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diff --git a/old/69062-0.txt b/old/69062-0.txt deleted file mode 100644 index f154af4..0000000 --- a/old/69062-0.txt +++ /dev/null @@ -1,3265 +0,0 @@ -The Project Gutenberg eBook of A bacteriological study of ham -souring, by C. N. McBryde - -This eBook is for the use of anyone anywhere in the United States and -most other parts of the world at no cost and with almost no restrictions -whatsoever. You may copy it, give it away or re-use it under the terms -of the Project Gutenberg License included with this eBook or online at -www.gutenberg.org. If you are not located in the United States, you -will have to check the laws of the country where you are located before -using this eBook. - -Title: A bacteriological study of ham souring - -Author: C. N. McBryde - -Release Date: September 28, 2022 [eBook #69062] - -Language: English - -Produced by: Charlene Taylor, Les Galloway and the Online Distributed - Proofreading Team at https://www.pgdp.net (This file was - produced from images generously made available by The - Internet Archive/American Libraries.) - -*** START OF THE PROJECT GUTENBERG EBOOK A BACTERIOLOGICAL STUDY OF -HAM SOURING *** - - Transcriber’s Notes - -Obvious typographical errors have been silently corrected. Several -tables have been divided with duplication of the first column because -of their excessive width. - -Italics are represented thus _italic_. - - - - - Issued March 17, 1911. - - U. S. DEPARTMENT OF AGRICULTURE, - BUREAU OF ANIMAL INDUSTRY.—BULLETIN 132. - A. D. MELVIN, CHIEF OF BUREAU. - - - A BACTERIOLOGICAL STUDY - OF HAM SOURING. - - - BY - - C. N. McBRYDE, M. D., - _Senior Bacteriologist, Biochemic Division_. - - [Illustration: UNITED STATES DEPARTMENT OF AGRICULTURE.] - - - WASHINGTON: - GOVERNMENT PRINTING OFFICE. - 1911. - - - - - THE BUREAU OF ANIMAL INDUSTRY. - - - _Chief_: A. D. MELVIN. - _Assistant Chief_: A. M. FARRINGTON. - _Chief Clerk_: CHARLES C. CARROLL. - _Animal Husbandry Division_: GEORGE M. ROMMEL, chief. - _Biochemic Division_: M. DORSET, chief. - _Dairy Division_: B. H. RAWL, chief. - _Inspection Division_: RICE P. STEDDOM, chief; R. A. RAMSAY, - MORRIS WOODEN, and ALBERT E. BEHNKE, associate chiefs. - _Pathological Division_: JOHN R. MOHLER, chief. - _Quarantine Division_: RICHARD W. HICKMAN, chief. - _Zoological Division_: B. H. RANSOM, chief. - _Experiment Station_: E. C. SCHROEDER, superintendent. - _Editor_: JAMES M. PICKENS. - - - - - LETTER OF TRANSMITTAL. - - - UNITED STATES DEPARTMENT OF AGRICULTURE, - BUREAU OF ANIMAL INDUSTRY, - _Washington, D. C., November 2, 1910_. - -SIR: I have the honor to transmit and to recommend for publication as -a bulletin of this Bureau a paper entitled “A Bacteriological Study -of Ham Souring,” by Dr. C. N. McBryde, senior bacteriologist in the -Biochemic Division of this Bureau. - -The souring of hams is a source of considerable loss in the -meat-packing industry, and the cause of this trouble has heretofore -been in doubt. Dr. McBryde’s paper presents the results of an -exhaustive study of the subject, from which it appears that he has -succeeded in discovering the true cause of the trouble. Besides a -description of the experimental work the paper discusses methods of -preventing the souring of hams and the proper disposal of those which -have become affected. - - Respectfully, A. D. MELVIN, - _Chief of Bureau_. - - Hon. JAMES WILSON, - _Secretary of Agriculture_. - - - - - CONTENTS. - - - Page. - - Introductory 7 - - Method of curing hams 8 - - Definition of souring 10 - - Classification of sour hams and location of sour areas 10 - - Method of detecting sour hams 12 - - Theories in regard to ham souring 12 - - Previous experimental work to determine cause of ham - souring 13 - - The present experiments 14 - - Media employed 14 - - Method of procedure in examining hams 15 - - Results of examination of sour and sound hams 16 - - Histological changes in sour hams 17 - - Chemical analyses of sour and sound hams 18 - - Bacteriological examination of sour and sound hams 20 - - Inoculation experiments with hams 21 - - Probable method by which ham-souring bacillus enters hams 33 - - Possibility of infection prior to slaughter 33 - - Possible infection from pickling fluids 34 - - Experiment to show whether infection takes place - from the curing pickle 34 - - Possible infection through manipulation or handling 35 - - Infection from ham thermometers 35 - - Experiment to show whether hams become infected - from ham thermometers 37 - - Infection from pumping needles 41 - - Infection from billhooks 42 - - Biological and morphological characteristics of the - ham-souring bacillus 43 - - Conditions favorable to growth 43 - - Growth on different culture media 43 - - Morphology 46 - - Spore formation 46 - - Resistance to heat and chemical agents 47 - - Gas production 47 - - Acid production 48 - - Pathogenic properties 48 - - Nature of the bacillus 48 - - Prevention of ham souring 50 - - General summary and conclusions 53 - - Acknowledgments 55 - - - - - ILLUSTRATIONS. - - - PLATES. - - Page. - - PLATE I. Fig. 1.—Section of muscular tissue from sound ham, - showing muscle fibers cut longitudinally; nuclei sharply - defined and cross striation distinct. Fig. 2.—Section of - muscular tissue from sour ham, showing muscle fibers cut - longitudinally; nuclei undergoing disintegration and cross - striation indistinct 16 - - II. Fig. 1.—Section through muscular tissue of ham which has - undergone natural or spontaneous souring, showing distribution - of bacilli between the muscle fibers, which are cut obliquely. - Fig. 2.—Section through muscular tissue of ham which has - undergone natural or spontaneous souring, showing individual - bacilli between the muscle fibers, which are cut somewhat - obliquely 18 - - 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. Fig. 2.—Section through - muscular tissue of artificially soured ham, showing individual - bacilli between the muscle fibers, which are cut longitudinally - 26 - - IV. Glucose bouillon culture in Smith fermentation tube at four - days 48 - - - TEXT FIGURES. - - FIG. 1. Cross section through body of ham, with sour areas - indicated by shading and dotted lines 11 - - 2. Cross section through body of ham to show method of sampling - for chemical analysis 18 - - 3. Cross section through body of artificially soured ham, - showing sour areas and points at which cultures were taken 25 - - 4. Diagrammatic views showing construction of ham thermometer 36 - - 5. Ham-souring bacillus (_Bacillus putrefaciens_), grown on - egg-pork medium 46 - - - - - A BACTERIOLOGICAL STUDY OF HAM SOURING. - - - - - INTRODUCTORY. - - -The souring of hams is a matter of considerable importance to those -engaged in the meat-packing industry, and has been the occasion of no -little worry, as in even the best-regulated packing establishments -the yearly losses it entails are considerable. The subject has given -rise to much speculation on the part of those engaged in the curing -of meats, as to the cause of the trouble and how it may be remedied, -and has received considerable attention in a practical way, but little -seems to have been done in a scientific way toward determining the -cause and nature of ham souring. - -In a well-regulated meat-packing establishment the loss from ham -souring is usually figured at about one-tenth of 1 per cent of the -total weight of hams cured. At first thought this would seem but a -small loss, but when one reflects that in a single large packing -establishment some 3,000,000 hams are cured during the year, the loss, -when figured out, is considerable. Taking 15 pounds as the average -weight of a ham, 3,000,000 hams would represent 45,000,000 pounds of -meat. Figuring the loss from souring on the basis mentioned, the amount -of meat condemned and destroyed during the year would be 45,000 pounds. -Assuming that hams sell at an average wholesale price of 15 cents a -pound, the yearly loss for a single plant which cures 3,000,000 hams a -year would be nearly $7,000. - -While one-tenth of 1 per cent of the total weight of hams cured would -represent the loss from souring in a well-regulated establishment, -statistics obtained through Government meat inspectors show that -0.25 per cent would more nearly represent the loss for the entire -country. During the fiscal year from July 1, 1908, to June 30, 1909, -some 670,000,000 pounds of hams were placed in cure in packing -establishments subject to Government inspection. Estimating the loss -from souring at 0.25 per cent, the total amount of meat condemned -and destroyed as sour would be 1,675,000 pounds. At 15 cents a pound -the total annual loss from ham souring in packing houses subject to -Government inspection would figure up something over a quarter of a -million of dollars. - -The problem of ham souring, therefore, is quite an important one from a -practical and financial standpoint; but aside from these considerations -it is also a subject of considerable scientific interest, and in -view of the fact that all sour meats are condemned under the Federal -regulations governing meat inspection it has seemed fitting that this -question should be made the subject of scientific investigation on the -part of the Bureau which is charged with the administration of this -inspection. - -The investigation reported in this paper has been conducted chiefly -along bacteriological lines, and has been confined entirely to the wet -method of curing hams, as this method is the one generally used in -American packing houses. - - - - - METHOD OF CURING HAMS. - - -In order to make clear certain points in regard to the nature and -occurrence of ham souring and to insure a better understanding of the -experiments which are to be described later, it would seem best to -begin with a brief outline of the method of curing hams as practiced in -the larger packing establishments of the country. This description is -merely a general outline of the method of preparing hams for cure and -the method of handling hams while in cure, and deals chiefly with those -points that bear on the question of souring. - -After the slaughtered animal has been cleaned, scraped, eviscerated, -washed, and split down the middle, the carcass is usually allowed to -hang for an hour or so in a large room open to the outside air, known -as the “hanging floor.” This is done with a view to getting rid of a -certain amount of the body heat before the carcass is run into the -chill rooms, and effects a saving in refrigeration. - -The carcasses are next run into “coolers” or chill rooms, and subjected -to refrigeration with a view to ridding them entirely of their body -heat. The coolers are large rooms fitted with brine pipes and capable -of accommodating several hundred carcasses. The temperature of the -coolers when the carcasses are run in is about 32° F. When filled, the -temperature of the cooler rises to about 45° F., owing to the heat -given off from the carcasses. The temperature is then gradually reduced -to 28 or 30° F. Hog carcasses are left in the coolers as a rule for -forty-eight hours, at the end of which time they are stiff and firm, -but not frozen. The temperature of the chill rooms is always carefully -watched, thermometer readings being made every few hours and duly -recorded. The temperature of the carcasses is always tested when they -leave the chill room. In those plants provided with a hanging floor, a -certain number of the carcasses are also tested before they are sent to -the chill rooms in order to determine the amount of heat lost on the -hanging floor. - -The carcasses are tested by means of an especially constructed -thermometer, known as a “ham thermometer,” which has a pointed metal -protector so that it can be thrust into the body of the ham. (See fig. -4.) The ham has been rightly selected as the proper portion of the -carcass at which to take the temperature, as it constitutes the largest -mass of muscular tissue in the carcass and holds the body heat longer -than any other portion. In taking the temperature, the thermometer -is thrust deep into the body of the ham so that the point of the -thermometer rests alongside or a little behind the upper portion of the -femur or middle bone, the latter being used as a guide in introducing -the thermometer. A certain number of the carcasses from each cooler -are tested in this way as a check on the refrigeration. The inside -temperature of the hams when they leave the chill rooms should be about -34° F. - -The carcasses are next cut up and the hams trimmed for pickling. In -some houses the hams are given an additional chilling of 48 hours after -they are cut from the carcasses, but this is not done as a rule, nor -does it seem to be necessary. - -The hams are now sent to the pickling rooms, or “sweet pickle -department,” as this branch of the packing house is designated, and -here a certain number are again tested with a thermometer, as described -above. This test is carried out by the foreman in charge of the sweet -pickle department in order that he may satisfy himself that the -hams are properly chilled before they go into the pickle and as an -additional check on the refrigeration. - -The hams are now ready to be “pumped,” and this pumping, as will be -shown later, constitutes an important step in a successful cure. -Pumping consists in forcing a strong brine solution containing -saltpeter into the muscular tissues of the ham, and is accomplished by -means of a large, hollow, fenestrated needle connected by means of a -rubber hose with a powerful hand pump. The needle is introduced along -the bone, the latter being used as a guide. - -In all of the larger packing establishments two general methods of -curing hams are followed, the two methods being designated as the -“fancy” or “mild cure” and the “regular cure,” the term “cure” being -used to designate the curing period. Various trade names are given -by the different packing establishments to the hams cured by these -methods. In the fancy cure the hams are pumped in the shank only, -whereas in the regular cure they are pumped in both body and shank. -The same pumping pickle is generally used for the two cures. It is a -significant fact that the greater proportion of the “sours” are found -among the fancy or mild cure hams. This point will be discussed farther -on in connection with some experiments to be described later. - -The actual curing is usually carried out in large vats which hold about -1,400 pounds of meat or some hundred hams. The hams are packed in the -vats in layers and are entirely covered with the pickling solution or -brine. A certain proportion is always observed between the weight of -the meat and the amount of the solution. The pickling solution, or -“pickle,” as it is termed, is a brine solution containing saltpeter -and sugar. The composition of the pickle varies somewhat with the -different packing establishments. The fancy-cure hams are usually cured -in a milder pickle, that is, one that contains less salt and saltpeter -than the pickle used in the regular cure, although in some packing -establishments the same curing pickle is used for the two cures, the -only difference being the additional pumping given the regular-cure -hams. The pickling rooms, or “cellars,” as they are called, are held at -a temperature of 34° to 36° F., and the pickling solutions are always -chilled to this temperature before being used. - -The hams are allowed to remain in cure for about 60 days, and during -this time are “overhauled” several times. Overhauling consists in -throwing the hams from the vat in which they are packed into a -neighboring empty vat, and then transferring the pickle to the new vat. -The pickle is not changed, and the same pickle follows the hams through -the entire curing process. The object in overhauling is to stir up the -pickle and expose fresh surfaces of the meat to its action. - -Hams are also cured in tierces which hold about 300 pounds of meat. -In the tierce cure, the hams are packed in the tierces, the latter -are then headed up, the pickling solution is next run in through the -bunghole, so as to fill the tierce entirely, and a wooden stopper is -finally driven into the bunghole. The tierces are rolled back and forth -across the floor on dates corresponding to the dates of overhauling in -the vat cure. The object of the rolling is to stir up the pickle, and -in this way it corresponds to overhauling in the vat cure. - - - - - DEFINITION OF SOURING. - - -To the meat inspector, a sour ham is one which has a tainted or “off” -odor, that is, any odor which deviates from the normal. The odor may be -very slight, so slight that at times only the trained meat inspector -can detect it. When slight, the odor is elusive and hard to define, but -when pronounced it has a distinctly putrefactive quality. When not very -pronounced, the odor possesses, as a rule, a slightly sour quality, -chemically speaking, and at times this sour quality may be quite -marked; hence the term “sour ham,” or “sour” has originated. In a badly -soured ham—using the term “sour” in the packing-house sense to denote -any ham that is tainted—the odor loses this sour quality and becomes -distinctly putrefactive in nature. - - - - - CLASSIFICATION OF SOUR HAMS AND LOCATION OF SOUR AREAS. - - -Sour hams are classed as “shank sours” and “body sours,” according to -the location of the souring, and these may be “light” or “heavy.” When -the souring is very pronounced, the ham is termed a “stinker.” - -Souring appears to start, as a rule, around the stifle joint -(femorotibial articulation), and extends upward into the body of the -ham. - -In quite a large proportion of the hams which are sour in the -body—probably from 40 to 50 per cent—the souring extends through to -the bone marrow of the femur or middle bone, and the sour odor is at -times more pronounced in the bone marrow than in the meat. The odor -of the bone marrow, when pronounced, is strongly suggestive of a -dissecting-room odor, and is distinctly putrefactive in quality. - -In the case of light body sours the sour odor is confined to a small -area immediately around the bone, and may be so slight that it is -detected only with difficulty. In such hams the bone marrow is apt to -be sweet, and it is not until the souring becomes more extensive that -the bone marrow becomes involved. - -[Illustration: FIG. 1.—Cross section through body of ham, with sour -areas indicated by shading and dotted lines.] - -The distribution of the sour area in the body of a well-developed sour -is shown in figure 1. - -In the case of a well-developed body sour the sour area is more -pronounced near the bone, as represented in figure 1 by the shaded -area, and may extend out into the body of the ham for a variable -distance, according to the degree of souring, as represented by the -dotted lines, gradually fading off toward the margins, where it may be -imperceptible or entirely wanting. - -In the pronounced sours, termed “stinkers,” the odor pervades the -entire ham, and is of a distinctly putrefactive quality. - -In shank sours, the souring is more or less confined to the shank, or -the region about the tibio-femoral articulation, but may extend upward -into the lower portion of the body of the ham. - - - - - METHOD OF DETECTING SOUR HAMS. - - -Souring is detected and located by means of a pointed metal instrument -known as a “ham trier,” which resembles a long, slightly flattened ice -pick. The trier is thrust into the ham at different points along the -bone, rapidly withdrawn, and the odor which clings to the metal noted. -The trained inspector works very rapidly, and is able to detect even -the slightest sour or off odor which might be imperceptible to one not -trained to the work. At the end of the cure all hams are tested with -the trier under the supervision of Government meat inspectors. - -Hams are also given what is called the “30-day inspection” by plant -inspectors during the process of curing. An average ham weighing -from 14 to 16 pounds requires about 60 days to cure, and at the end -of 30 days a certain number of hams in each run are usually tested -to see how the cure is progressing. If no sour hams are discovered -at this inspection the packer knows that the cure is progressing -satisfactorily, and moreover he feels sure that his hams will finish -satisfactorily, for experience has taught him that souring develops -within the first four weeks of the curing period, and if his hams are -sweet at the end of this time, he can feel practically sure that no -sours will develop later on. - - - - - THEORIES IN REGARD TO HAM SOURING. - - -The theories as to the cause of souring are many and varied. The -majority of them are pure speculation and have no foundation upon -observed facts. A few of these theories may be enumerated to show how -wide and varied has been the speculation upon this subject. - -A theory which is quite prevalent among packing-house employees -attributes souring to overheating of the animal previous to slaughter, -but tests were made by driving hogs to the point of exhaustion just -prior to slaughter and curing the hams from these animals in comparison -with hams taken from animals which had been rested prior to slaughter, -with no difference in the cured product; that is, the hams taken from -overheated hogs cured equally as well as those taken from rested hogs. - -Another theory attributes souring to a diseased condition of the meat. -Prior to the enforcement of the Federal regulations governing meat -inspection there might have been some ground for such a supposition, -but this theory could not hold at the present time, in view of the -thorough and efficient inspection now in force, for it can be safely -said that no diseased meat now passes the Government inspectors, and -therefore no diseased meat goes into cure in inspected houses. In -order to test this theory, however, hams were secured from a number -of condemned animals which showed various diseased conditions, such -as hog cholera, pyemia, septicemia, scirrhous chord, etc., and these -hams were cured in comparison with hams taken from normal hogs. It was -found that the hams taken from the diseased hogs cured equally as well -as those taken from healthy hogs. The hams from the diseased hogs were -destroyed after the experiment, as the meat taken from diseased animals -was of course not considered fit for consumption, the object of the -experiment being merely to determine whether or not souring is caused -by diseased conditions. - -Another theory attributes souring to imperfect or too rapid chilling -of the meat before it is put in pickle, and places the blame upon the -refrigeration. According to this theory, souring results when the meat -is chilled too suddenly, the idea being that by the rapid congealing of -the juices of the meat a coating is formed on the outside of the ham -whereby the animal heat is prevented from escaping from the interior, -leaving the meat next to the bone at a higher temperature than the -outside of the ham. - -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. - -[Illustration: - -BUL. 132, BUREAU OF ANIMAL INDUSTRY, U. S. DEPT. OF AGRICULTURE. - PLATE I. - - FIG. 1.—SECTION OF MUSCULAR TISSUE FROM SOUND HAM, SHOWING - MUSCLE FIBERS CUT LONGITUDINALLY; NUCLEI SHARPLY DEFINED AND - CROSS STRIATION DISTINCT. - -(Pen-and-ink drawing made with camera lucida from section stained with - hematoxylin and eosin to show histological structure. × 320.)] - - - 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.)] - -[Illustration: - - BUL. 132, BUREAU OF ANIMAL INDUSTRY, U. S. DEPT. OF AGRICULTURE. - PLATE II. - - FIG. 1.—SECTION THROUGH MUSCULAR TISSUE OF HAM WHICH HAS - UNDERGONE NATURAL OR SPONTANEOUS SOURING, SHOWING DISTRIBUTION - OF BACILLI BETWEEN THE MUSCLE FIBERS, WHICH ARE CUT OBLIQUELY. - THE DARK 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 HAM WHICH HAS - UNDERGONE NATURAL OR SPONTANEOUS SOURING, SHOWING INDIVIDUAL - BACILLI BETWEEN THE MUSCLE FIBERS, WHICH ARE CUT SOMEWHAT - OBLIQUELY. NUCLEI HAVE LOST SHARP OUTLINE AND CROSS STRIATION - IS INDISTINCT. - -(Pen-and-ink drawing made with camera lucida from section stained by the - Gram-Weigert method to show bacteria. × 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. - -[Illustration: FIG. 2.—Cross section through body of ham to show method -of sampling for chemical analysis. A, slice below bone; B, bone slice; -C, slice above bone; D, fat slice.] - -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. - -The first question to be decided was whether the bacillus isolated from -sour hams was actually capable of causing ham souring. The bacillus in -question had, when cultivated on the egg-pork medium, given rise to a -sour odor similar to that obtained from sour hams, but this was not -regarded as proof positive that the organism was the actual cause of -souring in hams. The proper way to decide this point seemed to be to -inoculate hams with the bacillus and then subject these hams to the -regular method of cure and see whether they became sour, just as the -pathogenic properties of a disease-producing organism are determined by -the inoculation of experiment animals. The first two experiments which -follow were designed to decide this point. - -It was regarded as important to conduct similar experiments at two -different establishments, in order to determine whether the same -results would be obtained under the somewhat different conditions -imposed by different methods of cure. The two experiments which follow -were carried out, therefore, at different establishments. - - - _Experiment I._ - -In carrying out this experiment four tierces of hams were “put down” -or “packed”—that is, placed in cure. Two of the tierces were given the -fancy or mild cure and two the regular or stronger cure. The hams in -two of the tierces, one mild and one regular cure, were injected with -a culture suspension of the bacillus; the other two tierces were not -injected with culture and were put down to serve as checks on the -cure. Hams weighing from 12 to 14 pounds were used for the mild cure, -while for the regular cure hams weighing from 14 to 16 pounds were -used. This was in accordance with the general rule which prevails in -packing houses, the lighter hams being subjected to the mild cure and -the heavier hams to the regular cure. The only difference between the -mild and the regular cure in this experiment lay in the pumping. The -hams which were given the mild cure were pumped in the shank only, -while those given the regular cure were pumped in the body as well as -in the shank. - -All of the hams had received the usual 48-hour chill. They were all -pumped with the same pumping pickle and cured in the same curing -pickle, and were in cure for the same length of time. The pumping and -curing pickles used were the regular pumping and curing pickles of the -establishment at which the experiment was carried out, and the hams -were cured in accordance with the fancy and regular cures as practiced -at this establishment. - -The hams were packed in new tierces which had been thoroughly scalded -with boiling water. The tierces were held in a curing room which was -kept at an average temperature of from 34° to 36° F., the temperature -occasionally going as high as 38° and 40° F., but never above 40° -F. The hams were left in cure for about 70 days, which is a little -longer than the usual cure. The tierces were rolled three times during -the cure. At the end of the cure the hams in all four tierces were -carefully tested by an expert meat inspector, who knew nothing of the -treatment which the hams had received. - -The hams in two of the tierces were inoculated with a culture -suspension prepared as follows: Ten tubes of egg-pork medium, each -tube containing approximately 10 cubic centimeters of the medium, -were inoculated with the bacillus and held at room temperature (20° -to 25° C.) for six days. The cultures were then filtered through -sterile gauze into a large sterile flask; this was done in order to -remove the particles of meat, which might otherwise have clogged the -syringes used in inoculating the hams. In transferring the contents -of the culture tubes to the filter the tubes were washed out with -sterile physiological salt solution (0.6 per cent sodium chlorid), -and the meat particles on the filter were afterwards washed with the -salt solution, a sufficient quantity of the latter being used to bring -the total volume of filtrate to 400 cubic centimeters. A microscopic -preparation from the filtrate showed the organisms in large numbers, -with an occasional rod showing a large terminal spore. This suspension -was used for the injection of 40 hams, each ham being given 10 cubic -centimeters, or the equivalent of 2.5 cubic centimeters of the original -culture. The hams were injected with the culture suspension by means of -a sterile syringe carrying a long 5-inch needle. The needle was thrust -well into the body of the ham at a point near the upper end of the -middle bone or femur, the latter being used as a guide in inserting -the needle and the injection being made into the tissues just behind -and a little to one side of the upper end of the femur. - -The details of the experiment were as follows: - - _Tierce No. 1 (fancy cure)._—This tierce contained 20 hams - weighing from 12 to 14 pounds each. These hams were pumped in - the shank only. Immediately after pumping they were injected - with 10 cubic centimeters each of the liquid culture or - suspension described above. After injection the hams were - immediately packed in the tierce, which was then headed up, - filled with the regular curing pickle, and placed in cure. - - Result: When tested at the end of the cure all of the hams - in this tierce save one were found to be sour. In 10 of them - the souring was very marked throughout the body of the ham - and extended into the shank as well. In six the souring was - very marked in the body of the ham, but did not extend into - the shank. In three there was slight but well-marked souring - in the body of the ham with no souring in the shank, and one - remained sweet. The probable explanation of the variation in - the degree and the extent of the souring will be discussed - later. The bone marrow of the femur or middle bone was tested - in all of the hams and found to be sour in 18. In one of the - hams which showed only slight souring in the body the souring - did not extend through to the bone marrow, and in the ham which - remained sweet the bone marrow was also sweet. The fact that - one ham in this tierce remained sweet was in all likelihood - due to an oversight in making the inoculations. In making the - inoculations the hams were spread out in a row on a table by a - packing-house assistant, who removed the hams as soon as they - were inoculated and placed them in tierces; and it is more than - probable that the assistant removed one of the hams before it - was inoculated in the interval when the writer was busy filling - the syringe for the next inoculation. - - _Tierce No. 2 (fancy cure)._—This tierce contained 20 hams of - the same average weight as the preceding. They were pumped in - the shank only, but were not injected with culture, being put - down to serve as checks on the hams in tierce No. 1. These - hams, therefore, were subjected to exactly the same cure and - were held under exactly the same conditions as those in tierce - No. 1, the only difference being that the hams in this tierce - were not injected with culture. - - Result: When tested at the end of the cure all of the hams in - this tierce were found to be perfectly sound and sweet, showing - that the curing in this instance was properly carried out and - that the souring of the hams in tierce No. 1 was undoubtedly - due to the injections of culture which they received. - - _Tierce No. 3 (regular cure)._—This tierce contained 20 hams - weighing from 14 to 16 pounds each. These hams were pumped in - the shank and also in the body. Immediately after pumping they - were each injected in the same manner as those in tierce No. 1 - with 10 cubic centimeters of culture. The hams were then packed - in tierce and placed in cure. - - Result: At the end of the cure 9 of the hams were found to be - sour, while 11 remained sweet. Of the 9 hams which became sour, - 1 showed very pronounced souring in the body and in the shank - as well, 3 showed very pronounced souring in the body, 1 showed - pronounced souring in the body, and 4 slight souring in the - body. The bone marrow of the femur was tested in all of the - sour hams and was found to be sour in 7. In 2 of the sour hams - which showed slight souring in the body the souring noted in - the meat had not extended through to the bone marrow. - - _Tierce No. 4 (regular cure)._—This tierce contained 20 hams - of the same average weight as those in tierce No. 3, and, like - the latter, were pumped in both shank and body, but were not - injected with culture. This tierce was put down to serve as - a check on tierce No. 3 and was held under exactly the same - conditions, the only difference being that these hams were not - injected with culture. - - Result: At the end of the cure the hams were carefully tested - and all were found to be perfectly sound and sweet. - - - _Results of Experiment I._ - - ───────┬────────┬─────────┬────────┬───────────── - No. of │ Number │ Average │ Cure. │ How pumped. - tierce.│of hams.│ weight │ │ - │ │ of hams.│ │ - │ │ │ │ - ───────┼────────┼─────────┼────────┼───────────── - │ │_Pounds._│ │ - 1 │ 20 │ 12-14 │ Fancy │Shank only - │ │ │ │ - │ │ │ │ - 2 │ 20 │ 12-14 │ do │ do - │ │ │ │ - │ │ │ │ - 3 │ 20 │ 14-16 │Regular │Shank and body - │ │ │ │ - │ │ │ │ - 4 │ 20 │ 14-16 │ do │ do - │ │ │ │ - ───────┴────────┴─────────┴────────┴───────────── - - ───────┬─────────────────┬───────────────────────── - │ │ Condition at end - │ │ of cure - │ ├───────────┬───────────── - No. of │ Treatment │ Number of │Percentage of - tierce.│ │ sour hams │ sour hams - ───────┼─────────────────┼───────────┼───────────── - 1 │Each ham injected│ 19 │ 95 - │with 10 c.c. of │ │ - │culture. │ │ - │ │ │ - 2 │Not injected with│ 0 │ 0 - │culture; check on│ │ - │tierce 1. │ │ - │ │ │ - 3 │Each ham injected│ 9 │ 45 - │with 10 c.c. of │ │ - │culture. │ │ - │ │ │ - 4 │Not injected with│ 0 │ 0 - │culture; check on│ │ - │tierce 3. │ │ - ───────┴─────────────────┴───────────┴───────────── - -Three hams from each tierce were selected for bacteriological and -histological examination. From tierces 1 and 3, which contained the -injected hams, three of the most pronounced “sours” were selected from -each tierce. In examining the hams bacteriologically the following -method was adopted: The hams were sectioned near the center of the body -and the larger or butt end turned up so as to expose the cut surface. A -cross section of a ham thus cut is shown in figure 3. - -Cultures were taken at the points indicated by the numbers and from the -exposed bone marrow of the femur by first searing the surface, and then -taking out plugs of the meat or marrow by means of sterile instruments. -The plugs of meat or marrow were dropped into tubes containing egg-pork -medium and pushed to the bottom of the tubes by means of a sterile -platinum wire. In the cultures made from the sour hams from tierces 1 -and 3, which were injected with culture, the bacillus with which these -hams were injected was found in practically every culture, although it -was sometimes absent in the cultures taken at points near the skinned -surfaces of the hams (i. e., at points 1, 4, and 5 in fig. 3). In the -cultures taken from the meat, the bacillus was not always present in -pure culture, but this is not to be wondered at when we remember that -the pickling fluids often contain large numbers of bacteria of various -kinds, and these, of course, find their way into the hams in the -pickling fluids. Especially is this true of the hams which are pumped -in the body, where bacteria are actually pumped into the bodies of the -hams in the pumping pickle. In the case of hams which are not pumped in -the body, the pickle bacteria do not appear to penetrate the body of -the ham to any great depth. - -In figure 3 the plus signs after the figures represent the distribution -of the sour-ham bacillus in one of the hams from tierce 1, and this -may be taken as a typical example of the other sour hams which were -examined in this experiment. It should be explained that the shaded -areas are not intended to represent the actual limits of souring, but -simply the areas in which the sour odor was most pronounced and from -which it could be readily obtained with the trier. In comparing the -regular and mild cure hams, it was found that the areas of souring as -defined with the trier were more restricted in the regular cure hams, -and this was undoubtedly due to the additional pumping which these hams -received, whereby the growth of the bacillus was partially inhibited. - -[Illustration: - - 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. - - -[Illustration: - - 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 │Number│ Average │ How pumped. - tierce.│ of │ weight │ - │hams. │ of hams.│ - │ │ │ - ───────┼──────┼─────────┼────────────────────── - │ │_Pounds._│ - │ │ │{ 10 in shank - 1 │ 20 │ 14-16 │{ - │ │ │{ - │ │ │{ 10 in body and shank - │ │ │ - │ │ │{ 10 in shank - 2 │ 20 │ 14-16 │{ - │ │ │{ - │ │ │{ 10 in body and shank - │ │ │ - 3 │ 20 │ 14-16 │{ 10 in shank - │ │ │{ 10 in body and shank - ───────┴──────┴─────────┴─────────────────────── - ───────┬───────────────────────────┬─────────────────── - │ │Condition at end of - No. of │ Treatment. │ cure. - tierce.│ ├————————┬—————————— - │ │ Number │Percentage - │ │of sour │ of sour - │ │ hams. │ hams. - ───────┼───────────────────────────┼────────┼─────────── - │ │ │ - │Each ham injected with 20 │ 10 │ 100 - 1 │c. c. of culture prior to │ │ - │chilling and pumping. │ │ - │ do │ 7 │ 70 - │ │ │ - │Each ham injected with 20 │ 10 │ 100 - 2 │c. c. of culture subsequent│ │ - │to chilling and pumping. │ │ - │ do │ 6 │ 60 - │ │ │ - 3 │Not injected with culture. │ 0 │ 0 - │ 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 2½ 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: - - _Tierce 1._—Contained 20 hams, half of which were pumped in - both body and shank and half in the shank only. As soon as they - were pumped the hams were packed in the tierce. Sufficient - curing pickle to fill the tierce was then measured out in a - clean barrel and to it was added the culture suspension. The - culture was thoroughly mixed with the pickle and the latter was - then run into the tierce containing the hams. - - Result: When tested at the end of the cure, two of the hams - which had been pumped in the shank only showed slight souring - in the body. The rest of the hams were sweet. - - _Tierce 2._—Contained 20 hams which were pumped in the same - manner as those in tierce 1. The curing pickle was the same as - that used for tierce 1, but without the addition of culture. - This tierce was put down as a check on tierce 1, the hams - being cured under exactly the same conditions, but without the - addition of culture to the curing pickle. - - Result: One of the hams which was pumped in the shank only - developed slight souring in the body. The remainder of the hams - were sweet. - -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. - -[Illustration: A. B. - - FIG. 4.—Diagrammatic views showing construction of ham - thermometer. A, front view, showing open space between metal - point and mercury bulb, which becomes filled with particles of - meat, grease, and dirt; B, side view.] - -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. - - _Lot 1._—The hams in this lot were tested with an ordinary ham - thermometer as they entered the hanging floor, as they left the - hanging floor, and as they left the coolers. The thermometer - used was borrowed from one of the plant attendants and was used - in the condition in which it was received from him; that is, it - was not cleaned or disinfected prior to use. - - _Lot 2._—The hams in this lot were tested as they entered the - hanging floor with a thermometer which had been previously - cleaned and disinfected and then dipped in a culture suspension - of the meat-souring bacillus which was isolated from sour - hams. The thermometer was dipped in the culture suspension - before each ham was tested. No further temperatures were taken - of these hams. The thermometer was carefully cleaned and - disinfected before it was returned to the attendant from whom - it was borrowed. - - _Lot 3._—The hams in this lot were not tested at all, and were - intended as checks on the cure. - -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: - - _Tierce 1._—Contained 20 hams, half of which were pumped in - both body and shank and half in the shank only. These hams were - taken from the carcasses in lot 1 and had been tested several - times with a ham thermometer, as already described. - - Result: At the end of the cure it was found that of the 10 hams - which were pumped in the shank, 5 showed well-marked souring in - the body, while of the 10 hams which were pumped in both body - and shank, 2 showed slight souring in the body. - - _Tierce 2._—Contained 20 hams, which were pumped in the same - manner as those in tierce 1. These hams were taken from the - carcasses in lot 2 and had been tested once with a thermometer - which was dipped in a culture suspension of the bacillus - isolated from sour hams. - - Result: The 10 hams which were pumped in the shank only all - became sour. When they were tried out at the end of the cure, - they showed pronounced souring throughout the entire body and - were classed as “stinkers” by the meat inspector who examined - them. The souring extended through to the bone marrow of the - femur in all of these hams. Of the 10 hams which were pumped in - both body and shank, 7 showed well-marked souring in the body, - but not as pronounced as in those pumped in the shank only; in - five of these hams the souring extended through to the bone - marrow of the femur, while in 2 the bone marrow remained sweet. - - _Tierce 3._—Contained 20 hams, which were pumped in the same - manner as those in the two preceding tierces. These hams were - not tested with a thermometer, and were put down as a check - on the cure. They were pumped with the same pumping pickle, - subjected to the same cure, and held under precisely the same - conditions as the hams in the two preceding tierces. - - Result: When tested at the end of the cure, all of these hams - were found to be perfectly sound and sweet. - - - _Results of experiment to show whether hams become infected from ham - thermometers._ - ────────┬────────┬───────────┬──────────────┐ - │ │ │ │ - │ │ │ │ - No. of │ Number │ Average │ How pumped. │ - tierce.│ of │ weight of │ │ - │ hams. │ hams. │ │ - ────────┼────────┼───────────┼──────────────┤ - │ │ _Pounds._ │ │ - │ │ │ │ - │ │ │{ 10 in shank │ - │ │ │{ │ - 1 │ 20 │ 12-14 │{ │ - │ │ │{ │ - │ │ │{ │ - │ │ │{ │ - │ │ │{ │ - │ │ │{ 10 in body │ - │ │ │{ and shank │ - │ │ │ │ - │ │ │{ 10 in shank │ - 2 │ 20 │ 12-14 │{ │ - │ │ │{ │ - │ │ │{ │ - │ │ │{ │ - │ │ │{ │ - │ │ │{ 10 in body │ - │ │ │{ and shank │ - │ │ │ │ - │ │ │{ 10 in shank │ - 3 │ 20 │ 12-14 │{ │ - │ │ │{ 10 in body │ - │ │ │{ and shank │ - ────────┴────────┴───────────┴──────────────┘ - - ────────┬─────────────────┬────────────────── - │ │ Condition at - │ │ end of cure. - No. of │ ├───────┬────────── - tierce.│ Treatment. │Number │Percentage - │ │of sour│ of sour - │ │ hams. │ hams. - ────────┼─────────────────┼───────┼────────── - │ │ │ - │Tested at several│ 5 │ 50 - │stages in │ │ - 1 │preparation for │ │ - │cure with ham │ │ - │thermometer which│ │ - │had not been │ │ - │cleaned. │ │ - │ do │ 2 │ 20 - │ │ │ - │ │ │ - │Tested once with │ 10 │ 100 - 2 │ham thermometer │ │ - │dipped in culture│ │ - │suspension of │ │ - │anaerobic │ │ - │bacillus isolated│ │ - │from sour hams. │ │ - │ do │ 7 │ 70 - │ │ │ - │ │ │ - 3 │Not tested with │ 0 │ 0 - │thermometer. │ │ - │ do │ 0 │ 0 - ────────┴─────────────────┴───────┴────────── - -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. - -[Illustration: - - 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. - - - SPORE FORMATION. - -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. At ice-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. - - - RESISTANCE TO HEAT AND CHEMICAL AGENTS. - -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. - - - GAS PRODUCTION. - -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: - - Cubic centimeters. - Total amount of gas collected 37.7 - Carbon dioxide, by absorption with NaOH 6.2 - Hydrogen, by difference 31.5 - -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. - - - ACID PRODUCTION. - -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._ - - ──────────────┬───────┬───────┬───────┬────────┬───────┬─────────── - Age of culture│Culture│Culture│Culture│Average.│Medium.│Acidity of - (days). │ A. │ B. │ C. │ │ │ culture. - ──────────────┼───────┼───────┼───────┼────────┼───────┼─────────── - │ │ │ │ │ │_Per cent._ - 2 │ 0.038 │ 0.030 │ 0.040 │ 0.036 │ 0.009 │ 0.027 - 4 │ .105 │ .100 │ .102 │ .102 │ .009 │ .093 - 6 │ .106 │ .110 │ .109 │ .108 │ .009 │ .099 - 8 │ .124 │ .115 │ .117 │ .119 │ .009 │ .110 - 10 │ .128 │ .130 │ .126 │ .128 │ .009 │ .119 - 12 │ .129 │ .120 │ .129 │ .126 │ .009 │ .117 - 19 │ .126 │ .125 │ .125 │ .125 │ .009 │ .116 - 33 │ .125 │ .123 │ .125 │ .124 │ .009 │ .115 - 47 │ .122 │ .120 │ .121 │ .121 │ .009 │ .112 - 61 │ .121 │ .116 │ .119 │ .118 │ .009 │ .109 - ──────────────┴───────┴───────┴───────┴────────┴───────┴─────────── - -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. - - - PATHOGENIC PROPERTIES. - -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. - - - NATURE OF THE BACILLUS. - -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. - -[Illustration: - - 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.] - -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_. - - - PREVENTION OF HAM SOURING. - -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 in contaminated 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 of sodium 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. - - - GENERAL SUMMARY AND CONCLUSIONS. - -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 pumping were 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. - - - ACKNOWLEDGMENTS. - -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. - -*** END OF THE PROJECT GUTENBERG EBOOK A BACTERIOLOGICAL STUDY OF HAM -SOURING *** - -Updated editions will replace the previous one--the old editions will -be renamed. - -Creating the works from print editions not protected by U.S. copyright -law means that no one owns a United States copyright in these works, -so the Foundation (and you!) can copy and distribute it in the -United States without permission and without paying copyright -royalties. Special rules, set forth in the General Terms of Use part -of this license, apply to copying and distributing Project -Gutenberg-tm electronic works to protect the PROJECT GUTENBERG-tm -concept and trademark. 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