Chapter I: Part 1

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_.

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.

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.

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.)]

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.

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._