A bacteriological study of ham souring

by Charles Neil McBryde

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 tierce.	Number of hams.	Average weight of hams Pounds.	Cure.	How pumped.	Treatment.	Condition at end of cure.
						Number of sour hams	Percentage of sour hams
1	20	12-14	Fancy	Shank only	Each ham injectedwith 10 c. c. of culture.	19	95
2	20	12-14	do	do	Not injected with culture;check on tierce 1.	0	0
3	20	14-16	Regular	Shank and body	Each ham injectedwith 10 c. c. of culture.	9	45
4	20	14-16	do	do	Not injected with culture;check on tierce 3.	0	0

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 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.

Bul. 132, Bureau of Animal Industry, U. S. Dept. of Agriculture. Plate III.

Fig. 1.—Section Through Muscular Tissue of Artificially Soured Ham, Showing Distribution of Bacilli Between the Muscle Fibers, which are Shown in Cross Section. The Dark Lines and Masses Between the Muscle Fibers Represent Clumps of Bacilli.

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

Fig. 2.—Section Through Muscular Tissue of Artificially Soured Ham, Showing Individual Bacilli Between the Muscle Fibers, which are Cut Longitudinally.

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

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

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

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

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

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

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

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

Experiment II.

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

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

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

The details of the experiment were as follows:

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

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

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

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

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

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

Results of Experiment II.

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

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

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

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

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

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

Summary of Experiments I and II.

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

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

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

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