Part 2

In order to test this last theory, a number of hog carcasses were run direct from the killing floor to a cooler at 28° F. and a like number of carcasses of the same average weight which had been allowed to stand for two hours at the outside temperature of the air (53° F.) were placed in the same cooler. The carcasses which had hung for two hours in the air had lost an average of 14 degrees in temperature before going to the cooler. The temperature of the cooler rose to 29° F. after the carcasses were put in, but was soon reduced to 28° F. and held at this temperature. The temperatures of the hams were taken at the end of 24 hours, and practically no difference was found in the inside temperatures of the two lots; that is, the hams on the hot carcasses which were subjected to a sudden chilling exhibited practically the same inside temperature (i. e., next to the bone) as those which had cooled for two hours at the temperature of the air before being placed in the cooler.

Still another theory attributes souring to lack of penetration of the pickling fluids, but analyses of sour and sound hams do not seem to bear out this theory. The rate of penetration of the pickling fluids, however, would seem to have some bearing on the subject, and this point will be discussed later in connection with some laboratory experiments on the inhibitory effects of sodium chlorid and potassium nitrate.

So much for the more commonly accepted theories which have been advanced to explain ham souring.

PREVIOUS EXPERIMENTAL WORK TO DETERMINE CAUSE OF HAM SOURING.

A review of the literature reveals but one article bearing directly on the subject of the cause of ham souring.

In June, 1908, Klein[1] published in the London Lancet an article on “miscured” hams. He describes a miscured ham as one which has a distinctly putrid smell, and the tainted areas he describes as varying in color from a dirty gray to a dirty green, the muscular tissues in the strongly tainted areas being swollen and soft, or jelly-like. From such hams he isolated a large nonmotile, nonspore-bearing, anaerobic bacillus which he calls _Bacillus fœdans_. He cultivated the organism on different media and obtained from the cultures a putrid odor resembling that of the ham from which the culture was obtained, but did not attempt to produce tainting by injecting sound hams with the bacillus.

[1] Klein, E. On the nature and causes of taint in miscured hams. The Lancet, vol. 174, London, June 27, 1908.

While there can be little doubt that Klein’s bacillus was the cause of the tainting in those hams which he examined, the proof would certainly have been stronger had he injected sound hams with cultures and thus proven that he could reproduce tainting experimentally by means of his bacillus. Klein examined only dry-cured hams and does not state the temperature at which they were cured. He fails to offer any explanation as to how the bacillus gained entrance into the hams.

THE PRESENT EXPERIMENTS.

MEDIA EMPLOYED.

After considerable experimentation as to a suitable culture medium for the bacteriological study of sour hams, a modification of the “egg-meat mixture” used by Rettger[2] in his studies on putrefaction was found to be the most satisfactory. This medium, which consists of chopped meat and egg albumen, furnishes an excellent medium for the growth of putrefactive organisms which rapidly break down the proteids of the meat, giving rise to the characteristic odors of putrid decomposition. Rettger used chopped beef and egg albumen, but for the present work chopped pork was substituted for the beef, as affording a more suitable medium for the growth of organisms accustomed to growth in pork hams. The modified medium is prepared as follows:

A. One-half pound of lean pork, freed from excess of fat and sinew, is finely chopped in a meat chopper, 250 cubic centimeters of water is then added, the meat acids are neutralized with sodium carbonate, and the mixture is heated in an Arnold sterilizer for 30 minutes, with occasional stirring. It is then set away in a cold place for several hours. A small amount of fat collects at the top in the form of a fatty scum, as it is impossible to remove all of the fat from the meat before it is chopped. The fatty scum, which hardens upon standing in the cold, is now removed.

B. The whites of three eggs are mixed with 250 cubic centimeters of water. The mixture is rendered neutral to phenolphthalein by means of dilute hydrochloric acid and heated for 30 minutes in the Arnold sterilizer, with occasional stirring.

A and B are now mixed and 2.5 grams (0.5 per cent) of powdered calcium carbonate added. The mixture is next run into large sterile test tubes, or sterile flasks, and sterilized in an Arnold sterilizer on three successive days.

[2] Rettger, L. F. Studies on putrefaction. Journal of Biological Chemistry, vol. 2, 1906.

In addition to the egg-pork mixture described above, culture tubes of agar and bouillon prepared from pork instead of beef, with the addition of 1 per cent of glucose, were also used; but the best results were obtained with the egg-pork medium, as with this medium, the early development of sour or putrefactive odors furnished a valuable indication as to the presence of organisms capable of producing sour or putrefactive changes in meat.

METHOD OF PROCEDURE IN EXAMINING HAMS.

The hams were sectioned through the body, the femur, or “middle bone,” as it is known in packing-house parlance, being cut at a point about 1½ or 2 inches below its head. A cross section of a ham thus cut is shown in figure 1. After sectioning, the hams were subjected to a microscopical, bacteriological, and chemical examination as follows:

_Microscopical examination._—Bits of muscular tissue, taken from various points, were teased out in salt solution and the condition of the muscle fibers noted. Smear preparations were also made from bits of muscular tissue and from the bone marrow, and these were stained and subjected to microscopical examination. Portions of the meat were also hardened and cut into microscopic sections, which were stained and mounted for histological and bacteriological study.

_Bacteriological examination._—In the bacteriological examination of sour hams, especial attention was directed to the detection of anaerobic species, as it seemed reasonable to suppose that if the changes taking place in sour hams were due to bacteria these bacteria would in all likelihood be anaerobes (i. e., organisms which develop in the absence of oxygen). This assumption was based upon the fact that, as a rule, souring begins in the interior of the ham next to the bone, and, furthermore, the hams are cured in large vats where they are completely submerged in the pickling fluids, so that any bacteria which develop within the bodies of the hams while they are in cure are probably restricted to practically anaerobic conditions.

Cultures were made from the interiors of the hams at various points by first searing the cut surface thoroughly with a heavy metal spatula and then cutting out, by means of sterile scissors and forceps, plugs of meat about 1 cm. square. The plugs of meat were then dropped into tubes containing the egg-pork medium and pushed down to the bottom of the tubes, where they were held in place by the chopped meat above; in this way conditions favorable for the development of anaerobic organisms were obtained. In inoculating the pork-agar tubes, the medium was first boiled to expel any inclosed air and cooled to 43° to 45° C; the plugs of meat were then dropped into the tubes and the agar rapidly solidified by plunging the tubes in cold water; in this way the bits of meat were inclosed in the agar at the bottom of the tubes, affording suitable conditions for anaerobic growth. Aerobic and anaerobic plates were also made from the meat, and in most cases bouillon tubes were also inoculated. Cultures were always taken from the bone marrow as well as from the meat. Novy jars were also used for obtaining anaerobic conditions in growing the cultures.

_Chemical examination._—In order to determine whether the souring was connected with or dependent upon a lack of penetration of the pickling fluids to the interior of the meat, the hams were further subjected to a chemical examination and the content of the meat in sodium chlorid and potassium nitrate determined at varying depths.

RESULTS OF EXAMINATION OF SOUR AND SOUND HAMS.

The sour hams examined were obtained from four different packing establishments. All of the hams studied were “sweet-pickle hams” which had not been smoked. The sour hams selected for examination were good typical body sours, in which the sour odor was well developed, but not of the very pronounced or putrefactive type.

The sour odor in every case was found to be more pronounced next to the bone, being usually rather more pronounced just behind the bone, that is, on the fat side of the bone. The sour odor in each instance was confined to an area of meat immediately surrounding the femur and extending out through the body of the ham for a variable distance, as shown by the dotted lines in figure 1, but in no case did the sour odor extend all the way to the margin of the meat, nor did it as a rule extend below the tibio-femoral articulation, the shank proper and the bone marrow of the shank (i. e., of the tibia) being usually sweet. The butt portion of the hams—that portion above and behind the hitch bone (symphasis pubis)—was also sweet.

Immediately after sectioning, the sour areas, as a rule, could be readily distinguished by a difference in color. In the freshly cut hams the muscular tissue near the bone, where the sour odor was more pronounced, exhibited a slight but distinct grayish hue, at times having a slight greenish tinge; in other words, the muscular tissue in the sour areas lacked the normal bright red color of the sound meat and was distinctly lighter in color than the surrounding tissues. Upon exposure to air, however, the lighter, grayish, sour areas tend to assume a reddish hue and become much less pronounced than in the freshly cut ham. After the cut surface of the ham has been exposed to the air for some time it may be difficult to distinguish the sour areas by any difference in color.

FIG. 2.—SECTION OF MUSCULAR TISSUE FROM SOUR HAM, SHOWING MUSCLE FIBERS CUT LONGITUDINALLY; NUCLEI UNDERGOING DISINTEGRATION AND CROSS STRIATION INDISTINCT.

(Pen-and-ink drawing made with camera lucida from section stained with hematoxylin and eosin to show histological structure. × 320.)]

In the sour areas near the bone the muscular tissue was distinctly softer; that is, it broke and cut more readily than the surrounding tissues. This was usually quite noticeable in cutting out plugs of the meat for making cultures. In a ham which shows pronounced souring the muscular tissues in the worst affected areas may become quite soft and even slightly gelatinous.

The sour areas, when tested with litmus paper, frequently showed a slight but distinct alkaline reaction. When aqueous extracts of the sour meat, however, were titrated with phenolphthalein they were found to be acid.

HISTOLOGICAL CHANGES IN SOUR HAMS.

In preparations made by teasing out bits of the meat in physiological salt solution, the cross striation of the muscle fibers from the sour areas was found to be much less distinct than in similar preparations taken from sound portions of the meat or from sound hams. At times it was found that the muscle fibers in the sour areas had completely lost their cross striæ, but the longitudinal striation could still be made out. In cases where the souring was pronounced there was sometimes complete loss of both longitudinal and cross striation; in these cases the muscle fibers appeared to have undergone slight swelling and the protoplasm exhibited a finely granular appearance.

In stained sections of the sour meat another striking change was noticed in the disintegration of the nuclei of the muscle fibers, which are at times completely broken up, appearing as bluish granular masses in sections stained with hematoxylin and eosin. (Compare figs. 1 and 2 of Pl. I.)

In sections stained by the Gram-Weigert method to show the presence of bacteria, a large Gram-staining bacillus was noted between the muscle fibers in the connective-tissue elements of the muscle. In some of the sections these bacilli were present in great numbers, sometimes in densely packed clumps or masses, while in other sections, or in other portions of the same section, they were only sparsely distributed between the muscle fibers. Where the bacteria were more numerous the histological changes in the muscle fibers, especially the breaking down of the nuclei, were more noticeable. The intermuscular connective tissue had apparently furnished paths of least resistance along which the organism followed. In Plate II, figures 1 and 2, the bacteria are shown between the muscle fibers under low and high power magnifications.

In Plate II, figure 1, under the low-power magnification, the bacteria appear as dark clumps or bands between the muscle bundles. Under the high power they are shown following along the sarcolemma sheaths between the muscle fibers.

CHEMICAL ANALYSES OF SOUR AND SOUND HAMS.

In order to determine whether there was any difference in regard to the penetration of the pickling fluids in the sour hams as compared with sound hams, a series of four sour hams were subjected to a chemical examination in comparison with four sound hams. All were sweet-pickle hams and were obtained from the same packing establishment. They were all of the same cure and the same approximate age (i. e., length of cure) and the same approximate weight.

In taking samples for chemical analysis, the following procedure was adopted: A section about 2½ inches wide was cut from the center of the body. The two ends of this section were then trimmed off along the lines L-M and N-O, as shown in figure 2. Beginning at the skinned surface, four slices, A, B, C, and D, were then made, as indicated by the dotted lines. Slice B contained the bone in each instance. Slice D was practically all fat. Each slice was ground separately in a meat chopper and the sample thoroughly mixed before taking out portions for analysis.

As all of the hams examined were mild-cure hams, that is, had been pumped in the shank only, the pickling fluids in order to reach the bodies of these hams had to penetrate chiefly from the skinned surface of the ham, as little if any penetration takes place through the thick skin of the ham.

The analyses[3] shown in the following tables therefore indicate the degree of penetration of the pickling fluids.

[3] These analyses were made by Mr. R. R. Henley, of the Biochemic Division, Bureau of Animal Industry.

_Analyses of sour hams._ ───┬────────────┬──────┬───────────┬─────────── No.│Description.│Slice.│ NaCl. │ KNO₃. ───┼────────────┼──────┼───────────┼─────────── │ │ │_Per cent._│_Per cent._ 1 │Sour body │ A │ 6.18 │ 0.175 │ │ B │ 4.83 │ .224 │ │ C │ 3.65 │ .299 │ │ D │ 1.03 │ .074 │ │ │ │ 2 │ do │ A │ 5.34 │ .174 │ │ B │ 3.70 │ .150 │ │ C │ 2.79 │ .174 │ │ D │ 1.12 │ .012 │ │ │ │ 3 │ do │ A │ 5.04 │ .125 │ │ B │ 4.08 │ .149 │ │ C │ 2.72 │ .099 │ │ D │ 1.19 │ .048 │ │ │ │ 4 │ do │ A │ 7.78 │ .250 │ │ B │ 5.31 │ .100 │ │ C │ 4.76 │ .200 │ │ D │ 1.96 │ .048 ───┴────────────┴──────┴───────────┴───────────

_Analyses of sound hams._ ───┬────────────┬──────┬───────────┬─────────── No.│Description.│Slice.│ NaCl. │ KNO₃. ───┼────────────┼──────┼───────────┼─────────── │ │ │_Per cent._│_Per cent._ 1 │Sound │ A │ 5.80 │ 0.211 │ │ B │ 4.83 │ .188 │ │ C │ 3.86 │ .221 │ │ D │ 1.33 │ .063 │ │ │ │ 2 │ do │ A │ 4.94 │ .197 │ │ B │ 4.08 │ .149 │ │ C │ 3.05 │ .223 │ │ D │ 1.56 │ .059 │ │ │ │ 3 │ do │ A │ 5.92 │ .173 │ │ B │ 4.29 │ .099 │ │ C │ 4.12 │ .139 │ │ D │ 2.32 │ .049 │ │ │ │ 4 │ do │ A │ 5.53 │ .119 │ │ B │ 4.89 │ .079 │ │ C │ 4.32 │ .099 │ │ D │ 2.19 │ .041 ───┴────────────┴──────┴───────────┴───────────

Taking an average of the four slices in each ham so as to get an average for the entire ham, and comparing the sour hams with the sound hams, we have the following comparison:

NaCl. KNO₃. Average for 4 sour hams (entire ham) per cent. 3.84 0.143 Average for 4 sound hams (entire ham) do 3.93 .131

These figures show practically no difference between the sour and the sound hams as regards the sodium chlorid and potassium nitrate content of the entire ham.

If, now, we compare the bone slices—and these afford really a better basis for comparison, as in sour-body hams the souring is always more pronounced around the bone—we have the following figures:

NaCl. KNO₃. Average for 4 sour hams (bone slice) per cent. 4.48 0.155 Average for 4 sound hams (bone slice) do 4.52 0.129

Here, again, we find no essential difference between the sour and the sound hams, and we must conclude from these analyses that souring does not depend upon or result from a lack of penetration of the pickling fluids.

It seems probable that in mild-cure hams, which are pumped in the shank only, the souring begins in the upper portion of the shank and extends upward along the bone into the body of the ham, and that it takes place before the pickling fluid has penetrated to the interior of the ham. When the pickling fluid reaches the interior of the ham it tends to inhibit the souring, which, as will be shown later, is due to the development of bacteria within the bodies of the hams. The growth of the bacteria, however, within the bodies of the hams and the histological changes in the muscle fibers do not seem to interfere with the penetration of the pickling fluids.

BACTERIOLOGICAL EXAMINATION OF SOUR AND SOUND HAMS.

In all of the sour hams which were examined bacteriologically a large anaerobic bacillus was found to be constantly present. From several of the hams this bacillus was obtained in pure culture; that is, it was the only organism present in cultures made from the sour meat and from the bone marrow of the femur. Such cultures, when held at room temperature, gave, at three days, a sour-meat odor exactly resembling that obtained from sour hams.

In many of the sour hams other bacteria were found in association with the anaerobic bacillus noted above. These other bacteria, however, were not constant, being sometimes present and sometimes absent. Among the other bacteria noted in the sour hams, the following forms occurred most frequently:

1. A nonmotile, gram-positive bacillus, measuring from 1.5 to 4 microns in length by 0.5 micron in breadth, sometimes in chains and filaments.

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

3. A large micrococcus.

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

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

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

INOCULATION EXPERIMENTS WITH HAMS.

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