A bacteriological study of ham souring

Results of Experiment II.

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.

PROBABLE METHOD BY WHICH HAM-SOURING BACILLUS ENTERS HAMS.

Regarding the question of the probable method by which the ham-souring bacillus enters hams, there were three possibilities to be taken into consideration:(1) That the bacillus is present in the flesh of hogs at the time of slaughter,(2) that the bacillus gains entrance through the pickling fluids,(3) that the bacillus is introduced into the bodies of the hams in the handling or manipulation which the hams undergo in preparation for, or during, the process of curing.

Possibility of Infection Prior to Slaughter.

In order to throw some light upon this point, a number of fresh hams—that is, hams which had been chilled but not pumped or subjected to any other manipulation—were examined bacteriologically, but in no case could the anaerobic bacillus which was isolated from sour hams be detected in any of them. The fact that in certain of the smaller packing establishments which cure their hams without pumping the percentage of souring is extremely low would also seem to negative this possibility, for if the bacillus which causes souring were present in the hams at the time of slaughter, sour hams would be as frequent at such establishments as at those establishments which make a practice of pumping. Furthermore, a laboratory study, biological and chemical, of the bacillus isolated from sour hams shows that this organism belongs to the class of putrefactive bacteria, and while such bacteria may be present in the intestines of healthy animals, as, for example, the bacillus of Bienstock (Bacillus putrificus), these bacteria do not invade the organs and tissues of the body until after the death of the animal, and the packing-house practice of rapidly eviscerating the hogs immediately after slaughter would certainly preclude this possibility.

Possible Infection from Pickling Fluids.

With regard to the second possibility, that the bacillus finds its way into the hams in the curing pickles, it was determined by laboratory experiment that the addition of 3 per cent of sodium chlorid or 3 per cent of potassium nitrate to laboratory media completely inhibits the growth of the bacillus. As the pickling solutions always contain considerably more than these percentages of sodium chlorid and potassium nitrate, it would be impossible for the bacillus to multiply in the pickles. Additional laboratory experiments demonstrated, however, that the bacillus or its spores may remain alive in the curing pickles for at least thirty days, and it seemed possible that the curing pickles might become contaminated at times with the bacilli, and that the bacilli, although incapable of multiplying in the pickles, might find their way into the bodies of the hams in the pickling fluids. In order to throw some light upon this point, the following experiment was carried out:

EXPERIMENT TO SHOW WHETHER INFECTION TAKES PLACE FROM THE CURING PICKLE.

In this experiment two tierces were put down, each containing 20 hams. The hams weighed from 14 to 16 pounds and had received the usual 48-hour chilling. The pickling solutions employed were the regular curing pickles of the establishment at which the experiment was carried out. The curing pickle in one tierce was inoculated with 400 cubic centimeters of a culture suspension of the bacillus, prepared in the same manner as that used for the injection of the hams in tierce 2 in Experiment II. A microscopic preparation made from a small drop of the culture suspension before adding it to the pickle showed the bacilli in large numbers, and in the 400 cubic centimeters of the suspension there were millions of the bacteria. The curing pickle in the other tierce was left untreated, the hams in this tierce serving as a check. The tierces used in this experiment, as in all of the experiments, were thoroughly cleaned with boiling water before the hams were placed in them. The experiment was conducted in a pickling room which was held at 33° to 36° F., and the tierces were rolled three times during the cure. The details of the experiment are as follows:

Comparing tierce 1, which contained the inoculated pickle, with tierce 2, the check tierce which contained uninoculated pickle, we find there was practically no difference in the final result. In tierce 1 two of the hams developed slight souring, while in tierce 2 one of the hams became slightly sour. All of these hams had been pumped in the shank only. The fact that one of the hams in the check tierce developed slight souring was undoubtedly due to bacterial contamination in pumping or in the handling which the hams underwent prior to pickling, and the slight souring of the two hams in tierce 1 must also be attributed to the same cause or causes, for had the souring in these last hams resulted from the penetration of the bacteria from the pickling solution a higher percentage should have become sour. Furthermore, if the souring of the two hams in tierce 1 had resulted from the penetration of the bacteria from the curing pickle, the souring should have been general throughout the bodies of these hams, whereas the souring was only evident around the bone and was slight in degree.

From this experiment the conclusion would seem justified that the bacillus which causes ham souring does not usually find its way into the bodies of the hams from the curing pickle, although it would be going too far, perhaps, to say that infection never takes place from the curing pickle. The experiment, however, indicates clearly that the curing pickles are certainly not the main channel through which the hams become infected. In referring to the curing pickles, it should be understood that we refer here to the pickling solutions in which the hams are immersed, and not to the pumping pickles. The possibility of infection through the pumping pickle will be discussed later.

Possible Infection through Manipulation or Handling.

There are at least three possible ways in which hams may become infected from the handling which they receive in preparation for, or during the process of curing, viz: From the thermometers used in taking the inside temperatures of the hams, from the pumping needles, and from the billhooks used in lifting the hams.

INFECTION FROM HAM THERMOMETERS.

The packing-house method of taking the temperatures of hams by means of a pointed, metal-capped thermometer which is thrust deep into the bodies of the hams has already been referred to, but deserves to be described somewhat more in detail, as it will be at once apparent that this manipulation furnishes a ready means whereby hams may become infected with putrefactive bacteria. The construction of a ham thermometer is shown in figure 4.

The instrument consists of a glass thermometer inclosed in a metal case, the front portions of the case being cut away so as to expose the scale above and the mercury bulb below. As was explained before, the thermometer is thrust deep into the body of the ham so that the pointed end containing the mercury bulb rests beside or a little behind the upper portion of the femur, the bone being used as a guide in introducing the thermometer.

Ham temperatures are taken at three stages in the preparation for cure—(1) on the hanging floor, just before the hams go to the chill rooms, in order to determine the amount of heat lost prior to chilling; (2) on leaving the chill rooms, in order to determine the thoroughness of the chill;(3) on the packing floor, just before the hams are placed in pickle, as a further check on the thoroughness of the chilling.

In taking the temperatures of hams which have been chilled—and most of the temperatures are taken subsequent to chilling—it is customary for the packing-house attendant who has this matter in charge to warm the thermometer by holding the pointed or bulb end in his hand, so as to force the mercury column up to about 60° F., or well above the temperature of hams. The thermometer is then thrust into the ham and allowed to remain for several minutes, by which time the mercury column will have fallen to the temperature of the ham. The thermometer is then slowly withdrawn so as to expose the top of the mercury column, and an accurate reading is thus obtained of the inside temperature of the ham. The thermometer is warmed by the hand before each ham is tested, and this undoubtedly insures more accurate readings than would result were the thermometer removed from one ham and plunged immediately into another, but the procedure is open to certain objections, for the open space between the metal point of the thermometer and the mercury bulb soon becomes filled with particles of meat and with grease and dirt from the attendant’s hands, and it is at once apparent that a thermometer in this condition would furnish a ready means whereby extraneous matter might be introduced into the bodies of the hams. In other words, a contaminated thermometer would furnish an excellent means whereby hams could be inoculated with putrefactive bacteria.

In order to determine whether hams actually become inoculated in this manner, the following experiment was carried out:

EXPERIMENT TO SHOW WHETHER HAMS BECOME INFECTED FROM HAM THERMOMETERS.

This experiment was designed to show (1) whether the usual packing-house method of taking ham temperatures was apt to induce souring in the hams thus tested, and (2) whether souring would result in hams which were tested with a thermometer purposely contaminated with the bacillus isolated from sour hams.

The experiment was carried out as follows: Thirty hog carcasses were selected as they entered the hanging floor from the killing floor. They had been cleaned, eviscerated, and split, and were of the same average weight and of sufficient size to yield hams weighing from 12 to 14 pounds. They were divided into three lots of 10 each and were allowed to remain on the hanging floor for two hours, after which they were given the usual 48-hour chilling.

The three lots of carcasses were carefully tagged and were chilled in a special cooler to themselves. Upon leaving the cooler the hams were cut from the carcasses and trimmed. The three lots of hams were then cured in separate tierces. All of the hams were subjected to exactly the same cure.

The pickles used were the regular pumping and regular curing pickles of the establishment at which the experiment was carried out.

The hams in lot 3 were pumped first and those in lot 1 were pumped next. The needle was then removed and a fresh, clean needle was used for lot 2. This was done in order to prevent the possibility of carrying over bacteria from one lot of hams to another on the pumping needle. The tierces were thoroughly cleaned with boiling water before being used. The curing was carried out in a pickling cellar which was held at 33° to 36° F., the temperature never rising above the latter figure. The tierces were rolled three times during the curing. The details and results were as follows:

Results of experiment to show whether hams become infected from ham thermometers.

Several hams from each tierce were examined bacteriologically. cultures being taken from the meat near the bone and from the bone marrow of the femur.

In the sour hams from tierce 1 cultures taken from the meat near the bone showed the same anaerobic bacillus noted in other sour hams (i. e., the same bacillus which caused souring in Experiments I and II), but these cultures were contaminated with other bacteria which were probably introduced on the thermometer along with the ham-souring bacillus. None of the contaminating bacteria were capable, however, of producing a sour-meat odor when grown on the egg-pork medium. Pure cultures of the ham-souring bacillus were obtained from the bone marrow of some of these hams, showing that this bacillus had penetrated through to the bone marrow while the other bacteria had not.

From the sour hams in tierce 2 the ham-souring bacillus was recovered readily, and often in pure culture, from the hams which had been pumped in the shank only, whereas it was usually contaminated with pickle bacteria in the hams which had been pumped in both body and shank.

In the case of the sound hams in tierce 3, cultures taken from the meat near the bone and from the bone marrow of the femur were negative in the hams which had been pumped in the shank only, while cultures taken from corresponding points in the hams pumped in both body and shank showed ordinary pickle bacteria, which had evidently been introduced into the bodies of these hams in the pumping pickles. None of these hams exhibited the slightest sour odor.

Summary of experiment.—In this experiment 20 hams (tierce 1) were tested with an ordinary ham thermometer in the usual packing-house manner. Half of these hams were subjected to the mild cure and half were given the regular cure, with the result that 50 per cent of those receiving the mild cure and 20 per cent of those receiving the regular cure became sour.

A second lot of 20 hams (tierce 2) were tested with a thermometer which had been purposely contaminated with a culture suspension of the ham-souring bacillus. These hams were cured in the same manner as the first lot, with the result that all of those receiving the mild cure and 70 per cent of those receiving the regular cure became sour.

A third lot of 20 hams (tierce 3) which had not been tested at all were cured in the same manner as the two preceding lots, as a check on the cure. All of these hams were sweet at the end of the cure.

Inasmuch as the three lots of hams were cured under precisely the same conditions and were handled in the same manner prior to pickling, the only difference being that the hams in tierces 1 and 2 were tested with the ham thermometer while those in tierce 3 were not, we must conclude that the souring of the hams in tierces 1 and 2 resulted from the testing which these hams received. In the case of tierce 1 the hams became infected from a thermometer which, in the ordinary routine use of the packing house, had become accidentally contaminated with the ham-souring bacillus. In the case of tierce 2 the hams became infected from a thermometer which had been artificially contaminated with the bacillus. The high percentage of sours in this last lot is due to the fact that these hams were heavily infected with the ham-souring bacillus, for owing to the construction of the ham thermometer many thousands of the bacilli were unquestionably introduced into each ham on the point of the thermometer. In the ordinary routine of ham testing, where hams become infected from foreign matter introduced on the thermometer, the percentage of souring, as shown in tierce 1, would be less, for it is not to be supposed that ham thermometers are always contaminated with the ham-souring bacillus, but that they only become so at times, and that probably only a few of the bacilli are then introduced.

This experiment, we think, proves conclusively (1) that the ham-souring bacillus may be introduced into the bodies of hams on the thermometers used in testing the hams, and (2) that the packing-house method of taking ham temperatures by means of a thermometer which is thrust deep into the bodies of the hams may cause souring in the hams thus tested.

As a further proof that hams may become contaminated in this manner, a series of cultures were made from scrapings taken from ham thermometers. The scrapings consisted of the accumulations of bits of meat, grease, and dirt that collect on the thermometers, and were taken from the thermometers while the latter were in ordinary routine use in the packing house. In a series of six cultures which were made from such scrapings at different times, the same bacillus which was isolated from sour hams and shown to cause meat souring was found three times. In other words, the ham-souring bacillus was present in 50 per cent of the cultures made from thermometer scrapings, and many hams undoubtedly become infected from the thermometers. Souring would be almost certain to result in mild-cure hams if these hams were tested with a thermometer which had become accidentally contaminated with the ham-souring bacillus, as the bacillus would have time to develop within the bodies of the hams before being inhibited by the curing pickle, which penetrates slowly into the bodies of these hams. In the case of regular cure hams—that is, hams which are pumped in both body and shank—souring would be much less apt to occur after the use of a contaminated thermometer, as these hams are more or less saturated with a strong pumping pickle at the beginning of the cure, which would tend to inhibit the growth of any bacteria that might be introduced on the thermometers.

The fact that souring may result in hams from the use of a contaminated thermometer would explain the occurrence of several sours in one vat, for in testing hams just before they go into cure several hams are usually tested in succession, and these would in all likelihood go into the same vat. Supposing the thermometer to have been contaminated with the ham-souring bacillus at the time these hams were tested, this would explain a fact which has been often noted, namely, the occurrence of several sours in one vat while other vats containing the same run of hams show no sours.

If souring resulted in all of the hams which are subjected to a thermometer test in the daily routine of the packing house, this manipulation alone might account for nearly all of the sours which occur, but the experiment which has been just described shows that all of these hams do not become sour. In tierce 1, where each ham was subjected to three thermometer tests at different times, souring resulted in 35 percent (this includes both mild and regular cure) of the hams thus tested, and in actual practice the percentage of sours in hams which have been subjected to the thermometer test would probably be somewhat less. Quite a large percentage of sour hams are thus left unaccounted for by the thermometer test, and we believe that these are chiefly the result of contamination carried in on the pumping needles or in the pumping pickles.

INFECTION FROM PUMPING NEEDLES.

In view of the results obtained in the last experiment, in which it was shown that hams may become infected from the use of ham thermometers, it seemed not improbable that hams might also become infected from the pumping needles, which, like the thermometers, are thrust deep into the bodies of the hams beside the bone. In order to throw some light upon this point, cultures were taken from the grease and dirt that accumulate on the shields at the bases of the pumping needles, as such material must undoubtedly be carried into the hams at times on the needles. The ham-souring bacillus was found several times in these cultures, and hence it is fair to infer that hams may also become infected at times from the pumping needles, just as they become infected from the thermometers. Bits of contaminated meat and grease and particles of dirt carried in on the pumping needles would be forced out into the hams by the pumping pickle, which passes out through small openings or fenestræ in the needles, and this probably affords one explanation as to why so many more body sours occur in the mild-cure hams. In the mild-cure hams, which are pumped in the shank only, the pumping needle is introduced near the femorotibial articulation, and the shank is saturated at the start with a strong brine solution, while the body of the ham is not. If the ham-souring bacillus were carried into these hams on the pumping needle, the growth of the bacillus in the shank would be inhibited by the strong brine solution with which the shank is saturated, but there would be nothing to prevent the bacillus from growing upward into the body of the ham, which has not been pumped and is free from pickle. This would also explain the fact that the souring often starts at the knee joint and extends upward into the body of the ham. In the case of the regular cure hams, where the ham is pumped in both body and shank, the entire ham is more or less saturated at the start with the strong brine of the pumping pickle, which tends to inhibit the growth of the ham-souring bacillus even if this bacillus should find its way into these hams on the pumping needles. It is in the mild-cure or partly pumped hams, where the body of the ham is left unpumped, that the ham-souring bacillus finds its best opportunity for development, and the greater proportion of the sours that occur in the packing house are found in these hams.

As regards the possibility of infection from the pumping pickle itself, it does not seem probable that this would often occur, for the pumping and curing pickles are always prepared on an upper floor of the pickling houses and are delivered to the pickle cellars in closed pipes, so the chances for the accidental contamination of these solutions from floating dust or dirt would not be great. Furthermore, the strong brine of the pumping pickle would completely inhibit the growth of the ham-souring bacillus, and the bacillus would be incapable of multiplying, even if it found its way into the pickle. On the other hand, laboratory experiments show that the bacillus or its spores may remain alive for a considerable length of time in the pumping pickle, so the possibility of infection from this source can not be overlooked.

INFECTION FROM BILLHOOKS.

After the hams are cut from the carcasses they are handled entirely by means of billhooks. In handling the hams the hooks are inserted beneath the skin of the shank at a point just above the tibio-femoral articulation. The hooks should be inserted in the connective tissue beneath the skin and should not penetrate the muscular tissue to any depth. When the hams lie in the right position, with the butt or large portion away from and the shank toward the operator, it is an easy matter to pick them up in the proper manner; but when they lie at different angles and are being rapidly handled it is almost impossible to prevent the hook from penetrating the muscular tissues, and if the hook should penetrate to the bone it might carry in foreign matter contaminated with the meat-souring bacillus. It is not probable that many hams become contaminated in this way, as the men who handle the hams are very skillful in manipulating their hooks; but the possibility that hams may become contaminated in this manner should not be entirely overlooked.

BIOLOGICAL AND MORPHOLOGICAL CHARACTERISTICS OF THE HAM-SOURING BACILLUS.

CONDITIONS FAVORABLE TO GROWTH.

The most favorable medium for the growth of the organism was found to be the modified egg-meat mixture of Rettger, which has been previously described. In this medium the organism develops rapidly at a temperature of 20° to 25° C., giving rise to the characteristic sour-meat odor. Like the bacillus described by Klein, it also grows readily on pork-agar and pork-bouillon containing glucose, but differs from Klein’s bacillus in that it will grow, though less luxuriantly, on ordinary nutrient media—agar, gelatin, and bouillon—without the addition of glucose.

The optimum temperature for growth is 20° to 25° C. The organism does not grow at incubator temperature (37.5° C.). At ice-box temperature (8° to 10° C.) it develops readily, although the growth is less rapid than at 20° to 25° C. That the organism will develop at even lower temperatures was shown in the inoculation experiments with hams, where it developed and multiplied extensively in the bodies of the hams at the temperature of the pickling cellars, which are held usually at 34° to 36° F.(1° to 2° C.).

The organism develops best in a neutral or slightly alkaline medium.

GROWTH ON DIFFERENT CULTURE MEDIA.

Growth on egg-pork medium.—At a temperature of 20° to 25° C. the cultures show a slight but distinct sour odor in from two to three days. This odor, as before stated, closely resembles the odor of a sour ham. Egg-pork cultures from three to five days old were given to a trained meat inspector, who knew nothing whatever as to the contents of the tubes, and he was asked to describe the odor; he described it as that of a sour ham.

At one week the albumins of the medium are gelatinized or partly coagulated and the odor is more pronounced. At ten days the albumins are completely coagulated except at the surface, where there is no apparent growth; the odor is more putrefactive in nature, and the reaction of the medium is slightly acid. At three weeks the coagulated albumin splits up into fragments and appears to undergo a slow digestion, gas bubbles form in the lower portion of the culture, and the odor becomes distinctly putrefactive in character. The slow digestion of the albumin is probably due to a proteolytic enzyme elaborated by the bacillus.

At the end of a week a dark zone usually appears at the surface of the coagulated albumin and gradually darkens until it becomes almost black. This zone is probably due to a pigment elaborated by the bacillus.

At ice-box temperature (8° to 10° C.) the same changes and the same odor were noted, but were somewhat slower in developing.

Glucose-pork-agar.—This medium was prepared from pork in the same manner as beef-agar, and contained 1 per cent of glucose. The organism grows readily on this medium and may be conveniently cultivated in deep stab cultures. The medium was always thoroughly boiled and then rapidly cooled in order to expel the inclosed air. The growth of the organism was found to vary considerably with the reaction.

When the reaction was +1.5, deep stab cultures at three days (20° to 25° C.) showed a well-marked arborescent growth, appearing as delicate filaments extending outward from the line of stab. The growth stopped within one-fourth or one-half inch of the surface of the agar on account of the presence of oxygen in the upper part of the culture medium. As the growth extended toward the walls of the test tube the agar became clouded, and there were sometimes gas bubbles in the depth of the agar, but the gas formation was not extensive.

When the reaction of the agar is neutral or slightly alkaline, extensive gas formation occurs and the agar is often much broken up.

The cultures developed a disagreeable, somewhat putrefactive odor, but did not give the characteristic sour-ham odor obtained from the egg-pork cultures.

The organism was also grown on anaerobic agar plates by Zinsser’s method, which is said to give absolutely anaerobic conditions. The colonies on agar have a cottony or woolly appearance at first, and spread slowly, with slightly irregular margins.

In glucose-pork-agar to which azolitmin was added the azolitmin in the lower portion of deep stab cultures was completely decolorized in five days at room temperature (20° to 25° C).

In glucose-pork-agar containing neutral red the red color in the lower portion of the tube was changed to yellow with the development of fluorescence.

Neutral gelatin.—Tubes of ordinary neutral gelatin without the addition of glucose were inoculated and held at ice-box temperature (8° to 10° C). At five days a delicate white growth appeared along the line of stab in the lower portion of the tube. At seven days the growth showed fine radial striæ, presenting an arborescent or tree-like appearance, and extended halfway from the line of stab to the walls of the test tube. At two weeks the growth had caused a delicate clouding of the medium in the lower portion of the tube. At three weeks the gelatin in the lower portion of the tube had become liquefied and the growth had settled to the bottom as a white precipitate.

In gelatin containing glucose, gas bubbles are formed in the depth of the medium through the splitting up of the glucose, and the characteristic arborescent growth is obscured.

Glucose-pork-bouillon.—This medium was prepared from pork instead of beef and contained 1 per cent of glucose. The best results were obtained when the reaction of the medium was neutral or slightly alkaline.

Culture tubes, which had been previously boiled to expel the contained air and then inoculated, were held in a Novy jar, in an atmosphere of hydrogen at a temperature of 20° to 25° C. At three days the tubes showed well-marked clouding. At one week the growth appeared as a heavy, white, flocculent, cottony precipitate in the bottom of the tubes with a slight flocculent precipitate above. When the culture was removed from the jar and shaken, the heavy, flocculent precipitate at the bottom of the tube broke up without much difficulty, giving rise to a heavy uniform clouding with some small floating masses, which soon settled to the bottom. On shaking the tube some evolution of gas in the form of very fine bubbles was noticed.

In Smith fermentation tubes containing neutral glucose-pork-bouillon the closed arm of the tube shows well-marked clouding with gas formation at three days at room temperature (20° to 25° C). The growth has a tufted, cottony appearance, and there are many filaments and threads. The growth settles to the bottom of the closed arm as a cottony, white precipitate (see Pl. IV). The organism splits the glucose vigorously, and at 10 days the tubes show from 40 to 50 per cent of gas. The bouillon in the open arm of the tube remains unclouded. The maximum gas production at room temperature is reached in from 10 to 14 days, by which time the growth in the closed arm has completely settled into the bend of the tube, leaving the bouillon in the closed arm clear. The gas formula, as determined by Smith’s method, was H/CO₂= 5/1. The reaction of the bouillon becomes acid to phenolphthalein.

The organism will grow on ordinary neutral bouillon without the addition of glucose, and in Smith tubes containing this medium a small amount of gas was formed, due to the splitting of the muscle sugar.

The bacillus also grows in a sugar-free broth—that is, a broth free from muscle sugar—and from cultures grown in this medium a well-marked indol test was obtained.

Litmus-milk.—The organism was grown in litmus-milk in Smith fermentation tubes at 20° to 25° C. At seven days the litmus in the lower portion of the closed arm had assumed a brownish-buff color. At two weeks the litmus in the closed arm had been reduced to a brownish-buff color except at the top of the tube, where a pale, bluish tinge remained, and the litmus in the open arm showed very slight reddening as compared with a check tube. At three weeks the litmus in the closed arm was entirely reduced to a light, brownish-buff color, and the litmus in the open arm showed a slight but distinct reddening as compared with the check. The reddening of the litmus in the open arm was evidently due to the transfusion of acids formed by the growth of the bacillus in the closed arm. After several weeks the milk is slowly peptonized, probably as a result of enzyme action.

MORPHOLOGY.

The organism is a large bacillus having an average size of 4 to 8 μ in length by 0.5 to 0.7 μ in thickness, but there are many longer forms measuring from 10 to 20 μ in length. It develops in long, irregular chains or filaments, which at times show a slightly spiral form.