THE PRESENT EXPERIMENTS.

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:

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

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.

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.

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 1-1/2 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-agartubes, 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.

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. 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.)

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 with hematoxylin and eosin to show histological structure.× 320.)

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

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 with hematoxylin and eosin to show histological structure.× 320.)

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

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. InPlate II, figures 1 and 2, the bacteria are shown between the muscle fibers under low and high power magnifications.

InPlate 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.

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 2-1/2 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.

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.

Analyses of sound hams.

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:

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:

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.

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 ofBacillus coliand 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 skinnedsurfaces 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.

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.

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 onthe 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 latterbeing 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.

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.

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.

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 inpure 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.

InPlate 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.

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.)

(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.

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. Thehams 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:

Back to Index Next