be determined preferably in the field; the
procedure as far as the final titration with sodium thiosulfate must be
carried out in the field.

_Reagents._—1. N/100 sodium thiosulfate.

2. Standard iodine. A N/100 solution containing potassium iodide
standardized against the N/100 sodium thiosulfate. To standardize, add
10 cc. of the iodine solution to 500 cc. of boiled distilled water. Add
about 1 gram of potassium iodide, and titrate with N/100 sodium
thiosulfate in the presence of starch indicator. One cc. of N/100 iodine
is equivalent to 0.17 mg. H_{2}S.

3. Potassium iodide. Crystals.

4. Starch. A freshly prepared solution for use as indicator.

_Procedure._—Add 500 cc. of the sample to 10 cc. of the standard iodine
solution and 1 gram of potassium iodide in a large glass-stoppered
bottle or flask. If the sample is to be collected from a tap lead the
water into the bottle through a rubber tube extending to the bottom of
the bottle so as to eliminate errors due to aeration. Shake the bottle,
allow it to stand for a few minutes, and then titrate the excess of
iodine with sodium thiosulfate in the presence of starch indicator.
Hydrogen sulfide (H_{2}S) in parts per million is equal to 0.34 times
the difference in cubic centimeters between the amount of iodine
solution added and the amount of N/100 thiosulfate used in the
titration.


                               CHLORINE.

In waters that have been treated with calcium hypochlorite or liquid
chlorine it is frequently advisable to ascertain the presence or absence
of chlorine. As the reagents which have been proposed for its detection
are not specific for chlorine but give similar or identical reactions
with oxidizing agents or reducible substances care must be exercised in
interpreting the results of such tests: nitrites and ferric salts are of
common occurrence, and chlorates also may lead to misinterpretation in
waters treated with calcium hypochlorite.

_Reagents._—1. Tolidin solution. One gram of o-tolidin, purified by
being recrystallized from alcohol, is dissolved in 1 liter of 10 per
cent hydrochloric acid.

2. Copper sulfate solution. Dissolve 1.5 grams of copper sulfate and 1
cc. of concentrated sulfuric acid in distilled water and dilute the
solution to 100 cc.

3. Potassium bichromate solution. Dissolve 0.025 gram of potassium
bichromate and 0.1 cc. of concentrated sulfuric acid in distilled water
and dilute the solution to 100 cc.

_Procedure._—Mix 1 cc. of the tolidin reagent with 100 cc. of the sample
in a Nessler tube and allow the solution to stand at least 5 minutes.
Small amounts of free chlorine give a yellow and larger amounts an
orange color.

For quantitative determination compare the color with that of standards
in similar tubes prepared from the solutions of copper sulfate and
potassium bichromate. The amounts of solution for various standards are
indicated in Table 13.

  Table 13.—PREPARATION OF PERMANENT STANDARDS FOR CONTENT OF CHLORINE.

 ───────────────────────┬───────────────────────┬───────────────────────
        Chlorine.       │  Solution of copper   │ Solution of potassium
                        │       sulfate.        │      bichromate.
 ───────────────────────┼───────────────────────┼───────────────────────
  _Parts per million._  │         _cc._         │         _cc._
                        │                       │
                    0.01│                    0.0│                    0.8
                     .02│                     .0│                    2.1
                     .03│                     .0│                    3.2
                     .04│                     .0│                    4.3
                     .05│                     .4│                    5.5
                     .06│                     .8│                    6.6
                     .07│                    1.2│                    7.5
                     .08│                    1.5│                    8.7
                     .09│                    1.7│                    9.0
                     .10│                    1.8│                   10.0
                     .20│                    1.9│                   20.0
                     .30│                    1.9│                   30.0
                     .40│                    2.0│                   38.0
                     .50│                    2.0│                   45.0
 ───────────────────────┴───────────────────────┴───────────────────────


           DISSOLVED OXYGEN.[16][65][68][71b][99][100c][120]

_Reagents._—1. Sulfuric acid, concentrated. (Sp. gr. 1.83–1.84.)

2. Potassium permanganate. Dissolve 6.32 grams of the salt in water and
dilute the solution to 1 liter.

3. Potassium oxalate. A 2 per cent solution.

4. Manganous sulfate. Dissolve 480 grams of the salt in water and dilute
the solution to 1 liter.

5. Alkaline potassium iodide. Dissolve 700 grams of potassium hydroxide
and 150 grams of potassium iodide in water and dilute the solution to 1
liter.

6. Hydrochloric acid. Concentrated (Sp. gr. 1.18–1.19).

7. Sodium thiosulfate. A N/40 solution. Dissolve 6.2 grams of chemically
pure recrystallized sodium thiosulfate in water and dilute the solution
to 1 liter with freshly boiled distilled water. Each cc. is equivalent
to 0.2 mg. of oxygen or to 0.1395 cc. of oxygen at 0°C. and 760 mm.
pressure. Inasmuch as this solution is not permanent it should be
standardized occasionally against a N/40 solution of potassium
bichromate. The keeping qualities of the thiosulfate solution are
improved by adding to each liter 5 cc. of chloroform and 1.5 grams of
ammonium carbonate before diluting to the prescribed volume.

8. Starch solution. Mix a small amount of clean starch with cold water
until it becomes a thin paste and stir this mass into 150 to 200 times
its weight of boiling water. Boil for a few minutes, then sterilize. It
may be preserved by adding a few drops of chloroform.

_Collection of sample._—Collect the sample in a narrow-necked
glass-stoppered bottle of 250 to 270 cc. capacity. The following
procedure should be followed in order to avoid entrainment or absorption
of atmospheric oxygen. In collecting from a tap fill the bottle through
a glass or rubber tube extending well into the tap and to the bottom of
the bottle. To avoid air bubbles allow the bottle to overflow for
several minutes, and then carefully replace the glass stopper so that no
air bubble is entrained. In collecting from the surface of a pond or
tank connect the sample bottle to a bottle of 1 liter capacity. Provide
each bottle with a two-hole rubber stopper having one glass tube
extending to the bottom and another glass tube entering but not
projecting into the bottle. Connect the short tube of the sample bottle
with the long tube of the liter bottle. Immerse the sample bottle in the
water and apply suction to the outlet of the liter bottle. To collect a
sample at any depth arrange the two bottles so that the outlet tube of
the liter bottle is at a higher elevation then the inlet tube of the
sample bottle. Lower the two bottles, in any convenient form of cage
properly weighted, to the desired depth. Water entering during the
descent will be flushed through into the liter bottle. When air bubbles
cease rising to the surface raise the bottles. Finally replace the
perforated stopper of the sample bottle with a glass stopper in such
manner as to avoid entraining bubbles of air.

_Procedure._—Remove the stopper from the bottle and add, first, 0.7 cc.
of the concentrated sulfuric acid, and then 1 cc. of the potassium
permanganate solution. These and all other reagents should be introduced
by pipette under the surface of the liquid. Insert the stopper and mix
by inverting the bottle several times. After 20 minutes have elapsed
destroy the excess of permanganate by adding 1 cc. of the potassium
oxalate solution, the bottle being at once restoppered and its contents
mixed. If a noticeable excess of potassium permanganate is not present
at the end of 20 minutes, again add 1 cc. of the potassium permanganate
solution. If this is still insufficient use a stronger potassium
permanganate solution. After the liquid has been decolorized by the
addition of potassium oxalate add 1 cc. of the manganous sulfate
solution and 3 cc. of the alkaline potassium iodide solution. Allow the
precipitate to settle. Add 2 cc. of the hydrochloric acid and mix by
shaking.

The procedure to this point must be carried out in the field, but after
the acid has been added and the stopper replaced there is no further
change, and the rest of the test may be performed within a few hours, as
convenient. Transfer 200 cc. of the contents of the bottle to a flask
and titrate with N/40 sodium thiosulfate, using a few cubic centimeters
of the starch solution as indicator toward the end of the titration. Do
not add the starch solution until the color has become faint yellow, and
titrate until the blue color disappears.

The use of potassium permanganate is made necessary by high nitrite or
organic matter. The procedure outlined must be followed in all work on
sewage and partly purified effluents or seriously polluted streams or
samples whose nitrite nitrogen exceeds 0.1 part per million. In testing
other samples the procedure may be shortened by beginning with the
addition of the manganous sulfate solution and proceeding from that
point as outlined, except that only 1 cc. of alkaline potassium iodide
need be added.

_Calculation of Results._—Oxygen shall be reported in parts per million
by weight. It is sometimes convenient to know the number of cubic
centimeters per liter of the gas at 0°C. temperature and 760 mm.
pressure and also to know the percentage which the amount of gas present
is of the maximum amount capable of being dissolved by distilled water
at the same temperature and pressure. If 200 cc. of the sample is taken
the number of cubic centimeters of N/40 thiosulfate used is equal to
parts per million of oxygen. Corrections for volume of reagents added
amount to less than 3 per cent and are not justified except in work of
unusual precision. To obtain the result in cubic centimeters per liter
multiply the number of cubic centimeters of thiosulfate used by 0.698.
To obtain the result in percentage of saturation divide the number of
cubic centimeters of thiosulfate by the figure in Table 14 opposite the
temperature of the water and under the proper chlorine figure. The last
column of Table 14 permits interpolation for intermediate chlorine
values. At elevations differing considerably from mean sea level and for
accurate work attention must be given to barometric pressure, the normal
pressure in the region being preferable to the specific pressure at the
time of sampling. The term “saturation” refers to a condition of
equilibrium between the solution and an oxygen pressure in the
atmosphere corresponding to 158.8 millimeters, or approximately
one-fifth atmosphere. The true saturation or equilibrium between the
solution and pure oxygen is nearly five times this value, and
consequently values in excess of 100 per cent saturation frequently
occur in the presence of oxygen-forming plants.

   Table 14.—SOLUBILITY OF OXYGEN IN FRESH WATER AND IN SEA WATER OF
 STATED DEGREES OF SALINITY AT VARIOUS TEMPERATURES WHEN EXPOSED TO AN
 ATMOSPHERE CONTAINING 20.9 PER CENT OF OXYGEN UNDER A PRESSURE OF 760
                                 MM.[F]

 (Calculated by G. C. Whipple and M. C. Whipple from measurements of C.
                           J. Fox.)[27][119]

 ─────────────┬────────────────────────────────────────────┬───────────
              │                                            │Difference
 Temperature. │   Chloride in sea water (milligrams per    │  per 100
              │                  liter).                   │ parts of
              │                                            │ chloride.
 ─────────────┼────────┬────────┬────────┬────────┬────────┼───────────
              │   0.   │ 5000.  │ 10000. │ 15000. │ 20000. │
 ─────────────┼────────┴────────┴────────┴────────┴────────┼───────────
     _°C._    │_Dissolved oxygen in milligrams per liter._ │_Parts per
              │                                            │ million._
             0│   14.62│   13.79│   12.97│   12.14│   11.32│     0.0165
             1│   14.23│   13.41│   12.61│   11.82│   11.03│      .0160
             2│   13.84│   13.05│   12.28│   11.52│   10.76│      .0154
             3│   13.48│   12.72│   11.98│   11.24│   10.50│      .0149
             4│   13.13│   12.41│   11.69│   10.97│   10.25│      .0144
             5│   12.80│   12.09│   11.39│   10.70│   10.01│      .0140
              │        │        │        │        │        │
             6│   12.48│   11.79│   11.12│   10.45│    9.78│      .0135
             7│   12.17│   11.51│   10.85│   10.21│    9.57│      .0130
             8│   11.87│   11.24│   10.61│    9.98│    9.36│      .0125
             9│   11.59│   10.97│   10.36│    9.76│    9.17│      .0121
            10│   11.33│   10.73│   10.13│    9.55│    8.98│      .0118
              │        │        │        │        │        │
            11│   11.08│   10.49│    9.92│    9.35│    8.80│      .0114
            12│   10.83│   10.28│    9.72│    9.17│    8.62│      .0110
            13│   10.60│   10.05│    9.52│    8.98│    8.46│      .0107
            14│   10.37│    9.85│    9.32│    8.80│    8.30│      .0104
            15│   10.15│    9.65│    9.14│    8.63│    8.14│      .0100
              │        │        │        │        │        │
            16│    9.95│    9.46│    8.96│    8.47│    7.99│      .0098
            17│    9.74│    9.26│    8.78│    8.30│    7.84│      .0095
            18│    9.54│    9.07│    8.62│    8.15│    7.70│      .0092
            19│    9.35│    8.89│    8.45│    8.00│    7.56│      .0089
            20│    9.17│    8.73│    8.30│    7.86│    7.42│      .0088
              │        │        │        │        │        │
            21│    8.99│    8.57│    8.14│    7.71│    7.28│      .0086
            22│    8.83│    8.42│    7.99│    7.57│    7.14│      .0085
            23│    8.68│    8.27│    7.85│    7.43│    7.00│      .0083
            24│    8.53│    8.12│    7.71│    7.30│    6.87│      .0083
            25│    8.38│    7.96│    7.56│    7.15│    6.74│      .0082
              │        │        │        │        │        │
            26│    8.22│    7.81│    7.42│    7.02│    6.61│      .0080
            27│    8.07│    7.67│    7.28│    6.88│    6.49│      .0079
            28│    7.92│    7.53│    7.14│    6.75│    6.37│      .0078
            29│    7.77│    7.39│    7.00│    6.62│    6.25│      .0076
            30│    7.63│    7.25│    6.86│    6.49│    6.13│      .0075
 ─────────────┴────────┴────────┴────────┴────────┴────────┴───────────

Footnote F:

  Under any other barometric pressure, B, the solubility can be obtained
  from the corresponding value in the table by the formula:

 S´ = S(B/760) = S(B´/29.92) in which S´ = Solubility at B or B´,
                                      S = Solubility at 760 mm. or 29.92
                                        inches,
                                      B = Barometric pressure in mm.,
                                  and B´ = Barometric pressure in
                                        inches.


                       ETHER-SOLUBLE MATTER.[44]

Evaporate 500 cc. of the sample in a porcelain evaporating dish to a
volume of about 50 cc. By means of a rubber-tipped glass rod remove to
the bottom of the dish the solid matter attached to the sides, and add
normal sulfuric acid to neutralize the alkalinity. Do not use an excess
of acid. Then evaporate the contents of the dish to dryness. Treat the
dry residue with boiling ether, rubbing the bottom and sides of the dish
to insure complete solution of fat. Three extractions with ether are
required. Filter the ether solution through a 5 cm. filter into a
weighed flask having a wide mouth. Evaporate the ether slowly, and dry
the flask at 100° C. for 30 minutes. The increase in weight of the flask
gives the amount of fats, or, in more precise language, the
ether-soluble matter.

An excess of acid gives too high results because of the formation of
fatty-acid residues.


                  RELATIVE STABILITY OF EFFLUENTS.[78]

_Reagent._—Methylene blue solution. A 0.05 per cent aqueous solution of
methylene blue, preferably the double zinc salt or commercial
variety.[60b]

_Collection of sample._—Collect the sample in a glass-stoppered bottle
holding approximately 150 cc. If the dissolved oxygen is low observe
precautions similar to those used in collecting samples for dissolved
oxygen (p. 66).

_Procedure._—Add 0.4 cc. of the methylene blue solution to the sample in
the 150 cc. bottle. As methylene blue has a slightly antiseptic property
be careful to add exactly 0.4 cc. Add the methylene blue solution
preferably below the surface of the liquid after filling the bottle with
the sample. If the methylene blue is added first do not allow the liquid
to overflow as coloring matter will thus be lost. Incubate the sample at
20° C. for ten days. Four days’ incubation may be considered sufficient
for all practical purposes in routine plant-control work. If quick
results are desired incubate the sample at 37° C. for five days using
suitable stoppers[1a][2a] to prevent the loss and reabsorption of
dissolved oxygen. The bacterial flora at 37° C. is different from that
at 20° C. The lower temperature is more nearly the average temperature
of surface waters and therefore the higher temperature should be used
only when quick approximate results are essential. Observe the sample at
least twice a day during incubation. Give a sample in which the
methylene blue becomes decolorized a relative stability corresponding to
the time required for reduction (see Table 15). For routine filter
control ordinary room or cellar temperature gives fairly satisfactory
results. For accurate studies, room temperature incubation is very
undesirable, as the fluctuations in temperature which are ordinarily not
noticed are responsible for appreciable deviations from the true values
of relative stability. If the samples are incubated less than 10 days at
20° C. and are not decolorized place a plus sign after the stability
value in order to indicate that the stability might have been higher if
more time had been allowed. In applying this test to river waters it
often happens that the blue coloring matter is removed either partly or
completely through absorption by the clay which many rivers carry in
suspension. True relative stabilities cannot be obtained for such waters
except by determining the initial available oxygen at the start and the
biochemical oxygen demand on incubation at 20° C. for 10 days (pp.
71–73). Germicides, such as hypochlorite of lime, if present in
sufficient quantity, vitiate the results. If a sample contains free
chlorine, therefore, store it about 2 hours, or until the chlorine is
gone, and then add methylene blue.

Table 15[78] gives the relation between the time in days to decolorize
methylene blue at 20° C. (_t__{20}) and the relative stability number or
ratio of available oxygen to oxygen required for equilibrium, expressed
in percentage (S).

                  Table 15.—RELATIVE STABILITY NUMBERS.

 ───────────────────────────────────┬───────────────────────────────────
 Time required for decolorization at│        Relative stability.
               20° C.               │
 ───────────────────────────────────┼───────────────────────────────────
               _Days._              │           _Percentage._
                                 0.5│                                 11
                                 1.0│                                 21
                                 1.5│                                 30
                                 2.0│                                 37
                                 2.5│                                 44
                                 3.0│                                 50
                                 4.0│                                 60
                                 5.0│                                 68
                                 6.0│                                 75
                                 7.0│                                 80
                                 8.0│                                 84
                                 9.0│                                 87
                                10.0│                                 90
                                11.0│                                 92
                                12.0│                                 94
                                13.0│                                 95
                                14.0│                                 96
                                16.0│                                 97
                                18.0│                                 98
                                20.0│                                 99
 ───────────────────────────────────┴───────────────────────────────────

The theoretical relation is, S = 100 (1 − 0.794_t__{20})

The relation between the time of reduction at 20° C. and that at 37° C.
is approximately two to one, but if an observer incubates at 37° C. he
should work out his own comparative 37° C. table or factor.

A relative stability of 75 signifies that the sample examined contains a
supply of available oxygen equal to 75 per cent of the amount of oxygen
which it requires in order to become perfectly stable. The available
oxygen is approximately equivalent to the dissolved oxygen plus the
available oxygen of nitrate and nitrite. Nitrite in sewage is usually so
low as to be negligible.


   BIOCHEMICAL OXYGEN DEMAND OF SEWAGE AND EFFLUENTS.[60a][60c][60d]


                       RELATIVE STABILITY METHOD.

The relative stability method may be employed to obtain a measure of the
putrescible material in sewages and effluents in terms of oxygen demand.

_Procedure for effluents._—Divide the total available oxygen, including
the oxygen of nitrite and nitrate, by the relative stability expressed
as a decimal.

_Procedure for sewages._—Make one or two dilutions with fully aerated
distilled water of known dissolved oxygen content. Tap water may be
employed if it is free from nitrates. Vary the relative proportions of
sewage and water to be employed to give a relative stability of 50 to
75. Unless seals[1b][2b][52a] are used bring the water as well as the
sewage to the temperature at which the mixtures are to be incubated
before preparing the dilutions. During the manipulation avoid aeration.
Having made the proper dilutions, determine the relative stability of
each.

Calculate the oxygen demand in parts per million by the formula:

                      Oxygen demand = O(1 − p)/Rp

In this formula, O is the initial dissolved oxygen of the diluting
water, p is the proportion of sewage; and R is the relative stability of
the mixture. Ordinarily the available oxygen in crude sewages, septic
tank effluents, settling tank effluents, and trade wastes can be
neglected.


                         SODIUM NITRATE METHOD.

For the determination of the biochemical oxygen demand the sodium
nitrate method may be used[60a][60c][60d][52a]. The method is based on
the biochemical consumption of oxygen from sodium nitrate by a sewage or
polluted water during an incubation period of ten days at 20° C. A
reasonable excess of sodium nitrate does not give a higher oxygen
demand, as do higher dilutions with aerated water. The oxygen absorbed
from the air in applying the method to sewages is negligible.

_Reagent._—Sodium nitrate solution. Dissolve 26.56 grams of pure sodium
nitrate in 1 liter of distilled water. One cc. of this solution in 250
cc. of sewage represents 50 parts per million of available oxygen. The
strength of the sodium nitrate solution may be varied to suit
conditions.

_Procedure for sewages._—Ordinarily disregard the initial available
oxygen as it is very small compared with the total biochemical oxygen
demand. Add measured amounts of the sodium nitrate solution to the
sewage in bottles holding approximately 250 cc. which have been
completely filled and stoppered. Incubate for 10 days at 20° C. A seal
is not required during incubation. The appearance of a black sediment
and the development of a putrid odor during incubation indicates that
too little sodium nitrate has been added. Methylene blue solution in
proper proportion may be added at the start to serve as an indicator
during the incubation. Domestic sewage usually varies in its oxygen
demand from 100 to 300 parts per million, approximately 30 per cent of
which is used up at 20° C. in the first 24 hours. At the end of the
incubation period determine the residual nitrite and nitrate. Determine
the nitrate by the aluminium reduction method and direct Nesslerization.
To convert the nitrogen into oxygen equivalents, multiply the nitrite
nitrogen by 1.7 and the nitrate nitrogen by 2.9. The difference between
the available oxygen added as sodium nitrate and that found as nitrite
and nitrate at the end of the incubation period is the biochemical
oxygen demand.

_Procedure for industrial wastes._—Employ the same procedure using
larger quantities of the sodium nitrate solution. Make the reaction
alkaline to methyl orange and acid to phenolphthalein. Adjust an acid
reaction with sodium bicarbonate and a caustic alkaline reaction with
weak hydrochloric acid. If the liquid is devoid of sewage bacteria seed
it with sewage after adjusting the reaction.

_Procedure for polluted river waters._—Determine the initial available
oxygen. Unless the river water is badly polluted add 10 parts per
million of sodium nitrate oxygen. Collect carefully, avoiding aeration,
three samples in 250 cc. bottles. To one sample add a definite quantity
of sodium nitrate solution and incubate. Incubate the other two samples
for the determination of the residual free oxygen, nitrite, and nitrate.
If there is free oxygen left, the bottle containing the sodium nitrate
solution may be discarded. If there is no free oxygen determine residual
nitrite and nitrate as directed under the procedure for sewage (p. 72)
and calculate the oxygen demand.




              ANALYSIS OF SEWAGE SLUDGE AND MUD DEPOSITS.


                         COLLECTION OF SAMPLE.

Collect a representative sample of the material. In general more than
one sample should be taken from a spot and a large number of samples
should be collected rather than a few large samples. If the surface
layer is darker and a lower layer consists of pure clay sample only the
surface layer. Samples may be analyzed either separately or as
composites of careful mixtures. After the sample has settled a few
minutes roughly drain or siphon the excess water. Allow sewage sludge to
stand for one hour before draining it free from excess water unless it
is essential to determine the moisture content of the sample originally
collected. If sludge cannot be analyzed within twenty-four hours it is
better not to use air-tight bottles and to add small quantities of
chloroform and keep in the ice box to retard decomposition. At the time
of collection carefully examine mud from the bottom of surface water for
evidence of sewage pollution and macroscopic and microscopic animal and
plant organisms. Record the predominant species. Note the physical
appearance of the material, particularly its color, odor, and
consistency. Express all analytical results in percentage on a dry
basis.


                               REACTION.

Determine the reaction by diluting a definite quantity of the wet sludge
and titrating by the methods given under alkalinity and acidity (pp.
35–39 and 39–41).


                           SPECIFIC GRAVITY.

Weigh to the nearest tenth of a gram a wide-mouthed flask of 100 to 300
cc. capacity, according to the quantity of material available. Then
completely fill the flask with distilled water to the brim and weigh it
again. Empty the flask and fill it completely with fresh sewage sludge
or mud. If the material is of such consistency that it flows readily
fill the flask to the brim and weigh. The specific gravity is equal to
the weight of the sludge or mud divided by the weight of an equal volume
of distilled water.

If the material does not flow readily fill the weighed flask as
completely as possible without exerting pressure during the procedure.
Weigh and then fill the flask to the brim with distilled water. Let it
stand for a few minutes, until trapped air has escaped, then add more
water if necessary and weigh. Subtract the weight of the added water
from the weight of the water that completely fills the flask; the
specific gravity is equal to the weight of the material divided by this
difference. Record the specific gravity only to the second decimal
place.


                               MOISTURE.

Heat approximately 25 grams of sludge or mud in a weighed nickel dish on
the water bath until it is fairly dry. Dry the residue in an oven at
100° C., cool, and weigh. Repeat to approximate constant weight. The
loss in weight is moisture.


                       VOLATILE AND FIXED MATTER.

Ignite, at dull red heat in a hood, the residue from the determination
of moisture until all the carbon has disappeared. Cool the residue in a
desiccator and weigh it. The residue is the fixed matter. The volatile
matter is the difference in weight between the original dried sludge and
the ignited sludge.


                        TOTAL ORGANIC NITROGEN.

_Preparation of sample._—For the determination of organic nitrogen and
fats dry approximately 50 to 75 grams of the sludge or mud in a
porcelain dish first on the water bath and finally in the hot-water oven
until all the moisture has disappeared. Grind the dry material to a fine
powder and keep it in a glass-stoppered bottle.

       _Reagents._—1. Sulfuric acid. Concentrated, nitrogen-free.
                   2. Copper sulfate solution. Ten per cent.
                   3. Potassium permanganate. Crystals.

_First procedure._—Weigh accurately 0.5 gram of dried sludge or 5.0
grams of dried mud and put it into a 500 cc. Kjeldahl flask. Digest it
with 20 cc. of sulfuric acid, or more if necessary, and 1 cc. of copper
sulfate solution to assist the oxidation. Boil for several hours until
the liquid becomes colorless or slightly yellow. Oxidize the residue
with 0.5 gram of potassium permanganate, and follow the “Procedure for
Sewage” (pp. 21–22).

_Second procedure._—The following method is convenient for routine work
at sewage disposal plants. After digestion as described in the first
procedure, cool, transfer to a glass-stoppered 100 cc. flask, dilute
with distilled water to 100 cc., and mix well. Transfer 50 cc. with a
pipette into another 100 cc. volumetric flask, and make this portion
alkaline with 50 per cent sodium hydroxide, testing a drop of the liquid
on a porcelain plate with phenolphthalein to insure neutralization. The
formation of a floc usually indicates that neutralization is complete.
Dilute the solution to 100 cc., pour it into a small glass-stoppered
bottle and permit it to stand until the next day. Nesslerize an aliquot
portion of the clear supernatant liquid, and calculate the percentage of
nitrogen in the material.


                         ETHER-SOLUBLE MATTER.

Fats are usually determined only on sewage sludge, but some mud deposits
contain small quantities due to the presence of trade wastes.

_Procedure._—Weigh 0.5 to 25 grams of dry material according to the
quality of the sludge or mud. Add water to the weighed portion in a
porcelain dish and acidify the mixture with N/50 sulfuric acid in the
presence of litmus tincture or azolitmin solution as indicator. Avoid
adding too much acid as an excess gives too high results on account of
fatty acid residues. Evaporate the acidified mixture to dryness on the
water bath, and heat it in the hot air oven at 100° C. two to three
hours. Extract the dry residue with boiling ether, rubbing the sides and
bottom of the dish to insure complete solution of the fat. Three
extractions with ether are usually sufficient. Filter the ether solution
through a 5 cm. filter paper into a small flask. Evaporate the ether
slowly, dry the fatty extract for half an hour at 100° C., cool in a
desiccator, and weigh. If it is desirable, particularly with certain
industrial wastes, to determine the quantity of saponified fat determine
the fats with and without the addition of acid. The difference between
the quantities found by the two determinations is the content of
saponified fat.


                            FERROUS SULFIDE.

The liberation of hydrogen sulfide on adding dilute hydrochloric acid to
a sludge indicates the presence of ferrous sulfide. As ferrous sulfide
quickly oxidizes on exposure to air a quantitative determination of this
constituent must be made immediately after collection of the sample.

_Procedure._—Heat a definite portion of the sludge with hydrochloric
acid in a flask. Pass the liberated gas through bromine water or
hydrogen peroxide. Determine gravimetrically the sulfate in the
oxidizing solution, and calculate the equivalent of ferrous sulfide by
multiplying the weight of barium sulfate by 0.376.


                       BIOCHEMICAL OXYGEN DEMAND.

The quantity of river mud most suitable for the determination of the
biochemical oxygen demand ranges within certain limits, largely
according to the amount of oxidizable matter present. For examinations
of river mud prepare a 1 per cent stock suspension in distilled water or
tap water saturated with oxygen and free from nitrate; use in the test a
dilution of this stock suspension equivalent to a concentration of 1 to
10 grams per liter of mud. For examinations of fresh sewage sludge
prepare a 1 per cent stock suspension in a similar manner, but use in
the test a dilution equivalent to only 0.1 to 1.0 gram per liter of wet
material. For examinations of dried sludges, which have undergone more
or less oxidation higher concentrations may be required.

_Procedure._—Place a measured portion of the sample, or the proper
amount of the 1 per cent stock suspension of the sample, in a 300 cc.
narrow-mouth glass-stoppered bottle, and dilute it to the desired
concentration with water saturated with oxygen. Determine the oxygen
content at 20° C. of the waters that are used for dilution. This
determination must be made before the mud or sludge is added because
iron sulfide in the mud or sludge rapidly consumes part of the dissolved
oxygen. Incubate at 20° C. for five days.

Shortly before the determination of the oxygen remaining in solution at
the end of five days rotate the bottle once or twice to mix its contents
and allow sedimentation for about 30 minutes. Siphon the greater part of
the liquid through a narrow-bore siphon into a 150 cc. bottle, which has
been filled with carbon dioxide. Reject the first 25 cc. of the siphoned
liquid and allow a little to overflow at the end of siphoning. Determine
the oxygen content of the solution in the bottle in the usual way (pp.
65–68). Report the oxygen demand in percentage of the dried mud or
sludge.




                         ANALYSIS OF CHEMICALS.


The following sections describe the accepted methods for the analysis of
the chemicals commonly used in the treatment of water.


                               REAGENTS.

1. Distilled water. In practically all the tests of chemicals it is
necessary to use exclusively distilled water that has been freshly
boiled to free it from carbon dioxide and oxygen.

2. Concentrated hydrochloric acid. Sp. gr. 1.20.

3. Hydrochloric acid, N/2.

4. Hydrochloric acid, N/10.

5. Ammonium hydroxide. Redistilled; sp. gr. 0.90.

6. Dilute sulfuric acid. Dilute 1 part of concentrated sulfuric acid
with 3 parts of freshly boiled distilled water.

7. Methyl orange indicator. See page 36.

8. Phenolphthalein indicator. See page 36.

9. Bromine water.

10. Stannous chloride, N/20. This should be frequently standardized by
titration against a standard iron solution. One cc. of N/20 stannous
chloride is equal to 0.0028 gram of iron (Fe) estimated in the ferrous
state.

11. Sodium hydroxide, N/1. Free from carbonate. This should be
frequently standardized by titration against a standard acid solution in
presence of phenolphthalein indicator. One cc. of N/1 sodium hydroxide
is equal to 0.049 gram of sulfuric acid (H_{2}SO_{4}), or to 0.03645
gram of hydrochloric acid (HCl).

12. Sodium hydroxide, N/20. Free from carbonate.

13. Standard potassium permanganate. A N/10 solution. One cc. of N/10
potassium permanganate is equal to 0.0056 gram of iron (Fe) estimated in
the ferrous state.

14. Alcohol. Ethyl alcohol, 95 per cent.

15. Sugar. Solid granulated cane sugar.


                         SULFATE OF ALUMINIUM.

Determine and report insoluble matter, aluminium oxide (Al_{2}O_{3}),
ferric oxide (Fe_{2}O_{3}), ferrous oxide (FeO), basicity ratio, and, if
present, free acid as H_{2}SO_{4}. If the material is what is known as
“granular” sulfate mix it well before sampling. If it is in lump form
crush it to ⅛ to ¼ inch size, mix, and sample it. It is unnecessary to
grind the sample to a fine powder, but it is preferable to have the
particles fairly uniform in size.


                           INSOLUBLE MATTER.

Treat 10 grams of the sample with 100 cc. of distilled water and digest
one hour at boiling temperature. Filter through a weighed Gooch crucible
and wash the insoluble matter with hot water freshly boiled to free it
from carbon dioxide. Dry the crucible to constant weight at 100° C.,
cool, and weigh. Report the percentage of insoluble matter.


                     OXIDES OF IRON AND ALUMINIUM.

Dilute the filtrate from the determination of insoluble matter to 500
cc. with water free from carbon dioxide and thoroughly mix the solution.
Transfer 50 cc. of the solution to a 250 cc. beaker, add about 150 cc.
of water and 5 cc. of concentrated hydrochloric acid, and heat to
boiling. Add ammonium hydroxide in slight excess; when the solution has
been almost neutralized it is convenient to add a drop of methyl orange
indicator and then to add about 0.5 cc. of ammonium hydroxide after the
solution is neutral to the indicator. Digest at about 100° C. for a few
minutes and filter. Some analysts prefer to wash this gelatinous
precipitate with hot water by decantation, and some to wash it evenly
distributed over the surface of a filter paper; either method may be
used. It is difficult to free it completely from impurities and it is
not necessary to do so unless unusual quantities of calcium, magnesium,
sodium, or potassium are present. While the precipitate is being washed
do not allow it to become dry, as it then packs and can not be washed
clean. After most of the water has drained drying the filter may be
hastened by placing it on a sheet of blotting paper. If much iron is
present completely dry the precipitate, remove it from the paper, and
ignite the paper separately. Finally, blast the precipitate, with free
access of air to the crucible, for five or ten minutes, cool, and weigh
as oxides of iron and aluminium (Fe_{2}O_{3} + Al_{2}O_{3}).

Subtract the content of total iron, expressed as ferric oxide
(Fe_{2}O_{3}), from the weight of the combined oxides and report the
difference as aluminium oxide (Al_{2}O_{3}), in percentage.


                              TOTAL IRON.

As filter alum usually contains 0.2 to 0.3 per cent of iron use a 10
gram sample for the determination of total iron. Treat the sample with
50 cc. of freshly boiled distilled water and add 5 cc. of concentrated
hydrochloric acid and 1 cc. of bromine water. Evaporate the solution to
dryness, dissolve the residue in water, and wash it into a flask with
sufficient water to make the volume about 50 cc. Add 50 cc. of
concentrated hydrochloric acid, boil to expel oxygen, and titrate, as
hot as possible, with N/20 stannous chloride.

If a 10 gram sample is used the percentage of iron (Fe) is equal to the
number of cubic centimeters of stannous chloride used multiplied by
0.028, and the percentage of iron expressed as ferric oxide is equal to
the number of cubic centimeters of stannous chloride used multiplied by
0.040.


                              FERRIC IRON.

As filter alum usually contains 0.02 to 0.04 per cent of ferric iron use
a 20 gram sample. Boil 50 cc. of distilled water to expel oxygen, add 50
cc. of concentrated hydrochloric acid, and add the sample while the
solution is boiling. Keep it boiling till the sample is dissolved. The
flask should be kept filled with carbon dioxide during this process by
dropping in occasionally small amounts of sodium carbonate. When
solution of the sample is complete titrate it hot immediately with N/20
stannous chloride.

If a 20 gram sample is used the percentage of ferric oxide (Fe_{2}O_{3})
is equal to the number of cubic centimeters of stannous chloride used
multiplied by 0.020.


                             FERROUS IRON.

The content of ferrous iron is the difference between total and ferric
iron. The percentage of ferrous oxide (FeO) is, therefore, equal to 0.90
times the difference between the percentage of total iron expressed as
ferric oxide and the percentage of ferric iron expressed as ferric
oxide. Report the percentage of ferrous oxide (FeO).


                            BASICITY RATIO.

Transfer 50 cc. of the filtrate from the determination of insoluble
matter to a 200 cc. casserole and dilute it to 100 cc. Boil the solution
and titrate it at boiling temperature with N/1 sodium hydroxide in
presence of phenolphthalein indicator. The percentage of acidity in
equivalent of sulfuric acid (H_{2}SO_{4}) is equal to the number of
cubic centimeters of sodium hydroxide used multiplied by 4.9. In this
titration iron and aluminium are precipitated as hydroxides and any free
acid is neutralized.

Calculate the percentage of sulfuric acid equivalent to the determined
percentages of aluminium oxide, ferric oxide, and ferrous oxide by the
following formula:

            2.88 Al_{2}O_{3} + 1.83 Fe_{2}O_{3} + 1.36 FeO.

If this percentage of acid equivalent is less than that found by
titration report the difference as percentage of free acid. If the
percentage of acid equivalent is greater than that found by titration
the difference divided by 2.88 is the percentage equivalent to the
excess of aluminium oxide present. Divide this excess by the percentage
of total aluminium oxide and report the quotient as the basicity ratio.


                                 LIME.

Mix well the sample, which should contain no lumps. If foreign matter is
present grind the sample to pass a 100–mesh sieve.

Place 20 grams of granulated cane sugar and 1 gram of the sample in a
250 cc. glass-stoppered bottle, tightly stopped, and mix the mass by
rolling. Do not shake hard as much of the lime could thus be lost as
dust. Then add 187.4 cc. of distilled water freshly boiled to expel
carbon dioxide. This makes 200 cc. of sugar solution. The lime is mixed
dry with the sugar and the water added later to keep the lime from
lumping. After shaking the sugar solution one hour titrate 50 cc. of it
with N/2 hydrochloric acid in presence of methyl orange indicator. The
acid used is equivalent to the carbonate and hydroxide in 0.25 gram of
the sample.

Filter the remainder of the sugar solution, discarding the first 25 cc.
of filtrate. Titrate 50 cc. of the filtrate with N/2 hydrochloric acid
in presence of methyl orange indicator. The acid used is equivalent to
the hydroxide in 0.25 gram of the sample.

If a 1 gram sample is used the percentage of calcium oxide (CaO) is
equal to 5.6 times the number of cubic centimeters of hydrochloric acid
used in the second titration; and the percentage of calcium carbonate
(CaCO_{3}) equivalent to the carbonate present is equal to 10 times the
difference in cubic centimeters between the results of the two
titrations.


                            SULFATE OF IRON.


                           INSOLUBLE MATTER.

Treat 10 grams of the sample with 100 cc. of freshly boiled distilled
water cooled to 30° C. or less. When solution is complete filter through
a weighed Gooch crucible, wash, dry, cool, and weigh. Report the weight
of the residue, in percentage, as insoluble matter.


                        IRON AS FERROUS SULFATE.

Dissolve 1 gram of the sample and dilute to 200 cc. with freshly boiled
distilled water cooled to 30° C. or less. Add 5 cc. of dilute sulfuric
acid (1 to 3) to a 50 cc. portion of the solution and titrate with N/10
potassium permanganate. The percentage of ferrous sulfate
(FeSO_{4}.7H_{2}O) is equal to 11.12 times the number of cubic
centimeters of potassium permanganate used.


                                ACIDITY.

Shake 12.25 grams of the sample in a 150 cc. bottle with 75 cc. of 95
per cent alcohol for ten minutes. Run a blank. Filter rapidly both
sample and blank and wash rapidly with alcohol sufficient to make 100
cc. of filtrate. Titrate with N/20 sodium hydroxide in presence of
phenolphthalein and subtract the result of titrating the blank from that
of titrating the solution of the sample. The percentage of acidity,
expressed as sulfuric acid (H_{2}SO_{4}), is equal to 0.02 times the
number of cubic centimeters of sodium hydroxide used.


                               SODA ASH.


                           INSOLUBLE MATTER.

Treat 5.305 grams of the sample with 200 cc. of freshly boiled and
cooled distilled water. When solution is complete filter through an
asbestos mat in a weighed Gooch crucible, dry, cool, and weigh. Report
the weight of the residue, in percentage, as insoluble matter.


                           AVAILABLE ALKALI.

Dilute the filtrate from the determination of insoluble matter to 1,000
cc. and thoroughly mix. Titrate 25 cc. of this dilution with N/10
hydrochloric acid in presence of methyl orange indicator. The percentage
of available alkali, expressed as sodium carbonate (Na_{2}CO_{3}), is
equal to 4 times the number of cubic centimeters of hydrochloric acid
used.




                         CHEMICAL BIBLIOGRAPHY.


The subjoined bibliography comprises the publications cited in the text
of this report. The references are arranged alphabetically by authors’
names and under each author in order of dates of publication. When
different pages of a single work are cited letters are used in
connection with the number that refers to the work.

Bibliography 1:

  ANDREWS, L. W. Sprengel’s method for colorimetric determination of
  nitrates: _J. Am. Chem. Soc._, Vol. 26, pp. 388–91, 1904.

Bibliography 1a:

  _a._ ASSOC. OFF. AG. CHEMISTS. Determination of iodine and bromine:
  _J. A. O. A. C._, Vol. 1, No. 4, pt. 1, pp. 47–8, 1916.

Bibliography 1b:

  _b._ BACHMANN, FRANK. A new seal for the prevention of aeration in
  deaerated liquids: _J. Ind. Eng. Chem._, Vol. 6, pp. 764–5, 1914.

Bibliography 2:

  BARTOW, EDWARD, and RODGERS, J. S. Determination of nitrates by
  reduction with aluminium: _Am. J. Public Hygiene_, new ser., Vol. 5,
  pp. 536–44, 1909; also _Illinois Univ. Bull._, Vol. 7, No. 2 (Water
  Survey Ser. No. 7), pp. 14–27, 1909.

Bibliography 2a:

  _a._ BLINN, WILLIAM. Determination of manganese as sulfate and by the
  sodium bismuthate method: _J. Am. Chem. Soc._, Vol. 34, pp. 1379–98,
  1912.

Bibliography 2b:

  _b._ BUSWELL, A. M. Modified apparatus for the putrescibility test:
  _J. Ind. Eng. Chem._, Vol. 6, p. 325, 1914.

Bibliography 3:

  CALDWELL, G. C. A method in part for the sanitary examination of water
  and for the statement of results, offered for general adoption: _J.
  Anal. Chem._, Vol. 3, pp. 398–403, 1889.

Bibliography 4:

  CALKINS, G. N. A study of odors observed in the drinking waters of
  Massachusetts: _Report Mass. State Board of Health_, pp. 355–80, 1892.

Bibliography 5:

  CHAMOT, E. M., and PRATT, D. S. A study on the phenoldisulfonic acid
  method for the determination of nitrates in water: _J. Am. Chem.
  Soc._, Vol. 31, pp. 922–8, 1909; Vol. 32, pp. 630–7, 1910; and
  REDFIELD, H. W., Vol. 33, pp. 366–81, 381–4, 1911.

Bibliography 6:

  CLARK, H. W. Experiments upon the purification of sewage and water at
  the Lawrence Experiment Station: _Report Mass. State Board of Health_,
  pp. 427–578, 1896.

Bibliography 7:

  CLARK, H. W., and FORBES, F. B. Methods for the determination of lead,
  tin, zinc, and copper in drinking waters: _Report Mass. State Board of
  Health_, pp. 577–85, 1898; pp. 498–506, 1900.

Bibliography 8:

  COHN, A. I. Tests and reagents, 1st ed., p. 216, John Wiley & Sons,
  New York, 1903.

Bibliography 9:

  DIBDIN, W. J. The purification of sewage and water, 3d ed., pp.
  345–51, D. Van Nostrand Co., New York, 1903.

Bibliography 10:

  DOLE, R. B. The quality of the surface waters in the United States:
  _U. S. Geol. Survey Water-Supply Paper_ 236, pp. 15–9, 1909.

Bibliography 11:

  DRAPER, H. N. Lacmoid and carminic acid as reagents for alkalies:
  _Chem. News_, Vol. 51, pp. 206–7, 1885.

Bibliography 13:

  DROWN, T. M., and MARTIN, HENRY. Determination of organic nitrogen in
  natural waters by the Kjeldahl method: _Tech. Quart._, Vol. 2, No. 3;
  _Chem. News_, Vol. 59, pp. 272–6, 1889.

Bibliography 14:

  DROWN, T. M. The chemical examination of waters and the interpretation
  of analyses: _Examinations by the State Board of Health of water
  supplies of Mass. 1887–90_, pt. 1, Examination of water supplies, pp.
  519–78, 1890.

Bibliography 15:

  ——. Report upon the examination of the outlets of sewers and the
  effect of sewage disposal in Massachusetts: _Report Mass. State Board
  of Health_, pp. 285–452, 1902.

Bibliography 16:

  ——, and HAZEN, ALLEN. A report of the chemical work done at the
  Lawrence Experiment Station: _Examinations by the State Board of
  Health of water supplies of Mass. 1887–90_, pt. 2, Purification of
  sewage and water, pp. 707–34, 1890.

Bibliography 17:

  DUPRE, Dr. Some observations on the permanganate test in water
  analysis: _Analyst_, Vol. 10, pp. 118–22, 1885.

Bibliography 18:

  ELLMS, J. W. A study of the relative value of lacmoid, phenacetolin,
  and erythrosine as indicators in the determination of the alkalinity
  of water by Hehner’s method: _J. Am. Chem. Soc._, Vol. 21, pp. 359–69,
  1899.

Bibliography 20:

  ——, and BENEKER, J. C. The estimation of carbonic acid in water: _J.
  Am. Chem. Soc._, Vol. 23, pp. 405–31, 1901.

Bibliography 21:

  FARNSTEINER, BUTTENBURG, and KORN, _Leitfaden für die chemische
  Untersuchung von Abwasser_, Berlin, p. 20, 1902.

Bibliography 22:

  FITZGERALD, and FOSS, _Report Boston Water Board_, p. 86, 1893.

Bibliography 23:

  FORBES, F. B., and PRATT, G. H. The determination of carbonic acid in
  drinking water: _J. Am. Chem. Soc._, Vol. 25, pp. 742–56, 1903.

Bibliography 24:

  FOWLER, G. J. Sewage works analyses, pp. 21–37, John Wiley & Sons, New
  York, 1902;

Bibliography 24a:

  pp. 31–4;

Bibliography 24b:

  pp. 58–60;

Bibliography 24c:

  pp. 86–9;

Bibliography 24d:

  pp. 89–95;

Bibliography 24e:

  pp. 96–7;

Bibliography 24f:

  pp. 98–100.

Bibliography 26:

  FOWLER, G. J. _Univ. of Manchester Lecture_, March, 1904, Pamphlet, p.
  7.

Bibliography 27:

  FOX, CHARLES J. J. On the coefficients of absorption of nitrogen and
  oxygen in distilled water and sea water and of atmospheric carbonic
  acid in sea water: _Trans. Faraday Soc._, Vol. 5, pp. 68–87, 1909.

Bibliography 29:

  ——. The composition of sewage in relation to problems of disposal:
  _Tech. Quart._, Vol. 16, pp. 132–160, 1903.

Bibliography 30:

  ——. Experiments upon the purification of sewage and water at the
  Lawrence Experiment Station: _Report Mass. State Board of Health_, pp.
  447–700, 1894.

Bibliography 31:

  HAYWOOD, J. K., and WARNER, H. J. Arsenic in papers and fabrics: _U.
  S. Agri. Dept. Bur. Chem. Bull. 86_, pp. 25–7, 1904.

Bibliography 32:

  GILL, A. H. On the determination of nitrates in potable water: _J. Am.
  Chem. Soc._, Vol. 16, pp. 122–32, 193–7, 1894.

Bibliography 33:

  GOOCH, F. A. A method for the separation and estimation of boric acid:
  _Am. Chem. J._, Vol. 9, pp. 23–33, 1887.

Bibliography 34:

  ——. A method for the separation of sodium and potassium from lithium
  by the action of amyl alcohol on the chlorides: _Am. Chem. J._, Vol.
  9, pp. 33–51, 1887; also _U. S. Geol. Survey Bull. 422_, p. 175; also
  _U. S. Agri. Dept. Bur. Chem._, _Bull. 152_, p. 80, 1911.

Bibliography 35:

  GOTTSCHALK, V. H., and ROESLER, H. A. Action of soap on calcium and
  magnesium solutions: _J. Am. Chem. Soc._, Vol. 26, pp. 851–6, 1904.

Bibliography 36:

  GRANDVAL, AL., and LAJOUX, H. Nouveau procédé pour la recherche et le
  dosage rapide de faibles quantités d’ acide nitrique dans l’air,
  l’eau, le sol, etc., _Comptes rend._, Vol. 101, pp. 62–5, 1885.

Bibliography 37:

  HANDY, J. O. Determination of acidity or alkalinity: _Proc. Engineers
  Soc. West. Pa._, Vol. 19, p. 705, 1903.

Bibliography 38:

  HARRINGTON, CHARLES, and RICHARDSON, M. W. A manual of practical
  hygiene, 5th ed., pp. 457–62, Lea & Febiger, Phila. and New York,
  1914.

Bibliography 39:

  HAZEN, ALLEN. On the determination of chlorine in water: _Am. Chem.
  J._, Vol. 11, pp. 409–14, 1889.

Bibliography 40:

  ——. Apparatus for the determination of ammonias in sand sewage: _Am.
  Chem. J._, Vol. 12, pp. 427–8, 1890.

Bibliography 42:

  ——. Report on the chemical precipitation of sewage: _Examinations by
  the State Board of Health of water supplies of Mass., 1887–90_, pt. 2,
  Purification of water and sewage, pp. 735–91, 1890.

Bibliography 43:

  ——. A new color standard for natural waters: _Am. Chem. J._, Vol. 14,
  pp. 300–10, 1892.

Bibliography 44:

  ——. Experiments on the purification of sewage at the Lawrence
  Experiment Station: _Report Mass. State Board of Health_, pp. 393–448,
  1892.

Bibliography 45:

  ——, and CLARK, H. W. On the effect of temperature upon the
  determination of ammonia by Nesslerization: _Am. Chem. J._, Vol. 12,
  pp. 425–6, 1890.

Bibliography 46:

  ——, and ——. On the determination of nitrates in water: _Chem. News_,
  Vol. 64, pp. 162–4, 1891.

Bibliography 47:

  HEHNER, OTTO. Estimation of hardness without soap solution: _Analyst_,
  Vol. 8, pp. 77–81, 1883.

Bibliography 48:

  HILLEBRAND, W. F. The analysis of silicate and carbonate rocks: _U. S.
  Geol. Survey Bull. 422_, pp. 113–8, 127–8, 141–6, 219–20, 221, 222,
  and 230, 1910.

Bibliography 49:

  HOLLIS, F. S. Methods for the determination of color and the relation
  of the color to the character of the water: _J. N. E. Water Works
  Assoc._, Vol. 13, pp. 94–118, 1898.

Bibliography 50:

  HOWE, FREELAND, JR. A new method for determining the color of the
  turbidity of water: _Eng. Rec._, Vol. 50, pp. 720–1, 1904.

Bibliography 51:

  ILOSVAY, L. L’acide azoteux dans la salive et dans l’air exhalé:
  _Bull. de la Sociéte Chimique_, ser. 3, Vol. 2, pp. 388–91, 1889.

Bibliography 52:

  JACKSON, D. D. Permanent standards for use in the analysis of water:
  _Tech. Quart._, Vol. 13, pp. 314–26, 1900.

Bibliography 52a:

  _a._ ——, and HORTON, W. A. Experiments on the putrescibility test for
  sewage and sewage effluents: _J. Ind. Eng. Chem._, Vol. 1, pp. 328–33,
  1909.

Bibliography 53:

  ——, and ELLMS, J. W. On odors and tastes of surface waters, with
  special reference to anabaena, a microscopical organism found in
  certain water supplies of Massachusetts: _Tech. Quart._, Vol. 10, pp.
  410–20, 1897.

Bibliography 54:

  JOHNSON, G. A. Report on sewage purification at Columbus, Ohio, made
  to the chief engineer of the Board of Public Service, p. 47, 1905.

Bibliography 55:

  KENDALL, L. M., and RICHARDS, E. H. Permanent standards in water
  analysis: _Tech. Quart._, Vol. 17, pp. 277–80, 1904.

Bibliography 56:

  KIMBERLEY, A. E., and HOMMON, H. B. The practical advantages of the
  Gooch crucible in the determination of the total and volatile
  suspended matter in sewage: _Pub. Health Papers and Repts._, _Am. Pub.
  Health Assoc._, Vol. 31, pt. 2, pp. 123–35, 1905.

Bibliography 57:

  KINNICUTT, L. P. Quoted by Gage, _J. Am. Chem. Soc._, Vol. 27, p. 339,
  1905.

Bibliography 58:

  KJELDAHL, J. Neue Methode zur Bestimmung des Stickstoffs in
  organischen Körpern: _Z. anal. Chem._, Vol. 22, pp. 366–82, 1883.

Bibliography 59:

  KLUT, _Mitt. a. d. König. Prüfungs_, Vol. 12, p. 186.

Bibliography 60:

  LEACH, A. E. Food inspection and analysis, pp. 493, 495, and 497, John
  Wiley & Sons, New York, 1904.

Bibliography 60a:

  _a._ LEDERER, ARTHUR. A new method for determining the relative
  stability of sewage, effluent, or polluted river water: _J. Infect.
  Diseases_, Vol. 14, pp. 482–97, 1914.

Bibliography 60b:

  _b._ ——. A serious fallacy of the “standard” methylene blue
  putrescibility test: _Am. J. Pub. Health_, Vol. 4 (old series Vol.
  10), pp. 241–8, 1914.

Bibliography 60c:

  _c._ ——. Notes on the practical application of the “saltpeter method”
  for determining the strength of sewages: _Am. J. Pub. Health_, Vol. 5,
  pp. 354–61, 1915.

Bibliography 60d:

  _d._ ——. Determination of the biochemical oxygen demand by the
  saltpeter method in stockyards, tannery, and corn products wastes: _J.
  Ind. Eng. Chem._, Vol. 7, pp. 514–6, 1915.

Bibliography 61:

  LEEDS, A. R. Estimation by titration of dissolved carbon dioxide in
  water: _J. Am. Chem. Soc._, Vol. 13, pp. 98–9, 1891.

Bibliography 62:

  ——. The alteration of standard ammonium solutions when kept in the
  dark: _Proc. Am. Chem. Soc._, Vol. 2, p. 1, 1878.

Bibliography 63:

  LEFFMAN, HENRY. Examination of water, 3d ed., pp. 46–50, P.
  Blakiston’s Son & Co., Philadelphia, 1895;

Bibliography 63a:

  pp. 44–6;

Bibliography 63b:

  pp. 57–8.

Bibliography 64:

  ——. Examination of water, 7th ed., pp. 35–7, P. Blakiston’s Son & Co.,
  Philadelphia, 1915;

Bibliography 64a:

  pp. 64–7.

Bibliography 65:

  LEVY, D. D. _Ann. de l’Observatoire de Mont-Souris_, 1883 _et seq._

Bibliography 66:

  LOVIBOND, J. W. A description of the tintometer with some remarks on
  its application to chemical analysis: _J. Soc. Chem. Ind._, Vol. 7,
  pp. 424–6, 1888.

Bibliography 67:

  MALLET, J. W. Water analysis: _Annual Report National Board of
  Health_, pp. 189–354, 1882.

Bibliography 68:

  MASON, W. P. Examination of water, 4th ed., pp. 85–9, John Wiley &
  Sons, New York, 1910;

Bibliography 68a:

  pp. 33–41;

Bibliography 68b:

  pp. 59–74;

Bibliography 68c:

  pp. 106–9.

Bibliography 69:

  MCGOWAN, GEORGE. Kjeldahl process for the estimation of total nitrogen
  and (indirectly) of total organic nitrogen: _Royal Commission on
  Sewage Disposal_, Vol. 4, pt. 5, pp. 24–31, 1904;

Bibliography 69a:

  pp. 37–41;

Bibliography 69b:

  pp. 47–8.

Bibliography 70:

  PALMER, A. W. Chemical survey of the waters of Illinois, Report for
  years 1897–1902, pp. 27–8, Univ. Ill., 1903.

Bibliography 71:

  ——. Report of the University of Illinois [in Report of streams
  examination, Sanitary Dist. Chicago], p. 60, Chicago, 1903;

Bibliography 71a:

  p. 56;

Bibliography 71b:

  pp. 61–4.

Bibliography 72:

  PARKER, G. H. Report of the biologist: _Examinations by the State
  Board of Health of water supplies of Mass., 1887–90_, pt. 1,
  Examination of water supplies, pp. 583–7, 1890.

Bibliography 73:

  PARMELEE, C. L., and ELLMS, J. W. On rapid methods for the estimation
  of the weight of suspended matters in turbid waters: _Tech. Quart._,
  Vol. 12, pp. 145–64, 1899.

Bibliography 74:

  PFEIFER, J., and WARTHA, PROF. Kritische Studien über Untersuchung und
  Reinigung des Kesselspeisewassers: _Z. angew. Chem._, Vol. 15, pp.
  193–207, 1902.

Bibliography 75:

  PHELPS, E. B. A critical study of the methods in current use for the
  determination of free and albuminoid ammonia in sewage, _Public Health
  Papers and Reports, Am. Pub. Health Assoc._, Vol. 29, p. 354, 1904; J.
  Infect. Dis., Vol. 1, p. 327, 1904.

Bibliography 76:

  ——. The determination of the organic nitrogen in sewage by the
  Kjeldahl process: _J. Infect. Dis._, Supp. 1, pp. 255–72, 1905.

Bibliography 77:

  ——. The determination of small quantities of copper in water: _J. Am.
  Chem. Soc._, Vol. 28, pp. 368–72, 1906.

Bibliography 78:

  ——. Putrescibility and stability of sewage effluents: _Contrib. Sanit.
  Research Lab., Mass. Inst. Tech._, Vol. 5, p. 87, 1909; also The
  disinfection of sewage and sewage filter effluents: _U. S. Geol.
  Survey Water-Supply Paper 229_, pp. 74–88, 1909.

Bibliography 79:

  Preface to report of committee on the pollution of water supplies:
  _Public Health Papers and Reports, Am. Pub. Health Assoc._, Vol. 23,
  pp. 56–7, 1897. Report, pp. 58–100.

Bibliography 80:

  Report of committee on standard methods of water analysis: _Public
  Health Papers and Reports, Am. Pub. Health Assoc._, Vol. 27, pp.
  377–91, 1901.

Bibliography 81:

  PROCTOR, H. R. Some recent methods of technical water analysis: _J.
  Soc. Chem. Ind._, Vol. 23, pp. 8–11, 1904.

Bibliography 82:

  ——. On a modified form of tintometer or colorimeter: _J. Soc. Chem.
  Ind._, Vol. 14, pp. 122–4, 1895.

Bibliography 83:

  RICHARDS, E. H., and ELLMS, J. W. The coloring matter of natural
  waters, its source, composition, and quantitative measurement: _J. Am.
  Chem. Soc._, Vol. 18, pp. 68–81, 1896.

Bibliography 84:

  RIDEAL, SAMUEL. Sewage, 2d ed., pp. 38–40, John Wiley & Sons, New
  York, 1901;

Bibliography 84a:

  pp. 31–4.

Bibliography 85:

  Royal Commission on Sewage Disposal, Testimony, Vol. 2, pp. 326–37,
  1902.

Bibliography 86:

  SCHOLL, CLARENCE. The perchloric method of determining potassium as
  applied to water analysis: _J. Am. Chem. Soc._, Vol. 36, pp. 2985–9,
  1914.

Bibliography 87:

  SEYLER, C. A. Notes on water analysis: _Chem. News_, Vol. 70, pp.
  82–3, 104–5, 112–4, 140–1, 151–2, and 187, 1894.

Bibliography 88:

  ——. The estimation of carbonic acid in natural waters: _Analyst_, Vol.
  22, pp. 312–9, 1897.

Bibliography 89:

  SMART, CHAS. Report of the committee on pollution of water supplies:
  _Public Health Papers and Reports, Am. Pub. Health Assoc._, Vol. 20,
  pp. 72–82, 1895;

Bibliography 89a:

  pp. 459–516.

Bibliography 90:

  SPRENGEL, HERMANN. Ueber die Erkennung der Salpetersaüre: _Ann. Physik
  und Chemie_, Vol. 121, pp. 188–91, 1864.

Bibliography 91:

  Standard methods of water analysis: _Science_, new ser., Vol. 12, pp.
  906–15, 1900.

Bibliography 92:

  STEARNS, F. P., and DROWN, T. M. Discussion of special topics relating
  to the quality of public water supplies: _Examinations by the State
  Board of Health of water supplies of Mass., 1887–90_, pt. 1,
  Examination of water supplies, pp. 740–9, 1890.

Bibliography 93:

  STREET, J. P. Report on nitrogen: [In _Proc. Assoc. Off. Agri.
  Chemists_]; _U. S. Agri. Dept. Bur. Chem. Bull. 49_, pp. 12–25, 1897.

Bibliography 94:

  SUTTON, FRANCIS. Volumetric analysis, 10th ed., pp. 72–4, P.
  Blakiston’s Son & Co., Philadelphia, 1911;

Bibliography 94a:

  pp. 99–101;

Bibliography 94b:

  pp. 239 and 477;

Bibliography 94c:

  pp. 470–1;

Bibliography 94d:

  pp. 479–83;

Bibliography 94e:

  p. 473, 479–83;

Bibliography 94f:

  pp. 484–8.

Bibliography 95:

  TATLOCK, R. R., and THOMPSON, R. T. The analysis of waters and their
  changes in composition when employed in steam raising: _J. Soc. Chem.
  Ind._, Vol. 23, pp. 428–31, 1904.

Bibliography 96:

  THOMAS, G. E., and HALL, C. A. New apparatus in water analysis: _J.
  Am. Chem. Soc._, Vol. 24, pp. 535–9, 1902.

Bibliography 97:

  THOMSON, R. T. Use of litmus, methyl orange, phenacetolin, and
  phenolphthalein as indicators: _Chem. News_, Vol. 47, pp. 123–7, 1883.

Bibliography 98:

  THOMSON, ANDREW. Colorimetric method for determining small quantities
  of iron: _J. Chem. Soc._, Vol. 47, pp. 493–7, 1885.

Bibliography 99:

  THRESH, J. C. A new method of estimating the oxygen dissolved in
  water: _J. Chem. Soc._, Vol. 57, pp. 185–95, 1890.

Bibliography 100:

  ——. The examination of water and water supplies, p. 200, Philadelphia,
  1904;

Bibliography 100a:

  p. 219;

Bibliography 100b:

  p. 195;

Bibliography 100c:

  p. 282.

Bibliography 101:

  TIDY, C. M. The process for determining the organic purity of potable
  waters: _J. Chem. Soc._, Vol. 35, pp. 46–106, 1879.

Bibliography 102:

  TIEMANN, FERDINAND, and GÄRTNER, AUGUST. Handbuch der Wässer, 4th ed.,
  pp. 255–8, Friedrich Vieweg und Sohn, Braunschweig, 1895.

Bibliography 103:

  TREADWELL, F. P. [translated by Hall, W. T.], Analytical Chemistry, 3d
  ed., Vol. 2, pp. 687–688, John Wiley & Sons, New York, 1911;

Bibliography 103a:

  pp. 50–3.

Bibliography 104:

  TROMMSDORFF, HUGO. Bestimmung der Organischen Substanzen: _Zeit. Anal.
  Chem._, Vol. 8, p. 344, 1869.

Bibliography 105:

  U. S. GEOLOGICAL SURVEY. Measurement of color and turbidity of water,
  Form 9–182, Washington, 1902.

Bibliography 106:

  WANKLYN, J. A. Verification of Wanklyn, Chapman, and Smith’s water
  analyses on a series of artificial waters: _J. Chem. Soc._, Vol. 20,
  pp. 591–5, 1867.

Bibliography 107:

  ——. Water analysis, 10th ed., pp. 33–5, Kegan, Paul, Trench, Trübner,
  & Co., Ltd., London, 1896;

Bibliography 107a:

  pp. 106–7.

Bibliography 108:

  WARINGTON, ROBERT. Note on the appearance of nitrous acid during
  evaporation of water: _J. Chem. Soc._, Vol. 39, pp. 229–34, 1881.

Bibliography 109:

  WARREN, H. E., and WHIPPLE, G. C. The thermophone, a new instrument
  for determining temperatures: _Tech. Quart._, Vol. 8, pp. 125–52,
  1895.

Bibliography 110:

  WEST, F. D. The preparation of standards for the determination of
  turbidity of water: _Proc. Ill. Water Supply Assoc._, Vol. 6, pp.
  49–51, 1914.

Bibliography 111:

  WESTON, R. S. Apparatus for the determination of ammonia in water by
  the Wanklyn method, and total nitrogen by the Kjeldahl method: _J. Am.
  Chem. Soc._, Vol. 22, pp. 468–73, 1900.

Bibliography 112:

  ——. The determination of nitrogen as nitrites in waters: _J. Am. Chem.
  Soc._, Vol. 27, pp. 281–7, 1905.

Bibliography 113:

  ——. The determination of manganese in water: _J. Am. Chem. Soc._, Vol.
  29, pp. 1074–8, 1907.

Bibliography 114:

  WHIPPLE, G. C. The observation of odor as an essential part of water
  analysis: _Public Health Papers and Reports, Am. Pub. Health Assoc._,
  Vol. 25, pp. 587–93, 1899.

Bibliography 115:

  ——. The microscopy of drinking water, 3d ed., pp. 186–205, John Wiley
  & Sons, New York, 1914.

Bibliography 116:

  ——, and JACKSON, D. D. A comparative study of the methods used for the
  measurement of the turbidity of water: _Tech. Quart._, Vol. 13, pp.
  274–94, 1900.

Bibliography 117:

  ——, and others. The decolorization of water: _Trans. Am. Soc. Civil
  Eng._, Vol. 46, pp. 141–81, 1901.

Bibliography 118:

  ——, and PARKER, H. N. On the amount of oxygen and carbonic acid
  dissolved in natural waters and the effect of these gases upon the
  occurrence of microscopic organisms: _Trans. Am. Microscopical Soc._,
  Vol. 23, pp. 103–44, 1901.

Bibliography 119:

  ——, and WHIPPLE, M. C. Solubility of oxygen in sea water: _J. Am.
  Chem. Soc._, Vol. 33, pp. 362–5, 1911.

Bibliography 120:

  WINKLER, L. W. Die Bestimmung des im Wasser gelösten Sauerstoffes:
  _Ber._, pp. 2843–54, 1888.

Bibliography 121:

  WOODMAN, A. G., and NORTON, J. F. Air, water, and food, 4th ed., pp.
  72–8, John Wiley & Sons, New York, 1914;

Bibliography 121a:

  pp. 85–7;

Bibliography 121b:

  pp. 90–1, 216, and 231;

Bibliography 121c:

  pp. 106–8.




                       MICROSCOPICAL EXAMINATION.


The microscopical examination of water consists of the enumeration of
the kinds of microscopic organisms (Plankton), and an estimation of
their quantity.

It may serve any one or more of the following purposes:

 (1) To explain the presence of objectionable odors and tastes.

 (2) To indicate the progress of the self purification of streams.

 (3) To indicate the presence of sewage contamination.

 (4) To explain the chemical analysis.

 (5) To identify the source of a water.

 (6) To aid in the study of the food of fish, shellfish, and other
       aquatic organisms.

The term “Microscopic Organisms” shall include all organisms microscopic
or barely visible to the naked eye, with the exception of the bacteria.
It includes the diatomaceae, chlorophyceae, cyanophyceae, fungi,
protozoa, rotifera, crustacea, bryophyta, and spongidae found in water.

Fragments of organic matter, silt, mineral matter, zoöglea, etc., shall
be considered as amorphous matter. The recording of amorphous matter
usually serves no useful purpose and shall not be considered a part of
the standard method.

_Apparatus._—1. A cylindrical funnel about two inches in diameter at the
top, with a straight side for nine inches, narrowed over a distance of
three inches to a bore of one-half inch in diameter, and terminating in
a straight portion of this diameter two and one-half inches in length.
The capacity of this funnel is 500 cc. It shall be provided at the
bottom with a tightly fitting rubber stopper with a single perforation
and a disk of silk bolting cloth over the hole about three eighths of an
inch in diameter.

2. A counting cell consisting of a brass rim closely cemented to a plate
of optical glass. The shape and size of this cell are not essential but
its depth shall be one millimeter. A convenient capacity is about one
cubic centimeter.

3. An ocular micrometer ruled as follows: The ocular micrometer is
commonly of such a size that with a 16 mm. objective and a suitable tube
length, the largest square cuts off one square millimeter on the stage.

_Procedure._—Filter 250 cc. of the water (more or less according to the
clearness of the sample) through a one-half inch layer of quartz sand
(washed and screened between 60 and 120 mesh sieves) supported by the
disk of bolting cloth and rubber stopper at the bottom of the funnel.
Suction may be applied to hasten the filtration.

Remove the stopper and catch the plug of sand and its entrained
organisms in a small beaker or test tube, washing down the inside of the
funnel into the beaker with 5 cc. of clean (preferably distilled) water.
Agitate the mixture of sand, water, and organisms to detach the latter
from the sand grains, and quickly decant the water and the organisms in
suspension to a test tube. If desired the sand may then be again washed
with 5 cc. water and the wash water added to the first portion.

Cover the cell partially with a cover glass, and by means of a pipette
run the concentrate under the cover glass until the cell is completely
filled.

Cover and place on the microscope stage in a horizontal position for
examination.

Count the organisms in twenty fields, i. e., twenty cubic millimeters,
estimating their areas in terms of Standard Units.

_The Standard Unit is the smallest square in the ocular micrometer, and
represents an area 20µ × 20µ, or 400 square microns on the stage._

Results shall be expressed in the number of Standard Units of each kind
of micro-organism per cc. and also the total number of standard units of
all kinds per cc. The general directions as to significant figures given
under Turbidity shall apply also to the microscopical examination.

_Caution._—Many micro-organisms, especially some of those causing odors,
are so fragile that they are broken up in filtration, especially if the
agitation of the filtrate is too vigorous. A direct examination of a
fresh sample is therefore a useful supplementary procedure. For the same
reason the concentrate should not stand long before examination. Also
some organisms are carried by specific gravity to the top of the cell
which should be scrutinized as well as the bottom layer each time.

It is always better to examine the micro-organisms in the field when
possible, and for this purpose the sling filter has been devised
consisting of a metal funnel slung to a pivoted handle, with a disk of
wire gauze in the detachable lower end to support the sand. Filtration
is hastened by imparting a whirling motion to the whole and utilizing
the centrifugal force thus generated.

[Illustration: THE OCULAR MICROMETER.]


                      MICROSCOPICAL BIBLIOGRAPHY.

 _a._ KEAN, A. L. A new method for the microscopical examination of
        water: _Science_, Vol. 13, p. 132, 1889; _Eng. News_, pp. 21,
        276, 1889.

 _b._ SEDGWICK, W. T. Recent progress in biological water analysis: _J.
        N. E. Water Works Assoc._, Vol. 4, pp. 50–64, 1889.

 _c._ ——. A report of the biological work of the Lawrence Experiment
        Station: _Examinations by the State Board of Health of water
        supplies of Mass., 1887–90_, pt. 2, Purification of sewage and
        water, pp. 793–862, 1890.

 _d._ PARKER, G. H. Report upon the organisms, excepting the bacteria
        found in the waters of the State: _Examinations by the State
        Board of Health of water supplies of Mass., 1887–90_, pt. 1,
        Examination of water supplies, pp. 579–620, 1890.

 _e._ RAFTER, G. W. The microscopical examination of potable water, D.
        Van Nostrand Co., New York, 1910. (Contains bibliography.)

 _f._ CALKINS, G. N. The microscopical examination of water: _Report
        Mass. State Board of Health_, pp. 397–421, 1892.

 _g._ JACKSON, D. D. On an improvement in the Sedgwick-Rafter method for
        the microscopical examination of drinking water: _Tech. Quart._,
        Vol. 9, pp. 271–4, 1896.

 _h._ WHIPPLE, G. C. Experience with the Sedgwick-Rafter method at the
        Biological Laboratory of the Boston Water Works: _Tech. Quart._,
        Vol. 9, pp. 275–9, 1896.

 _i._ ——. Microscopy of drinking water, 3d ed., John Wiley & Sons, New
        York, 1914. (Contains bibliography.)




                      BACTERIOLOGICAL EXAMINATION.


                             I. APPARATUS.

1. _Sample Bottles._—Any size, shape or quality of bottle may be used
for a bacterial sample, provided it holds a sufficient amount to carry
out all the tests required and is such that it may be properly washed
and sterilized and will keep the sample uncontaminated until the
analysis is made. Four- or eight-ounce, ground-glass-stoppered bottles
are recommended. These should be protected by being wrapped in paper, or
their necks covered with tin foil, and should be placed in proper boxes
for transportation.

2. _Pipettes._—Pipettes may be of any convenient size or shape provided
it is found by actual test that they deliver accurately the required
amount in the manner in which they are used. The error of calibration
shall in no case exceed 2 per cent. Protecting the pipettes with a
cotton stopper is recommended.

3. _Dilution Bottles._—Bottles for use in making dilutions should
preferably be of tall form, of such capacity as to hold at least twice
the volume of water actually used. Close-fitting ground-glass stoppers
are preferable, but tight fitting cotton stoppers may be used, provided
due care is taken to prevent contamination and to avoid loss of volume
through wetting of the stopper before mixing has been accomplished.

4. _Petri Dishes._—Petri dishes ten centimeters in diameter shall be
used with glass or porous tops[211] as preferred. The bottoms of the
dishes shall be as flat as possible so that the medium shall be of
uniform thickness throughout the plate.

5. _Fermentation Tubes._—Any type of fermentation tube[203] may be used
provided it holds at least three times as much medium as the amount of
water to be tested.


                             II. MATERIALS.

1. _Water._—Distilled water shall be used in the preparation of all
culture media and reagents.

2. _Meat Extract._—Liebig’s meat extract shall be used in place of meat
infusion. Other brands may be substituted for Liebig’s when comparative
tests have shown that they give equivalent results.

3. _Peptone._—Armour’s, Digestive Ferments Company’s, Fairchild’s, or
any other peptone which gives equivalent results may be used.

4. _Sugars._—All sugars used shall be of the highest purity obtainable.

5. _Agar._—The agar used shall be of the best quality and shall be dried
for one-half hour at 105° C. before weighing. Much of the agar on the
market contains considerable amounts of sea salts.[221][225][228] These
may be removed by soaking in water and draining before use.

6. _Gelatin._—The gelatin used shall be of light color, shall contain
not more than a trace of arsenic, copper, sulfides, and shall be free
from preservatives, and of such a melting point that a 10 per cent.
standard nutrient gelatin shall have a melting point of 25° C. or over.
Gelatin shall be dried for one-half hour at 105° C. before weighing.

7. _Litmus._—Reagent litmus of highest purity (not litmus cubes) or
azolitmin (Kahlbaum) shall be used for all media requiring a litmus
indicator.

8. _General Chemicals._—Special effort shall be made to have all the
other ingredients used for culture media chemically pure.


                             III. METHODS.


                    1. PREPARATION OF CULTURE MEDIA.


                      a. _Adjustment of Reaction._

_aa._ _Phenol Red Method for adjustment to a hydrogen-ion concentration
of P_{H+} = 6.8–8.4._ Withdraw 5 cc. of the medium, dilute with 5 cc. of
distilled water, and add 5 drops of a solution of phenol red (phenol
sulphone phthalein). This solution is made by dissolving 0.04 grams of
phenol red in 30 cc. of alcohol and diluting to 100 cc. with distilled
water.

Titrate with a 1:10 dilution of a standard solution of NaOH (which need
not be of known normality) until the phenol red shows a slight but
distinct pink color. Calculate the amount of the standard NaOH solution
which must be added to the medium to reach this reaction. After the
addition check the reaction by adding 5 drops of phenol red to 5 cc. of
the medium and 5 cc. of water.

_bb._ _Titration with phenolphthalein._ (For the convenience of those
who wish to retain the use of this method for the present it is given
here, but it is recommended that as soon as possible the more accurate
method of determining the hydrogen-ion concentration be substituted.)

In a white porcelain dish put 5 cc. of the medium to be tested, add 45
cc. of distilled water. Boil briskly for one minute. Add 1 cc. of
phenolphthalein solution (5 grams of commercial salt to one liter of 50
per cent. alcohol). Titrate immediately with a n/20 solution of sodium
hydrate. A faint but distinct pink color marks the true end-point. This
color may be precisely described as a combination of 25 per cent. of red
(wave length approximately 658) with 75 per cent. of white as shown by
the disks of the standard color top made by the Milton Bradley
Educational Co., Springfield, Mass.

All reactions shall be expressed with reference to the phenolphthalein
neutral point and shall be stated in percentages of normal acid or
alkali solutions required to neutralize them. Alkaline media shall be
recorded with a minus (-) sign before the percentage of normal acid
needed for their neutralization and acid media with a plus (+) sign
before the percentage of normal alkali solution needed for their
neutralization.

The standard reaction for culture media for water analysis shall be +1.0
per cent., as determined by tests of the sterilized medium. As
ordinarily prepared, broth and agar will be found to have a reaction
between +0.5 and +1.0. For such media no adjustment shall be made. The
reaction of media containing sugar shall be neutral to phenolphthalein.
Whenever reactions other than the standard are used, it shall be so
stated.


                          b. _Sterilization._

All media and dilution water shall be sterilized in the autoclav at 15
lbs. (120° C.) for 15 minutes after the pressure reaches 15 lbs. All air
must be forced out of the autoclav before the pressure is allowed to
rise. As soon as possible after sterilization the media shall be removed
from the autoclav and cooled rapidly. Rapid and immediate cooling of
gelatin is imperative.

Media shall be sterilized in small containers, and these must not be
closely packed together. No part of the medium shall be more than 2.5
cm. from the outside surface of the glass. All glassware shall be
sterilized in the dry oven at 170° C. for at least 1½ hours.


                c. _Nutrient Broth. To Make One Liter._

1. Add 3 grams of beef extract and 5 grams of peptone to 1,000 cc. of
distilled water.

2. Heat slowly on a steam bath to at least 65° C.

3. Make up lost weight and adjust the reaction to a faint pink with
phenol red, or if the phenolphthalein titration is used, and the
reaction is not already between +0.5 and +1, adjust to +1.

4. Cool to 25° C. and filter through filter paper until clear.

5. Distribute in test-tubes, 10 cc. to each tube.

6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after
the pressure reaches 15 lbs.


                           d. _Sugar Broths._

Sugar broths shall be prepared in the same general manner as nutrient
broth with the addition of 0.5 per cent. of the required carbohydrate
just before sterilization. The removal of muscle sugar is unnecessary as
the beef extract and peptone are free from any fermentable
carbohydrates. The reaction of sugar broths shall be a faint pink with
phenol red or, if on titration with phenolphthalein the reaction is not
already between neutral and +1, adjust to neutral. Sterilization shall
be in the autoclav at 15 lbs. (120° C.) for 15 minutes after the
pressure reaches 15 lbs., provided the total time of exposure to heat is
not more than one-half hour; otherwise a 10 per cent. solution of the
required carbohydrate shall be made in distilled water and sterilized at
100° C. for 1½ hours, and this solution shall be added to sterile
nutrient broth in amounts sufficient to make a 0.5 per cent. solution of
the carbohydrate and the mixture shall then be tubed and sterilized at
100° C. for 30 minutes, or it is permissible to add by means of a
sterile pipette directly to a tube of sterile neutral broth enough of
the carbohydrate to make the required 0.5 per cent. The tubes so made
shall be incubated at 37° C. for 24 hours as a test for sterility.


               e. _Nutrient Gelatin. To Make One Liter._

1. Add 3 grams of beef extract and 5 grams of peptone to 1,000 cc. of
distilled water and add 100 grams of gelatin dried for one-half hour at
105° C. before weighing.

2. Heat slowly on a steam bath to 65° C. until all gelatin is
dissolved.[G]

Footnote G:

  The solution of the gelatin will be facilitated by allowing it to soak
  in the cold one-half hour before heating.

3. Make up lost weight and adjust the reaction to a faint pink with
phenol red, or if the phenolphthalein titration is used, and the
reaction is not already between +0.5 and +1, adjust to +1.

4. Filter through cloth and cotton until clear.

5. Distribute in test-tubes, 10 cc. to each tube, or in larger
containers as desired.

6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after
the pressure reaches 15 lbs.


                 f. _Nutrient Agar. To Make One Liter._

1. Add 3 grams of beef extract, 5 grams of peptone and 12 grams of agar,
dried for one-half hour at 105° C. before weighing, to 1,000 cc. of
distilled water. Boil over a water bath until all agar is dissolved, and
then make up the loss by evaporation.

2. Cool to 45° C. in a cold water bath, then warm to 65° C. in the same
bath, without stirring.

3. Make up lost weight and adjust the reaction to a faint pink with
phenol red, or if the phenolphthalein titration is used, and the
reaction is not already between +0.5 and +1, adjust to +1.

4. Filter through cloth and cotton until clear.

5. Distribute in test-tubes, 10 cc. to each tube, or in larger
containers, as desired.

6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after
the pressure reaches 15 lbs.


                   g. _Litmus or Azolitmin Solution._

The standard litmus solution shall be a 2 per cent. aqueous solution of
reagent litmus. Powder the litmus, add to the water and boil for five
minutes. The solution usually needs no correction in reaction and may be
at once distributed in flasks or test-tubes and sterilized as is culture
media. It should give a distinctly blue plate when 1 cc. is added to 10
cc. of neutral culture medium in a Petri dish.

The standard azolitmin solution shall be a 1 per cent. solution of
Kahlbaum’s azolitmin. Add the azolitmin powder to the water and boil for
five minutes. The solution may need to be corrected in reaction by the
addition of sodium hydrate solution so that it will be approximately
neutral and will give a distinctly blue plate when 1 cc. is added to 10
cc. of neutral culture medium in a Petri dish. It may be distributed in
flasks or test-tubes and sterilized as is culture media.


                       h. _Litmus-lactose-agar._

Litmus-lactose-agar shall be prepared in the same manner as nutrient
agar with the addition of 1 per cent. of lactose just before
sterilization. The reaction shall be a faint pink with phenol red, or,
if on titration with phenolphthalein the reaction is not already between
neutral and +1, adjust to neutral. One cc. of sterilized litmus or
azolitmin solution shall be added to each 10 cc. of the medium just
before it is poured into the Petri dish, or the mixture may be made in
the dish itself.


        i. _Endo’s Medium._[209][214][215] _To Make One Liter._

1. Add 5 grams of beef extract, 10 grams of peptone and 30 grams of agar
dried for one-half hour at 105° C. before weighing, to 1,000 cc. of
distilled water. Boil on a water bath until all the agar is dissolved
and then make up the loss by evaporation.

2. Cool the mixture to 45° C. in a cold water bath, then warm to 65° C.
in the same bath without stirring.

3. Make up lost weight, titrate, and if the reaction is not already
between neutral and +1 adjust to neutral.

4. Filter through cloth and cotton until clear.

5. Distribute 100 cc. or larger known quantities in flasks large enough
to hold the other ingredients which are to be added later.

6. Sterilize in the autoclav at 15 lbs. (120° C.) for 15 minutes after
the pressure reaches 15 lbs.

7. Prepare a 10 per cent. solution of basic fuchsin in 95 per cent.
alcohol, allow to stand 20 hours, decant and filter the supernatant
fluid. This is a stock solution.

8. When ready to make plates melt 100 cc. of agar in streaming steam or
on a water bath. Dissolve 1 gram of lactose in 15 cc. of distilled
water, using heat if necessary. Dissolve 0.25 gram anhydrous sodium
sulphite in 10 cc. water. To the sulphite solution add 0.5 cc. of the
fuchsin stock solution. Add the fuchsin-sulphite solution to the lactose
solution and then add the resulting solution to the melted agar. The
lactose used must be chemically pure and the sulphite solution must be
made up fresh.

9. Pour plates and allow to harden thoroughly in the incubator before
use.


                        2. COLLECTION OF SAMPLE.

Samples for bacterial analysis shall be collected in bottles which have
been cleansed with great care, rinsed in clean water, and sterilized
with dry heat for at least one hour and a half at 170° C., or in the
autoclav at 15 lbs. (120° C.) for 15 minutes or longer after the
pressure reaches 15 lbs.

Great care must be exercised to have the samples representative of the
water to be tested and to see that no contamination occurs at the time
of filling the sample bottles.


               3. STORAGE AND TRANSPORTATION OF SAMPLES.

Because of the rapid and often extensive changes which may take place in
the bacterial flora of bottled samples when stored even at temperatures
as low as 10° C., it is urged, as of importance, that all samples be
examined as promptly as possible after collection.

The time allowed for storage or transportation of a bacterial sample
between the filling of the sample bottle and the beginning of the
analysis should be not more than six hours for impure waters and not
more than twelve hours for relatively pure waters. During the period of
storage, the temperature shall be kept as near 10° C. as possible. Any
deviation from the above limits shall be so stated in making reports.


                             4. DILUTIONS.

Dilution bottles shall be filled with the proper amount of tap water so
that after sterilization they shall contain exactly 9 cc. or 99 cc. as
desired. The exact amount of water can only be determined by experiment
with the particular autoclav in use. If desired, the 9 cc. dilution may
be measured out from a flask of sterile water with a sterile pipette.

Dilution bottles shall be sterilized in the autoclav at 15 lbs. (120°
C.) for 15 minutes after the pressure reaches 15 lbs.

The sample bottle shall be shaken vigorously 25 times and 1 cc.
withdrawn and added to the proper dilution bottles as required. Each
dilution bottle after the addition of the 1 cc. of the sample, shall be
shaken vigorously 25 times before a second dilution is made from it or
before a sample is removed for plating.


                              5. PLATING.

All sample and dilution bottles shall be shaken vigorously 25 times
before samples are removed for plating. Plating shall be done
immediately after the dilutions are made. One cc. of the sample or
dilution shall be used for plating and shall be placed in the Petri
dish, first. Ten cc. of liquefied medium at a temperature of 40° C.
shall be added to the 1 cc. of water in the Petri dish. The cover of the
Petri dish shall be lifted just enough for the introduction of the
pipette or culture medium, and the lips of all test-tubes or flasks used
for pouring the medium shall be flamed. In making litmus-lactose-agar
plates, 1 cc. of sterile litmus or azolitmin solution shall be added to
each 10 cc. of culture medium either in the Petri dish or before pouring
into the Petri dish. The medium and sample in the Petri dish shall be
thoroughly mixed and uniformly spread over the bottom of the Petri dish
by tilting or rotating the dish. All plates shall be solidified as
rapidly as possible after pouring and gelatin plates shall be placed
immediately in the 20° C. incubator and the agar plates in the 37° C.
incubator. Endo plates shall be made by placing one loopful of the
material to be tested on the surface of the plate and distributing the
material with a sterile loop or glass rod.


                             6. INCUBATION.

All gelatin plates shall be incubated for 48 hours at 20 C. in a dark,
well-ventilated incubator in an atmosphere practically saturated with
moisture.[227]

All agar plates shall be incubated for 24 hours at 37° C. in a dark,
well-ventilated incubator in an atmosphere practically saturated with
moisture. Glass covered plates shall be inverted in the incubator. Any
deviation from the above described method shall be stated in making
reports.


                              7. COUNTING.

In preparing plates, such amounts of the water under examination shall
be planted as will give from 25 to 250 colonies on a plate;[202] and the
aim should be always to have at least two plates giving colonies between
these limits. Where it is possible to obtain plates showing colonies
within these limits, only such plates should be considered in recording
results, except where the same amount of water has been planted in two
or more plates, of which one gives colonies within these limits, while
the others give less than 25 or more than 250. In such case, the result
recorded should be the average of all the plates planted with this
amount of water. Ordinarily it is not desirable to plant more than 1 cc.
of water in a plate; therefore, when the total number of colonies
developing from 1 cc. is less than 25, it is obviously necessary to
record the results as observed, disregarding the general rule given
above.

Counting shall in all cases be done with a lens of 2½ diameter’s
magnification, 3½X. The Engraver’s Lens No. 146 made by the Bausch &
Lomb Optical Company fills the requirements, and is a convenient lens
for the purpose.


     8. THE TEST FOR THE PRESENCE OF MEMBERS OF THE B. COLI GROUP.

It is recommended that the B. coli group be considered as including all
non-spore-forming bacilli which ferment lactose with gas formation and
grow aërobically on standard solid media.

The formation of 10 per cent. or more of gas in a standard lactose broth
fermentation tube within 24 hours at 37° C. is presumptive evidence of
the presence of members of the B. coli group, since the majority of the
bacteria which give such a reaction belong to this group.

The appearance of aërobic lactose-splitting colonies on
lactose-litmus-agar or Endo’s medium plates made from a lactose broth
fermentation tube in which gas has formed confirms to a considerable
extent the presumption that gas-formation in the fermentation tube was
due to the presence of members of the B. coli group.

To complete the demonstration of the presence of B. coli as above
defined, it is necessary to show that one or more of these aërobic plate
colonies consists of non-spore-forming bacilli which, when inoculated
into a lactose broth fermentation tube, form gas.

It is recommended that the standard tests for the B. coli group be
either (A) the _Presumptive_, (B) the _Partially Confirmed_, or (C) the
_Completed_ test as hereafter defined, each test being applicable under
the circumstances specified.


                          A. PRESUMPTIVE TEST.

1. Inoculate a series of fermentation tubes with appropriate graduated
quantities of the water to be tested. In every fermentation tube there
must always be at least three times as much medium as the amount of
water to be tested. When necessary to examine larger amounts than 10 cc.
as many tubes as necessary shall be inoculated with 10 cc. each.

2. Incubate these tubes at 37° C. for 48 hours. Examine each tube at 24
and 48 hours, and record gas-formation. The records should be such as to
distinguish between:

(a) Absence of gas-formation.

(b) Formation of gas occupying less than ten per cent. (10%) of the
closed arm.

(c) Formation of gas occupying more than ten per cent. (10%) of the
closed arm.

More detailed records of the amount of gas formed, though desirable for
purposes of study, are not necessary for carrying out the standard tests
prescribed.

3. The formation within 24 hours of gas occupying more than ten per
cent. (10%) of the closed arm of fermentation tube constitutes _a
positive presumptive test_.

4. If no gas is formed in 24 hours, or if the gas formed is less than
ten per cent. (10%), the incubation shall be continued to 48 hours. The
presence of gas in any amount in such a tube at 48 hours constitutes _a
doubtful test_, which in all cases requires confirmation.

5. The absence of gas formation after 48 hours’ incubation constitutes
_a negative test_. (An arbitrary limit of 48 hours’ observation
doubtless excludes from consideration occasional members of the B. coli
group which form gas very slowly, but for the purposes of a standard
test the exclusion of these occasional slow gas-forming organisms is
considered immaterial.)


                      B. PARTIALLY CONFIRMED TEST.

1. Make one or more Endo’s medium or lactose-litmus-agar plates from the
tube which, after 48 hours’ incubation, shows gas formation from the
smallest amount of water tested. (For example, if the water has been
tested in amounts of 10 cc., 1 cc., and 0.1 cc., and gas is formed in 10
cc., and 1 cc., not in 0.1 cc., the test need be confirmed only in the 1
cc. amount.)

2. Incubate the plates at 37° C., 18 to 24 hours.

3. If typical colon-like red colonies have developed upon the plate
within this period, the confirmed test may be considered positive.

4. If, however, no typical colonies have developed within 24 hours, the
test cannot yet be considered definitely negative, since it not
infrequently happens that members of the B. coli group fail to form
typical colonies on Endo’s medium or lactose-litmus-agar plates, or that
the colonies develop slowly. In such case, it is always necessary to
complete the test as directed under “C” 2 and 3.


                           C. COMPLETED TEST.

1. From the Endo’s medium or lactose-litmus-agar plate made as
prescribed under “B,” fish at least two typical colonies, transferring
each to an agar slant and a lactose broth fermentation tube.

2. If no typical colonies appear upon the plate within 24 hours, the
plate should be reincubated another 24 hours, after which at least two
of the colonies considered to be most likely B. coli, whether typical or
not, shall be transferred to agar slants and lactose broth fermentation
tubes.

3. The lactose broth fermentation tubes thus inoculated shall be
incubated until gas formation is noted; the incubation not to exceed 48
hours. The agar slants shall be incubated at 37° C. for 48 hours, when a
microscopic examination shall be made of at least one culture, selecting
one which corresponds to one of the lactose broth fermentation tubes
which has shown gas-formation.

The formation of gas in lactose broth and the demonstration of
non-spore-forming bacilli in the agar culture shall be considered a
satisfactory completed test, demonstrating the presence of a member of
the B. coli group.

The absence of gas-formation in lactose broth or failure to demonstrate
non-spore-forming bacilli in a gas-forming culture constitutes a
negative test.


 APPLICATION OF PRESUMPTIVE, PARTIALLY CONFIRMED, AND COMPLETED TESTS.


                        A. The Presumptive Test.

  1. When definitely positive, that is showing more than 10 per cent.
    (10%) of gas in 24 hours, is sufficient:

      (a) As applied to all except the smallest gas-forming portion of
        each sample in all examinations.

      (b) As applied to the smallest gas-forming portion in the
        examination of sewage or of water showing relatively high
        pollution, such that its fitness for use as drinking water does
        not come into consideration. This applies to the routine
        examinations of raw water in connection with control of the
        operation of purification plants.

  2. When definitely negative, that is showing no gas in 48 hours, is
    final and therefore sufficient in all cases.

  3. When doubtful, that is showing gas less than 10 per cent. (10%) (or
    none) in 24 hours, with gas either more or less than 10 per cent. in
    48 hours, must always be confirmed.


                    B. The Partially Confirmed Test.

  1. When definitely positive, that is, showing typical plate colonies
    within 24 hours, is sufficient:

      (a) When applied to confirm a doubtful presumptive test in cases
        where the latter, if definitely positive, would have been
        sufficient.

      (b) In the routine examination of water supplies where a
        sufficient number of prior examinations have established a
        satisfactory index of the accuracy and significance of this test
        in terms of the completed test.

  2. When doubtful, that is, showing colonies of doubtful or negative
    appearance in 24 hours, must always be completed.


                         C. The Completed Test.

  The completed test is required as applied to the smallest gas-forming
    portion of each sample in all cases other than those noted as
    exceptions under the “presumptive” and the “partially confirmed”
    tests.

  The completed test is required in _all_ cases where the result of the
    confirmed test has been doubtful.


                       9. EXPRESSION OF RESULTS.

In order to avoid fictitious accuracy and yet to express the numerical
results by a method consistent with the precision of the work, the
numbers of colonies of bacteria per cubic centimeter shall be recorded
as follows:[212]

                       Number of bacteria per cc.
 From         1 to         50    shall be recorded as found
  "          51 "         100    "    "   "      to the nearest       5
  "         101 "         250    "    "   "      "   "    "          10
  "         251 "         500    "    "   "      "   "    "          25
  "         501 "       1,000    "    "   "      "   "    "          50
  "       1,001 "      10,000    "    "   "      "   "    "         100
  "      10,001 "      50,000    "    "   "      "   "    "         500
  "      50,001 "     100,000    "    "   "      "   "    "       1,000
  "     100,001 "     500,000    "    "   "      "   "    "      10,000
  "     500,001 "   1,000,000    "    "   "      "   "    "      50,000
  "   1,000,001 "  10,000,000    "    "   "      "   "    "     100,000

This applies to the gelatin count at 20° C. and to the agar count at 37°
C.


SUMMARY OF STEPS INVOLVED IN MAKING PRESUMPTIVE, PARTIALLY CONFIRMED AND
                      COMPLETED TESTS FOR B. COLI.

 ────────────────────────────────────────────────────────────┬─────────
                     Steps in procedure.                     │ Further
                                                             │procedure
                                                             │required.
 ────────────────────────────────────────────────────────────┼─────────
 I. Inoculate lactose broth fermentation tubes; incubate 24  │
   hours at 37° C.; observe gas-formation in each tube.      │
      1. Gas-formation, 10 per cent. or more; constitutes    │
        positive presumptive test.                           │
           (a) For other than smallest portion of any sample │
             showing gas at this time, and for all portions, │
             including smallest, of sewage and raw water this│
             test is sufficient.                             │None
           (b) For smallest gas-forming portion, except in   │
             examinations of sewage and raw water.           │III
      2. Gas-formation less than 10 per cent. in 24 hours;   │
        inconclusive.                                        │II
 II. Incubate an additional 24 hours, making a total of 48   │
   hours’ incubation; observe gas-formation.                 │
      1. Gas-formation, any amount; constitutes doubtful     │
        test, which must always be carried further.          │III
      2. No gas-formation in 48 hours; constitutes final     │
        negative test.                                       │None
 III. Make plate from smallest gas-forming portion of sample │
   under examination; incubate 18 to 24 hours; observe       │
   colonies.                                                 │
      1. One or more colonies typical in appearance.         │
           (a) If only “partially confirmed” test is required│None
           (b) If completed test is required, select two     │
             typical colonies for identification.            │V
      2. No typical colonies.                                │IV
 IV. Replace plate in incubator for an additional 18 to 24   │
   hours; then, whether colonies appear typical or not,      │
   select at least two of those which most nearly resemble B.│
   coli.                                                     │V
 V. Transfer each colony fished to:                          │
      1. Lactose broth fermentation tube; incubate not more  │
        than 48 hours at 37° C. Observe gas-formation.       │None
      2. Agar slant; incubate 48 hours at 37° C.             │
           (a) If gas formed in lactose broth tube inoculated│
             with corresponding culture                      │VI
           (b) If no gas formed in corresponding lactose     │
             broth tube, test is completed and negative.     │None
 VI. Make stained cover-slip or slide preparation, and       │
   examine microscopically.                                  │
      1. If preparation shows non-spore-forming bacilli in   │
        apparently pure culture, demonstration of B. coli is │
        completed.                                           │None
      2. If preparation fails to show non-spore-forming      │
        bacilli or shows them mixed with spore-bearing forms │
        or bacteria of other morphology.                     │VII
 VII. Replate, to obtain assuredly pure culture, select      │
   several colonies of bacilli and repeat steps V and VI.    │
 ────────────────────────────────────────────────────────────┴─────────

In order that tests for B. coli may have quantitative significance, the
following general principles and rules should be observed:

Ordinarily not less than three portions of each sample should be tested,
the portions being even decimal multiples or fractions of a cubic
centimeter; for example, 10 cc., 1 cc., 0.1 cc., .01 cc., etc. It is
essential that the dilutions should be such that the largest amount
gives a positive test (unless the water is such as to give negative
tests in 10 cc.), and the smallest dilution, a negative result. To
insure this result, it is often necessary to plant four or five
dilutions, especially in the examination of a sample of entirely unknown
quality. The quantitative value of a series of tests is lost, unless all
or at least a large proportion of the smallest dilutions tested have
given negative results.

In reporting a single test, it is preferable merely to record results as
observed, indicating the amounts tested and the result in each, rather
than to attempt expression of the result in numbers of B. coli per cc.
In summarizing the results of a series of tests, however, it is
desirable, for the sake of simplicity, to express the results in terms
of the numbers of B. coli per cc., or per 100 cc. To convert results of
fermentation tests to this form, the result of each test is recorded as
indicating a number of B. coli per cc. equal to the reciprocal of the
smallest decimal or multiple fraction of a cubic centimeter giving a
positive result. For example, the result: 10 cc. +; 1 cc. +; 0.1 cc. -;
would be recorded as indicating one B. coli per cc. An exception should
be made in the case where a negative result is obtained in an amount
larger than the smallest portion giving a positive result; for example,
in a result such as: 10 cc. +; 1 cc. -; 0.1 cc. +. In such case, the
result should be recorded as indicating a number of B. coli per cc.
equal to the reciprocal of the dilution next larger than the smallest
one giving a positive test, this being a more probable result.

Where tests are made in amounts larger than 1 cc., giving average
results less than one B. coli per cc., it is more convenient to express
results in terms of the numbers of B. coli per 100 cc.

The following table illustrates the method of recording and averaging
results of B. coli tests:

     Result of Tests in Amounts Designated.      Indicated Number of B.
                                                          coli.
   10 cc.       1 cc.      0.1 cc.     .01 cc.     per cc.   per 100 cc.
      +           −           −           −              0.1         10.
      +           +           −           −              1.0        100.
      +           +           +           −             10.0      1,000.
      +           +           +           +            100.0     10,000.
      +           +           −           +             10.0      1,000.
                                                       —————       —————
 Totals (for estimating averages)                      121.1     12,110.
 Average of 5 tests                                     24.0      2,422.

The above method of expressing results is not mathematically altogether
correct. The average number of B. coli per cc., as thus estimated, is
not precisely the most probable number calculated by application of the
theory of probability.[220] To apply this theory to a correct
mathematical solution of any considerable series of results involves,
however, mathematical calculations so complex as to be impracticable of
application in general practice. The simpler method given is therefore
considered preferable, since it is easily applied and the results so
expressed are readily comprehensible.

In order that results as reported may be checked and carefully valuated,
it is necessary that the report should show not only the average number
of B. coli per cc., but also the number of samples examined; and, for
each dilution, the total number of tests made, and the number (or per
cent.) positive.


                     10. INTERPRETATION OF RESULTS.

While it is not within the province of this report to suggest the proper
interpretation of results obtained by the use of the methods herein
specified as standard, the committee feels that a word of caution should
be given regarding the significance of the presence in a water of
members of the B. coli group as defined in this report. Recent work
seems to indicate that the B. coli group as herein defined consists of
organisms of both fecal and non-fecal origin. Therefore care must be
exercised in judging the sanitary quality of a water solely from the
determination of the presence of members of the group.


   11. DIFFERENTIATION OF FECAL FROM NON-FECAL MEMBERS OF THE B. COLI
                                 GROUP.

(1) At least 10 cultures should be used. If possible these should be
subcultured from plates made direct from the water since all of the
cultures obtained by plating from fermentation tubes may be descendants
of a single cell in the water. If cultures from water plates are not
available those obtained from plates made as prescribed under B (p. 101)
may be used.

(2) Inoculate each culture into dextrose potassium phosphate broth,[H]
adonite broth, and gelatin. For additional confirmatory evidence
inoculation may be made into tryptophane broth,[I] and saccharose broth.
The dextrose broth must be incubated at 30°. Other sugar broths may be
incubated at 30° or 37° as convenient. Gelatin should be incubated at
20°.

Footnote H:

  (a) _Peptone Medium for the Methyl Red Test. To Make One Liter._

  1. To 800 cc. of distilled water add 5 grams of Proteose-Peptone,
  Difco., or Witte’s Peptone (other peptones should not be substituted),
  5 grams c. p. dextrose, and 5 grams dipotassium hydrogen phosphate
  (K_{2}HPO_{4}). A dilute solution of the K_{2}HPO_{4} should give a
  distinct pink with phenolphthalein.

  2. Heat with occasional stirring over steam for twenty minutes.

  3. Filter through folded filter paper, cool to 20° C. and dilute to
  1,000 cc. with distilled water.

  4. Distribute 10 cc. portions in sterilized test-tubes.

  5. Sterilize by the intermittent method for 20 minutes on three
  successive days.

Footnote I:

  _Tryptophane Broth for Indol Test._

  To 1,000 cc. of distilled water add 0.3 gram tryptophane, 5 grams
  dipotassium hydrogen phosphate (K_{2}HPO_{4}), and 1 gram peptone.
  Heat until ingredients are thoroughly dissolved, tube (6 to 8 cc.),
  and sterilize in autoclave for 15 minutes after the pressure reaches
  15 pounds. Some American peptones are standardized to contain a
  uniform amount of tryptophane. If such peptone is used the tryptophane
  in the above formula may be omitted and the peptone increased to 5
  grams.

(3) After 48 hours record gas formation in adonite and saccharose
broths. Determine indol formation in tryptophane broth by adding drop by
drop, to avoid mixing with the medium, about 1 cc. of a 2 per cent.
alcoholic solution of p-dimethyl amido-benzaldehyd, then a few drops of
concentrated hydrochloric acid. The presence of indol is indicated by a
red color which is soluble in chloroform. There may be some unconverted
tryptophane still present which will give a distinctly blue color which
is insoluble in chloroform. A mixture of the two will be either blue or
violet. If from such a mixture of colors the red of indol be extracted
with chloroform proof of the presence of indol will be complete.

(4) After 5 days apply methyl red test and Voges-Proskauer test to
dextrose broth.


                         _Methyl Red Test._[J]

Indicator solution.—Dissolve 0.1 gram methyl red in 300 cc. alcohol and
dilute to 500 cc. with distilled water.

Footnote J:

  (b) _Synthetic Medium for the Methyl Red Test._ To Make One Liter.
  Dissolve 7 grams Na_{2}HPO_{4} (anhydrous) or 8.8 grams
  Na_{2}HPO_{4}.2H_{2}O, 2 grams KHphthalate, 1 gram aspartic acid, and
  4 grams dextrose in about 800 cc. of warm distilled water. When
  solution is complete, cool and make up to 1 liter at room temperature.
  Heat in an autoclave for 15 minutes after the pressure has reached 15
  pounds, provided the total time of exposure to heat is not more than
  one-half hour. The hydrogen-ion concentration of the medium is fixed
  by the composition. It should be very close to P_{H} 7.0, slightly red
  with phenol red. All materials should be recrystallized or if used
  from stock furnished by manufacturers, should be carefully examined.
  The di-sodium hydrogen phosphate may be used either as the anhydrous
  salt obtained by dessication in vacuo at 100° C. or else as the salt
  containing two molecules of water of crystallization. This is obtained
  by exposing the recrystallized Na_{2}HPO_{4}.12H_{2}O for two weeks.
  Use 0.88 per cent. of Na_{2}HPO_{4}.2H_{2}O.

Procedure in test.—1. To 5 cc. of each culture add 5 drops of methyl red
solution.

2. Record distinct red color as methyl red +, distinct yellow color as
methyl red -, and intermediate colors as ?.


                      _Voges-Proskauer Test._[216]

To the remaining 5 cc. of medium add 5 cc. of a 10 per cent. solution of
potassium hydroxide. Allow to stand over night. A positive test is
indicated by an eosin pink color.

(5) Gelatin tubes should not be pronounced negative until they have been
incubated at least 15 days.

The following group reactions indicate the source of the culture with a
high degree of probability:

    Methyl red +         │B. coli of fecal origin.
    Voges-Proskauer −    │
    Gelatin −            │
    Adonite −            │
    Indol, usually +     │
    Saccharose, usually −│

    Methyl red −         │B. aërogenes of fecal origin.
    Voges-Proskauer +    │
    Gelatin −            │
    Adonite +            │
    Indol, usually −     │
    Saccharose +         │

    Methyl red −         │B. aërogenes, probably not of fecal
                         │  origin.
    Voges-Proskauer +    │
    Gelatin −            │
    Adonite −            │
    Indol, usually −     │
    Saccharose +         │

    Methyl red −         │B. cloacae, may or may not be of fecal
                         │  origin.
    Voges-Proskauer +    │
    Gelatin +            │
    Adonite +            │
    Indol, usually −     │
    Saccharose +         │


       12. ROUTINE PROCEDURE FOR EXAMINATION OF SAMPLES OF WATER.

_First Day_:

    1. Prepare dilutions as required.

    2. Make two (2) gelatin plates from each dilution, and incubate at
      20° C.

    3. Make two (2) agar plates from each dilution, and incubate at 37°
      C.

    4. Inoculate lactose broth fermentation tubes with appropriate
      amounts for B. coli tests, inoculating two (2) tubes with each
      amount.

Note:—Where repeated tests are made of water from the same source, as is
customary in the control of public supplies, it is not necessary to make
duplicate plates or fermentation tubes in each dilution. It is
sufficient, in such circumstances, to make duplicate plates only from
the dilution which will most probably give from 25 to 250 colonies per
plate.

_Second Day_:

    1. Count the agar plates made on the first day.

    2. Record the number of lactose broth fermentation tubes which show
      10 per cent. (10%) or more of gas.

Note:—In case only the presumptive test for B. coli is required,
fermentation tubes showing more than 10 per cent. (10%) of gas at this
time may be discarded.

_Third Day_:

    1. Count gelatin plates made on first day.

    2. Record the number of additional fermentation tubes which show 10
      per cent. (10%) or more of gas.

    3. Make a lactose-litmus-agar or Endo’s medium plate from the
      smallest portion of each sample showing gas. Incubate plate at 37°
      C.

Note:—In case the smallest portion in which gas has been formed shows
less than 10 per cent. (10%) of gas, it is well to make a plate also
from the next larger portion, so that, in case the smallest portion
gives a negative end result it may still be possible to demonstrate B.
coli in the next larger dilution.

_Fourth Day_:

    1. Examine Endo’s medium or lactose-litmus-agar plates. If typical
      colonies have developed, select two and transfer each to a lactose
      broth fermentation tube and an agar slant, both of which are to be
      incubated at 37° C.

    2. If no typical B. coli colonies are found, incubate the plates
      another 24 hours.

_Fifth Day_:

    1. Select at least two colonies, whether typical or not, from the
      Endo’s medium or lactose-litmus-agar plates which have been
      incubated an additional 24 hours; transfer each to a lactose broth
      fermentation tube and an agar slant, and complete the test as for
      typical colonies.

    2. Examine lactose broth fermentation tubes inoculated from plates
      on the previous day. Tubes in which gas has been formed may be
      discarded after the result has been recorded. Those in which no
      gas has formed should be incubated an additional 24 hours.

_Sixth Day_:

    1. Examine lactose broth fermentation tubes reincubated the previous
      day.

    2. Examine microscopically agar slants corresponding to lactose
      fermentation tubes inoculated from plate colonies and showing
      gas-formation.


                     BACTERIOLOGICAL BIBLIOGRAPHY.

Bibliography 201:

  BOVIE, W. T. A Direct Reading Potentiometer for Measuring and
  Recording both the Actual and the Total Reaction of Solutions. _Jour.
  Med. Research_, 33, 1915–16, 295.

Bibliography 202:

  BREED, R. S. and DOTTERRER, W. D. The Number of Colonies Allowable on
  Satisfactory Agar Plates. _Jour. of Bact._, 1, 1916, 321.

Bibliography 203:

  BROWNE, W. W. A Comparative Study of the Smith Fermentation Tube and
  the Inverted Vial in the Determination of Sugar Fermentation. _Amer.
  Jour. of Public Health_, 3, 1913, 701.

Bibliography 204:

  CLARK, W. M. An Hydrogen Electrode Vessel. _Jour. Biol. Chem._, 23,
  1915, 475.

Bibliography 205:

  CLARK, W. M. The “Reaction” of Bacteriological Culture Media. _Jour.
  of Inf. Diseases_, 17, 1915, 109.

Bibliography 206:

  CLARK, W. M. The Final Hydrogen Ion Concentrations of Cultures of
  Bacillus Coli. _Science_, n. s. 42, 1915, 71.

Bibliography 207:

  CLARK, W. M. and LUBS, H. A. Hydrogen Electrode Potentials of
  Phthalate, Phosphate, and Borate Buffer Mixtures. _Jour. Biol. Chem._,
  25, 1916, 479.

Bibliography 208:

  CLARK, W. M. and LUBS, H. A. The Differentiation of Bacteria of the
  Colon-Aërogenes Family by the Use of Indicators. _Jour. of Inf.
  Diseases_, 17, 1915, 160.

Bibliography 209:

  ENDO, S. Ueber ein Verfahren zum Nachweis der Typhusbacillen Centbl.
  f. Bakt. _Erste Abt._, 35, 1903–4, 109.

Bibliography 210:

  GILLESPIE, L. J. The Reaction of Soil and Measurements of Hydrogen Ion
  Concentration. _Jour. Wash. Acad. of Sciences_, 6, 1916, 7.

Bibliography 211:

  HILL, H. W. Porous Tops for Petri Dishes. _Jour. Med. Research_, 13,
  1904, 93.

Bibliography 212:

  HILL, H. W. The Mathematics of the Bacterial Count. _Public Health
  Reports and Papers_, 33, 1907, 110.

Bibliography 213:

  ITANO, A. The Relation of Hydrogen Ion Concentration of Media to the
  Proteolytic Activity of Bacillus Subtilis. _Bulletin 167_, 1916, Mass.
  Agric. Ex. Station.

Bibliography 214:

  KENDALL, A. I. and WALKER, A. W. The Isolation of Bacillus Dysenteriae
  from Stools. _Jour. Med. Research_, 23, 1910, 481.

Bibliography 215:

  KINYOUN, J. J. and DEITER, L. V. On the Preparation of Endo’s Medium.
  _Amer. Jour. Public Health_, n. s. 2, 1912, 979.

Bibliography 216:

  LEVINE, M. On the Significance of the Voges-Proskauer Reaction. _Jour.
  of Bacteriology_, 1, 1916, 153.

Bibliography 217:

  LUBS, H. A. and CLARK, W. M. On Some New Indicators for the
  Colorimetric Determination of Hydrogen-ion Concentration. _Jour. Wash.
  Acad. of Sciences_, 5, 1915, 609.

Bibliography 218:

  MCCLENDON, J. F. New Hydrogen Electrodes and Rapid Methods of
  Determining Hydrogen Ion Concentrations. _Amer. Jour. of Physiology_,
  38, 1915, 180.

Bibliography 219:

  MCCLENDON, J. F. A Direct Reading Potentiometer for Measuring Hydrogen
  Ion Concentrations. _Amer. Jour. of Physiology_, 38, 1915, 186.

Bibliography 220:

  MCCRADY, M. H. The Numerical Interpretation of Fermentation-tube
  Results. _Jour. Inf. Diseases_, 17, 1915, 183.

Bibliography 221:

  NOYES, H. A. Agar Agar for Bacteriological Use. _Science_, n. s. 44,
  1916, 797.

Bibliography 222:

  ROGERS, L. A., CLARK, W. M. and DAVIS, B. J. The Colon Group of
  Bacteria. _Jour. of Inf. Diseases_, 14, 1914, 411.

Bibliography 223:

  ROGERS, L. A., CLARK, W. M. and EVANS, A. C. The Characteristics of
  Bacteria of the Colon Type Found in Bovine Feces. _Jour. of Inf.
  Diseases_, 15, 1914, 99.

Bibliography 224:

  ROGERS, L. A., CLARK, W. M. and EVANS, A. C. The Characteristics of
  Bacteria of the Colon Type Occurring on Grains. _Jour. of Inf.
  Diseases_, 17, 1915, 137.

Bibliography 225:

  SMITH, H. M. The Seaweed Industries of Japan. _Bulletin of the Bureau
  of Fisheries_, 24, 1904, 135.

Bibliography 226:

  SÖRENSEN, S. P. L. Enzymstudien. _Biochem. Ztschr._, 21, 1909, 131 and
  201.

Bibliography 227:

  WHIPPLE, G. C. On the Necessity of Cultivating Water Bacteria in an
  Atmosphere Saturated with Moisture. _Tech. Quart._, 12, 1899, 276.

Bibliography 228:

  WHITTAKER, H. A. The Source, Manufacture and Composition of Commercial
  Agar-agar. _Jour. Amer. Pub. Health Assoc._, n. s. 1, 1911, 632.

Bibliography 229:

  CLARK, W. M. and LUBS, H. A. The colorimetric determination of the
  hydrogen-ion concentration of bacteriological culture media. _Jour.
  Wash. Acad. Sciences_, 6, 1916, 483.

Bibliography 230:

  CLARK, W. M. and LUBS, H. A. The colorimetric determination of
  hydrogen-ion concentration and its application in bacteriology. _Jour.
  of Bact._, 2, 1919, 1 and 109.

Bibliography 231:

  COHEN, B. and CLARK, W. M. The growth of certain bacteria in media of
  different hydrogen-ion concentrations. _Jour. of Bact._, 4, 1919, 409.

Bibliography 232:

  FENNEL, E. A. and FISHER, M. A. Adjustment of culture medium
  reactions. _Jour. of Inf. Diseases_, 25, 1919, 444.

Bibliography 233:

  JONES, H. M. A rapid hydrogen-ion electrode method for the
  determination of hydrogen-ion concentrations in bacterial cultures or
  other turbid or colored solutions. _Jour. of Inf. Diseases_, 25, 1919,
  262.




                                 INDEX.


                                   A.

 Acidity, determination of, 39.

 Acids, mineral, 41.

 Agar, nutrient, 94, 96.
   lactose-litmus, 97.

 Alkali carbonates, 39.

 Alkalinity, determination of, 35.

 Albuminoid nitrogen, 20.

 Aluminium sulfate, determination of, 41.
   analysis of, 78.

 Aluminium and iron, determination of, 57.

 Ammonia nitrogen, determination of, 15.

 Apparatus, bacteriological, 93.

 Application of colon group tests, 102.

 Arsenic, determination of, 63.

 Azolitmin solution, 96.


                                   B.

 Bacillus aërogenes, reactions, 108.
   cloacae, reactions, 108.
   coli, reactions, 108.

 B. coli group, tests, 100.
   application of, 102.
   fecal and non-fecal, 106.
   summary of tests, 104.

 Bacteriological examination, 93.
   bibliography, 110.

 Basicity ratio, 80.

 Bibliography,
   bacteriological, 110.
   chemical, 82.
   microscopical, 91.

 Biochemical oxygen demand, 71.
   in sludge and mud, 76.

 Bismuthate method (Mn), 49.

 Boric acid, 63.

 Bottles, sample, 1, 93.
   dilution, 93.

 Bromine and iodine, determination of, 61.

 Broth, nutrient, 95.
   sugar, 95.


                                   C.

 Calcium, determination of, 57.

 Carbon dioxide, determination of, 40.

 Chemical analysis, water and sewage, 1.
   bibliography, 82.

 Chemicals, analysis of, 77.

 Chloride, determination of, 41.

 Chlorine, determination of, 64.

 Coefficient of fineness, 8.

 Collection of samples, bacteriological, 93.
   chemical, 1.

 Colon group, tests (see “B. coli”), 100.

 Color, determination of, 9.

 Copper, determination of, 53, 55.

 Counting (bacterial), 99.

 Cultural characters of colon group, 108.

 Culture media, 94.
   azolitmin solution, 96.
   Endo’s medium, 97.
   litmus-lactose-agar, 97.
   litmus solution, 96.
   methyl red test, 107.
   nutrient agar, 96.
   nutrient broth, 95.
   nutrient gelatin, 96.
   sterilization, 95.
   sugar broth, 95.
   titration, 94.
   tryptophane broth, 107.


                                   D.

 Dilution (bacteriological), 98.

 Dissolved oxygen, 65.


                                   E.

 Effluents, relative stability of, 69.
   biochemical, oxygen, demand of, 71.

 Endo’s medium, 97.

 Erythrosine indicator, 36.

 Ether—soluble matter, 69.
   in sludge and mud, 75.

 Evaporation, 29.

 Expression of results (see under “Results”).


                                   F.

 Fat, determination of, 69, 75.

 Fecal and non-fecal members, colon group, 106.

 Fermentation tubes, 93.

 Ferrous sulfide in sludge and mud, 76.

 Ferrous iron, determination of, 47.

 Ferric iron, determination of, 48.

 Fineness, coefficient of, 8.


                                   G.

 Gelatin media, 94, 96.


                                   H.

 Hardness, determination of, 30.
   bicarbonate, 37.
   carbonate, 38.
   hydroxide, 38.
   non-carbonate, 34.
   temporary, 34.

 Hydrogen sulfide, determination of, 63.

 Hydrogen-ion determination, 94.


                                   I.

 Ignition, loss on, 30.

 Incubation, 99.

 Indol test, broth for, 107.

 Indicators, 36, 94, 107.

 Iodine and bromine, determination of, 61.

 Iron and Aluminium, separation, 57.
   analysis of, 79.

 Iron, determination of, 43.
   standards, 45.
   sulfate, determination of, 41.
   analysis of, 81.


                                   L.

 Lacmoid indicator, 36.

 Lead, determination of, 51, 55.

 Lime, analysis of, 80.

 Lithium, determination of, 60.

 Litmus reagent, 94.
   lactose-agar, 97.
   solution, 96.


                                   M.

 Manganese, determination of, 48.

 Materials, bacteriological, 93.

 Meat extract, 93.

 Media, culture (see “Culture media”), 94–7, 107.

 Methyl orange indicator, 37.

 Methyl red media, 107.
   test, 107.

 Microscopical bibliography, 91.
   examination, 89.

 Mineral analysis, 56.

 Moisture in sludge and mud, 74.

 Mud deposits, analysis, 73.


                                   N.

 Nessler’s reagent,
   color standards, 10.
   ammonia determination, 19.

 Nitrogen, 15.
   ammonia, 15.
   albuminoid, 20.

 Nitrogen, in sludge and mud, 74.
   nitrate, 23.
   nitrite, 22.
   organic, 21.
   total, 25.

 Nutrient media (see “Culture media”), 94, 107.


                                   O.

 Odor, 12.

 Organic nitrogen, 21.

 Oxygen consumed, 25.
   demand, biochemical, 71.
   dissolved, 65.
   in fresh and sea water (table), 68.


                                   P.

 Peptone, authorized brands, 93.

 Persulfate method (Mn), 48.

 Petri dishes, 93.

 Phenoldisulfonic acid method (nitrate), 23.

 Phenolphthalein indicator, 36, 94.

 Physical examination, 4.

 Pipettes, bacteriological, 93.

 Plating, bacteriological, 99.

 Platinum-cobalt color standard, 9.
   wire turbidity, 5.

 Potassium, determination of, 59.

 Presumptive tests, colon group, 102.


                                   R.

 Reactions of colon group, 108.

 Reaction of culture media, 94.
   of sludge and mud, 73.

 Reduction method (nitrate), 24.

 Relative stability method, 71.

 Residue on evaporation, 29.

 Results, expression of,
   bacteriological, 103.
   chemical examination, 14.
   color, 8.
   odor, 12.

 Results, interpretation of (bacteriological), 106.

 Routine procedure (bacteriological), 108.


                                   S.

 Samples,
   bacterial, 93.
   bottles, 1.
   chemical, 1.
   interval before analysis of, 2.
   quantity required, 1.
   representative, 3.
   sludge and mud, 73.

 Sewage sludge, analysis, 73.

 Silica, determination of, 56.

 Soda ash, analysis of, 82.

 Soap method (hardness), 31.

 Sodium and potassium, 58.

 Solids, total, fixed, volatile, 29.

 Specific gravity of sludge and mud, 74.

 Stability, relative, of effluents, 69.
   method, relative, 71.

 Standards,
   ammonia, 17.
   chlorine, 65.
   color, 9.
   hardness, 32.
   iron, 45.
   Nessler, color, 10.
   platinum-cobalt, 10.
   turbidity, 4.

 Sterilization of media, 95.

 Storage of samples, 2, 98.

 Sugars for media, 94.

 Sugar broths, 95.

 Sulfate, K and Na, 58.

 Suspended matter, 30.


                                   T.

 Tin, determination of, 54, 55.

 Tintometer, Lovibond, 11.

 Titration of media, 94.

 Total nitrogen, 25.
   residue on evaporation, 29.

 Tryptophane broth, 107.

 Turbidity, 4.
   coefficient of fineness, 8.
   platinum wire method, 5.
   rod, graduation, 6.
   standard, 4.
   turbidimetric method, 7.
   turbidometer, graduation, 8.


                                   V.

 Voges-Proskauer test, 107.

 Volatile matter, 29.
   in sludge and mud, 74.


                                   Z.

 Zinc, 52.

------------------------------------------------------------------------




                          TRANSCRIBER’S NOTES


 1. Silently corrected typographical errors and variations in spelling.
 2. Archaic, non-standard, and uncertain spellings retained as printed.
 3. The Chemical Bibliography was reformatted in footnote style.
 4. The Bacteriological Bibliography was reformatted in footnote style
      and the numbering was increased by 200.
 5. Enclosed italics font in _underscores_.