Transcriber’s Notes

Obvious typographical errors have been silently corrected. Several
tables have been divided with duplication of the first column because
of their excessive width.

Italics are represented thus _italic_.




                                                 Issued March 17, 1911.

                   U. S. DEPARTMENT OF AGRICULTURE,
               BUREAU OF ANIMAL INDUSTRY.—BULLETIN 132.
                    A. D. MELVIN, CHIEF OF BUREAU.


                        A BACTERIOLOGICAL STUDY
                            OF HAM SOURING.


                                  BY

                         C. N. McBRYDE, M. D.,
             _Senior Bacteriologist, Biochemic Division_.

       [Illustration: UNITED STATES DEPARTMENT OF AGRICULTURE.]


                              WASHINGTON:
                      GOVERNMENT PRINTING OFFICE.
                                 1911.




                    THE BUREAU OF ANIMAL INDUSTRY.


    _Chief_: A. D. MELVIN.
    _Assistant Chief_: A. M. FARRINGTON.
    _Chief Clerk_: CHARLES C. CARROLL.
    _Animal Husbandry Division_: GEORGE M. ROMMEL, chief.
    _Biochemic Division_: M. DORSET, chief.
    _Dairy Division_: B. H. RAWL, chief.
    _Inspection Division_: RICE P. STEDDOM, chief; R. A. RAMSAY,
      MORRIS WOODEN, and ALBERT E. BEHNKE, associate chiefs.
    _Pathological Division_: JOHN R. MOHLER, chief.
    _Quarantine Division_: RICHARD W. HICKMAN, chief.
    _Zoological Division_: B. H. RANSOM, chief.
    _Experiment Station_: E. C. SCHROEDER, superintendent.
    _Editor_: JAMES M. PICKENS.




                        LETTER OF TRANSMITTAL.


    UNITED STATES DEPARTMENT OF AGRICULTURE,
                         BUREAU OF ANIMAL INDUSTRY,
                          _Washington, D. C., November 2, 1910_.

SIR: I have the honor to transmit and to recommend for publication as
a bulletin of this Bureau a paper entitled “A Bacteriological Study
of Ham Souring,” by Dr. C. N. McBryde, senior bacteriologist in the
Biochemic Division of this Bureau.

The souring of hams is a source of considerable loss in the
meat-packing industry, and the cause of this trouble has heretofore
been in doubt. Dr. McBryde’s paper presents the results of an
exhaustive study of the subject, from which it appears that he has
succeeded in discovering the true cause of the trouble. Besides a
description of the experimental work the paper discusses methods of
preventing the souring of hams and the proper disposal of those which
have become affected.

    Respectfully,       A. D. MELVIN,
                          _Chief of Bureau_.

    Hon. JAMES WILSON,
    _Secretary of Agriculture_.




                               CONTENTS.


                                                             Page.

    Introductory                                                       7

    Method of curing hams                                              8

    Definition of souring                                             10

    Classification of sour hams and location of sour areas            10

    Method of detecting sour hams                                     12

    Theories in regard to ham souring                                 12

    Previous experimental work to determine cause of ham
      souring                                                         13

    The present experiments                                           14

        Media employed                                                14

        Method of procedure in examining hams                         15

        Results of examination of sour and sound hams                 16

        Histological changes in sour hams                             17

        Chemical analyses of sour and sound hams                      18

        Bacteriological examination of sour and sound hams            20

        Inoculation experiments with hams                             21

        Probable method by which ham-souring bacillus enters hams     33

            Possibility of infection prior to slaughter               33

            Possible infection from pickling fluids                   34

                Experiment to show whether infection takes place
                  from the curing pickle                              34

            Possible infection through manipulation or handling       35

                Infection from ham thermometers                       35

                Experiment to show whether hams become infected
                  from ham thermometers                               37

                Infection from pumping needles                        41

                Infection from billhooks                              42

        Biological and morphological characteristics of the
          ham-souring bacillus                                        43

             Conditions favorable to growth                           43

             Growth on different culture media                        43

             Morphology                                               46

             Spore formation                                          46

             Resistance to heat and chemical agents                   47

             Gas production                                           47

             Acid production                                          48

             Pathogenic properties                                    48

             Nature of the bacillus                                   48

    Prevention of ham souring                                         50

    General summary and conclusions                                   53

    Acknowledgments                                                   55




                            ILLUSTRATIONS.


                                PLATES.

                                                                   Page.

    PLATE I. Fig. 1.—Section of muscular tissue from sound ham,
    showing muscle fibers cut longitudinally; nuclei sharply
    defined and cross striation distinct. Fig. 2.—Section of
    muscular tissue from sour ham, showing muscle fibers cut
    longitudinally; nuclei undergoing disintegration and cross
    striation indistinct                                              16

    II. Fig. 1.—Section through muscular tissue of ham which has
    undergone natural or spontaneous souring, showing distribution
    of bacilli between the muscle fibers, which are cut obliquely.
    Fig. 2.—Section through muscular tissue of ham which has
    undergone natural or spontaneous souring, showing individual
    bacilli between the muscle fibers, which are cut somewhat
    obliquely                                                         18

    III. Fig. 1.—Section through muscular tissue of artificially soured
    ham, showing distribution of bacilli between the muscle fibers,
    which are shown in cross section. Fig. 2.—Section through
    muscular tissue of artificially soured ham, showing individual
    bacilli between the muscle fibers, which are cut longitudinally
                                                                      26

    IV. Glucose bouillon culture in Smith fermentation tube at four
    days                                                              48


                             TEXT FIGURES.

    FIG. 1. Cross section through body of ham, with sour areas
    indicated by shading and dotted lines                             11

    2. Cross section through body of ham to show method of sampling
    for chemical analysis                                             18

    3. Cross section through body of artificially soured ham,
    showing sour areas and points at which cultures were taken        25

    4. Diagrammatic views showing construction of ham thermometer     36

    5. Ham-souring bacillus (_Bacillus putrefaciens_), grown on
    egg-pork medium                                                   46




                A BACTERIOLOGICAL STUDY OF HAM SOURING.




                             INTRODUCTORY.


The souring of hams is a matter of considerable importance to those
engaged in the meat-packing industry, and has been the occasion of no
little worry, as in even the best-regulated packing establishments
the yearly losses it entails are considerable. The subject has given
rise to much speculation on the part of those engaged in the curing
of meats, as to the cause of the trouble and how it may be remedied,
and has received considerable attention in a practical way, but little
seems to have been done in a scientific way toward determining the
cause and nature of ham souring.

In a well-regulated meat-packing establishment the loss from ham
souring is usually figured at about one-tenth of 1 per cent of the
total weight of hams cured. At first thought this would seem but a
small loss, but when one reflects that in a single large packing
establishment some 3,000,000 hams are cured during the year, the loss,
when figured out, is considerable. Taking 15 pounds as the average
weight of a ham, 3,000,000 hams would represent 45,000,000 pounds of
meat. Figuring the loss from souring on the basis mentioned, the amount
of meat condemned and destroyed during the year would be 45,000 pounds.
Assuming that hams sell at an average wholesale price of 15 cents a
pound, the yearly loss for a single plant which cures 3,000,000 hams a
year would be nearly $7,000.

While one-tenth of 1 per cent of the total weight of hams cured would
represent the loss from souring in a well-regulated establishment,
statistics obtained through Government meat inspectors show that
0.25 per cent would more nearly represent the loss for the entire
country. During the fiscal year from July 1, 1908, to June 30, 1909,
some 670,000,000 pounds of hams were placed in cure in packing
establishments subject to Government inspection. Estimating the loss
from souring at 0.25 per cent, the total amount of meat condemned
and destroyed as sour would be 1,675,000 pounds. At 15 cents a pound
the total annual loss from ham souring in packing houses subject to
Government inspection would figure up something over a quarter of a
million of dollars.

The problem of ham souring, therefore, is quite an important one from a
practical and financial standpoint; but aside from these considerations
it is also a subject of considerable scientific interest, and in
view of the fact that all sour meats are condemned under the Federal
regulations governing meat inspection it has seemed fitting that this
question should be made the subject of scientific investigation on the
part of the Bureau which is charged with the administration of this
inspection.

The investigation reported in this paper has been conducted chiefly
along bacteriological lines, and has been confined entirely to the wet
method of curing hams, as this method is the one generally used in
American packing houses.




                        METHOD OF CURING HAMS.


In order to make clear certain points in regard to the nature and
occurrence of ham souring and to insure a better understanding of the
experiments which are to be described later, it would seem best to
begin with a brief outline of the method of curing hams as practiced in
the larger packing establishments of the country. This description is
merely a general outline of the method of preparing hams for cure and
the method of handling hams while in cure, and deals chiefly with those
points that bear on the question of souring.

After the slaughtered animal has been cleaned, scraped, eviscerated,
washed, and split down the middle, the carcass is usually allowed to
hang for an hour or so in a large room open to the outside air, known
as the “hanging floor.” This is done with a view to getting rid of a
certain amount of the body heat before the carcass is run into the
chill rooms, and effects a saving in refrigeration.

The carcasses are next run into “coolers” or chill rooms, and subjected
to refrigeration with a view to ridding them entirely of their body
heat. The coolers are large rooms fitted with brine pipes and capable
of accommodating several hundred carcasses. The temperature of the
coolers when the carcasses are run in is about 32° F. When filled, the
temperature of the cooler rises to about 45° F., owing to the heat
given off from the carcasses. The temperature is then gradually reduced
to 28 or 30° F. Hog carcasses are left in the coolers as a rule for
forty-eight hours, at the end of which time they are stiff and firm,
but not frozen. The temperature of the chill rooms is always carefully
watched, thermometer readings being made every few hours and duly
recorded. The temperature of the carcasses is always tested when they
leave the chill room. In those plants provided with a hanging floor, a
certain number of the carcasses are also tested before they are sent to
the chill rooms in order to determine the amount of heat lost on the
hanging floor.

The carcasses are tested by means of an especially constructed
thermometer, known as a “ham thermometer,” which has a pointed metal
protector so that it can be thrust into the body of the ham. (See fig.
4.) The ham has been rightly selected as the proper portion of the
carcass at which to take the temperature, as it constitutes the largest
mass of muscular tissue in the carcass and holds the body heat longer
than any other portion. In taking the temperature, the thermometer
is thrust deep into the body of the ham so that the point of the
thermometer rests alongside or a little behind the upper portion of the
femur or middle bone, the latter being used as a guide in introducing
the thermometer. A certain number of the carcasses from each cooler
are tested in this way as a check on the refrigeration. The inside
temperature of the hams when they leave the chill rooms should be about
34° F.

The carcasses are next cut up and the hams trimmed for pickling. In
some houses the hams are given an additional chilling of 48 hours after
they are cut from the carcasses, but this is not done as a rule, nor
does it seem to be necessary.

The hams are now sent to the pickling rooms, or “sweet pickle
department,” as this branch of the packing house is designated, and
here a certain number are again tested with a thermometer, as described
above. This test is carried out by the foreman in charge of the sweet
pickle department in order that he may satisfy himself that the
hams are properly chilled before they go into the pickle and as an
additional check on the refrigeration.

The hams are now ready to be “pumped,” and this pumping, as will be
shown later, constitutes an important step in a successful cure.
Pumping consists in forcing a strong brine solution containing
saltpeter into the muscular tissues of the ham, and is accomplished by
means of a large, hollow, fenestrated needle connected by means of a
rubber hose with a powerful hand pump. The needle is introduced along
the bone, the latter being used as a guide.

In all of the larger packing establishments two general methods of
curing hams are followed, the two methods being designated as the
“fancy” or “mild cure” and the “regular cure,” the term “cure” being
used to designate the curing period. Various trade names are given
by the different packing establishments to the hams cured by these
methods. In the fancy cure the hams are pumped in the shank only,
whereas in the regular cure they are pumped in both body and shank.
The same pumping pickle is generally used for the two cures. It is a
significant fact that the greater proportion of the “sours” are found
among the fancy or mild cure hams. This point will be discussed farther
on in connection with some experiments to be described later.

The actual curing is usually carried out in large vats which hold about
1,400 pounds of meat or some hundred hams. The hams are packed in the
vats in layers and are entirely covered with the pickling solution or
brine. A certain proportion is always observed between the weight of
the meat and the amount of the solution. The pickling solution, or
“pickle,” as it is termed, is a brine solution containing saltpeter
and sugar. The composition of the pickle varies somewhat with the
different packing establishments. The fancy-cure hams are usually cured
in a milder pickle, that is, one that contains less salt and saltpeter
than the pickle used in the regular cure, although in some packing
establishments the same curing pickle is used for the two cures, the
only difference being the additional pumping given the regular-cure
hams. The pickling rooms, or “cellars,” as they are called, are held at
a temperature of 34° to 36° F., and the pickling solutions are always
chilled to this temperature before being used.

The hams are allowed to remain in cure for about 60 days, and during
this time are “overhauled” several times. Overhauling consists in
throwing the hams from the vat in which they are packed into a
neighboring empty vat, and then transferring the pickle to the new vat.
The pickle is not changed, and the same pickle follows the hams through
the entire curing process. The object in overhauling is to stir up the
pickle and expose fresh surfaces of the meat to its action.

Hams are also cured in tierces which hold about 300 pounds of meat.
In the tierce cure, the hams are packed in the tierces, the latter
are then headed up, the pickling solution is next run in through the
bunghole, so as to fill the tierce entirely, and a wooden stopper is
finally driven into the bunghole. The tierces are rolled back and forth
across the floor on dates corresponding to the dates of overhauling in
the vat cure. The object of the rolling is to stir up the pickle, and
in this way it corresponds to overhauling in the vat cure.




                        DEFINITION OF SOURING.


To the meat inspector, a sour ham is one which has a tainted or “off”
odor, that is, any odor which deviates from the normal. The odor may be
very slight, so slight that at times only the trained meat inspector
can detect it. When slight, the odor is elusive and hard to define, but
when pronounced it has a distinctly putrefactive quality. When not very
pronounced, the odor possesses, as a rule, a slightly sour quality,
chemically speaking, and at times this sour quality may be quite
marked; hence the term “sour ham,” or “sour” has originated. In a badly
soured ham—using the term “sour” in the packing-house sense to denote
any ham that is tainted—the odor loses this sour quality and becomes
distinctly putrefactive in nature.




        CLASSIFICATION OF SOUR HAMS AND LOCATION OF SOUR AREAS.


Sour hams are classed as “shank sours” and “body sours,” according to
the location of the souring, and these may be “light” or “heavy.” When
the souring is very pronounced, the ham is termed a “stinker.”

Souring appears to start, as a rule, around the stifle joint
(femorotibial articulation), and extends upward into the body of the
ham.

In quite a large proportion of the hams which are sour in the
body—probably from 40 to 50 per cent—the souring extends through to
the bone marrow of the femur or middle bone, and the sour odor is at
times more pronounced in the bone marrow than in the meat. The odor
of the bone marrow, when pronounced, is strongly suggestive of a
dissecting-room odor, and is distinctly putrefactive in quality.

In the case of light body sours the sour odor is confined to a small
area immediately around the bone, and may be so slight that it is
detected only with difficulty. In such hams the bone marrow is apt to
be sweet, and it is not until the souring becomes more extensive that
the bone marrow becomes involved.

[Illustration: FIG. 1.—Cross section through body of ham, with sour
areas indicated by shading and dotted lines.]

The distribution of the sour area in the body of a well-developed sour
is shown in figure 1.

In the case of a well-developed body sour the sour area is more
pronounced near the bone, as represented in figure 1 by the shaded
area, and may extend out into the body of the ham for a variable
distance, according to the degree of souring, as represented by the
dotted lines, gradually fading off toward the margins, where it may be
imperceptible or entirely wanting.

In the pronounced sours, termed “stinkers,” the odor pervades the
entire ham, and is of a distinctly putrefactive quality.

In shank sours, the souring is more or less confined to the shank, or
the region about the tibio-femoral articulation, but may extend upward
into the lower portion of the body of the ham.




                    METHOD OF DETECTING SOUR HAMS.


Souring is detected and located by means of a pointed metal instrument
known as a “ham trier,” which resembles a long, slightly flattened ice
pick. The trier is thrust into the ham at different points along the
bone, rapidly withdrawn, and the odor which clings to the metal noted.
The trained inspector works very rapidly, and is able to detect even
the slightest sour or off odor which might be imperceptible to one not
trained to the work. At the end of the cure all hams are tested with
the trier under the supervision of Government meat inspectors.

Hams are also given what is called the “30-day inspection” by plant
inspectors during the process of curing. An average ham weighing
from 14 to 16 pounds requires about 60 days to cure, and at the end
of 30 days a certain number of hams in each run are usually tested
to see how the cure is progressing. If no sour hams are discovered
at this inspection the packer knows that the cure is progressing
satisfactorily, and moreover he feels sure that his hams will finish
satisfactorily, for experience has taught him that souring develops
within the first four weeks of the curing period, and if his hams are
sweet at the end of this time, he can feel practically sure that no
sours will develop later on.




                  THEORIES IN REGARD TO HAM SOURING.


The theories as to the cause of souring are many and varied. The
majority of them are pure speculation and have no foundation upon
observed facts. A few of these theories may be enumerated to show how
wide and varied has been the speculation upon this subject.

A theory which is quite prevalent among packing-house employees
attributes souring to overheating of the animal previous to slaughter,
but tests were made by driving hogs to the point of exhaustion just
prior to slaughter and curing the hams from these animals in comparison
with hams taken from animals which had been rested prior to slaughter,
with no difference in the cured product; that is, the hams taken from
overheated hogs cured equally as well as those taken from rested hogs.

Another theory attributes souring to a diseased condition of the meat.
Prior to the enforcement of the Federal regulations governing meat
inspection there might have been some ground for such a supposition,
but this theory could not hold at the present time, in view of the
thorough and efficient inspection now in force, for it can be safely
said that no diseased meat now passes the Government inspectors, and
therefore no diseased meat goes into cure in inspected houses. In
order to test this theory, however, hams were secured from a number
of condemned animals which showed various diseased conditions, such
as hog cholera, pyemia, septicemia, scirrhous chord, etc., and these
hams were cured in comparison with hams taken from normal hogs. It was
found that the hams taken from the diseased hogs cured equally as well
as those taken from healthy hogs. The hams from the diseased hogs were
destroyed after the experiment, as the meat taken from diseased animals
was of course not considered fit for consumption, the object of the
experiment being merely to determine whether or not souring is caused
by diseased conditions.

Another theory attributes souring to imperfect or too rapid chilling
of the meat before it is put in pickle, and places the blame upon the
refrigeration. According to this theory, souring results when the meat
is chilled too suddenly, the idea being that by the rapid congealing of
the juices of the meat a coating is formed on the outside of the ham
whereby the animal heat is prevented from escaping from the interior,
leaving the meat next to the bone at a higher temperature than the
outside of the ham.

In order to test this last theory, a number of hog carcasses were run
direct from the killing floor to a cooler at 28° F. and a like number
of carcasses of the same average weight which had been allowed to stand
for two hours at the outside temperature of the air (53° F.) were
placed in the same cooler. The carcasses which had hung for two hours
in the air had lost an average of 14 degrees in temperature before
going to the cooler. The temperature of the cooler rose to 29° F. after
the carcasses were put in, but was soon reduced to 28° F. and held at
this temperature. The temperatures of the hams were taken at the end
of 24 hours, and practically no difference was found in the inside
temperatures of the two lots; that is, the hams on the hot carcasses
which were subjected to a sudden chilling exhibited practically the
same inside temperature (i. e., next to the bone) as those which had
cooled for two hours at the temperature of the air before being placed
in the cooler.

Still another theory attributes souring to lack of penetration of the
pickling fluids, but analyses of sour and sound hams do not seem to
bear out this theory. The rate of penetration of the pickling fluids,
however, would seem to have some bearing on the subject, and this point
will be discussed later in connection with some laboratory experiments
on the inhibitory effects of sodium chlorid and potassium nitrate.

So much for the more commonly accepted theories which have been
advanced to explain ham souring.




     PREVIOUS EXPERIMENTAL WORK TO DETERMINE CAUSE OF HAM SOURING.


A review of the literature reveals but one article bearing directly on
the subject of the cause of ham souring.

In June, 1908, Klein[1] published in the London Lancet an article
on “miscured” hams. He describes a miscured ham as one which has a
distinctly putrid smell, and the tainted areas he describes as varying
in color from a dirty gray to a dirty green, the muscular tissues in
the strongly tainted areas being swollen and soft, or jelly-like. From
such hams he isolated a large nonmotile, nonspore-bearing, anaerobic
bacillus which he calls _Bacillus fœdans_. He cultivated the organism
on different media and obtained from the cultures a putrid odor
resembling that of the ham from which the culture was obtained, but
did not attempt to produce tainting by injecting sound hams with the
bacillus.

[1] Klein, E. On the nature and causes of taint in miscured hams. The
Lancet, vol. 174, London, June 27, 1908.

While there can be little doubt that Klein’s bacillus was the cause of
the tainting in those hams which he examined, the proof would certainly
have been stronger had he injected sound hams with cultures and thus
proven that he could reproduce tainting experimentally by means of his
bacillus. Klein examined only dry-cured hams and does not state the
temperature at which they were cured. He fails to offer any explanation
as to how the bacillus gained entrance into the hams.




                       THE PRESENT EXPERIMENTS.


                            MEDIA EMPLOYED.

After considerable experimentation as to a suitable culture medium for
the bacteriological study of sour hams, a modification of the “egg-meat
mixture” used by Rettger[2] in his studies on putrefaction was found
to be the most satisfactory. This medium, which consists of chopped
meat and egg albumen, furnishes an excellent medium for the growth of
putrefactive organisms which rapidly break down the proteids of the
meat, giving rise to the characteristic odors of putrid decomposition.
Rettger used chopped beef and egg albumen, but for the present work
chopped pork was substituted for the beef, as affording a more suitable
medium for the growth of organisms accustomed to growth in pork hams.
The modified medium is prepared as follows:

A. One-half pound of lean pork, freed from excess of fat and sinew,
is finely chopped in a meat chopper, 250 cubic centimeters of water
is then added, the meat acids are neutralized with sodium carbonate,
and the mixture is heated in an Arnold sterilizer for 30 minutes, with
occasional stirring. It is then set away in a cold place for several
hours. A small amount of fat collects at the top in the form of a fatty
scum, as it is impossible to remove all of the fat from the meat before
it is chopped. The fatty scum, which hardens upon standing in the cold,
is now removed.

B. The whites of three eggs are mixed with 250 cubic centimeters of
water. The mixture is rendered neutral to phenolphthalein by means
of dilute hydrochloric acid and heated for 30 minutes in the Arnold
sterilizer, with occasional stirring.

A and B are now mixed and 2.5 grams (0.5 per cent) of powdered calcium
carbonate added. The mixture is next run into large sterile test tubes,
or sterile flasks, and sterilized in an Arnold sterilizer on three
successive days.

[2] Rettger, L. F. Studies on putrefaction. Journal of Biological
Chemistry, vol. 2, 1906.

In addition to the egg-pork mixture described above, culture tubes
of agar and bouillon prepared from pork instead of beef, with the
addition of 1 per cent of glucose, were also used; but the best results
were obtained with the egg-pork medium, as with this medium, the
early development of sour or putrefactive odors furnished a valuable
indication as to the presence of organisms capable of producing sour or
putrefactive changes in meat.


                METHOD OF PROCEDURE IN EXAMINING HAMS.

The hams were sectioned through the body, the femur, or “middle bone,”
as it is known in packing-house parlance, being cut at a point about
1½ or 2 inches below its head. A cross section of a ham thus cut
is shown in figure 1. After sectioning, the hams were subjected to a
microscopical, bacteriological, and chemical examination as follows:

_Microscopical examination._—Bits of muscular tissue, taken from
various points, were teased out in salt solution and the condition of
the muscle fibers noted. Smear preparations were also made from bits
of muscular tissue and from the bone marrow, and these were stained
and subjected to microscopical examination. Portions of the meat were
also hardened and cut into microscopic sections, which were stained and
mounted for histological and bacteriological study.

_Bacteriological examination._—In the bacteriological examination
of sour hams, especial attention was directed to the detection of
anaerobic species, as it seemed reasonable to suppose that if the
changes taking place in sour hams were due to bacteria these bacteria
would in all likelihood be anaerobes (i. e., organisms which develop
in the absence of oxygen). This assumption was based upon the fact
that, as a rule, souring begins in the interior of the ham next to the
bone, and, furthermore, the hams are cured in large vats where they
are completely submerged in the pickling fluids, so that any bacteria
which develop within the bodies of the hams while they are in cure are
probably restricted to practically anaerobic conditions.

Cultures were made from the interiors of the hams at various points by
first searing the cut surface thoroughly with a heavy metal spatula and
then cutting out, by means of sterile scissors and forceps, plugs of
meat about 1 cm. square. The plugs of meat were then dropped into tubes
containing the egg-pork medium and pushed down to the bottom of the
tubes, where they were held in place by the chopped meat above; in this
way conditions favorable for the development of anaerobic organisms
were obtained. In inoculating the pork-agar tubes, the medium was
first boiled to expel any inclosed air and cooled to 43° to 45° C; the
plugs of meat were then dropped into the tubes and the agar rapidly
solidified by plunging the tubes in cold water; in this way the bits of
meat were inclosed in the agar at the bottom of the tubes, affording
suitable conditions for anaerobic growth. Aerobic and anaerobic plates
were also made from the meat, and in most cases bouillon tubes were
also inoculated. Cultures were always taken from the bone marrow as
well as from the meat. Novy jars were also used for obtaining anaerobic
conditions in growing the cultures.

_Chemical examination._—In order to determine whether the souring was
connected with or dependent upon a lack of penetration of the pickling
fluids to the interior of the meat, the hams were further subjected to
a chemical examination and the content of the meat in sodium chlorid
and potassium nitrate determined at varying depths.


            RESULTS OF EXAMINATION OF SOUR AND SOUND HAMS.

The sour hams examined were obtained from four different packing
establishments. All of the hams studied were “sweet-pickle hams” which
had not been smoked. The sour hams selected for examination were good
typical body sours, in which the sour odor was well developed, but not
of the very pronounced or putrefactive type.

The sour odor in every case was found to be more pronounced next to
the bone, being usually rather more pronounced just behind the bone,
that is, on the fat side of the bone. The sour odor in each instance
was confined to an area of meat immediately surrounding the femur and
extending out through the body of the ham for a variable distance, as
shown by the dotted lines in figure 1, but in no case did the sour odor
extend all the way to the margin of the meat, nor did it as a rule
extend below the tibio-femoral articulation, the shank proper and the
bone marrow of the shank (i. e., of the tibia) being usually sweet. The
butt portion of the hams—that portion above and behind the hitch bone
(symphasis pubis)—was also sweet.

Immediately after sectioning, the sour areas, as a rule, could be
readily distinguished by a difference in color. In the freshly cut
hams the muscular tissue near the bone, where the sour odor was more
pronounced, exhibited a slight but distinct grayish hue, at times
having a slight greenish tinge; in other words, the muscular tissue in
the sour areas lacked the normal bright red color of the sound meat
and was distinctly lighter in color than the surrounding tissues.
Upon exposure to air, however, the lighter, grayish, sour areas tend
to assume a reddish hue and become much less pronounced than in the
freshly cut ham. After the cut surface of the ham has been exposed to
the air for some time it may be difficult to distinguish the sour areas
by any difference in color.

[Illustration:

BUL. 132, BUREAU OF ANIMAL INDUSTRY, U. S. DEPT. OF AGRICULTURE.
                         PLATE I.

    FIG. 1.—SECTION OF MUSCULAR TISSUE FROM SOUND HAM, SHOWING
    MUSCLE FIBERS CUT LONGITUDINALLY; NUCLEI SHARPLY DEFINED AND
    CROSS STRIATION DISTINCT.

(Pen-and-ink drawing made with camera lucida from section stained with
    hematoxylin and eosin to show histological structure. × 320.)]


    FIG. 2.—SECTION OF MUSCULAR TISSUE FROM SOUR HAM, SHOWING
    MUSCLE FIBERS CUT LONGITUDINALLY; NUCLEI UNDERGOING
    DISINTEGRATION AND CROSS STRIATION INDISTINCT.

(Pen-and-ink drawing made with camera lucida from section stained with
    hematoxylin and eosin to show histological structure. × 320.)]

[Illustration:

    BUL. 132, BUREAU OF ANIMAL INDUSTRY, U. S. DEPT. OF AGRICULTURE.
                               PLATE II.

    FIG. 1.—SECTION THROUGH MUSCULAR TISSUE OF HAM WHICH HAS
    UNDERGONE NATURAL OR SPONTANEOUS SOURING, SHOWING DISTRIBUTION
    OF BACILLI BETWEEN THE MUSCLE FIBERS, WHICH ARE CUT OBLIQUELY.
    THE DARK MASSES BETWEEN THE MUSCLE FIBERS REPRESENT CLUMPS OF
    BACILLI.

 (Pen-and-ink drawing made with camera lucida from section stained by
           the Gram-Weigert method to show bacteria. × 85.)


    FIG. 2.—SECTION THROUGH MUSCULAR TISSUE OF HAM WHICH HAS
    UNDERGONE NATURAL OR SPONTANEOUS SOURING, SHOWING INDIVIDUAL
    BACILLI BETWEEN THE MUSCLE FIBERS, WHICH ARE CUT SOMEWHAT
    OBLIQUELY. NUCLEI HAVE LOST SHARP OUTLINE AND CROSS STRIATION
    IS INDISTINCT.

(Pen-and-ink drawing made with camera lucida from section stained by the
             Gram-Weigert method to show bacteria. × 320.)]

In the sour areas near the bone the muscular tissue was distinctly
softer; that is, it broke and cut more readily than the surrounding
tissues. This was usually quite noticeable in cutting out plugs of the
meat for making cultures. In a ham which shows pronounced souring the
muscular tissues in the worst affected areas may become quite soft and
even slightly gelatinous.

The sour areas, when tested with litmus paper, frequently showed a
slight but distinct alkaline reaction. When aqueous extracts of the
sour meat, however, were titrated with phenolphthalein they were found
to be acid.


                  HISTOLOGICAL CHANGES IN SOUR HAMS.

In preparations made by teasing out bits of the meat in physiological
salt solution, the cross striation of the muscle fibers from the sour
areas was found to be much less distinct than in similar preparations
taken from sound portions of the meat or from sound hams. At times it
was found that the muscle fibers in the sour areas had completely lost
their cross striæ, but the longitudinal striation could still be made
out. In cases where the souring was pronounced there was sometimes
complete loss of both longitudinal and cross striation; in these cases
the muscle fibers appeared to have undergone slight swelling and the
protoplasm exhibited a finely granular appearance.

In stained sections of the sour meat another striking change was
noticed in the disintegration of the nuclei of the muscle fibers, which
are at times completely broken up, appearing as bluish granular masses
in sections stained with hematoxylin and eosin. (Compare figs. 1 and 2
of Pl. I.)

In sections stained by the Gram-Weigert method to show the presence of
bacteria, a large Gram-staining bacillus was noted between the muscle
fibers in the connective-tissue elements of the muscle. In some of the
sections these bacilli were present in great numbers, sometimes in
densely packed clumps or masses, while in other sections, or in other
portions of the same section, they were only sparsely distributed
between the muscle fibers. Where the bacteria were more numerous the
histological changes in the muscle fibers, especially the breaking
down of the nuclei, were more noticeable. The intermuscular connective
tissue had apparently furnished paths of least resistance along which
the organism followed. In Plate II, figures 1 and 2, the bacteria are
shown between the muscle fibers under low and high power magnifications.

In Plate II, figure 1, under the low-power magnification, the bacteria
appear as dark clumps or bands between the muscle bundles. Under the
high power they are shown following along the sarcolemma sheaths
between the muscle fibers.


               CHEMICAL ANALYSES OF SOUR AND SOUND HAMS.

In order to determine whether there was any difference in regard to the
penetration of the pickling fluids in the sour hams as compared with
sound hams, a series of four sour hams were subjected to a chemical
examination in comparison with four sound hams. All were sweet-pickle
hams and were obtained from the same packing establishment. They were
all of the same cure and the same approximate age (i. e., length of
cure) and the same approximate weight.

In taking samples for chemical analysis, the following procedure was
adopted: A section about 2½ inches wide was cut from the center of
the body. The two ends of this section were then trimmed off along
the lines L-M and N-O, as shown in figure 2. Beginning at the skinned
surface, four slices, A, B, C, and D, were then made, as indicated by
the dotted lines. Slice B contained the bone in each instance. Slice
D was practically all fat. Each slice was ground separately in a meat
chopper and the sample thoroughly mixed before taking out portions for
analysis.

[Illustration: FIG. 2.—Cross section through body of ham to show method
of sampling for chemical analysis. A, slice below bone; B, bone slice;
C, slice above bone; D, fat slice.]

As all of the hams examined were mild-cure hams, that is, had been
pumped in the shank only, the pickling fluids in order to reach the
bodies of these hams had to penetrate chiefly from the skinned surface
of the ham, as little if any penetration takes place through the thick
skin of the ham.

The analyses[3] shown in the following tables therefore indicate the
degree of penetration of the pickling fluids.

[3] These analyses were made by Mr. R. R. Henley, of the Biochemic
Division, Bureau of Animal Industry.

                  _Analyses of sour hams._
    ───┬────────────┬──────┬───────────┬───────────
    No.│Description.│Slice.│   NaCl.   │  KNO₃.
    ───┼────────────┼──────┼───────────┼───────────
       │            │      │_Per cent._│_Per cent._
     1 │Sour body   │  A   │   6.18    │   0.175
       │            │  B   │   4.83    │    .224
       │            │  C   │   3.65    │    .299
       │            │  D   │   1.03    │    .074
       │            │      │           │
     2 │   do       │  A   │   5.34    │    .174
       │            │  B   │   3.70    │    .150
       │            │  C   │   2.79    │    .174
       │            │  D   │   1.12    │    .012
       │            │      │           │
     3 │   do       │  A   │   5.04    │    .125
       │            │  B   │   4.08    │    .149
       │            │  C   │   2.72    │    .099
       │            │  D   │   1.19    │    .048
       │            │      │           │
     4 │   do       │  A   │   7.78    │    .250
       │            │  B   │   5.31    │    .100
       │            │  C   │   4.76    │    .200
       │            │  D   │   1.96    │    .048
    ───┴────────────┴──────┴───────────┴───────────


                 _Analyses of sound hams._
    ───┬────────────┬──────┬───────────┬───────────
    No.│Description.│Slice.│   NaCl.   │  KNO₃.
    ───┼────────────┼──────┼───────────┼───────────
       │            │      │_Per cent._│_Per cent._
     1 │Sound       │  A   │   5.80    │   0.211
       │            │  B   │   4.83    │    .188
       │            │  C   │   3.86    │    .221
       │            │  D   │   1.33    │    .063
       │            │      │           │
     2 │   do       │  A   │   4.94    │    .197
       │            │  B   │   4.08    │    .149
       │            │  C   │   3.05    │    .223
       │            │  D   │   1.56    │    .059
       │            │      │           │
     3 │   do       │  A   │   5.92    │    .173
       │            │  B   │   4.29    │    .099
       │            │  C   │   4.12    │    .139
       │            │  D   │   2.32    │    .049
       │            │      │           │
     4 │   do       │  A   │   5.53    │    .119
       │            │  B   │   4.89    │    .079
       │            │  C   │   4.32    │    .099
       │            │  D   │   2.19    │    .041
    ───┴────────────┴──────┴───────────┴───────────

Taking an average of the four slices in each ham so as to get an
average for the entire ham, and comparing the sour hams with the sound
hams, we have the following comparison:

                                                      NaCl.   KNO₃.
    Average for 4 sour hams (entire ham)   per cent.  3.84    0.143
    Average for 4 sound hams (entire ham)     do      3.93     .131

These figures show practically no difference between the sour and the
sound hams as regards the sodium chlorid and potassium nitrate content
of the entire ham.

If, now, we compare the bone slices—and these afford really a better
basis for comparison, as in sour-body hams the souring is always more
pronounced around the bone—we have the following figures:

                                                      NaCl.   KNO₃.
    Average for 4 sour hams (bone slice)   per cent.  4.48    0.155
    Average for 4 sound hams (bone slice)     do      4.52    0.129

Here, again, we find no essential difference between the sour and the
sound hams, and we must conclude from these analyses that souring does
not depend upon or result from a lack of penetration of the pickling
fluids.

It seems probable that in mild-cure hams, which are pumped in the
shank only, the souring begins in the upper portion of the shank and
extends upward along the bone into the body of the ham, and that it
takes place before the pickling fluid has penetrated to the interior
of the ham. When the pickling fluid reaches the interior of the ham
it tends to inhibit the souring, which, as will be shown later, is
due to the development of bacteria within the bodies of the hams. The
growth of the bacteria, however, within the bodies of the hams and the
histological changes in the muscle fibers do not seem to interfere with
the penetration of the pickling fluids.


          BACTERIOLOGICAL EXAMINATION OF SOUR AND SOUND HAMS.

In all of the sour hams which were examined bacteriologically a large
anaerobic bacillus was found to be constantly present. From several
of the hams this bacillus was obtained in pure culture; that is, it
was the only organism present in cultures made from the sour meat and
from the bone marrow of the femur. Such cultures, when held at room
temperature, gave, at three days, a sour-meat odor exactly resembling
that obtained from sour hams.

In many of the sour hams other bacteria were found in association with
the anaerobic bacillus noted above. These other bacteria, however, were
not constant, being sometimes present and sometimes absent. Among the
other bacteria noted in the sour hams, the following forms occurred
most frequently:

1. A nonmotile, gram-positive bacillus, measuring from 1.5 to 4 microns
in length by 0.5 micron in breadth, sometimes in chains and filaments.

2. A small, nonmotile, gram-negative bacillus, about the size of
_Bacillus coli_ and usually in pairs.

3. A large micrococcus.

Sometimes one and sometimes all of these bacteria were present in a
given ham. They were encountered most frequently in hams which had
been pumped in both body and shank, and were probably ordinary pickle
bacteria. They were not strict anaerobes, but belonged to the class of
facultative or optional anaerobes; that is, organisms which will grow
either with or without free oxygen. These bacteria were isolated and
grown on the egg-pork medium, but failed to give any characteristic
sour or putrefactive odors, and were therefore discarded.

A series of sound hams, all of them of mild cure—that is, hams which
had been pumped in the shank only—were also examined bacteriologically.
In examining these hams cultures were taken at varying depths,
beginning at the skinned surface and going backward toward the fat.
Cultures were also taken from the bone marrow of the femur. In the
cultures taken near the skinned surfaces the ordinary pickle bacteria
were obtained, but these did not, as a rule, extend beyond a depth of
3 centimeters below the skinned surface. The cultures taken from the
deeper portions of the hams and from the bone marrow of the femur were
entirely negative—that is, failed to show any growth—and the anaerobic
bacillus noted in the sour hams was not encountered in any of the
cultures made from these hams.

The anaerobic bacillus isolated from the sour hams was found to
correspond in morphology with the organism noted in the microscopic
sections made from the muscular tissue. In view of this fact and the
fact that it was constantly present in the sour hams examined, and was
capable of producing in egg-pork cultures a sour-meat odor of the same
nature as that obtained from sour hams, this organism was subjected to
further study and experimentation.


                  INOCULATION EXPERIMENTS WITH HAMS.

The experiments which follow were conducted at two different packing
establishments in one of the larger packing centers of the country.
The officials at each of these establishments showed great interest in
the experiments and were most courteous and obliging in supplying the
necessary materials.

The first question to be decided was whether the bacillus isolated from
sour hams was actually capable of causing ham souring. The bacillus in
question had, when cultivated on the egg-pork medium, given rise to a
sour odor similar to that obtained from sour hams, but this was not
regarded as proof positive that the organism was the actual cause of
souring in hams. The proper way to decide this point seemed to be to
inoculate hams with the bacillus and then subject these hams to the
regular method of cure and see whether they became sour, just as the
pathogenic properties of a disease-producing organism are determined by
the inoculation of experiment animals. The first two experiments which
follow were designed to decide this point.

It was regarded as important to conduct similar experiments at two
different establishments, in order to determine whether the same
results would be obtained under the somewhat different conditions
imposed by different methods of cure. The two experiments which follow
were carried out, therefore, at different establishments.


                            _Experiment I._

In carrying out this experiment four tierces of hams were “put down”
or “packed”—that is, placed in cure. Two of the tierces were given the
fancy or mild cure and two the regular or stronger cure. The hams in
two of the tierces, one mild and one regular cure, were injected with
a culture suspension of the bacillus; the other two tierces were not
injected with culture and were put down to serve as checks on the
cure. Hams weighing from 12 to 14 pounds were used for the mild cure,
while for the regular cure hams weighing from 14 to 16 pounds were
used. This was in accordance with the general rule which prevails in
packing houses, the lighter hams being subjected to the mild cure and
the heavier hams to the regular cure. The only difference between the
mild and the regular cure in this experiment lay in the pumping. The
hams which were given the mild cure were pumped in the shank only,
while those given the regular cure were pumped in the body as well as
in the shank.

All of the hams had received the usual 48-hour chill. They were all
pumped with the same pumping pickle and cured in the same curing
pickle, and were in cure for the same length of time. The pumping and
curing pickles used were the regular pumping and curing pickles of the
establishment at which the experiment was carried out, and the hams
were cured in accordance with the fancy and regular cures as practiced
at this establishment.

The hams were packed in new tierces which had been thoroughly scalded
with boiling water. The tierces were held in a curing room which was
kept at an average temperature of from 34° to 36° F., the temperature
occasionally going as high as 38° and 40° F., but never above 40°
F. The hams were left in cure for about 70 days, which is a little
longer than the usual cure. The tierces were rolled three times during
the cure. At the end of the cure the hams in all four tierces were
carefully tested by an expert meat inspector, who knew nothing of the
treatment which the hams had received.

The hams in two of the tierces were inoculated with a culture
suspension prepared as follows: Ten tubes of egg-pork medium, each
tube containing approximately 10 cubic centimeters of the medium,
were inoculated with the bacillus and held at room temperature (20°
to 25° C.) for six days. The cultures were then filtered through
sterile gauze into a large sterile flask; this was done in order to
remove the particles of meat, which might otherwise have clogged the
syringes used in inoculating the hams. In transferring the contents
of the culture tubes to the filter the tubes were washed out with
sterile physiological salt solution (0.6 per cent sodium chlorid),
and the meat particles on the filter were afterwards washed with the
salt solution, a sufficient quantity of the latter being used to bring
the total volume of filtrate to 400 cubic centimeters. A microscopic
preparation from the filtrate showed the organisms in large numbers,
with an occasional rod showing a large terminal spore. This suspension
was used for the injection of 40 hams, each ham being given 10 cubic
centimeters, or the equivalent of 2.5 cubic centimeters of the original
culture. The hams were injected with the culture suspension by means of
a sterile syringe carrying a long 5-inch needle. The needle was thrust
well into the body of the ham at a point near the upper end of the
middle bone or femur, the latter being used as a guide in inserting
the needle and the injection being made into the tissues just behind
and a little to one side of the upper end of the femur.

The details of the experiment were as follows:

    _Tierce No. 1 (fancy cure)._—This tierce contained 20 hams
    weighing from 12 to 14 pounds each. These hams were pumped in
    the shank only. Immediately after pumping they were injected
    with 10 cubic centimeters each of the liquid culture or
    suspension described above. After injection the hams were
    immediately packed in the tierce, which was then headed up,
    filled with the regular curing pickle, and placed in cure.

    Result: When tested at the end of the cure all of the hams
    in this tierce save one were found to be sour. In 10 of them
    the souring was very marked throughout the body of the ham
    and extended into the shank as well. In six the souring was
    very marked in the body of the ham, but did not extend into
    the shank. In three there was slight but well-marked souring
    in the body of the ham with no souring in the shank, and one
    remained sweet. The probable explanation of the variation in
    the degree and the extent of the souring will be discussed
    later. The bone marrow of the femur or middle bone was tested
    in all of the hams and found to be sour in 18. In one of the
    hams which showed only slight souring in the body the souring
    did not extend through to the bone marrow, and in the ham which
    remained sweet the bone marrow was also sweet. The fact that
    one ham in this tierce remained sweet was in all likelihood
    due to an oversight in making the inoculations. In making the
    inoculations the hams were spread out in a row on a table by a
    packing-house assistant, who removed the hams as soon as they
    were inoculated and placed them in tierces; and it is more than
    probable that the assistant removed one of the hams before it
    was inoculated in the interval when the writer was busy filling
    the syringe for the next inoculation.

    _Tierce No. 2 (fancy cure)._—This tierce contained 20 hams of
    the same average weight as the preceding. They were pumped in
    the shank only, but were not injected with culture, being put
    down to serve as checks on the hams in tierce No. 1. These
    hams, therefore, were subjected to exactly the same cure and
    were held under exactly the same conditions as those in tierce
    No. 1, the only difference being that the hams in this tierce
    were not injected with culture.

    Result: When tested at the end of the cure all of the hams in
    this tierce were found to be perfectly sound and sweet, showing
    that the curing in this instance was properly carried out and
    that the souring of the hams in tierce No. 1 was undoubtedly
    due to the injections of culture which they received.

    _Tierce No. 3 (regular cure)._—This tierce contained 20 hams
    weighing from 14 to 16 pounds each. These hams were pumped in
    the shank and also in the body. Immediately after pumping they
    were each injected in the same manner as those in tierce No. 1
    with 10 cubic centimeters of culture. The hams were then packed
    in tierce and placed in cure.

    Result: At the end of the cure 9 of the hams were found to be
    sour, while 11 remained sweet. Of the 9 hams which became sour,
    1 showed very pronounced souring in the body and in the shank
    as well, 3 showed very pronounced souring in the body, 1 showed
    pronounced souring in the body, and 4 slight souring in the
    body. The bone marrow of the femur was tested in all of the
    sour hams and was found to be sour in 7. In 2 of the sour hams
    which showed slight souring in the body the souring noted in
    the meat had not extended through to the bone marrow.

    _Tierce No. 4 (regular cure)._—This tierce contained 20 hams
    of the same average weight as those in tierce No. 3, and, like
    the latter, were pumped in both shank and body, but were not
    injected with culture. This tierce was put down to serve as
    a check on tierce No. 3 and was held under exactly the same
    conditions, the only difference being that these hams were not
    injected with culture.

    Result: At the end of the cure the hams were carefully tested
    and all were found to be perfectly sound and sweet.


                      _Results of Experiment I._

    ───────┬────────┬─────────┬────────┬─────────────
    No. of │ Number │ Average │ Cure.  │ How pumped.
    tierce.│of hams.│ weight  │        │
           │        │ of hams.│        │
           │        │         │        │
    ───────┼────────┼─────────┼────────┼─────────────
           │        │_Pounds._│        │
       1   │   20   │  12-14  │ Fancy  │Shank only
           │        │         │        │
           │        │         │        │
       2   │   20   │  12-14  │   do   │    do
           │        │         │        │
           │        │         │        │
       3   │   20   │  14-16  │Regular │Shank and body
           │        │         │        │
           │        │         │        │
       4   │   20   │  14-16  │   do   │    do
           │        │         │        │
    ───────┴────────┴─────────┴────────┴─────────────

    ───────┬─────────────────┬─────────────────────────
           │                 │    Condition at end
           │                 │        of cure
           │                 ├───────────┬─────────────
    No. of │    Treatment    │ Number of │Percentage of
    tierce.│                 │ sour hams │  sour hams
    ───────┼─────────────────┼───────────┼─────────────
       1   │Each ham injected│    19     │    95
           │with 10 c.c. of  │           │
           │culture.         │           │
           │                 │           │
       2   │Not injected with│     0     │     0
           │culture; check on│           │
           │tierce 1.        │           │
           │                 │           │
       3   │Each ham injected│     9     │    45
           │with 10 c.c. of  │           │
           │culture.         │           │
           │                 │           │
       4   │Not injected with│     0     │     0
           │culture; check on│           │
           │tierce 3.        │           │
    ───────┴─────────────────┴───────────┴─────────────

Three hams from each tierce were selected for bacteriological and
histological examination. From tierces 1 and 3, which contained the
injected hams, three of the most pronounced “sours” were selected from
each tierce. In examining the hams bacteriologically the following
method was adopted: The hams were sectioned near the center of the body
and the larger or butt end turned up so as to expose the cut surface. A
cross section of a ham thus cut is shown in figure 3.

Cultures were taken at the points indicated by the numbers and from the
exposed bone marrow of the femur by first searing the surface, and then
taking out plugs of the meat or marrow by means of sterile instruments.
The plugs of meat or marrow were dropped into tubes containing egg-pork
medium and pushed to the bottom of the tubes by means of a sterile
platinum wire. In the cultures made from the sour hams from tierces 1
and 3, which were injected with culture, the bacillus with which these
hams were injected was found in practically every culture, although it
was sometimes absent in the cultures taken at points near the skinned
surfaces of the hams (i. e., at points 1, 4, and 5 in fig. 3). In the
cultures taken from the meat, the bacillus was not always present in
pure culture, but this is not to be wondered at when we remember that
the pickling fluids often contain large numbers of bacteria of various
kinds, and these, of course, find their way into the hams in the
pickling fluids. Especially is this true of the hams which are pumped
in the body, where bacteria are actually pumped into the bodies of the
hams in the pumping pickle. In the case of hams which are not pumped in
the body, the pickle bacteria do not appear to penetrate the body of
the ham to any great depth.

In figure 3 the plus signs after the figures represent the distribution
of the sour-ham bacillus in one of the hams from tierce 1, and this
may be taken as a typical example of the other sour hams which were
examined in this experiment. It should be explained that the shaded
areas are not intended to represent the actual limits of souring, but
simply the areas in which the sour odor was most pronounced and from
which it could be readily obtained with the trier. In comparing the
regular and mild cure hams, it was found that the areas of souring as
defined with the trier were more restricted in the regular cure hams,
and this was undoubtedly due to the additional pumping which these hams
received, whereby the growth of the bacillus was partially inhibited.

[Illustration:

    FIG. 3.—Cross section through body of artificially soured ham,
    showing sour areas and points at which cultures were taken.
    Darker shading indicates sour area in hams pumped in body and
    shank; light shading indicates sour area in hams pumped in
    shank only; figures indicate points at which cultures were
    taken; plus signs indicate presence of bacillus; minus sign
    indicates absence of bacillus; X indicates point of inoculation.]

It will be noticed that the sour-ham bacillus was present in cultures
taken at points outside the shaded areas, indicating that the organism
had extended generally throughout the bodies of the hams. As the
hams were inoculated at a point just to one side of and a little
behind the femur (i. e., at the point X in the figure), the presence
of the bacillus generally throughout the hams would indicate a very
extensive multiplication of the original bacilli with which the hams
were injected. In view of the fact that the bacillus in question is
nonmotile, the spread of the bacilli throughout the hams must result
simply from subdivision and growth by extension, and in spreading
throughout the hams the bacilli appear to follow along the connective
tissue bands which afford paths of least resistance. In the cultures
made from the bone marrow the bacillus was recovered in pure culture
from each of the hams examined, and it is probable that the bacillus
finds its way into the bone marrow from the meat by following along the
small arteries which pass through the bone. The fact that the bacillus
was found in pure culture (i. e., uncontaminated) in the cultures
made from the bone marrow is explained probably by its capacity for
growth by extension, and also by the fact that the pickling solutions
probably do not reach the bone marrow until late in the curing and
then only to a limited extent. The bacteria which ordinarily occur in
pickling fluids are not strict anaerobes and are not placed under the
most suitable conditions for growth when they reach the interior of
the ham, for it seems probable that in the interior of hams which are
totally submerged in pickling fluids the amount of available oxygen
must be extremely small. The ordinary pickle bacteria, therefore, would
not multiply as rapidly in the interior of the hams and would not find
their way into the bone marrow as soon as would a strictly anaerobic
organism.

Pure cultures of the sour-ham bacillus, recovered from the meat and
bone marrow of the injected hams, were compared with cultures of the
original bacillus used for inoculating the hams, and were found to be
identical. Furthermore, the bacillus with which the hams were injected
was recovered from the injected hams at points far removed from the
original point of injection, showing that the organism had multiplied
and extended throughout the bodies of the hams and that it was clearly
responsible for the souring which the hams had undergone.

Sound hams from tierces 2 and 4 were examined bacteriologically in the
same manner as the injected hams, and some of the cultures showed the
ordinary pickle bacteria, but in not a single instance did egg-pork
cultures yield a sour odor, and in no case could the sour-ham bacillus
be demonstrated in any of these hams.

Microscopic sections and teased preparations of the muscle fibers in
salt solution were prepared from several of the sour hams in this
experiment, and these preparations showed the same histological changes
and the same distribution of bacilli as noted in the natural sours.

In Plate III, figures 1 and 2, sections are shown of artificially
soured hams, that is, hams which were artificially soured by injections
of culture; and if these figures be compared with the sections made
from hams which had undergone spontaneous souring (see Pl. II, figs. 1
and 2) the similarity in the form and distribution of the bacilli will
be at once apparent.


[Illustration:

    BUL. 132, BUREAU OF ANIMAL INDUSTRY, U. S. DEPT. OF AGRICULTURE.
                              PLATE III.

    FIG. 1.—SECTION THROUGH MUSCULAR TISSUE OF ARTIFICIALLY SOURED
    HAM, SHOWING DISTRIBUTION OF BACILLI BETWEEN THE MUSCLE FIBERS,
    WHICH ARE SHOWN IN CROSS SECTION. THE DARK LINES AND MASSES
    BETWEEN THE MUSCLE FIBERS REPRESENT CLUMPS OF BACILLI.

 (Pen-and-ink drawing made with camera lucida from section stained by
           the Gram-Weigert method to show bacteria. × 85.)]

    FIG. 2.—SECTION THROUGH MUSCULAR TISSUE OF ARTIFICIALLY SOURED
    HAM, SHOWING INDIVIDUAL BACILLI BETWEEN THE MUSCLE FIBERS,
    WHICH ARE CUT LONGITUDINALLY.

 (Pen-and-ink drawing made with camera lucida from section stained by
          the Gram-Weigert method to show bacteria. × 320.)]

_Summary and discussion of Experiment I._—Comparing tierces 1 and 2,
where the hams were pumped in the shank only, the only difference being
that the hams in tierce 1 were inoculated with culture while those in
tierce 2 were not, we find that in tierce 1 nineteen out of twenty, or
95 per cent, of the hams became sour, whereas in tierce 2 all of the
hams remained sweet. In view of the fact that these tierces were held
under exactly the same conditions, we must conclude that the souring
of the hams in tierce 1 was due to the injection of culture which they
received.

Comparing tierces 3 and 4, where the hams were pumped in both shank and
body, the hams in tierce 3 being injected with culture while those in
tierce 4 were not, we find that in tierce 3 nine out of twenty, or 45
per cent, of the hams became sour, whereas in tierce 4 all of the hams
remained sweet. As the conditions of cure were the same for all four
tierces, we must again conclude that the souring of the hams in tierce
3 was directly attributable to the injections of culture which they
received.

If now we compare tierces 1 and 3, the two tierces which were injected
with culture, we find that in the case of tierce 1, where the hams were
pumped in the shank only, 95 per cent became sour; whereas in the case
of tierce 3, where the hams were pumped in both shank and body, only
45 per cent became sour. In other words, the percentage of souring in
those hams which were pumped in the body as well as in the shank was 50
percent less than in those hams which were pumped in the shank only.
Inasmuch as the only difference in the treatment accorded tierces 1
and 3 lay in the additional pumping given the hams in tierce 3, we
must conclude that the marked diminution in the percentage of souring
in the case of tierce 3 was undoubtedly due to the additional pumping
which these hams received, the hams being saturated at the start with
the pumping pickle. It will be shown later that both sodium chlorid and
potassium nitrate exert an inhibitory effect upon the bacillus with
which the hams were injected, which directly bears out the foregoing
conclusion.

In tierces 2 and 4, the two check tierces which were not injected with
culture, all of the hams were sweet at the end of the cure, showing
that the conditions under which the experiment was carried out were
entirely favorable to a successful cure.

The sour odor obtained from the artificially soured hams in this
experiment was pronounced by the meat inspector who tested the hams,
and who was entirely unaware of the treatment they had received, to be
identical with the usual sour odor which characterizes hams that have
undergone spontaneous souring; in other words, there was no difference
in odor between these artificially soured hams and natural sours.

With regard to the variation in the degree and the extent of the
souring exhibited by the individual hams in the two inoculated tierces,
where some of the hams showed pronounced souring throughout the body
and shank, while others which had been injected with the same amount of
culture showed only slight souring in the body, several factors must
be considered, viz: (1) Differences in the reaction of the meat of the
individual hams which may have exerted an influence on the growth of
the bacteria with which the hams were injected. (2) Variations in the
texture of the muscle fibers and connective tissue of the individual
hams, permitting in some cases a more rapid and thorough penetration of
the pickling fluids to the interior of the hams, whereby the inhibitory
effect of the sodium chlorid and the potassium nitrate on the bacteria
would come into play earlier. (3) Variations in pumping, whereby more
of the pickling solution was forced into some of the hams than into
others. Probably all three of these factors would have to be taken into
account in explaining the variation in the degree and extent of the
souring exhibited by the injected hams.

With regard to the souring of the bone marrow, we find that of nineteen
sour hams in tierce 1 eighteen showed sour marrows, while in tierce 3
nine sour hams showed seven sour marrows. The high proportion of marrow
sours is not surprising when it is recalled that of the nineteen sour
hams in tierce 1 the meat was markedly sour in sixteen, while of the
nine sour hams in tierce 3 the meat was markedly sour in five. In the
case of the four sour hams in tierce 3 which showed slight souring in
the body, two of these showed sour marrows, while in two the marrows
were sweet. In this experiment the percentage of sour hams showing sour
marrows corresponds with the percentage of marrow-sour hams found in
the packing house, where, as has been pointed out before, a ham which
is markedly sour in the body will practically always show sour marrow,
while in hams which show only slight souring in the body the marrow is
involved in about 50 per cent of the cases.


                           _Experiment II._

This experiment was essentially a repetition of Experiment I, but was
carried out at a different packing establishment and under somewhat
different conditions.

Two lots of hams were injected with a culture suspension of the
bacillus at different stages of the cure, or rather at different stages
in the preparation for cure, i. e., (1) on the hanging floor, previous
to chilling, and (2) after chilling and pumping and immediately before
packing. Three tierces, each containing 20 hams, were put down. Two of
the tierces contained the hams injected with culture, while the third
tierce contained check hams which had not been treated with culture.
Half of the hams in each tierce were pumped in the shank, while the
other half were pumped in both body and shank. The same pumping and
curing pickles were used for all three tierces, and were the regular
pumping and regular curing pickles of the establishment at which the
experiment was carried out. The hams used were all 14 to 16 pounds
in weight and were subjected to the usual 48-hour chill with an
additional chill of 48 hours after they were cut from the carcass. They
were packed in tierces which had been thoroughly scrubbed and cleaned
with boiling water. The tierces were held in a pickling room at a
temperature of 33° to 36° F., the temperature never rising above 36°
F., and were rolled three times during the curing period. The hams were
in cure for about eighty days. At the end of the cure the hams were
carefully tested by a trained meat inspector, who knew nothing of the
treatment they had received.

The culture suspension was prepared from 20 tubes of egg-pork medium
in the same manner as that used in Experiment I, the cultures being
diluted with sufficient salt solution to give 400 cubic centimeters of
suspension. The cultures from which the suspension was prepared had
grown at room temperature for ten days. The suspension was examined
microscopically and showed large numbers of the bacilli in the form
of filaments or long chains, with many of the individual organisms
showing large terminal spores. The hams were injected with the culture
suspension in the same manner as those in Experiment I.

The details of the experiment were as follows:

    _Tierce No. 1._—Contained 20 hams, each ham being injected
    with 20 cubic centimeters of the suspension or the equivalent
    of 10 cubic centimeters of the original culture. The hams were
    injected while on the hanging floor, before they had been cut
    from the carcasses and previous to chilling. The carcasses were
    still quite warm—that is, had lost but little of their body
    heat when the injections were made. The carcasses, which had
    been carefully tagged, were then run into coolers and given the
    usual 48-hour chill, after which the hams were severed from the
    carcasses and given an additional 48-hour chill in accordance
    with the custom of the packing house at which the experiment
    was carried out. The hams were next pumped with regular pumping
    pickle, 10 being pumped in both body and shank and 10 in shank
    only. They were finally packed in a tierce, which was then
    headed up, filled with regular curing pickle, and placed in
    cure.

    Result: When tested at the end of the cure it was found that
    the 10 hams which were pumped in the shank only were all sour.
    In each of them the souring extended throughout the entire ham,
    in the shank as well as in the body, and was very pronounced,
    so much so that they were characterized as “stinkers” by the
    meat inspector who assisted in testing them. The bone marrow
    of the femur or middle bone was sour in all of these hams. Of
    the 10 hams which were pumped in both body and shank 7 showed
    well-marked souring throughout the body, but the souring did
    not extend into the shank. The bone marrow of the femur was
    found to be sour in 6 of these hams, while in 1 the souring had
    not extended through to the bone marrow.

    _Tierce No. 2._—Contained 20 hams which were chilled and pumped
    in exactly the same manner as those in tierce No. 1. These hams
    were injected with culture after they had been chilled and
    pumped, or just before they were placed in cure. The hams in
    this tierce, therefore, were injected with culture four days
    later than those in tierce 1. The hams were injected with a
    bacterial suspension prepared in the same manner as that used
    for tierce 1, except that the egg-pork cultures from which
    the suspension was prepared were 7 days instead of 10 days
    old. Each ham was injected with 20 cubic centimeters of the
    suspension or the equivalent of 10 cubic centimeters of the
    original culture. The hams were injected in the same manner as
    those in tierce 1.

    Result: When tested at the end of the cure, it was found that
    of the 10 hams which were pumped in the shank all were sour; in
    8 of these the souring was very marked throughout the body of
    the ham and extended into the shank; in all of these hams the
    souring had extended through to the bone marrow of the middle
    bone or femur. Of the 10 hams which were pumped in both body
    and shank 6 were sour in the body. These hams were classed by
    the meat inspector who examined them as “light body sours,”
    and in none of them did the souring extend into the shank or
    through the bone into the bone marrow of the femur.

    _Tierce No. 3._—Contained 20 hams which were chilled and pumped
    in the same manner as those in the two preceding tierces. These
    hams were not injected with culture and were put down to serve
    as checks on the cure. In other words, they were pumped with
    the same pickling fluids, were subjected to exactly the same
    cure, and were held under precisely the same conditions as
    those in the preceding tierces, the only difference being that
    the hams in this tierce were not injected with culture.

    Result: When tested at the end of the cure, all of the hams in
    this tierce were found to be perfectly sound and sweet.


                      _Results of Experiment II._

    ───────┬──────┬─────────┬──────────────────────
           │      │         │
    No. of │Number│ Average │     How pumped.
    tierce.│  of  │ weight  │
           │hams. │ of hams.│
           │      │         │
    ───────┼──────┼─────────┼──────────────────────
           │      │_Pounds._│
           │      │         │{ 10 in shank
       1   │  20  │  14-16  │{
           │      │         │{
           │      │         │{ 10 in body and shank
           │      │         │
           │      │         │{ 10 in shank
       2   │  20  │  14-16  │{
           │      │         │{
           │      │         │{ 10 in body and shank
           │      │         │
       3   │  20  │  14-16  │{ 10 in shank
           │      │         │{ 10 in body and shank
    ───────┴──────┴─────────┴───────────────────────
    ───────┬───────────────────────────┬───────────────────
           │                           │Condition at end of
    No. of │        Treatment.         │      cure.
    tierce.│                           ├————————┬——————————
           │                           │ Number │Percentage
           │                           │of sour │ of sour
           │                           │  hams. │  hams.
    ───────┼───────────────────────────┼────────┼───────────
           │                           │        │
           │Each ham injected with 20  │   10   │   100
       1   │c. c. of culture prior to  │        │
           │chilling and pumping.      │        │
           │    do                     │    7   │    70
           │                           │        │
           │Each ham injected with 20  │   10   │   100
       2   │c. c. of culture subsequent│        │
           │to chilling and pumping.   │        │
           │    do                     │    6   │    60
           │                           │        │
       3   │Not injected with culture. │    0   │     0
           │    do                     │    0   │     0
    ───────┴───────────────────────────┴────────┴──────────

Four hams were selected from each tierce for bacteriological and
histological examination. From tierces 1 and 2, in which the hams
were injected with culture, 4 of the sourest hams were selected from
each tierce. Cultures were made from these hams in the same manner
as described under Experiment I and with the same result—that is,
the sour-ham bacillus was found throughout the bodies of the hams.
Microscopic sections were also prepared from these hams and showed the
same histological changes and the same distribution of bacilli as noted
for the hams in Experiment I.

_Summary and discussion of Experiment II._—Comparing tierces 1 and 2,
in which the hams were injected with culture, with tierce 3, where the
hams were not injected with culture, we find that in tierce 1 seventeen
hams (85 per cent) became sour and in tierce 2 sixteen hams (80 per
cent) became sour, whereas in tierce 3 all of the hams were sweet. The
fact that all of the hams in tierce 3, the check tierce, were sweet
indicates that the conditions were favorable for a successful cure; and
as all three tierces were cured under exactly the same conditions, the
only difference being that the hams in tierces 1 and 2 were injected
with culture, whereas those in tierce 3 were not injected with culture,
we must conclude that the souring of the hams in tierces 1 and 2 was
due to the injections of culture which they received.

Comparing tierce 1 with tierce 2, we find that the hams in tierce 1
showed more extensive souring than did those in tierce 2, this being
especially noticeable in the case of the hams which were pumped in both
body and shank. This difference in the extent or degree of souring was
probably due to the fact that the hams in tierce 1 were injected while
they were still warm and before they had lost their animal heat, the
bacterial suspension thus having a better chance to become disseminated
through the meat. The hams in tierce 2 were injected with culture after
they had been chilled, when the tissues were more or less contracted
and the conditions less favorable for the dissemination of the
suspension throughout the meat. The hams in tierce 1 were also injected
four days earlier than those in tierce 2, and prior to pumping; and
this would explain the greater difference in the extent of the souring
in the case of the hams which were pumped in both body and shank, as in
tierce 1 the bacteria had four days in which to develop before coming
in contact with the pickling fluids, whereas in tierce 2 the bacteria
were injected after the hams were pumped with pickle and were thus
brought into immediate contact with the pickling fluids, which, as will
be shown later, have a distinct inhibitory action upon the bacillus in
question. In the case of the hams which were pumped in the shank but
not in the body there was not this difference, as in these hams the
pickling fluids must penetrate into the bodies of the hams from the
outside. As it requires some time for the pickling fluids to reach the
interior of a ham, the bacteria were thus afforded quite an interval
in which to develop before being exposed to the inhibitory action of
the pickling fluids. A chemical study of the processes involved in
ham curing has been carried out in the Biochemic Division and the
approximate rate of penetration of the curing pickle determined,
and it was found that it required about four weeks for the interior
of a 10-pound ham which had not been pumped to acquire its maximum
percentage of sodium chlorid.

To recapitulate: In this experiment 40 hams were injected with culture,
half of this number being pumped in the shank only and half in both
body and shank. Of the 20 which were pumped in the shank only, every
ham without exception, or 100 per cent, became sour. Of those which
were pumped in both body and shank, 13, or 65 per cent, became sour.
The reduction in the percentage of sours in the last lot was clearly
due to the additional pumping which these hams received.

If now we compare tierce 2 in this experiment with tierces 1 and 3 in
Experiment I—these three tierces being comparable, as they were all
injected with culture at the same stage in their preparation for cure,
that is, subsequent to chilling and pumping—we find, in the case of the
hams pumped in both body and shank, 65 per cent of sours in Experiment
II as against 45 per cent in Experiment I, and this difference is
undoubtedly due to the heavier dose of culture used in Experiment
II, where the hams were given the equivalent of 10 cubic centimeters
of egg-pork culture as against 2½ cubic centimeters in Experiment
I. In the case of the hams which were pumped in the shank but not in
the body, the percentage of sours was practically the same in the two
experiments—in Experiment I all but one of these hams became sour,
while in Experiment II all of them became sour. The degree or extent of
the souring in these last hams, however, was greater in Experiment II,
a result of the heavier injections of culture which they received.


                  _Summary of Experiments I and II._

Summarizing the results obtained in Experiments I and II, we find that
culture suspensions of the anaerobic bacillus isolated from sour hams
caused souring with great uniformity when injected into the bodies of
sound hams which were pumped in the shank only. In the two experiments,
40 sound hams which were pumped in the shank only were injected with
culture suspensions of the bacillus, with the result that 39, or 97.5
per cent, became sour during the process of cure; and it is quite
probable, as we have pointed out before, that one of these hams was
overlooked in making the inoculations, otherwise the entire lot would
have become sour.

The inhibitory action of the pickling fluids upon the bacillus is well
shown in the case of those hams which were pumped in both body and
shank. Out of 40 hams which were pumped in both body and shank, 22, or
55 per cent, became sour in the process of curing. Inasmuch as these
hams were cured under precisely the same conditions as the hams which
were pumped in the shank only, we must conclude that the diminution in
souring in these hams was undoubtedly due to the additional pumping
which they received, whereby the bacteria with which these hams were
injected were brought into immediate contact with the strong pumping
pickle and their development thereby inhibited.

In these two experiments it was proven beyond doubt that the anaerobic
bacillus isolated from sour hams was capable of producing souring
when introduced into the bodies of sound hams; and in view of the fact
that this bacillus was constantly present in hams which had undergone
spontaneous or natural souring, and was the only organism that could
be isolated from such hams that was capable of producing in egg-pork
cultures the characteristic sour-ham odor, the conclusion seems
justifiable that this bacillus is an undoubted cause of the ham souring
which occurs in the packing house; and the results thus far obtained
indicate that it is an important, if not the only, factor concerned in
ham souring.

Having established the etiological relation of the bacillus isolated
from sour hams with ham souring, the next point to be considered was
the manner in which this bacillus finds its way into the bodies of the
hams.


      PROBABLE METHOD BY WHICH HAM-SOURING BACILLUS ENTERS HAMS.

Regarding the question of the probable method by which the ham-souring
bacillus enters hams, there were three possibilities to be taken into
consideration: (1) That the bacillus is present in the flesh of hogs at
the time of slaughter, (2) that the bacillus gains entrance through the
pickling fluids, (3) that the bacillus is introduced into the bodies
of the hams in the handling or manipulation which the hams undergo in
preparation for, or during, the process of curing.


             POSSIBILITY OF INFECTION PRIOR TO SLAUGHTER.

In order to throw some light upon this point, a number of fresh
hams—that is, hams which had been chilled but not pumped or subjected
to any other manipulation—were examined bacteriologically, but in no
case could the anaerobic bacillus which was isolated from sour hams
be detected in any of them. The fact that in certain of the smaller
packing establishments which cure their hams without pumping the
percentage of souring is extremely low would also seem to negative this
possibility, for if the bacillus which causes souring were present in
the hams at the time of slaughter, sour hams would be as frequent at
such establishments as at those establishments which make a practice of
pumping. Furthermore, a laboratory study, biological and chemical, of
the bacillus isolated from sour hams shows that this organism belongs
to the class of putrefactive bacteria, and while such bacteria may be
present in the intestines of healthy animals, as, for example, the
bacillus of Bienstock (_Bacillus putrificus_), these bacteria do not
invade the organs and tissues of the body until after the death of the
animal, and the packing-house practice of rapidly eviscerating the hogs
immediately after slaughter would certainly preclude this possibility.


               POSSIBLE INFECTION FROM PICKLING FLUIDS.

With regard to the second possibility, that the bacillus finds its way
into the hams in the curing pickles, it was determined by laboratory
experiment that the addition of 3 per cent of sodium chlorid or 3 per
cent of potassium nitrate to laboratory media completely inhibits
the growth of the bacillus. As the pickling solutions always contain
considerably more than these percentages of sodium chlorid and
potassium nitrate, it would be impossible for the bacillus to multiply
in the pickles. Additional laboratory experiments demonstrated,
however, that the bacillus or its spores may remain alive in the curing
pickles for at least thirty days, and it seemed possible that the
curing pickles might become contaminated at times with the bacilli, and
that the bacilli, although incapable of multiplying in the pickles,
might find their way into the bodies of the hams in the pickling
fluids. In order to throw some light upon this point, the following
experiment was carried out:


EXPERIMENT TO SHOW WHETHER INFECTION TAKES PLACE FROM THE CURING PICKLE.

In this experiment two tierces were put down, each containing 20 hams.
The hams weighed from 14 to 16 pounds and had received the usual
48-hour chilling. The pickling solutions employed were the regular
curing pickles of the establishment at which the experiment was carried
out. The curing pickle in one tierce was inoculated with 400 cubic
centimeters of a culture suspension of the bacillus, prepared in the
same manner as that used for the injection of the hams in tierce 2 in
Experiment II. A microscopic preparation made from a small drop of the
culture suspension before adding it to the pickle showed the bacilli
in large numbers, and in the 400 cubic centimeters of the suspension
there were millions of the bacteria. The curing pickle in the other
tierce was left untreated, the hams in this tierce serving as a check.
The tierces used in this experiment, as in all of the experiments, were
thoroughly cleaned with boiling water before the hams were placed in
them. The experiment was conducted in a pickling room which was held at
33° to 36° F., and the tierces were rolled three times during the cure.
The details of the experiment are as follows:

    _Tierce 1._—Contained 20 hams, half of which were pumped in
    both body and shank and half in the shank only. As soon as they
    were pumped the hams were packed in the tierce. Sufficient
    curing pickle to fill the tierce was then measured out in a
    clean barrel and to it was added the culture suspension. The
    culture was thoroughly mixed with the pickle and the latter was
    then run into the tierce containing the hams.

    Result: When tested at the end of the cure, two of the hams
    which had been pumped in the shank only showed slight souring
    in the body. The rest of the hams were sweet.

    _Tierce 2._—Contained 20 hams which were pumped in the same
    manner as those in tierce 1. The curing pickle was the same as
    that used for tierce 1, but without the addition of culture.
    This tierce was put down as a check on tierce 1, the hams
    being cured under exactly the same conditions, but without the
    addition of culture to the curing pickle.

    Result: One of the hams which was pumped in the shank only
    developed slight souring in the body. The remainder of the hams
    were sweet.

Comparing tierce 1, which contained the inoculated pickle, with tierce
2, the check tierce which contained uninoculated pickle, we find
there was practically no difference in the final result. In tierce 1
two of the hams developed slight souring, while in tierce 2 one of
the hams became slightly sour. All of these hams had been pumped in
the shank only. The fact that one of the hams in the check tierce
developed slight souring was undoubtedly due to bacterial contamination
in pumping or in the handling which the hams underwent prior to
pickling, and the slight souring of the two hams in tierce 1 must
also be attributed to the same cause or causes, for had the souring
in these last hams resulted from the penetration of the bacteria from
the pickling solution a higher percentage should have become sour.
Furthermore, if the souring of the two hams in tierce 1 had resulted
from the penetration of the bacteria from the curing pickle, the
souring should have been general throughout the bodies of these hams,
whereas the souring was only evident around the bone and was slight in
degree.

From this experiment the conclusion would seem justified that the
bacillus which causes ham souring does not usually find its way into
the bodies of the hams from the curing pickle, although it would be
going too far, perhaps, to say that infection never takes place from
the curing pickle. The experiment, however, indicates clearly that the
curing pickles are certainly not the main channel through which the
hams become infected. In referring to the curing pickles, it should be
understood that we refer here to the pickling solutions in which the
hams are immersed, and not to the pumping pickles. The possibility of
infection through the pumping pickle will be discussed later.


         POSSIBLE INFECTION THROUGH MANIPULATION OR HANDLING.

There are at least three possible ways in which hams may become
infected from the handling which they receive in preparation for, or
during the process of curing, viz: From the thermometers used in taking
the inside temperatures of the hams, from the pumping needles, and from
the billhooks used in lifting the hams.


                   INFECTION FROM HAM THERMOMETERS.

The packing-house method of taking the temperatures of hams by means
of a pointed, metal-capped thermometer which is thrust deep into the
bodies of the hams has already been referred to, but deserves to be
described somewhat more in detail, as it will be at once apparent
that this manipulation furnishes a ready means whereby hams may
become infected with putrefactive bacteria. The construction of a ham
thermometer is shown in figure 4.

[Illustration: A.   B.

    FIG. 4.—Diagrammatic views showing construction of ham
    thermometer. A, front view, showing open space between metal
    point and mercury bulb, which becomes filled with particles of
    meat, grease, and dirt; B, side view.]

The instrument consists of a glass thermometer inclosed in a metal
case, the front portions of the case being cut away so as to expose
the scale above and the mercury bulb below. As was explained before,
the thermometer is thrust deep into the body of the ham so that the
pointed end containing the mercury bulb rests beside or a little behind
the upper portion of the femur, the bone being used as a guide in
introducing the thermometer.

Ham temperatures are taken at three stages in the preparation for
cure—(1) on the hanging floor, just before the hams go to the chill
rooms, in order to determine the amount of heat lost prior to chilling;
(2) on leaving the chill rooms, in order to determine the thoroughness
of the chill; (3) on the packing floor, just before the hams are placed
in pickle, as a further check on the thoroughness of the chilling.

In taking the temperatures of hams which have been chilled—and most
of the temperatures are taken subsequent to chilling—it is customary
for the packing-house attendant who has this matter in charge to warm
the thermometer by holding the pointed or bulb end in his hand, so
as to force the mercury column up to about 60° F., or well above the
temperature of hams. The thermometer is then thrust into the ham and
allowed to remain for several minutes, by which time the mercury column
will have fallen to the temperature of the ham. The thermometer is then
slowly withdrawn so as to expose the top of the mercury column, and
an accurate reading is thus obtained of the inside temperature of the
ham. The thermometer is warmed by the hand before each ham is tested,
and this undoubtedly insures more accurate readings than would result
were the thermometer removed from one ham and plunged immediately into
another, but the procedure is open to certain objections, for the open
space between the metal point of the thermometer and the mercury bulb
soon becomes filled with particles of meat and with grease and dirt
from the attendant’s hands, and it is at once apparent that a thermometer
in this condition would furnish a ready means whereby extraneous matter
might be introduced into the bodies of the hams. In other words, a
contaminated thermometer would furnish an excellent means whereby hams
could be inoculated with putrefactive bacteria.

In order to determine whether hams actually become inoculated in this
manner, the following experiment was carried out:


EXPERIMENT TO SHOW WHETHER HAMS BECOME INFECTED FROM HAM THERMOMETERS.

This experiment was designed to show (1) whether the usual
packing-house method of taking ham temperatures was apt to induce
souring in the hams thus tested, and (2) whether souring would result
in hams which were tested with a thermometer purposely contaminated
with the bacillus isolated from sour hams.

The experiment was carried out as follows: Thirty hog carcasses were
selected as they entered the hanging floor from the killing floor. They
had been cleaned, eviscerated, and split, and were of the same average
weight and of sufficient size to yield hams weighing from 12 to 14
pounds. They were divided into three lots of 10 each and were allowed
to remain on the hanging floor for two hours, after which they were
given the usual 48-hour chilling.

    _Lot 1._—The hams in this lot were tested with an ordinary ham
    thermometer as they entered the hanging floor, as they left the
    hanging floor, and as they left the coolers. The thermometer
    used was borrowed from one of the plant attendants and was used
    in the condition in which it was received from him; that is, it
    was not cleaned or disinfected prior to use.

    _Lot 2._—The hams in this lot were tested as they entered the
    hanging floor with a thermometer which had been previously
    cleaned and disinfected and then dipped in a culture suspension
    of the meat-souring bacillus which was isolated from sour
    hams. The thermometer was dipped in the culture suspension
    before each ham was tested. No further temperatures were taken
    of these hams. The thermometer was carefully cleaned and
    disinfected before it was returned to the attendant from whom
    it was borrowed.

    _Lot 3._—The hams in this lot were not tested at all, and were
    intended as checks on the cure.

The three lots of carcasses were carefully tagged and were chilled in a
special cooler to themselves. Upon leaving the cooler the hams were cut
from the carcasses and trimmed. The three lots of hams were then cured
in separate tierces. All of the hams were subjected to exactly the same
cure.

The pickles used were the regular pumping and regular curing pickles of
the establishment at which the experiment was carried out.

The hams in lot 3 were pumped first and those in lot 1 were pumped
next. The needle was then removed and a fresh, clean needle was used
for lot 2. This was done in order to prevent the possibility of
carrying over bacteria from one lot of hams to another on the pumping
needle. The tierces were thoroughly cleaned with boiling water before
being used. The curing was carried out in a pickling cellar which was
held at 33° to 36° F., the temperature never rising above the latter
figure. The tierces were rolled three times during the curing. The
details and results were as follows:

    _Tierce 1._—Contained 20 hams, half of which were pumped in
    both body and shank and half in the shank only. These hams were
    taken from the carcasses in lot 1 and had been tested several
    times with a ham thermometer, as already described.

    Result: At the end of the cure it was found that of the 10 hams
    which were pumped in the shank, 5 showed well-marked souring in
    the body, while of the 10 hams which were pumped in both body
    and shank, 2 showed slight souring in the body.

    _Tierce 2._—Contained 20 hams, which were pumped in the same
    manner as those in tierce 1. These hams were taken from the
    carcasses in lot 2 and had been tested once with a thermometer
    which was dipped in a culture suspension of the bacillus
    isolated from sour hams.

    Result: The 10 hams which were pumped in the shank only all
    became sour. When they were tried out at the end of the cure,
    they showed pronounced souring throughout the entire body and
    were classed as “stinkers” by the meat inspector who examined
    them. The souring extended through to the bone marrow of the
    femur in all of these hams. Of the 10 hams which were pumped in
    both body and shank, 7 showed well-marked souring in the body,
    but not as pronounced as in those pumped in the shank only; in
    five of these hams the souring extended through to the bone
    marrow of the femur, while in 2 the bone marrow remained sweet.

    _Tierce 3._—Contained 20 hams, which were pumped in the same
    manner as those in the two preceding tierces. These hams were
    not tested with a thermometer, and were put down as a check
    on the cure. They were pumped with the same pumping pickle,
    subjected to the same cure, and held under precisely the same
    conditions as the hams in the two preceding tierces.

    Result: When tested at the end of the cure, all of these hams
    were found to be perfectly sound and sweet.


 _Results of experiment to show whether hams become infected from ham
                            thermometers._
   ────────┬────────┬───────────┬──────────────┐
           │        │           │              │
           │        │           │              │
    No. of │ Number │  Average  │ How pumped.  │
    tierce.│   of   │ weight of │              │
           │  hams. │   hams.   │              │
   ────────┼────────┼───────────┼──────────────┤
           │        │ _Pounds._ │              │
           │        │           │              │
           │        │           │{ 10 in shank │
           │        │           │{             │
       1   │   20   │   12-14   │{             │
           │        │           │{             │
           │        │           │{             │
           │        │           │{             │
           │        │           │{             │
           │        │           │{ 10 in body  │
           │        │           │{   and shank │
           │        │           │              │
           │        │           │{ 10 in shank │
       2   │   20   │   12-14   │{             │
           │        │           │{             │
           │        │           │{             │
           │        │           │{             │
           │        │           │{             │
           │        │           │{ 10 in body  │
           │        │           │{   and shank │
           │        │           │              │
           │        │           │{ 10 in shank │
       3   │   20   │   12-14   │{             │
           │        │           │{ 10 in body  │
           │        │           │{   and shank │
   ────────┴────────┴───────────┴──────────────┘

   ────────┬─────────────────┬──────────────────
           │                 │  Condition at
           │                 │   end of cure.
    No. of │                 ├───────┬──────────
    tierce.│    Treatment.   │Number │Percentage
           │                 │of sour│  of sour
           │                 │ hams. │   hams.
   ────────┼─────────────────┼───────┼──────────
           │                 │       │
           │Tested at several│   5   │    50
           │stages in        │       │
       1   │preparation for  │       │
           │cure with ham    │       │
           │thermometer which│       │
           │had not been     │       │
           │cleaned.         │       │
           │     do          │   2   │    20
           │                 │       │
           │                 │       │
           │Tested once with │  10   │   100
       2   │ham thermometer  │       │
           │dipped in culture│       │
           │suspension of    │       │
           │anaerobic        │       │
           │bacillus isolated│       │
           │from sour hams.  │       │
           │     do          │   7   │    70
           │                 │       │
           │                 │       │
       3   │Not tested with  │   0   │     0
           │thermometer.     │       │
           │     do          │   0   │     0
   ────────┴─────────────────┴───────┴──────────

Several hams from each tierce were examined bacteriologically. cultures
being taken from the meat near the bone and from the bone marrow of the
femur.

In the sour hams from tierce 1 cultures taken from the meat near the
bone showed the same anaerobic bacillus noted in other sour hams (i.e.,
the same bacillus which caused souring in Experiments I and II),
but these cultures were contaminated with other bacteria which were
probably introduced on the thermometer along with the ham-souring
bacillus. None of the contaminating bacteria were capable, however,
of producing a sour-meat odor when grown on the egg-pork medium. Pure
cultures of the ham-souring bacillus were obtained from the bone marrow
of some of these hams, showing that this bacillus had penetrated
through to the bone marrow while the other bacteria had not.

From the sour hams in tierce 2 the ham-souring bacillus was recovered
readily, and often in pure culture, from the hams which had been pumped
in the shank only, whereas it was usually contaminated with pickle
bacteria in the hams which had been pumped in both body and shank.

In the case of the sound hams in tierce 3, cultures taken from the meat
near the bone and from the bone marrow of the femur were negative in
the hams which had been pumped in the shank only, while cultures taken
from corresponding points in the hams pumped in both body and shank
showed ordinary pickle bacteria, which had evidently been introduced
into the bodies of these hams in the pumping pickles. None of these
hams exhibited the slightest sour odor.

_Summary of experiment._—In this experiment 20 hams (tierce 1) were
tested with an ordinary ham thermometer in the usual packing-house
manner. Half of these hams were subjected to the mild cure and half
were given the regular cure, with the result that 50 per cent of those
receiving the mild cure and 20 per cent of those receiving the regular
cure became sour.

A second lot of 20 hams (tierce 2) were tested with a thermometer
which had been purposely contaminated with a culture suspension of the
ham-souring bacillus. These hams were cured in the same manner as the
first lot, with the result that all of those receiving the mild cure
and 70 per cent of those receiving the regular cure became sour.

A third lot of 20 hams (tierce 3) which had not been tested at all were
cured in the same manner as the two preceding lots, as a check on the
cure. All of these hams were sweet at the end of the cure.

Inasmuch as the three lots of hams were cured under precisely the same
conditions and were handled in the same manner prior to pickling, the
only difference being that the hams in tierces 1 and 2 were tested with
the ham thermometer while those in tierce 3 were not, we must conclude
that the souring of the hams in tierces 1 and 2 resulted from the
testing which these hams received. In the case of tierce 1 the hams
became infected from a thermometer which, in the ordinary routine use
of the packing house, had become accidentally contaminated with the
ham-souring bacillus. In the case of tierce 2 the hams became infected
from a thermometer which had been artificially contaminated with the
bacillus. The high percentage of sours in this last lot is due to
the fact that these hams were heavily infected with the ham-souring
bacillus, for owing to the construction of the ham thermometer many
thousands of the bacilli were unquestionably introduced into each
ham on the point of the thermometer. In the ordinary routine of ham
testing, where hams become infected from foreign matter introduced on
the thermometer, the percentage of souring, as shown in tierce 1, would
be less, for it is not to be supposed that ham thermometers are always
contaminated with the ham-souring bacillus, but that they only become
so at times, and that probably only a few of the bacilli are then
introduced.

This experiment, we think, proves conclusively (1) that the ham-souring
bacillus may be introduced into the bodies of hams on the thermometers
used in testing the hams, and (2) that the packing-house method of
taking ham temperatures by means of a thermometer which is thrust deep
into the bodies of the hams may cause souring in the hams thus tested.

As a further proof that hams may become contaminated in this manner,
a series of cultures were made from scrapings taken from ham
thermometers. The scrapings consisted of the accumulations of bits
of meat, grease, and dirt that collect on the thermometers, and were
taken from the thermometers while the latter were in ordinary routine
use in the packing house. In a series of six cultures which were made
from such scrapings at different times, the same bacillus which was
isolated from sour hams and shown to cause meat souring was found three
times. In other words, the ham-souring bacillus was present in 50 per
cent of the cultures made from thermometer scrapings, and many hams
undoubtedly become infected from the thermometers. Souring would be
almost certain to result in mild-cure hams if these hams were tested
with a thermometer which had become accidentally contaminated with
the ham-souring bacillus, as the bacillus would have time to develop
within the bodies of the hams before being inhibited by the curing
pickle, which penetrates slowly into the bodies of these hams. In the
case of regular cure hams—that is, hams which are pumped in both body
and shank—souring would be much less apt to occur after the use of a
contaminated thermometer, as these hams are more or less saturated with
a strong pumping pickle at the beginning of the cure, which would tend
to inhibit the growth of any bacteria that might be introduced on the
thermometers.

The fact that souring may result in hams from the use of a contaminated
thermometer would explain the occurrence of several sours in one vat,
for in testing hams just before they go into cure several hams are
usually tested in succession, and these would in all likelihood go into
the same vat. Supposing the thermometer to have been contaminated with
the ham-souring bacillus at the time these hams were tested, this would
explain a fact which has been often noted, namely, the occurrence of
several sours in one vat while other vats containing the same run of
hams show no sours.

If souring resulted in all of the hams which are subjected to a
thermometer test in the daily routine of the packing house, this
manipulation alone might account for nearly all of the sours which
occur, but the experiment which has been just described shows that
all of these hams do not become sour. In tierce 1, where each ham
was subjected to three thermometer tests at different times, souring
resulted in 35 percent (this includes both mild and regular cure) of
the hams thus tested, and in actual practice the percentage of sours in
hams which have been subjected to the thermometer test would probably
be somewhat less. Quite a large percentage of sour hams are thus left
unaccounted for by the thermometer test, and we believe that these are
chiefly the result of contamination carried in on the pumping needles
or in the pumping pickles.


                    INFECTION FROM PUMPING NEEDLES.

In view of the results obtained in the last experiment, in which it was
shown that hams may become infected from the use of ham thermometers,
it seemed not improbable that hams might also become infected from
the pumping needles, which, like the thermometers, are thrust deep
into the bodies of the hams beside the bone. In order to throw some
light upon this point, cultures were taken from the grease and dirt
that accumulate on the shields at the bases of the pumping needles,
as such material must undoubtedly be carried into the hams at times
on the needles. The ham-souring bacillus was found several times in
these cultures, and hence it is fair to infer that hams may also
become infected at times from the pumping needles, just as they become
infected from the thermometers. Bits of contaminated meat and grease
and particles of dirt carried in on the pumping needles would be forced
out into the hams by the pumping pickle, which passes out through small
openings or fenestræ in the needles, and this probably affords one
explanation as to why so many more body sours occur in the mild-cure
hams. In the mild-cure hams, which are pumped in the shank only, the
pumping needle is introduced near the femorotibial articulation, and
the shank is saturated at the start with a strong brine solution, while
the body of the ham is not. If the ham-souring bacillus were carried
into these hams on the pumping needle, the growth of the bacillus in
the shank would be inhibited by the strong brine solution with which
the shank is saturated, but there would be nothing to prevent the
bacillus from growing upward into the body of the ham, which has not
been pumped and is free from pickle. This would also explain the fact
that the souring often starts at the knee joint and extends upward into
the body of the ham. In the case of the regular cure hams, where the
ham is pumped in both body and shank, the entire ham is more or less
saturated at the start with the strong brine of the pumping pickle,
which tends to inhibit the growth of the ham-souring bacillus even
if this bacillus should find its way into these hams on the pumping
needles. It is in the mild-cure or partly pumped hams, where the body
of the ham is left unpumped, that the ham-souring bacillus finds its
best opportunity for development, and the greater proportion of the
sours that occur in the packing house are found in these hams.

As regards the possibility of infection from the pumping pickle
itself, it does not seem probable that this would often occur, for
the pumping and curing pickles are always prepared on an upper floor
of the pickling houses and are delivered to the pickle cellars in
closed pipes, so the chances for the accidental contamination of these
solutions from floating dust or dirt would not be great. Furthermore,
the strong brine of the pumping pickle would completely inhibit the
growth of the ham-souring bacillus, and the bacillus would be incapable
of multiplying, even if it found its way into the pickle. On the other
hand, laboratory experiments show that the bacillus or its spores may
remain alive for a considerable length of time in the pumping pickle,
so the possibility of infection from this source can not be overlooked.


                       INFECTION FROM BILLHOOKS.

After the hams are cut from the carcasses they are handled entirely
by means of billhooks. In handling the hams the hooks are inserted
beneath the skin of the shank at a point just above the tibio-femoral
articulation. The hooks should be inserted in the connective tissue
beneath the skin and should not penetrate the muscular tissue to any
depth. When the hams lie in the right position, with the butt or large
portion away from and the shank toward the operator, it is an easy
matter to pick them up in the proper manner; but when they lie at
different angles and are being rapidly handled it is almost impossible
to prevent the hook from penetrating the muscular tissues, and if the
hook should penetrate to the bone it might carry in foreign matter
contaminated with the meat-souring bacillus. It is not probable that
many hams become contaminated in this way, as the men who handle the
hams are very skillful in manipulating their hooks; but the possibility
that hams may become contaminated in this manner should not be entirely
overlooked.


    BIOLOGICAL AND MORPHOLOGICAL CHARACTERISTICS OF THE HAM-SOURING
                               BACILLUS.


                    CONDITIONS FAVORABLE TO GROWTH.

The most favorable medium for the growth of the organism was found
to be the modified egg-meat mixture of Rettger, which has been
previously described. In this medium the organism develops rapidly
at a temperature of 20° to 25° C., giving rise to the characteristic
sour-meat odor. Like the bacillus described by Klein, it also grows
readily on pork-agar and pork-bouillon containing glucose, but differs
from Klein’s bacillus in that it will grow, though less luxuriantly,
on ordinary nutrient media—agar, gelatin, and bouillon—without the
addition of glucose.

The optimum temperature for growth is 20° to 25° C. The organism does
not grow at incubator temperature (37.5° C.). At ice-box temperature
(8° to 10° C.) it develops readily, although the growth is less rapid
than at 20° to 25° C. That the organism will develop at even lower
temperatures was shown in the inoculation experiments with hams, where
it developed and multiplied extensively in the bodies of the hams at
the temperature of the pickling cellars, which are held usually at 34°
to 36° F. (1° to 2° C.).

The organism develops best in a neutral or slightly alkaline medium.


                  GROWTH ON DIFFERENT CULTURE MEDIA.

_Growth on egg-pork medium._—At a temperature of 20° to 25° C. the
cultures show a slight but distinct sour odor in from two to three
days. This odor, as before stated, closely resembles the odor of a
sour ham. Egg-pork cultures from three to five days old were given to
a trained meat inspector, who knew nothing whatever as to the contents
of the tubes, and he was asked to describe the odor; he described it as
that of a sour ham.

At one week the albumins of the medium are gelatinized or partly
coagulated and the odor is more pronounced. At ten days the albumins
are completely coagulated except at the surface, where there is
no apparent growth; the odor is more putrefactive in nature, and
the reaction of the medium is slightly acid. At three weeks the
coagulated albumin splits up into fragments and appears to undergo a
slow digestion, gas bubbles form in the lower portion of the culture,
and the odor becomes distinctly putrefactive in character. The slow
digestion of the albumin is probably due to a proteolytic enzyme
elaborated by the bacillus.

At the end of a week a dark zone usually appears at the surface of the
coagulated albumin and gradually darkens until it becomes almost black.
This zone is probably due to a pigment elaborated by the bacillus.

At ice-box temperature (8° to 10° C.) the same changes and the same
odor were noted, but were somewhat slower in developing.

_Glucose-pork-agar._—This medium was prepared from pork in the same
manner as beef-agar, and contained 1 per cent of glucose. The organism
grows readily on this medium and may be conveniently cultivated in deep
stab cultures. The medium was always thoroughly boiled and then rapidly
cooled in order to expel the inclosed air. The growth of the organism
was found to vary considerably with the reaction.

When the reaction was +1.5, deep stab cultures at three days (20° to
25° C.) showed a well-marked arborescent growth, appearing as delicate
filaments extending outward from the line of stab. The growth stopped
within one-fourth or one-half inch of the surface of the agar on
account of the presence of oxygen in the upper part of the culture
medium. As the growth extended toward the walls of the test tube the
agar became clouded, and there were sometimes gas bubbles in the depth
of the agar, but the gas formation was not extensive.

When the reaction of the agar is neutral or slightly alkaline,
extensive gas formation occurs and the agar is often much broken up.

The cultures developed a disagreeable, somewhat putrefactive odor,
but did not give the characteristic sour-ham odor obtained from the
egg-pork cultures.

The organism was also grown on anaerobic agar plates by Zinsser’s method,
which is said to give absolutely anaerobic conditions. The colonies on
agar have a cottony or woolly appearance at first, and spread slowly,
with slightly irregular margins.

In glucose-pork-agar to which azolitmin was added the azolitmin in the
lower portion of deep stab cultures was completely decolorized in five
days at room temperature (20° to 25° C).

In glucose-pork-agar containing neutral red the red color in the lower
portion of the tube was changed to yellow with the development of
fluorescence.

_Neutral gelatin._—Tubes of ordinary neutral gelatin without the
addition of glucose were inoculated and held at ice-box temperature
(8° to 10° C). At five days a delicate white growth appeared along
the line of stab in the lower portion of the tube. At seven days the
growth showed fine radial striæ, presenting an arborescent or tree-like
appearance, and extended halfway from the line of stab to the walls of
the test tube. At two weeks the growth had caused a delicate clouding
of the medium in the lower portion of the tube. At three weeks the
gelatin in the lower portion of the tube had become liquefied and the
growth had settled to the bottom as a white precipitate.

In gelatin containing glucose, gas bubbles are formed in the depth
of the medium through the splitting up of the glucose, and the
characteristic arborescent growth is obscured.

_Glucose-pork-bouillon._—This medium was prepared from pork instead
of beef and contained 1 per cent of glucose. The best results were
obtained when the reaction of the medium was neutral or slightly
alkaline.

Culture tubes, which had been previously boiled to expel the contained
air and then inoculated, were held in a Novy jar, in an atmosphere of
hydrogen at a temperature of 20° to 25° C. At three days the tubes
showed well-marked clouding. At one week the growth appeared as a
heavy, white, flocculent, cottony precipitate in the bottom of the
tubes with a slight flocculent precipitate above. When the culture was
removed from the jar and shaken, the heavy, flocculent precipitate at
the bottom of the tube broke up without much difficulty, giving rise to
a heavy uniform clouding with some small floating masses, which soon
settled to the bottom. On shaking the tube some evolution of gas in the
form of very fine bubbles was noticed.

In Smith fermentation tubes containing neutral glucose-pork-bouillon
the closed arm of the tube shows well-marked clouding with gas
formation at three days at room temperature (20° to 25° C). The
growth has a tufted, cottony appearance, and there are many filaments
and threads. The growth settles to the bottom of the closed arm as
a cottony, white precipitate (see Pl. IV). The organism splits the
glucose vigorously, and at 10 days the tubes show from 40 to 50
per cent of gas. The bouillon in the open arm of the tube remains
unclouded. The maximum gas production at room temperature is reached
in from 10 to 14 days, by which time the growth in the closed arm has
completely settled into the bend of the tube, leaving the bouillon
in the closed arm clear. The gas formula, as determined by Smith’s
method, was H/CO₂ = 5/1. The reaction of the bouillon becomes acid to
phenolphthalein.

The organism will grow on ordinary neutral bouillon without the
addition of glucose, and in Smith tubes containing this medium a small
amount of gas was formed, due to the splitting of the muscle sugar.

The bacillus also grows in a sugar-free broth—that is, a broth free
from muscle sugar—and from cultures grown in this medium a well-marked
indol test was obtained.

_Litmus-milk._—The organism was grown in litmus-milk in Smith
fermentation tubes at 20° to 25° C. At seven days the litmus in the
lower portion of the closed arm had assumed a brownish-buff color.
At two weeks the litmus in the closed arm had been reduced to a
brownish-buff color except at the top of the tube, where a pale, bluish
tinge remained, and the litmus in the open arm showed very slight
reddening as compared with a check tube. At three weeks the litmus in
the closed arm was entirely reduced to a light, brownish-buff color,
and the litmus in the open arm showed a slight but distinct reddening
as compared with the check. The reddening of the litmus in the open arm
was evidently due to the transfusion of acids formed by the growth of
the bacillus in the closed arm. After several weeks the milk is slowly
peptonized, probably as a result of enzyme action.


                              MORPHOLOGY.

The organism is a large bacillus having an average size of 4 to 8 μ in
length by 0.5 to 0.7 μ in thickness, but there are many longer forms
measuring from 10 to 20 μ in length. It develops in long, irregular
chains or filaments, which at times show a slightly spiral form.

[Illustration:

    FIG. 5.—Ham-souring bacillus (_Bacillus putrefaciens_) grown
    on egg-pork medium, showing tendency to form chains. Partly
    developed and fully developed spores are shown at ends of rods;
    also free spores. (Pen-and-ink drawing made with camera lucida
    from preparation stained by Gram’s method. × 640.)]

The individual organisms show at times a widely open, slightly spiral
form, which was more apparent in hanging-drop preparations made
from bouillon cultures, where the organisms had been comparatively
undisturbed. This appearance was also noted at times in the stained
sections of soured muscular tissue, where the organisms were stained in
place. The organism possesses no motility. It stains with the ordinary
aniline dyes and by Gram’s method.


                           SPORE FORMATION.

The organism develops large, terminal spores, which are at first oval,
but when fully developed are perfectly round and measure from 1.5 to 2
μ in diameter.

Spores develop rapidly in the egg-pork medium at 20° to 25° C., fully
developed spores being noted in from five to seven days. At ice-box
temperature (8° to 10° C.) partly developed spores were noted in the
egg-pork medium at 10 days and fully developed spores at 2 weeks.

Occasional spores were noted in old agar and gelatin cultures, but
abundant spore formation was seen only in the egg-pork medium. No
spores were noted in bouillon cultures, even at 10 weeks.


                RESISTANCE TO HEAT AND CHEMICAL AGENTS.

In its vegetative form the bacillus is killed at 55° C. in 10 minutes.
The spores survive a temperature of 80° C. for 20 minutes, but are
killed at 100° C. in 10 minutes.

When sodium chlorid and potassium nitrate were added to glucose-pork
broth in varying amounts, it was found that 3 per cent of sodium
chlorid or 3 per cent of potassium nitrate was sufficient to inhibit
completely the growth of the bacillus at room temperature (20° to 25°
C.).

While the growth of the bacillus was inhibited by sodium chlorid and
potassium nitrate as just stated, it was found that very much stronger
solutions of the two salts failed to destroy the bacillus. Thus it was
found that the bacillus or its spores retained their vitality after
an exposure of 30 days in a solution containing 23 per cent of sodium
chlorid and 6 per cent of potassium nitrate.


                            GAS PRODUCTION.

The organism splits glucose, but not lactose or saccharose. That
it possesses the power of splitting muscle sugar was shown by the
formation of gas in Smith fermentation tubes containing ordinary
neutral bouillon without the addition of any sugar.

The formation of gas in glucose bouillon varies considerably with
the reaction of the medium. The largest amount of gas was formed
when the broth was neutral or slightly alkaline. When the reaction
of the broth was distinctly acid or distinctly alkaline the amount
of gas was diminished. The gas which is formed in bouillon cultures
consists chiefly of hydrogen and carbon dioxide. In order to collect a
sufficient amount of the gas for analysis, two large fermentation tubes
capable of holding 150 cubic centimeters each were constructed. These
tubes were filled with pork-bouillon and inoculated with the bacillus.
After 20 days at room temperature (20° to 25° C.) the gas was collected
and the carbon dioxide and hydrogen determined, with the following
result:

                                            Cubic centimeters.
    Total amount of gas collected                 37.7
    Carbon dioxide, by absorption with NaOH        6.2
    Hydrogen, by difference                       31.5

This analysis gives an approximate gas formula of H/CO₂ = 5/1, which
agrees with the gas formula as determined in the small fermentation
tubes by Smith’s method.

In hams which had undergone spontaneous souring and in hams which had
been artificially soured by inoculation, hydrogen-sulphid was often
noted when the sour portions of the meat were tested with lead-acetate
paper, but no distinct odor of the gas could be obtained. Hydrogen
sulphid was also noted in egg-pork cultures of the bacillus.


                           ACID PRODUCTION.

In glucose-bouillon, butyric and lactic acids are formed and the
reaction of the medium becomes distinctly acid. Butyric and lactic
acids were also noted in the egg-pork cultures.

A series of Smith fermentation tubes containing 10 c. c. each of
glucose-pork broth medium was inoculated with the bacillus and held
at room temperature (20° to 25° C.). These cultures were titrated
against [N/40]NaOH, with phenolphthalein as an indicator at intervals
of two days up to nineteen days, and then at two-week intervals up to
sixty-one days. Three of the cultures were titrated each time so as to
give a fair average of the acidity of the cultures, and an uninoculated
check tube was also titrated each time to see if there was any change
in the reaction of the medium. The results of the titrations are shown
in the following table:


       _Acidity determinations in glucose-pork broth cultures._

    ──────────────┬───────┬───────┬───────┬────────┬───────┬───────────
    Age of culture│Culture│Culture│Culture│Average.│Medium.│Acidity of
       (days).    │   A.  │   B.  │   C.  │        │       │ culture.
    ──────────────┼───────┼───────┼───────┼────────┼───────┼───────────
                  │       │       │       │        │       │_Per cent._
           2      │ 0.038 │ 0.030 │ 0.040 │  0.036 │ 0.009 │   0.027
           4      │  .105 │  .100 │  .102 │   .102 │  .009 │    .093
           6      │  .106 │  .110 │  .109 │   .108 │  .009 │    .099
           8      │  .124 │  .115 │  .117 │   .119 │  .009 │    .110
          10      │  .128 │  .130 │  .126 │   .128 │  .009 │    .119
          12      │  .129 │  .120 │  .129 │   .126 │  .009 │    .117
          19      │  .126 │  .125 │  .125 │   .125 │  .009 │    .116
          33      │  .125 │  .123 │  .125 │   .124 │  .009 │    .115
          47      │  .122 │  .120 │  .121 │   .121 │  .009 │    .112
          61      │  .121 │  .116 │  .119 │   .118 │  .009 │    .109
    ──────────────┴───────┴───────┴───────┴────────┴───────┴───────────

From the above table it will be seen that the maximum acidity was
reached at ten days, after which there was a gradual reduction in the
acidity, due probably to the formation of ammonia compounds.


                        PATHOGENIC PROPERTIES.

Rabbits, guinea pigs, and white mice were inoculated and fed with
cultures of the bacillus without effect, from which it would appear
that the bacillus possesses no pathogenic or disease-producing
properties.


                        NATURE OF THE BACILLUS.

The bacillus is essentially a saprogenic bacterium with zymogenic
properties. A preliminary study of the chemical changes which take
place in sour hams shows that these changes are of a putrefactive
nature. Hams which had undergone spontaneous souring were compared
with hams which had been artificially soured by inoculation, and the
chemical changes were found to be identical. A chemical study was also
made of the changes taking place in egg-pork cultures of the bacillus
at different stages of growth, and these changes were found to be of a
putrefactive nature and similar in character to the changes which occur
in sour hams. Among the putrefactive products formed by the growth of
the bacillus in the egg-pork medium were indol, skatol, volatile fatty
acids, skatol-carbonic acid, and hydrogen sulphid.[4]

[4] The tests for the putrefactive products formed by the growth
of the bacillus in the egg-pork medium were made by P. Castleman,
of the Biochemic Division, who also determined the percentage
composition of the gas formed by the growth of the bacillus in the
glucose-pork-bouillon medium.

[Illustration:

    BUL. 132, BUREAU OF ANIMAL INDUSTRY, U. S. DEPT. OF AGRICULTURE.
                               PLATE IV.

    GLUCOSE BOUILLON CULTURE IN SMITH FERMENTATION TUBE AT FOUR
    DAYS. CULTURE GROWN AT ROOM TEMPERATURE (20° TO 25° C.). GROWTH
    CONFINED ENTIRELY TO CLOSED ARM, WITH GAS COLLECTING AT TOP.]

A more extended study is now being carried on in the Biochemic Division
of the chemical changes which take place in hams during the process of
souring, together with a further study of the chemical changes which
result from the growth of the bacillus in the egg-pork medium. The
results of this investigation will be given in a later paper.

The bacillus described in this paper belongs to the class of
putrefactive anaerobes, which are widely distributed in nature in dust,
soil, and excrementitious matters. This group of bacteria contains
both pathogenic and nonpathogenic forms. The former have received
considerable attention, but the latter have never been thoroughly
cleared up. The bacillus isolated from sour hams belongs in the latter
category, being possessed of no pathogenic or disease-producing
properties. It occurs in the dust and dirt of the packing house and
finds its way into the hams in the various manipulations to which the
hams are subjected.

The bacillus described in this paper does not seem to correspond
with any forms heretofore described. It differs from Klei bacillus
(_Bacillus fœdans_) in the following important particulars: (1) It
forms large terminal spores, whereas Klein’s bacillus formed no spores;
(2) it will grow at a temperature of 34° F., while Klei bacillus did
not grow below 50° F.; (3) it produces an acid reaction in culture
media, while Klei bacillus gave a distinctly alkaline reaction;
(4) it will grow on the ordinary nutrient media—gelatin, agar, and
broth—without the addition of glucose, while Klein’s bacillus did not;
(5) it peptonizes the casein in milk, whereas Klein’s bacillus had no
action on milk; (6) it liquefies gelatin more rapidly, causing complete
liquefaction after three weeks at 8° to 10° C., whereas Klein’s bacillus
caused only partial liquefaction after eight weeks at 20° C.; (7) it
can be conveyed
from turbid broth cultures to new culture material by means of the
platinum loop, whereas Klein’s bacillus could not be thus conveyed.

For the bacillus described in the present paper the following name is
proposed: _Bacillus putrefaciens_.


                      PREVENTION OF HAM SOURING.

As it has been shown that souring in hams results from the growth
of a bacterium which is introduced into the bodies of the hams in
the various manipulations which the hams undergo, the only way to
eliminate souring in hams, as they are cured in the larger packing
establishments, would be to cure the hams under aseptic or sterile
conditions, which would, of course, be a physical impossibility.

While it will probably be impossible, therefore, to eliminate souring
entirely under the methods of ham curing which are at present employed
in the larger packing establishments, much can undoubtedly be done
toward reducing the percentage of sours. In the matter of taking ham
temperatures, for instance, if the thermometers used were thoroughly
cleaned and disinfected and the surfaces of the hams seared at the
point where the thermometer is introduced, infection from this source
could be entirely prevented; or it might be possible so to regulate
the temperature of the chill rooms that the taking of ham temperatures
could be discontinued.

The elimination of the souring that results from the introduction of
foreign matter on the pumping needles could be effected in two ways
only, (1) by not pumping the hams at all, or (2) by pumping them under
sterile or aseptic conditions. As has been stated before, some of the
smaller packing establishments cure their hams without pumping, and in
these establishments the percentage of sours runs very low. When hams
are cured without pumping, however, the period of curing has to be
materially lengthened in order to give the curing pickles sufficient
time to penetrate thoroughly, and this is what the larger plants wish
to avoid because of the greater space and greater number of vats which
would be necessitated. The object of pumping in the larger plants,
where the number of hams handled daily runs into the thousands, is to
hasten the cure and thus prevent the accumulation of a great number of
hams at one time. It is doubtful, therefore, whether the larger packing
houses could conveniently discontinue pumping.

To pump the hams under aseptic conditions would necessitate a technique
far too elaborate for routine use in the packing house; in fact,
anything like complete asepsis would be out of the question. Certain
measures might be adopted, however, that would tend to prevent the
possible introduction of ham-souring bacilli in the process of pumping.
It would undoubtedly be safer, for instance, to boil the pumping pickle
before use, and the chances of carrying in contaminated foreign matter
on the pumping needles could be lessened by sterilizing the pumps and
needles with boiling water and by frequently dipping the needles, while
in use, in boiling water. If the hams were sprayed with clean water
just prior to pumping, there would be less likelihood of carrying in
foreign matter on the needles. The danger of introducing contaminated
foreign matter on the needles might be further obviated by searing the
surfaces of the hams at the points where the needles are introduced;
but such a procedure would be hardly practicable in the larger packing
houses, where the great number of hams cured necessitates rapid
handling.

While the danger of possible contamination in pumping, through the
introduction of contaminated foreign matter on the pumping needles,
can not well be avoided, this danger is partly counterbalanced by the
inhibitory action of the pumping pickle, which is strikingly shown in
the experiments which have been described. In these experiments, 100
hams received large doses of the ham-souring bacillus, half of these
hams being subjected to the mild cure and half to the regular cure,
with the following result: In the case of the mild-cure hams, which
were pumped in the shank only, the percentage of sours was practically
100 per cent, every ham with possibly one exception becoming sour;
whereas in the regular-cure hams, which were pumped in both body and
shank, only 58 per cent of the hams became sour. In other words, the
additional pumping which the regular-cure hams received served to
prevent souring in 42 per cent of these hams. In these experiments the
number of bacteria introduced into the hams was very great, thousands
and even millions of the bacilli being introduced into each ham,
whereas in the routine of the packing house it is not likely that
more than a few of the bacilli are ever introduced at one time on the
thermometers and pumping needles. In view of these results it is safe
to say that in the larger packing houses, where pumping seems to be
necessary, the number of sours could be reduced fully 50 per cent if
all hams were pumped in the body as well as in the shank.

At present the usual procedure is to pump all hams, both mild and
regular cure, with the same pumping pickle, the mild-cure hams being
pumped in the shank only and the regular-cure hams at two additional
points in the body. The experiments quoted above show that the
additional pumping which the regular-cure hams receive undoubtedly
tends to prevent the development of souring in these hams, and this
result is unquestionably due to the inhibitory action of the salts
contained in the pumping pickle, as it was found by laboratory
experiment that the addition of 3 per cent of sodium chlorid to
culture media is sufficient to inhibit the growth of the ham-souring
bacillus. The pumping pickles consist of strong brine solutions and
always contain considerably more than 3 per cent of sodium chlorid.
If, therefore, the pumping of regular-cure hams were made more
thorough than at present, and all of the deeper portions of the ham
were thoroughly saturated with the strong brine solution, souring
could be largely eliminated, if not entirely prevented, in these hams,
as an unfavorable medium or soil would thus be created in which the
ham-souring bacillus could not develop. The ham-souring bacillus is
able to develop within the bodies of the regular-cure hams because the
pumping of these hams is not always thorough and there are certain
areas in the inner or deeper portions of the hams in which the tissues
are not thoroughly saturated with the pumping pickle.

Under the present methods of curing, the greater proportion of the
sours occur among the partly pumped or mild-cure hams. These hams are
pumped in the shank only, and the growth of the ham-souring bacillus
within the bodies of these hams is not interfered with until the curing
pickle has penetrated from the outside. As it requires several weeks
for the curing pickle to penetrate thoroughly into the deeper portions
of these hams, the bacillus is thus afforded a considerable interval
in which to develop before it is exposed to the inhibitory action of
the pickle. If these hams could be thoroughly pumped in the body at
the beginning of the cure in the same manner as the regular-cure hams,
the chief loss from ham souring would be eliminated. It would not do,
however, to pump these hams in the body with the same pumping pickle
used in the regular cure, as the meat would be rendered too salty and
the mild flavor of the ham would be lost. There is undoubtedly a demand
for mild-cure hams, otherwise they would not be on the market; and the
question then arises how to pump these hams and still retain a mild
cure. This might be accomplished by pumping these hams with their own
curing pickle, which is usually a milder pickle than that employed in
the regular cure, or an even milder pumping pickle might be used. If
mild-cure hams were pumped in this way, the percentage of souring in
these hams could undoubtedly be greatly diminished without materially
affecting the flavor of the ham.

To recapitulate briefly, the prevention of ham souring is to be sought
in two ways: (1) Through greater care in handling the hams and the
adoption of precautionary measures to prevent the introduction of the
ham-souring bacillus into the bodies of the hams, and (2) through more
thorough pumping of the deeper or inner portions of the hams, so as to
create an unfavorable soil or medium in which the ham-souring bacillus
can not develop even if it should gain entrance into the bodies of the
hams.

From what has been said it will be apparent that ham souring can
probably never be entirely eliminated from the packing house under the
present methods of curing, but the adoption of precautionary measures
in testing and pumping hams, together with a more thorough pumping
of all hams in ways similar to those suggested, would unquestionably
reduce very materially the losses from this source.


                   GENERAL SUMMARY AND CONCLUSIONS.

1. In this paper it has been shown that ham souring, as encountered in
the wet cure where the hams are entirely submerged in pickling fluids,
is due to the growth of an anaerobic bacillus within the bodies of the
hams. This bacillus (_B. putrefaciens_) was found in sour hams obtained
from four different packing establishments. It was isolated and grown
in various laboratory media, in one of which, the egg-pork medium, it
gave rise to the characteristic sour-ham odor. This bacillus was the
only organism that could be isolated from sour hams that was capable of
producing the characteristic sour-ham odor in the egg-pork medium.

2. When injected into the bodies of sound hams, the bacillus caused
these hams to sour in the process of curing. In hams which had been
inoculated with the bacillus and thus artificially soured, the bacillus
was recovered in cultures taken at points far removed, relatively
speaking, from the point of inoculation, indicating that the bacillus
had multiplied and progressed by extension throughout the bodies of the
hams.

3. The bacillus possesses no motility, and its extension throughout
the bodies of the hams is a result of multiplication. In its growth it
follows along the connective-tissue bands between the muscle bundles,
which are composed of comparatively loose tissue and afford paths of
least resistance. When it invades the muscle tissue proper, it follows
along the sarcolemma sheaths between the muscle fibers. As a result of
this growth the muscular tissue becomes softer and tends to break more
easily.

4. The bacillus belongs to the class of putrefactive anaerobes which
are widely distributed in nature in dust, soil, and excrementitious
matters. The bacillus or its spores is present in the dust and dirt
of packing houses and finds its way into the hams in the various
manipulations to which they are subjected.

5. The bacillus or its spores may be introduced into hams on the
thermometers used in testing the hams, on the pumping needles, and
possibly on the billhooks used in handling the hams. It may also be
carried into the hams in the pumping pickle, and may even find its way
into the hams from the curing pickle, although infection through the
latter channel probably does not often occur.

6. The bacillus develops in the deeper portions of the ham because of
the anaerobic conditions there prevailing, and souring is most often
encountered, therefore, in the deeper portions of the ham near the
bone.

7. A preliminary study of the chemical changes which take place in
the process of souring shows that these changes are of a putrefactive
nature, and ham souring, as ordinarily encountered, is to be regarded
as an incipient putrefaction. Hams which had been artificially soured
by injections of culture were compared with sour hams obtained from the
packing house, and the putrefactive changes were found to be identical.

8. Hams which have once become sour can never be restored to a sound
condition, because of the chemical changes which result from the growth
of the bacillus. In other words, the tissues of the ham undergo certain
chemical changes in the process of souring, and when these changes
have once taken place the tissues can never be restored to a sound
condition. The repumping of slightly soured hams with a strong pumping
pickle will check further souring, by inhibiting the growth of the
bacillus, but will not restore to a sound condition those portions of
the ham which have become sour.

9. The salts of the pickling fluids have a marked inhibitory action
on the ham-souring bacillus, and sours occur less frequently in
regular-cure hams.

10. In regular-cure hams the growth of the ham-souring bacillus is
restricted and often completely inhibited as a result of the additional
pumping which these hams receive, whereby they are more or less
saturated with pickle at the beginning of the cure.

11. If the pumping of regular-cure hams were more thorough and all
of the deeper portions of the ham were thoroughly saturated with the
pumping pickle, souring could be largely eliminated if not entirely
prevented in the hams, as an unfavorable medium or soil would thus
be created, in which the ham-souring bacillus could not develop. The
reason that souring does develop in regular-cure hams is because the
pumping is not always thorough and there are certain areas in the
deeper portions of these hams which are not saturated with the pumping
pickle.

12. Under the present methods of curing, the partly pumped or mild-cure
hams furnish the greater proportion of the sours, as these hams are not
pumped in the body and the growth of the ham-souring bacillus within
the bodies of these hams is not interfered with until the curing pickle
has penetrated from the outside. As it requires several weeks for the
curing pickle to penetrate thoroughly into the deeper portions of these
hams, the bacillus is thus afforded a considerable interval in which to
develop.

13. The percentage of souring in the mild-cure hams could be greatly
reduced without materially affecting the cure by pumping these hams
with their own curing pickle, which is usually a milder pickle than
that employed in the regular cure; and if the pumping were thorough
the number of sours in these hams could be reduced to a small figure.

14. The only way by which ham souring could be entirely eliminated from
the larger packing establishments under the present methods of curing
would be to handle the hams throughout under aseptic conditions, and
this, for obvious reasons, would be an impossibility. The losses from
ham souring may be materially reduced, however, by greater care in
handling the hams and the adoption of precautionary measures designed
to prevent the introduction of contaminated foreign matter into the
bodies of the hams, together with more thorough methods of pumping.


                           ACKNOWLEDGMENTS.

In conclusion, the writer desires to express his obligations to Dr. S.
E. Bennett, of the Inspection Division, inspector in charge at Chicago,
for the assignment of trained meat inspectors to assist in the work,
as well as for kind assistance in obtaining data and material for
laboratory study, and to Dr. L. E. Day, of the Pathological Division,
who kindly prepared the sections which are figured and described in the
present article.