Part 7

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

3. A large micrococcus.

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

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

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

INOCULATION EXPERIMENTS WITH HAMS.

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

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

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

Experiment I.

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

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

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

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

The details of the experiment were as follows:

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

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

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

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

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

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

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

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

Results of Experiment I.

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

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

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

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