Fig. 5.—Ham-souring bacillus (Bacillus putrefaciens) grown on egg-pork medium, showing tendency to form chains. Partly developed and fully developed spores are shown at ends of rods; also free spores. (Pen-and-ink drawing made with camera lucida from preparation stained by Gram’s method.× 640.)
Fig. 5.—Ham-souring bacillus (Bacillus putrefaciens) grown on egg-pork medium, showing tendency to form chains. Partly developed and fully developed spores are shown at ends of rods; also free spores. (Pen-and-ink drawing made with camera lucida from preparation stained by Gram’s method.× 640.)
Fig. 5.—Ham-souring bacillus (Bacillus putrefaciens) grown on egg-pork medium, showing tendency to form chains. Partly developed and fully developed spores are shown at ends of rods; also free spores. (Pen-and-ink drawing made with camera lucida from preparation stained by Gram’s method.× 640.)
The individual organisms show at times a widely open, slightly spiral form, which was more apparent in hanging-drop preparations made from bouillon cultures, where the organisms had been comparatively undisturbed. This appearance was also noted at times in the stained sections of soured muscular tissue, where the organisms were stained in place. The organism possesses no motility. It stains with the ordinary aniline dyes and by Gram’s method.
The organism develops large, terminal spores, which are at first oval, but when fully developed are perfectly round and measure from 1.5 to 2 μ in diameter.
Spores develop rapidly in the egg-pork medium at 20° to 25° C., fully developed spores being noted in from five to seven days. Atice-box temperature (8° to 10° C.) partly developed spores were noted in the egg-pork medium at 10 days and fully developed spores at 2 weeks.
Occasional spores were noted in old agar and gelatin cultures, but abundant spore formation was seen only in the egg-pork medium. No spores were noted in bouillon cultures, even at 10 weeks.
In its vegetative form the bacillus is killed at 55° C. in 10 minutes. The spores survive a temperature of 80° C. for 20 minutes, but are killed at 100° C. in 10 minutes.
When sodium chlorid and potassium nitrate were added to glucose-pork broth in varying amounts, it was found that 3 per cent of sodium chlorid or 3 per cent of potassium nitrate was sufficient to inhibit completely the growth of the bacillus at room temperature (20° to 25° C.).
While the growth of the bacillus was inhibited by sodium chlorid and potassium nitrate as just stated, it was found that very much stronger solutions of the two salts failed to destroy the bacillus. Thus it was found that the bacillus or its spores retained their vitality after an exposure of 30 days in a solution containing 23 per cent of sodium chlorid and 6 per cent of potassium nitrate.
The organism splits glucose, but not lactose or saccharose. That it possesses the power of splitting muscle sugar was shown by the formation of gas in Smith fermentation tubes containing ordinary neutral bouillon without the addition of any sugar.
The formation of gas in glucose bouillon varies considerably with the reaction of the medium. The largest amount of gas was formed when the broth was neutral or slightly alkaline. When the reaction of the broth was distinctly acid or distinctly alkaline the amount of gas was diminished. The gas which is formed in bouillon cultures consists chiefly of hydrogen and carbon dioxide. In order to collect a sufficient amount of the gas for analysis, two large fermentation tubes capable of holding 150 cubic centimeters each were constructed. These tubes were filled with pork-bouillon and inoculated with the bacillus. After 20 days at room temperature (20° to 25° C.) the gas was collected and the carbon dioxide and hydrogen determined, with the following result:
This analysis gives an approximate gas formula of H/CO2= 5/1, which agrees with the gas formula as determined in the small fermentation tubes by Smith’s method.
In hams which had undergone spontaneous souring and in hams which had been artificially soured by inoculation, hydrogen-sulphid was often noted when the sour portions of the meat were tested with lead-acetate paper, but no distinct odor of the gas could be obtained. Hydrogen sulphid was also noted in egg-pork cultures of the bacillus.
In glucose-bouillon, butyric and lactic acids are formed and the reaction of the medium becomes distinctly acid. Butyric and lactic acids were also noted in the egg-pork cultures.
A series of Smith fermentation tubes containing 10 c. c. each of glucose-pork broth medium was inoculated with the bacillus and held at room temperature (20° to 25° C.). These cultures were titrated against [N/40]NaOH, with phenolphthalein as an indicator at intervals of two days up to nineteen days, and then at two-week intervals up to sixty-one days. Three of the cultures were titrated each time so as to give a fair average of the acidity of the cultures, and an uninoculated check tube was also titrated each time to see if there was any change in the reaction of the medium. The results of the titrations are shown in the following table:
Acidity determinations in glucose-pork broth cultures.
From the above table it will be seen that the maximum acidity was reached at ten days, after which there was a gradual reduction in the acidity, due probably to the formation of ammonia compounds.
Rabbits, guinea pigs, and white mice were inoculated and fed with cultures of the bacillus without effect, from which it would appear that the bacillus possesses no pathogenic or disease-producing properties.
The bacillus is essentially a saprogenic bacterium with zymogenic properties. A preliminary study of the chemical changes which take place in sour hams shows that these changes are of a putrefactive nature. Hams which had undergone spontaneous souring were compared with hams which had been artificially soured by inoculation, and the chemical changes were found to be identical. A chemical study was also made of the changes taking place in egg-pork cultures of the bacillus at different stages of growth, and these changes were found to be of a putrefactive nature and similar in character to the changes which occur in sour hams. Among the putrefactive products formed by the growth of the bacillus in the egg-pork medium were indol, skatol, volatile fatty acids, skatol-carbonic acid, and hydrogen sulphid.[4]
[4]The tests for the putrefactive products formed by the growth of the bacillus in the egg-pork medium were made by P. Castleman, of the Biochemic Division, who also determined the percentage composition of the gas formed by the growth of the bacillus in the glucose-pork-bouillon medium.
[4]The tests for the putrefactive products formed by the growth of the bacillus in the egg-pork medium were made by P. Castleman, of the Biochemic Division, who also determined the percentage composition of the gas formed by the growth of the bacillus in the glucose-pork-bouillon medium.
Bul. 132, Bureau of Animal Industry, U. S. Dept. of Agriculture.Plate IV.
Glucose Bouillon Culture in Smith Fermentation Tube at Four Days. Culture Grown at Room Temperature (20° to 25° C.). Growth Confined Entirely to Closed Arm, with Gas Collecting at Top.
Glucose Bouillon Culture in Smith Fermentation Tube at Four Days. Culture Grown at Room Temperature (20° to 25° C.). Growth Confined Entirely to Closed Arm, with Gas Collecting at Top.
Glucose Bouillon Culture in Smith Fermentation Tube at Four Days. Culture Grown at Room Temperature (20° to 25° C.). Growth Confined Entirely to Closed Arm, with Gas Collecting at Top.
A more extended study is now being carried on in the Biochemic Division of the chemical changes which take place in hams during the process of souring, together with a further study of the chemical changes which result from the growth of the bacillus in the egg-pork medium. The results of this investigation will be given in a later paper.
The bacillus described in this paper belongs to the class of putrefactive anaerobes, which are widely distributed in nature in dust, soil, and excrementitious matters. This group of bacteria contains both pathogenic and nonpathogenic forms. The former have received considerable attention, but the latter have never been thoroughly cleared up. The bacillus isolated from sour hams belongs in the latter category, being possessed of no pathogenic or disease-producing properties. It occurs in the dust and dirt of the packing house and finds its way into the hams in the various manipulations to which the hams are subjected.
The bacillus described in this paper does not seem to correspond with any forms heretofore described. It differs from Klei bacillus (Bacillus fœdans) in the following important particulars:(1) It forms large terminal spores, whereas Klein’s bacillus formed no spores;(2) it will grow at a temperature of 34° F., while Klei bacillus did not grow below 50° F.;(3) it produces an acid reaction in culture media, while Klei bacillus gave a distinctly alkaline reaction;(4) it will grow on the ordinary nutrient media—gelatin, agar, and broth—without the addition of glucose, while Klein’s bacillus did not;(5) it peptonizes the casein in milk, whereas Klein’s bacillus had no action on milk; (6) it liquefies gelatin more rapidly, causing complete liquefaction after three weeks at 8° to 10° C., whereas Klein’s bacillus caused only partial liquefaction after eight weeks at 20° C.;(7) it can be conveyed
from turbid broth cultures to new culture material by means of the platinum loop, whereas Klein’s bacillus could not be thus conveyed.
For the bacillus described in the present paper the following name is proposed:Bacillus putrefaciens.
As it has been shown that souring in hams results from the growth of a bacterium which is introduced into the bodies of the hams in the various manipulations which the hams undergo, the only way to eliminate souring in hams, as they are cured in the larger packing establishments, would be to cure the hams under aseptic or sterile conditions, which would, of course, be a physical impossibility.
While it will probably be impossible, therefore, to eliminate souring entirely under the methods of ham curing which are at present employed in the larger packing establishments, much can undoubtedly be done toward reducing the percentage of sours. In the matter of taking ham temperatures, for instance, if the thermometers used were thoroughly cleaned and disinfected and the surfaces of the hams seared at the point where the thermometer is introduced, infection from this source could be entirely prevented; or it might be possible so to regulate the temperature of the chill rooms that the taking of ham temperatures could be discontinued.
The elimination of the souring that results from the introduction of foreign matter on the pumping needles could be effected in two ways only,(1) by not pumping the hams at all, or (2) by pumping them under sterile or aseptic conditions. As has been stated before, some of the smaller packing establishments cure their hams without pumping, and in these establishments the percentage of sours runs very low. When hams are cured without pumping, however, the period of curing has to be materially lengthened in order to give the curing pickles sufficient time to penetrate thoroughly, and this is what the larger plants wish to avoid because of the greater space and greater number of vats which would be necessitated. The object of pumping in the larger plants, where the number of hams handled daily runs into the thousands, is to hasten the cure and thus prevent the accumulation of a great number of hams at one time. It is doubtful, therefore, whether the larger packing houses could conveniently discontinue pumping.
To pump the hams under aseptic conditions would necessitate a technique far too elaborate for routine use in the packing house; in fact, anything like complete asepsis would be out of the question. Certain measures might be adopted, however, that would tend to prevent the possible introduction of ham-souring bacilli in the process of pumping. It would undoubtedly be safer, for instance, to boil the pumping pickle before use, and the chances of carrying incontaminated foreign matter on the pumping needles could be lessened by sterilizing the pumps and needles with boiling water and by frequently dipping the needles, while in use, in boiling water. If the hams were sprayed with clean water just prior to pumping, there would be less likelihood of carrying in foreign matter on the needles. The danger of introducing contaminated foreign matter on the needles might be further obviated by searing the surfaces of the hams at the points where the needles are introduced; but such a procedure would be hardly practicable in the larger packing houses, where the great number of hams cured necessitates rapid handling.
While the danger of possible contamination in pumping, through the introduction of contaminated foreign matter on the pumping needles, can not well be avoided, this danger is partly counterbalanced by the inhibitory action of the pumping pickle, which is strikingly shown in the experiments which have been described. In these experiments, 100 hams received large doses of the ham-souring bacillus, half of these hams being subjected to the mild cure and half to the regular cure, with the following result: In the case of the mild-cure hams, which were pumped in the shank only, the percentage of sours was practically 100 per cent, every ham with possibly one exception becoming sour; whereas in the regular-cure hams, which were pumped in both body and shank, only 58 per cent of the hams became sour. In other words, the additional pumping which the regular-cure hams received served to prevent souring in 42 per cent of these hams. In these experiments the number of bacteria introduced into the hams was very great, thousands and even millions of the bacilli being introduced into each ham, whereas in the routine of the packing house it is not likely that more than a few of the bacilli are ever introduced at one time on the thermometers and pumping needles. In view of these results it is safe to say that in the larger packing houses, where pumping seems to be necessary, the number of sours could be reduced fully 50 per cent if all hams were pumped in the body as well as in the shank.
At present the usual procedure is to pump all hams, both mild and regular cure, with the same pumping pickle, the mild-cure hams being pumped in the shank only and the regular-cure hams at two additional points in the body. The experiments quoted above show that the additional pumping which the regular-cure hams receive undoubtedly tends to prevent the development of souring in these hams, and this result is unquestionably due to the inhibitory action of the salts contained in the pumping pickle, as it was found by laboratory experiment that the addition of 3 per cent of sodium chlorid to culture media is sufficient to inhibit the growth of the ham-souring bacillus. The pumping pickles consist of strong brine solutions and always contain considerably more than 3 per cent ofsodium chlorid. If, therefore, the pumping of regular-cure hams were made more thorough than at present, and all of the deeper portions of the ham were thoroughly saturated with the strong brine solution, souring could be largely eliminated, if not entirely prevented, in these hams, as an unfavorable medium or soil would thus be created in which the ham-souring bacillus could not develop. The ham-souring bacillus is able to develop within the bodies of the regular-cure hams because the pumping of these hams is not always thorough and there are certain areas in the inner or deeper portions of the hams in which the tissues are not thoroughly saturated with the pumping pickle.
Under the present methods of curing, the greater proportion of the sours occur among the partly pumped or mild-cure hams. These hams are pumped in the shank only, and the growth of the ham-souring bacillus within the bodies of these hams is not interfered with until the curing pickle has penetrated from the outside. As it requires several weeks for the curing pickle to penetrate thoroughly into the deeper portions of these hams, the bacillus is thus afforded a considerable interval in which to develop before it is exposed to the inhibitory action of the pickle. If these hams could be thoroughly pumped in the body at the beginning of the cure in the same manner as the regular-cure hams, the chief loss from ham souring would be eliminated. It would not do, however, to pump these hams in the body with the same pumping pickle used in the regular cure, as the meat would be rendered too salty and the mild flavor of the ham would be lost. There is undoubtedly a demand for mild-cure hams, otherwise they would not be on the market; and the question then arises how to pump these hams and still retain a mild cure. This might be accomplished by pumping these hams with their own curing pickle, which is usually a milder pickle than that employed in the regular cure, or an even milder pumping pickle might be used. If mild-cure hams were pumped in this way, the percentage of souring in these hams could undoubtedly be greatly diminished without materially affecting the flavor of the ham.
To recapitulate briefly, the prevention of ham souring is to be sought in two ways:(1) Through greater care in handling the hams and the adoption of precautionary measures to prevent the introduction of the ham-souring bacillus into the bodies of the hams, and (2) through more thorough pumping of the deeper or inner portions of the hams, so as to create an unfavorable soil or medium in which the ham-souring bacillus can not develop even if it should gain entrance into the bodies of the hams.
From what has been said it will be apparent that ham souring can probably never be entirely eliminated from the packing house under the present methods of curing, but the adoption of precautionary measures in testing and pumping hams, together with a more thorough pumping of all hams in ways similar to those suggested, would unquestionably reduce very materially the losses from this source.
1. In this paper it has been shown that ham souring, as encountered in the wet cure where the hams are entirely submerged in pickling fluids, is due to the growth of an anaerobic bacillus within the bodies of the hams. This bacillus (B. putrefaciens) was found in sour hams obtained from four different packing establishments. It was isolated and grown in various laboratory media, in one of which, the egg-pork medium, it gave rise to the characteristic sour-ham odor. This bacillus was the only organism that could be isolated from sour hams that was capable of producing the characteristic sour-ham odor in the egg-pork medium.
2. When injected into the bodies of sound hams, the bacillus caused these hams to sour in the process of curing. In hams which had been inoculated with the bacillus and thus artificially soured, the bacillus was recovered in cultures taken at points far removed, relatively speaking, from the point of inoculation, indicating that the bacillus had multiplied and progressed by extension throughout the bodies of the hams.
3. The bacillus possesses no motility, and its extension throughout the bodies of the hams is a result of multiplication. In its growth it follows along the connective-tissue bands between the muscle bundles, which are composed of comparatively loose tissue and afford paths of least resistance. When it invades the muscle tissue proper, it follows along the sarcolemma sheaths between the muscle fibers. As a result of this growth the muscular tissue becomes softer and tends to break more easily.
4. The bacillus belongs to the class of putrefactive anaerobes which are widely distributed in nature in dust, soil, and excrementitious matters. The bacillus or its spores is present in the dust and dirt of packing houses and finds its way into the hams in the various manipulations to which they are subjected.
5. The bacillus or its spores may be introduced into hams on the thermometers used in testing the hams, on the pumping needles, and possibly on the billhooks used in handling the hams. It may also be carried into the hams in the pumping pickle, and may even find its way into the hams from the curing pickle, although infection through the latter channel probably does not often occur.
6. The bacillus develops in the deeper portions of the ham because of the anaerobic conditions there prevailing, and souring is most often encountered, therefore, in the deeper portions of the ham near the bone.
7. A preliminary study of the chemical changes which take place in the process of souring shows that these changes are of a putrefactive nature, and ham souring, as ordinarily encountered, is to be regarded as an incipient putrefaction. Hams which had been artificially soured by injections of culture were compared with sour hams obtained from the packing house, and the putrefactive changes were found to be identical.
8. Hams which have once become sour can never be restored to a sound condition, because of the chemical changes which result from the growth of the bacillus. In other words, the tissues of the ham undergo certain chemical changes in the process of souring, and when these changes have once taken place the tissues can never be restored to a sound condition. The repumping of slightly soured hams with a strong pumping pickle will check further souring, by inhibiting the growth of the bacillus, but will not restore to a sound condition those portions of the ham which have become sour.
9. The salts of the pickling fluids have a marked inhibitory action on the ham-souring bacillus, and sours occur less frequently in regular-cure hams.
10. In regular-cure hams the growth of the ham-souring bacillus is restricted and often completely inhibited as a result of the additional pumping which these hams receive, whereby they are more or less saturated with pickle at the beginning of the cure.
11. If the pumping of regular-cure hams were more thorough and all of the deeper portions of the ham were thoroughly saturated with the pumping pickle, souring could be largely eliminated if not entirely prevented in the hams, as an unfavorable medium or soil would thus be created, in which the ham-souring bacillus could not develop. The reason that souring does develop in regular-cure hams is because the pumping is not always thorough and there are certain areas in the deeper portions of these hams which are not saturated with the pumping pickle.
12. Under the present methods of curing, the partly pumped or mild-cure hams furnish the greater proportion of the sours, as these hams are not pumped in the body and the growth of the ham-souring bacillus within the bodies of these hams is not interfered with until the curing pickle has penetrated from the outside. As it requires several weeks for the curing pickle to penetrate thoroughly into the deeper portions of these hams, the bacillus is thus afforded a considerable interval in which to develop.
13. The percentage of souring in the mild-cure hams could be greatly reduced without materially affecting the cure by pumping these hams with their own curing pickle, which is usually a milder pickle than that employed in the regular cure; and if the pumpingwere thorough the number of sours in these hams could be reduced to a small figure.
14. The only way by which ham souring could be entirely eliminated from the larger packing establishments under the present methods of curing would be to handle the hams throughout under aseptic conditions, and this, for obvious reasons, would be an impossibility. The losses from ham souring may be materially reduced, however, by greater care in handling the hams and the adoption of precautionary measures designed to prevent the introduction of contaminated foreign matter into the bodies of the hams, together with more thorough methods of pumping.
In conclusion, the writer desires to express his obligations to Dr. S. E. Bennett, of the Inspection Division, inspector in charge at Chicago, for the assignment of trained meat inspectors to assist in the work, as well as for kind assistance in obtaining data and material for laboratory study, and to Dr. L. E. Day, of the Pathological Division, who kindly prepared the sections which are figured and described in the present article.