Part 3

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

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

_Experiment I._

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

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

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

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

The details of the experiment were as follows:

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

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

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

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

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

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

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

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

_Results of Experiment I._

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

_Experiment II._

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