STUDIES ON THE INTESTINAL BACTERIAL FLORA OF THE CHICK I. II. Studies on the effect of certain antibiotics upon bacterial populations* Studies on the effect of penicillin on the production of certain B-vitamins. Robert J f Driesens A N ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology and Public Health 1952 Approved A3 3'151ACT S3U3IJS OIT 013 IIIBlSTIdrI BACTillAI FLORA 33 ILL; CHICII. I. Studies on the effect of certain antibiotics u p o n bacterial populations. II. Studies on the effect of penicillin on the p r o d u c t i o n of certain B-vitamins. A thesis for the degree of Doctor o f Philosplrjby Robert J. Driesens Michigan State College Bast Lansing, Michigan 1952 Bari" vorhers who studied the intestinal flora o f chicirens failed so relate the nature of the bacteria to tne health. o r nutritional condition of eroerirental fowl. More r e c e n t studies tend to minimize the concept of tonic metabolic i n f l u e n c e s urpon the host in favor of tne idea that the status of v i t a m i n a s s i m i ­ lation is affected. -o sterilization of animal intestines b p b & c t e r i o s t a t s antibiotics nas been noted. or Growth stimulation by c e r t a i n a n t i ­ biotics has been reported when they were ea.pl oyed in c o n c e n t r a t ­ ions of a low order or when bacteriostatic activity w a s by treatment with heat or chemicals. eliminated The characteristics os' the bacterial flora are apparently affected sy the nature of the dietary components. Growth stimu­ lation by procaine penicillin G and streptomycin does not appear to be invariably related to the total numbers of bacteria nor to the quantity of any particular type. Suppression of enterococci by penicillin is of questionable significance, because of their comparatively low concentration in the intestinal areas involved. Bacterial numbers tend to be highest within the intestines of chicks exhibiting maximal or minimal growth rates. JDnterococci were most numerous throughout the inoestinal tract of chicks which gave evidence of inferior growth. Estimation of certain B-vitanins in intestines reveals marked increase of cecal folic acid when penicillin is incorporated into the diet. Dialysis of chick feed supplemented with pancreatin and cecal feces reveals no alteration in dialysis rates at 4 to 6 hours. Production of folic acid was favored in vitro by penicillin. STUDIES ON THE INTESTINAL BACTERIAL FLORA OF THE CHICK I, II. Studies on the effect of certain antibiotics upon bacterial populations. Studies on the effect of penicillin on the production of certain B-vitamins. By Robert J . Driesens A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology and Public Health 1952 Robert James Driesens candidate for the degree of Doctor of Philosophy Pinal examination, May 21, 1952, 9:00 A. M . , Ward Giltner Hall, Room 32'6. Dissertation: Studies on the Intestinal Bacterial Flora of the Chick* I. Studies on the effect of certain anti­ biotics upon bacterial populations. II. Studies on the effect of penicillin on production of certain B-vitamins. Outline of Studies: Major subject: Minor subject: Bacteriology Biochemistry Biographical Items Born, January 12, 1918, Redford, Michigan Undergraduate Studies, Calvin College, 1935-39 Graduate Studies, Michigan State College, 1946-52 Experience: Graduate research assistant, Michigan State College, 1949-52 Member U. S. Army, 1942-46 Member of the Society of American Bacteriologists, the Society Of Sigma Xi ACKNOWLEDGEMENTS The author wishes to express his appreciation to Dr. W. L. Mallmann under whose supervision the investigation was under­ taken and to Dr. A. C. Groschke for advice and guidance given* He is also indebted to Dr. B. J. Evans and Mrs. Helen A. Butts for their suggestions and assistance, especially in respect to microbiological vitamin assays. To Miss Harriet Hall, Mr. Nazar El Shawi and Mr. Wendell Claxton for their technical assistance and cooperation the writer wishes to express his thanks. The project was supported in part by funds supplied by Merck and Company, Rahway, New Jersey, administered through the Poultry Husbandry Department, Michigan State College. TABLE OF CONTENTS STUDIES ON THE INTESTINAL BACTERIAL FLORA OF TEE CHICK STUDIES ON THE EFFECT OF CERTAIN ANTIBIOTICS UI?ON BACTERIAL’ POPULATIONS. Page 1 INTRODUCTION HISTORICAL REVIEW 2 THE FUNCTIONS OF DIETARY ANTIBIOTICS 12 EXPERIMENTAL 16 Methods of study 16 Review of media 16 Techniques 20 ResiULts 24- Discussion 30 THE EFFECT OF DIETARY PENICILLIN ON PRODUCTION OF CERTAIN B VITAMINS. INTRODUCTION 35 EXPERIMENTAL 35 RESULTS 37 CONCLUSIONS 40 ’EREHCES 42 L .D3 49 ’ENDIX i INTRODUCTION The interest of specialists and laymen alike has been captured by the spectacular effects of antibiotic feed supplements upon certain do­ mestic animals. Periodically, the daily newspapers, Sunday supplements and trade papers carry accounts of economies gained by their use. Fortune magazine (l) estimates that the use of antibiotics in feeds may ultimately become worth a half-billion dollars to the United States farm economy. This is in terms of the values of the antibiotics as sales items and also in terms of reduction of animal mortality rates and savings in the use of expensive protein food supplements. In the summer o f 1950 the investigation to be described was begun as a part of a broad program instituted by the Poultry Husbandry Department of Michigan State College. It was a cooperative effort involving studies by physiologists, chemists and nutritionists. The purpose o f the bacterio­ logical studies was to estimate, if possible, the relationship of the bacterial populations to the growth rates of chicks as reflecting the use of antibiotics in feeds. HISTORICAL REVIEW Early Studies on Chicken Flora The Information concerning the bacterial flora of the chicken is rather limited. Apparently, few workers explored the complete flora of poultry until recently. Interest in the intestinal flora of man, particularly the patho­ genic types, led to speculation as to the relationship of the normal flora to health and longevity. Pasteur (£7), in 1855, expressed the opinion that higher forms of life depended on the lower forms including intestinal bacteria. In his historic studies on beri-beri, Eijkman (20) was among the first to discover what was later to be called "refection". Hens fed raw potato starch failed to develop polyneuritis contrary to expectation. Eijkman recognized the possibility of formation of chemical products from the diet as a result of bacterial action. G-eim-free Studies In an attempt to understand the role of the microflora of the gut, Schottelius (78,79,80,81) raised normal and germ-free chickens on diets which today would be considered totally inadequate. The slow growth and abnormalities of his chicks were such that he concluded that normal chickens could not be raised germ-free. He concluded (81) that the func­ tion of the normal, nonpathogenic flora was to aid in preparing ingesta for absorption, to stimulate the intestinal wall and promote peristalsis, to oppose the establishment of foreign pathogens and to aid the defenses of the host in combating pathogens and bacterial toxins. In 1913 Schottelius (81) reviewed work on the general subject, especially that of his former associate, Cohendy. He took issue with a longevity theory of the Metchnikoff group, namely, that the total bacteria in the intestinal tract (a function of the size of the gut) as related to the size of the animal was in inverse proportion to the life span. Schot­ telius pointed to the age and size of the elephant and remarked that the concentration or amount per unit volume was more significant. Cohendy in 1918 reported his results on g e m - f r e e work. too, were protein and vitamin deficient. chicks beyond five weeks. His diets He was unable to rear sterile Neither Cohendy nor Schottelius were concerned with the systematic classification of the bacteria in their studies. Quan­ titative assays were not made. Balzam ( 5 ) , cited by Reyniers (74), reared five chickens germ-free up to two months. fed the same diets. Their development was similar to that of control birds Deficiency-inducing diets fed to control and germ-free birds revealed no essential differences in the degrees of avitaminosis or body development. Balzam concluded that the intestinal flora of chickens did not exert any appreciable effect on the assimilation of food. of flora had no effect on the requirements for vitamins. The lack This work was re­ ported in 1937. Reyniers, loc. cit., was able to grow chickens germ-free for four months on commercial feeds. Results were inconclusive as to whether the growth rate was materially affected by absence or presence of intestinal - microbes. 4- In some cases (experiment 4) the germ-free birds grew better. A principal difficulty was the alteration of the germ-free mashes due to autoclaving. In the germ-free studies cited, the workers did not attempt to enumerate or classify the bacteria of the control birds. Earliest studies on the flora of birds were not quantitative in the sense that there were no tallies of populations of general types, e.g., conforms, enterococci, aerobes, and anaerobes. Enrichment or differential media had not been developed to the extent available today. Identification of Species In 1897 Kern (43) studied 24 birds and identified 88 species of which 32 were motile bacilli, 28 micrococci, 8 sarcinae and 20 species of "bacteria". The work, cited by Gage (29) was not available for study. Rahner (73) found that the population o f Bacterium coli gallinarum increased as the cloaca was approached. Bergey's Manual 6th Edition (7), does not list this name nor is it found as a synonym for Escherichia coll. which the writer assunes it to be. Bacillus megatherium, gram-positive cocci, and lactic acid bacteria were found in smaller numbers. Gage, l o c . c i t .. reported that King (44) found anaerobes, e.g., Clostridium welchil (or perfringens) to be extremely rare. anaerobes found were obligate. Eew of the Bacterium coll. now called Escherichia coli. predominated throughout the digestive tract, but rarely occured in the duodenum. (Hereafter, the names of bacteria given are the present- day equivalents, if known, of the species listed by the authors cited.) Gage (29) studied birds which were allowed to peck at the earth floors of their pens. His finding Nocardia asteroides. Bacillus mycoides. - 5- Bacillus subtills, micrococci and psuedomonads is not unexpected. diplococci (enterococci?) or obligate anaerobes were noted. Few Escherichia coli predominated, but varied in numbers with the environmental conditions and the age of chickens. Gage (29) estimated that about sixty percent of the bacteria were gram-negative. Some difficulty was encountered in distinguishing bacteria from fiber and debris, much of which stained gram-positively. It was assumed that not all bacteria found on the stained (direct) smears of in­ testinal material were alive at the time of sampling and that many were un­ cult ivat able with media available at that time. In 1929 Ifenes and Rochlin (59) found the same bacterial species in all parts of the intestine, differences being in quantity. This uniformity of flora was explained on the basis of acid production which suppressed putrefactive Bacteria and their associated decomposition processes. flora producing lactic acid were: The Streptococcus faecalis. Escherichia coll. and Lactobacillus plantarum. the latter being the most prominent anaerobe. Emmel (27) did not attempt to detect anaerobes in the droppings of 20 adult hens. Twenty chickens suffering from enteritis showed a decrease in Escherichia coli. lyphoid-paratyphoid types appeared. Salmonella pul- lorum was invariably found in the intestines of chicks suffering from pullorum disease. The quantitative assay of the predominating bacterial types found in animal,, intestines and feces is fairly recent. Research in this direction was stimulated by the discovery that bacteriostats, especially sulfonamides, when fed to experimental animals in amounts sufficient to depress certain types of flora, e.g., the coliforms, resulted in the appearance of vitamin - deficiency symptoms. 6- (17, 18, 19. See also reviews 23, 24, 64, 70.) These symptoms were relieved by B-vitamin therapy. Although Lewis et al. (48) showed that the mortality rate for chicks was least when Escherichia coli existed at levels over 3,000,000 per gram of feces; deaths were greatest when one percent sulfaguanidine* alone or together with an Escherichia coli suspension or vitamin supple­ ment was added to the diet. Groschks and Evans (33) initiated chick growth studies on aureomycin and streptomycin after Stokstad (90) reported a mash fermented by Streptomyces aureofaciens contained chick growth factors. No bacterial studies were made, although it was surmised that antibiotics aided in establishing intestinal bacteria favoring greater utilization of nutrients by the chicks. Growth stimulation was reported to be less than that ob­ tained when a basal diet was supplemented by five B vitamins without vita­ min B 12, or with five B vitamins and vitamin B 12. The contribution of intestinal flora to the nutrition of animals have been reviewed by Daft and Sebrell, Elvehjem, Johansson. (19, 21, 23, 39.) Because the scope and complexity of the subject are very broad, insufficient data are n o w available to make quantitative estimates of the positive or negative contributions of the normal bacterial flora. This review is limited chiefly to studies on poultry flora as related to bacteriostats and growth-promoting agents. * One percent of body weight per day - 7- Growth Stimulation by Bacteriostats A very surprising phenomenon is the growth stimulation of chicks and pigs by the addition of extremely small concentrations of bacteriostats to diets properly balanced. Quantities are from one-tenth to one- hundredth the therapeutic doses. Moore et al. (62) established tolerance levels for chicks for streptothricin and streptomycin and sulfasuxidine. These additives re­ duced the eoliform bacteria in the ceca, but did not sterilize the diges­ tive tracts. At non-toxic levels sulfasuxidine and streptomycin, when folic acid was added to the diet, resulted in a growth stimulation not attributable to folic acid alone. The addition of folic acid, required for normal chick development, replaced that which was ordinarily supplied by the bacteria. Morehouse and Mayfield (63) found that 3 nitro-4 hydroxy phenylarsonic acid o r its sodium salt stimulated the growth of turkey poults when fed at one-fourth the concentrations required to prevent coccidiosis. Bird et_ al. (9) noted that phenyl arsonic acid and the last mentioned compound stimulated chick growth. Other derivatives were not effective. Groschke (32) confirmed the stimulatory effect on turkey poults, but found that antibiotics were far more stimulatory than arsonic acid deri­ vatives on experimentally raised chicks. Scott and Glista (82) fed individual chicks and found that aureomycin and 3 nitro-4 hydroxy phenylarsonic acid resulted in greater feed consumption, so that feed efficiency was not improved. consumption. That is to say, weight gains reflected increased feed - 8- Stokstad and Jukes (91) also obtained accelerated growth by feeding 3 nitro-4 hydroxy phenyl arsonic acid. Growth resembled that obtained with streptomycin and succinyl sulfathiazole, which were less effective than aureomyein. Severens (83) et al. gave two percent sulfamerazine to pulloruminfected chicks. This resulted in great reduction in mortality and a growth increase over the untreated controls. However, healthy chicks weighed 28 percent more than the sulfa-treated birds. A proprietary anti-coccidial agent, Megasul nitrophenide, at a concentration of 0.0125 percent served to increase weight gains in large flocks of chickens tested throughout the United States. laboratory animals. These were not Growth differences between treated and untreated birds may be due to suppression of intestinal parasites (42). Ingram and Edgar (37) tested streptomycin, aureomyein, penicillin, sulfaquinoxyline, sulfadiazine, nitrophenide, and phenylarsonic acid in diets fed to chicks infected and uninfected with Eimeria tanella. At four weeks the antibiotics gave more than 20 percent growth advantage over the uninfected controls. The other drugs were less stinulatory. therapeutic value in controlling the disease. No drug had Growth of infected survivors paralleled that of the uninfected birds when both were fed antibiotics. The growth stimulation was not due to suppression of cecal coecidiosis. Polymyxin D was tested on a small number of chicks by Peppier et a l . (68). Polymyxin, toxic to humans when given parenterally, greatly affects gram-negative organisms and has a negligible effect on grampositives. levels. It was fed as a charcoal adsorbate at one and one-half percent Although the antibiotic was eluted in the digestive processes, - 9- no polymyxin was detectable i n the blood. Coliforms were markedly suppressed, even for several days following removal of polymyxin from the diet. Streptococcus faecalis was the most resistant type of bacteria. Other lactic acid producers were not affected. Riedel, and others (76) tested quaternary ammonium compounds for their effectiveness in combating ascarids. Alkyl dimethyl benzyl ammonium chloride and alkyl dimethyl dichlor benzyl ammonitm chloride proved to be optimal growth stimulators at 1:2000 levels. pressed growth. Higher concentrations de­ The intestinal parasites were also stimulated. No bac­ terial studies were reported. Ely (25) reported that in two of three experiments terramyein, bacitracin and aureomyein were effective in producing more accelerated growth than fat acid-ethylene oxide condensates produced. experiment surfactants appeared to be more effective. gistic effect between surfactant and aureomyein. tinal bacteria In the third There was no syner­ No enumeration of intes­ was reported. Within the last two years the reports of substantial growth stimu­ lation by aureomyein hydrochloride, procaine penicillin G-, bacitracin and terramyein have been numerous. less efficient. Streptomycin and other antibiotics proved Jukes (41) mentioned favorable response of chicks to Chloromycetin and neomycin. Crude preparations derived from fermentation residues are customarily used in commercial feeds and may give results enhanced by nutrilites present in the concentrates. The majority of the reports credit the growth enhancing effect of antibiotics to a supposed alteration of the bacterial flora. But few « - 10- bacterial analyses were made to substantiate the opinions (3, 8, 18, 21, 22, 34, 40), Although not reported in every instance for each antibiotic, the effects common to growth-promoting antibiotics may be summarized as follows: 1, Some water soluble vitamins may be required in lower amounts when antibiotics are incorporated into the diet. Oleson and associates (66) reported mutual sparing action between vitamin B 12 and aureomyein. Biely and March (8) noted a lowered requirement for folacin, niacin and riboflavin. Davis and Briggs (18) found no diminution of vitamin B 12 requirement when four antibiotics were tested. Stokstad et al. (92) found no marked differences in the required levels of water-soluble vitamins when aureomyein was a part of the diet. Blaylock and associates (11) found penicillin and aureomyein to be growth-promoting only when their semi-synthetic diets contained adequate amounts of vitamins. Growth rate in­ creased when suboptimal quantities of folacin, riboflavin, choline and vitamin B 12 were in the diet. Inadequate amounts of pyridoxine, pantothenic acid or niacin resulted in failure of the antibiotics to promote accelerated growth. I - 2. 11- The animal protein may be eliminated from feeds when small amounts of vitamin B 12 and aureomyein or large amounts of vitamin B 12 are present with adequate plant-proteins in the diet. 3. Total proteins in feeds may be reduced slightly without sacrificing dietary efficiency. This is reported by Maehlin et al. (50), and McGinnis (57). Slinger et al.. failed to confirm this (87). 4. Aureomyein hastened the depletion of vitamin B 12 in hens on experimental "depletion" diets of Halick andCouch, 5. (34). Growth rate of chicks is enhanced up to eight weeks, possibly ten weeks. A summary of the effect of antibiotics on turkey poults was given by McGinnis (58). chicks. These effects apply also to Birds taken off antibiotic feed at eight weeks of age were the same weight at 20 weeks as birds kept on anti­ biotic feed. 6. The number of runts in a flock was reduced, (58). 7. Mortality rates were reduced, 8. Hatchability of eggs from hens fed aureomyein was improved. (58). Elam et a l . , (21) and Mariakulandai et a l . , (56) were not in agreement with Peterson et al., (71) who failed to show im­ provement in hatchability of eggs laid by hens receiving suboptimal amounts of vitamin B 12. 12- 9. The numbers and types of intestinal bacteria may be altered tinder some circumstances. At the time when the present study was initiated there was insufficient evi­ dence to warrant specific conclusions. The Functions of Dietary Antibiotics A characteristic common to reported growth stimulants is their antibacterial activity. However, this activity is manifested at levels considerably higher than are used in feeding experiments. Certainly these compounds, of diverse chemical constitution, do not contribute any common nutrient materials to the experimental animals. Kramke and Fritz (45) found that sulfamethiazine and sulfathiazole did not stimulate growth under conditions in which aureomyein, bacitracin, penicillin and terramyein promoted growth. From this information, and other data, we can infer, first, that the mere ability to suppress bac­ terial growth (at proper concentrations) is not significant. Secondly, the types of bacteria affected by each of the above-mentioned antibiotics (the antibacterial spectra) are not at all similar. For example, penicillin (at practical levels) affects the gram-positive organisms. Aureomyein and terramyein have broader spectra including both gram-positive and gram-nega­ tive bacteria. Or, in tennis of intestinal flora, terramyein, in vitro suppressed gram-positive cocci, Escherichia coli and most Aerobacter aerogenes strains. This was reported by Baker and Pulaski (4). fed to rats by Johansson et al. and increased in enterococci. Aureomyein (40) reduced Clostridia and lactobacilli The eoliforms are less susceptible to peni­ cillin than to the other antibiotics except bacitracin, which does not affect them. - Peppler, et, al. 13- (68), in a limited experiment on chicks, found polymyxin D to depress coliforms markedly but did not affect other lactic acid producers at one-half percent and one percent dietary levels. terococci were resistant. En- Growth was stimulated. March and Biely (55) noted considerable bacterial variations in day to day samples of feces taken from chicks fed diets with and without aureomyein. These enumerations of various aerobic bacteria were not taken from identical samples. Therefore, ratios of types of organisms at a given time cannot be estimated. in numbers. Lactobacilli were the first to be reduced Not until 40 to 80 mg of antibiotics were added per kilogram of feed, was there a significant alteration in total counts or colifoxm indices. This is higher by two to four times the amount ordinarily re­ quired for growth stimulation. Coliforms were initially depressed in rats and mice fed 2,500 units of streptomycin per gram of rations, as they also were by succimyl sulfathiazole, neither of which were growth promoting. Bnerson and Smith (26) noted that streptomycin had no significant effect on the bacterial numbers in rat and mouse feces. Coliforms reestablished themselves. Mice sustained a reduction in total bacteria in the intestines. A study by Elam and associates (21) revealed stimulation of total anaerobic counts, enterococci and penicillin-resistant bacteria in the feces of hens and chicks fed 33 mg of procaine penicillin G. This is more than the amount required to affect growth stimulation, i.e. ten to twenty mg per kg of feed. Workers in Washington (86) found Clostridium oerfringena in the cecal contents of turkey poults. Potassium penicillin G and terramyein I - 14- (100 ppm each) reduced these Clostridia to negligible numbers. Results were not so well defined with respect to enterococci, total anaerobes or aerobes. Larson and Carpenter (46) fed procaine penicillin to pigs. Fecal bacteria were diminished, particularly Clostridia. However, the growth of the pigs was not related to the numbers of Clostridia detected. Growth stimulation seems not always to be due to antibiotic effects, i.e. the suppressing of some intestinal inhabitants or encourag­ ing the growth of non-suseeptibles. Elam, Gee and Couch (22) presented evidence suggesting other mechanisms may be involved. caine penicillin G has no antibacterial potency. Autoclaved pro­ When this penicillin was injected or fed orally to chicks, there was no alteration of fecal flora. But growth rates were increased over those of controls. Parenteral injections of bacitracin also increased rate of growth without affecting fecal flora. Earlier work by Stokstad and Jukes (90) noted that alkali- treated aureomyein residues possessed groth-promoting power but no anti­ bacterial activity. Whitlock (95) summarized the theories concerning the mode of action of antibiotic feed supplements which were held early in 1951. These theories may be listed as follows: 1. The suppression of Clostridium perfringens may prevent enterotoxemia. 2. Alteration of flora to permit greater activity by bacteria favorable to the welfare of the host. 3. A synthesis of the latter two theories suggests that the former may occur only when high levels of antibiotics are utilized. 4. Antibiotics stimulate the appetite. - 5. 15- Antibiotics increase the availability of nutrients or stimulate intestinal synthesis of these nutrients* 6. Antibiotics may suppress colifonm organisms which utilize nutrients at the expense of the host. 7. Trace minerals present in antibiotic supplements may affect the activity of intestinal flora. 8. Antibiotics effect may be systemic. 9. Effective antibiotics decrease surface tension of the gut. This may result in more effective absorption of nutrients than when antibiotics are absent. 10. Antibiotics prevent the favorable balance existing between animal and pathogens from becoming upset. A low ’’disease level" is maintained. A review of these theories follows in the discussion of experi­ mental findings. 16- EXFERIMENTAL I. THE EFFECT OF CERTAIN ANTIBIOTICS ON THE INTESTINAL BACTERIAL FLORA OF THE CHICK Methods of Enumeration.: A Review of Media The purpose of enumerating the major groups was not taxonomic with respect to shapes, sizes or species identifications of the bacteria. Interest was slanted toward the grouping of bacteria with respect to their biochemical or biological activities. Earlier workers estimated bacterial species on the basis of fre­ quency of isolation: Gage (29), Rahner (73), King (44), Menes and Rochlin (59) and Emmel (27). Advances in the study of nutritional requirements of bacteria and the development of diagnostic media - either differential or enrichment has permitted, at least to a limited degree, the estimation of the total number of certain types of bacteria. Laborious and time-con­ suming fermentation or serological tests are eliminated. The major groups by which intestinal bacteria have been numbered are the aerobic, anaerobic, gram-positive, gram-negative, coliform, lac­ tic acid group, yeasts and enterococci. Spore forming, non-sporulating and Proteus-like groups have also been mentioned. The sporulating bacteris, Proteus-like rods and yeasts have not been found to exist in large numbers in the chicks as well as in other experimental birds raised in brooders, free from gross contamination from 17- soil and extraneous feed. No media favoring their enumeration war0 following preliminary tests, Typical starter diets seemed not to ed ®r the appearance of large numbers of yeasts. The total count of aerobes has been made on several media. Evenson (2s)» studying rodent flora, used a liver infusion broth chunks of liver settled at the bottom of tubes to permit anaerobes g row also. xro- Thioglycollate medium, a broth which enhances the growth of aerophils was utilized by Elam, e t . al. tract, agar, (2l) • Tryptose glucose ye00** e x ­ (hereafter referred to as TGE) was employed b y Shapir0 Sarles (85), Johansson, e t . a l . (38) and March and Biely ( 55), S i a ^ V l i et al. ( 86). Harrison and Hansen. ( 5 5 ) utilized Eugonagar ( 5 ), a urn designed to obtain growth of fastidious organisms of which Lactoba0*^- i. are the only bacteria found in noticeable amounts in the intestinal The numbers of intestinal bacteria estimated by microscopi0 a ^ u d y of smears are usually far in excess of those tallied by aerobic Discounting the possibility of large numbers of dead organisms bei*^ tallied with those viable, it appears that many fail to develop on ^ i a in current use. Anaerobic media, besides favoring the growth of o h ^ ^ & t e anaerobes, tend to favor growth of some fastidious organisms which best at low oxidation-reduction potentials but which are not neee80a* t ^iy obligate anaerobes. (Of. 6.) Anaerobes resembling Clostridium perfringens have been isolfl^ ^ a . from animal intestines by several workers (8b) (86). Cecal areas 0P^^a.r to harbor far more anaerobes than do the upper portions of the int0fl^i_nal tract. Crecelius and Rettger (16) used a cysteine deep-tube agar timating anaerobes in guinea pig guts. Evenson et al. es­ (l o c . clt.) I - 18- suspended chunks of liver in tubefuls of a liver infusion broth. The particulate matter serves as minute "nests” wherein some anaerobes may flourish even when the redox potential of the medium may not be at an­ aerobic levels. Shapiro, et a l . (l o c . cit.) used TGE in 100 percent hydrogen atmosphere. Sieburth, et al. (loc. cit.) employed Eugonagar under anaerobic conditions. thioglycollate medium. Johansson and associates employed BBL Harrison and Hansen (35) noting that many lacto- bacilli grow better under anaerobic conditions used a modified tomato juice agar and trypticase-soy-glucose agar (BEL) under anaerobic condi­ tions. Anaerobic agar with methylene blue (BBL) was used in this study. The coliform bacteria are of historic interest, particularly be­ cause they were reported by early workers as being the dominant flora in the gut and also because many animal nutrition studies suggested that suppression of coliform bacteria was related to vitamin deficiencies. The medium chosen by a majority of workers is Levine’s Eosin-Methylene Blue agar. Sieburth, loo* cit., used Difco Violet Red Bile Agar and Evenson, lo c . cit.. the *E. C. medium’1 of Perry and Hajna. A lauryl-tryptose-lactose broth (LTLB) studied by Mallmann and Darby (52) has become an accepted standard medium for evaluating sewage and water supplies (88). A greater index of coliforms is obtained upon confirmatory use of LTLB than when the more inhibitory dyes or bile salts are used. A high degree of agreement between "presumptive” tests and confirmatory tests led the writer to utilize this broth as sole medium for estimation of coliform organisms* The enterococci also have been utilized for estimation of fecal pollution of water, particularly in England. The estimation of these bacteria f l within the intestines has been by the use of Periy-Hajna’s (69) wSFn medium. Researches by Mallmann and Seiigmann(53) show that a lower con­ centration of sodium azide than that used by Perry and Hajna or Winter and Sandholzer (97) will suppress gram-negative organisms but allow for higher indices of streptococci. When lactose fermentation in the presence of azide is the sole criterion, nearly 99 percent of the portions showing (positives) acid production contain streptococci. Occasionally broth contains gram-positive rods which seldom produce acid under test condition** The appearance of the bacterial mass is such that tubes containing other than streptococci may be easily recognized and discarded for pur­ pose of establishing probable numbers calculations. The writer chose Azide-dextrose medium (Difco) which is primarily a result of the studies of Mallmann and Seligmann. (See Appendix, p.iii) A large group, not well defined as to nutritional significance in the gut, is the lactic acid group which includes the enterococci and c o n ­ forms as well as lactobacilli. Many grow in anaerobic media, such as a tomato juice agar which contains dextrose and a low agar content. Tryp- ticase-soy-glucose agar (BEL) was used by Harrison and Hansen, (35) and March and Biely(55). Evenson (q.v.) used an acetic acid broth for growing lactobacilli, depending upon low pH to discourage growth of other types. workers (3B), (39), and Sieburth et al. (86) and Elam The Wisconsin et al0 (21) made use of carrot-liver (CL) medium of Garey (30) as shake-cultures. The writer found a 1.2 percent agar adaptation of this medium to be superior to TGE medium. The majority of aerobic assays were made on CL. It was found that adding one of Squibbs’ B-complex tablets (Squibbs’ B-complex formula) which contains 2 mg thiamin hydrochloride, 3 mg riboflavin, 20 mg 20- niacin, 5 grains Natuplex-B, a yeast extract complex, to one liter of carrot-liver medium enhanced counts from ten to more than a hundred per­ cent. Dehydrated liver (Difco) was used in place of fresh liver which may account for additional vitamin requirement. It was necessary to clarify the carrot and liver extracts by centrifugation immediately be­ fore preparing the medium. This was to eliminate the confusing of floe with minute sub-surface colonies. Methods of Study; Techniques Gram stains were made of 10”^ or 10“® dilutions of the fecal samples and also of about one hundred of the intestinal samples. The method was that described in Manual of Methods for Pure Culture Study of Bacteria (54), leaflet I V - 51 p. 9, Burke's Modification. Fecal material was obtained by killing chicks within 15 to 30 minutes after removal from the brooders. The birds killed by neck frac­ ture were dipped in a 1:3000 solution of quaternary ammonium compound to reduce the possibility of sample contamination from loose down. The appropriate sections of gut were manually removed and placed into indi­ vidual sterile Petri dishes and refrigerated at once. Within 45 minutes the appropriate gut portions were withdrawn from refrigeration and their contents manually expressed into previously weighed containers, i.e. sterile wrapped 50 ml beakers. the gut contents. Care was taken to avoid contamination of From weighed samples, portions approximating 1 to 1.5 grams were removed to sterile mortars by means of a flamed microspatula. Containers were re-weighed and the weights of the assay portions calculated c by difference to the nearest milligram. for two days at 104-110 The remaining material was dried C in an electric oven for calculation of moisture content. The dilution water was added by means of 2 ml pipets in small amounts (l to 2 ml ) in order to obtain homogeneous suspensions. Addition of larger portions of water made it difficult to disperse small clumps which formed. From the primary dilution appropriate volumes were trans­ ferred to standard milk-dilution bottles which contained 90 or 99 ml of sterile saline solutions. refrigerated until The 10 -2 and 10 -3 ■.ilution oottles were all samples were treated similarly. Subsequent ten­ fold dilutions were made and portions of each introduced into the various media. Ho common or standard procedures are given by workers assaying in­ testinal bacteria. livenson bottles to disperse clumps. et_ al. (28) used glass beads in the shaking Bottles were shaken 200 times. Shapiro and Sarles (85) reported using 6 oz bottles with glass beads and occasional use of a haring blondor. Harrison and Hansen (35) used sterile sand when grinding samples. T he w a t e r found sand to aid in dispersing material from small intestines or feces. Cecal and duodenal material, being free from gritty matter, was easier to grind without sand. Careful addition of water and rapid grinding resulted in absence of visible clumps. It is believed that microscopic bacterial clumps were of minor significance in the lower dilutions which were transferred only to liquid media. Staking each dilution 25 times results in con­ siderable dispersal by the time the higher dilutions are obtained. Dilution and shaking techniques conformed to standard methods for -22- examining water and sewage (88). The method of obtaining feces v/as as follows: group were held and the anal area palpated. Birds in each dietary Feces were collected in sterile wide-mouth tubes (seeding pots) and refrigerated in transit from the noultry plant to the laboratory. Weighing and dilution of feces were as described above. Media were incubated at 37 0 for two days and plates were enumerated with the a i d of a Quebec colony counter. Most probable numbers of c o n ­ forms and enterococci were estimated from tables obtained from Buchanan and Fulmer (12). Except in the preliminary tests, three 1-ml portions of each sample dilution were made. An exception to standard platings v/as the use of about 20 ml instead of 10 ml of the agar were placed in the Petri plates and allowed to harden, after w h i c h the sample portions were added. of war: Approximately 10-ml amounts medium were mired with the sample aid allowed to gel. hardened, an overlay of 5 ml of hot medium v/as made. appearance of surface colonies. As soon as T.iis prevented ‘ The large amount of water of syneresis produced in the confinement of the anaerobic jars made this necessary. Calcium sulfate (Drierite") in three or four Petri plates placed among the culture plates reduced the appearance of excess surface v/ater. Plates could not be inverted because evacuation of gas resulted in displacement of the a g a r . Three anaerobic jars were constructed from SO - qt pressure earners ("All A m erican"). These were heavy aluminum vessels equipped with screw lochs w h i c h effected an air-tight seal of the ground-joint between cover and vessel. Safety valves and pressure gauges were removed and replaced 1 -23- v:itii brass gas cocks. To the inlet cocks pieces of rubber ttibing were taped to facilitate removal of air from all parts of the vessel. Tests on reduced litmus milk, reduced methylene blue (water solution) and ana -rooic agars with Eh indicators revealed that two washings with natural gas followed "by vacuum pump evacuation to pressure equal to about 30 microns of mercury v/ere sufficient to prevent onidation to the indi­ cators. Ana robic plates were incubated at 3? C , the anaerobic .jars containing natural gas at initial pressures slightly less than atmospheric. Media in plates, which at the time of introduction to the jars appeared slightly oxidized, v/ere invariably found to be in the reduced state when examined two days later. Agar plates were made in duplicate from three appropriate dilutions, viz.; 10 -4 , 10 -6 , 10 -7 (small intestines). averages of two "valid" pirates obtained. Reported data represent By "valid11 is meant plates in which not less than 30 or more than 350 colonies appeared. Occasionally these limits were necessarily extended. In vitro tests of blood serum, arsanilic acid, and intestinal contents were made by means of paper discs or penicylinders on penassay seed agar (Lifco). Cultures used were 24-hour suspensions made from slants seeded with laboratory stock cultures used for antibiotic assays. Bacillus sub- tilis spore suspensions were heat treated (80 C) and used according to procedures commonly accepted by workers assaying antibiotics. Chicks used in the nutrition studies were of the fast-growing large types: Rhode Island Reds and, in some cases, ITew Ha pshires. Chicks selected for studies were those whose weights approached the mean weight of the nrouos. Runts and especially larne birds were excluded from tests I -24- at the end of the preliminary vitamin depletion period. HESULTS Prelimiimry Assays of Intestines of Individual Chicks Table I presents data obtained from examination of four-week chicks taken from larger groups whose average weight gains in two weeks are listed. These chicks were representative of the test groups which were not fed the test diets for the first two-week depletion period. In this sampling only, a single-tube series of broths was employed to estimate the ranges required for future assays. It appears that streptomycin (0.01 percent) tended to depress colifor.ns but not enterococci in the upper part of the intestines, and possioly in the ceca. However the birds in Group 7 (Basal, vitamin 3 12 and strepto­ mycin) harbored bacteria of widely divergent power to grow on TGE agar. Vitamin ±5 12 tends to favor an increase of coliforns in the duodena and lower small intestines, but with a corresponding reduction in enterococci. It did not increase the flora of the cecum. The significance of these trends is questionable because of the low number of samples tested. Effect on Streptomycin on fecal Flora. fresh, non-cecal feces obtained aseptic.ally from four birds per group were assayed and are reported in Tables II ana III. The groups 1, 2, S, and 7 map- be compared with their streptomycin-supplemented counterparts, groups 8, 9, 10 and 14 respectively. (Table II.) Gram stained preparations reveal considerable increase in grampositive organisms in the chicks fed streptomycin and a decrease in gramnegatives. Ho correlation exists between the numbers estimated by stained I - 25- smears and the tallies of bacteria able to grow on the media employed. It is possible that debris or particulate matter may have been included in the former estimates. Estimates of stained preparations were discontinued in favor of media designed to grow the major groups found by Shapiro and Sarles (loc. cit.) and Harrison and Hansen (loc. cit.). Bacteria were generally depressed by streptomycin. However, this depression was not marked by reciprocal growth rate increase. Effect of Various Growth Stimulants on Bacteria Tests at Michigan State College showed arsanilie acid to be capable of stimulating the growth of turkey poults (32), Chicks fed a similar diet did not respond as did the turkeys. Eeces from two groups of turkey poults were collected from several birds (6) in each group and examined according to the routine method pre­ viously adopted. Table IV lists the results obtained. appeared to stimulate the growth of enterococci. pressed. Arsanilie acid The coliforras were de­ The total populations of bacteria were increased by the arsanilie acid. An in-vitro test of the activity of arsanilie acid solutions was made according to accepted procedures for antibiotic assay. Concentrations from 0.1 percent to 0.001 percent haa no effect on a virulent strain of Escheri­ chia coli. The concentrations approximating those fed to the turkeys stimu­ lated Bacillus subtilis. (Cf. Table V.) Several organic chemicals were tested for their ability to enhance the growth rate of chicks: cetic acid. tested. indolacetic acid, diphenol indol, ethyl ester of indola- In addition, crude olivacein (from S1?reptomyces olivaceus) was Tests on chicks showed only indolacetic acid to be stimulatory; the 1 - 26- the populations v/ere generally higher at all levels of the intestinal tract than are usually encountered. Sspecially noteworthy (Table VII pp. 7,8) is the high level of enterococci at all levels. The group of indol compounds which were tested tended to raise bacterial levels, particularly in the upper portion of the intestines. Porter (72) cites work in which indolacetic acid v/as shown to have been utilized in synthetic media for Neisseria gonorrheae (p. 715). Tscherichia coli is capable of forming indolacetic acid whereas Proteus vulgaris and Oidium lactis produce B-indolacetic acid from tryptophane. The latter acid is heteroauxin, a plant hormone which, in 1/50,000 to 1/30,000,000 dilutions, is capable of stimulating Bschorichia coli and Salmonella tvohosa. Indolacetic acid has been shown to inhibit the enzyme penicillinase (p. 596). This suggests that its use in conjunction wit... penicillin might be synergistic. substitute for tryptophane. Its effect 0.1 0 ns seems to be as a Tryptophane is required for niacin synthesis. However, all the feeds containing indol-compounds failed to elicit growth as great as chat obtained from the control diet (Basal plus 3y percent vitamin 3 12). Olivacein obtained from Streptomyces olivaceus was used in crude form.' It did not enhance the growth rate of chicks nor did it seem to inhibit the bacteria], flora, of their guts. Compared to populations of the birds fed Penicillin or basal-plus-vitamin B 12 diets, the olivacein-fed birds showed considerable enhancement of populations (Table VIII, p. 8). Noteworthy is the large number of enterococci in the small intestine. An in vitro disk assay by the following organisms on 0.1 to 0.001 per­ cent solutions of the cru e olivacein showed neither antibiotic nor stimula- I -27- tory activity: Lactobacilluscasei, L. arabinosus, L. leichmanii, Leuconostoc-mesenteroidesi Aerobaoter aerogenes, Proteus vulgaris, Bacillus subtilis, Escherichia coli and Streptococcus faecalis. mentioned do not grow well on Difco Penassay Seed Agar. (That first four Second trials v/ere made using a modified Tomato Juice Agar for the lactobacilli.) Effect on Procaine Penicillin on Intestinal Bacteria Procaine penicillin G v/as found to promote growth of chicks at levels one-fifth to one-tenth the amounts required for equivalent response to streptomycin. Chicks v/ere selected from groups fed basal diets supplemented with graded amounts of vitamin B 12 in crystalline form and also in crude form referred to in fable VIII as "P.P. B 12", a concentrate of dried fermentation solubles. An inspection of Table VI reveals a tendency of penicillin to restrain development of bacteria in the upper intestine, especially the enterococci which are knovrn to be penicillin-sensitive. Cecal flora v/as not so markedly affected, excepting the cocci. Fecal samples v/ere obtained from several birds in each group. Because it v/as impossible to obtain intestinal samples large enough to assay for both water content and bacteria the remaining experiments v/ere based upon pooled one samples from four chicks per dietary group. (Samples approximating ram v/ere employed to reduce the degree of error introduced by assay operations.) This also tends to minimize the wide fluctuations in counts between individual birds. (Cf. Shapiro and Sarles, 64.) Table VIII and Fig. 1 reveal no consistent maintenance of bacterial levels within groups although cecal flora tend to be less variable. This DIET BASAL BASAL 6VITAMIN B-12 BAS.&PEN. BASALB-I2& PEN m Ui S A M P L IN G D A TE LEGEND FIG.I-A DUODENA -O COLIFORW _E ENTEROCOCCI EFFECT OF PENICILLIN ON FLORA X PLATE c o u n t s DIET BASAL SAMPLING BASAL&VITAMIN B-12 BAS.a PEN. BASAL,JB-12 a PEN DATE ^ l' e g e n d FIG.I-B LOWER SMALLl-NTESTINES 0 COLIFORM E ENTEROCOCCI E F F E C T OF PENICILLIN ON FLORA X PLATE COUNTS B A S A L S V IT A M IN BASAL D IE T B -1 2 B A S A L ,B I2 a BAS. a PEN. P E N . 10 • \ N . / .» • -■ * * ‘ ••X 9 m M -J 1 < 0) o u -J o ? O) w e K UJ > « e' o _j * M / f a • A . ‘ J) f t . - X . .* A * *•« / > & \ # ' t V e \ 1 a f / < & V « \ e r \ h n* / \ # % I '6 * r i •fc 4 /• i \ * 1 SAMPLING 1 / V 1 DATE i* M \ u v * y t LEG E N D FIG.I-C C E C A ---------O C O L IF O R M S ---------E E N T E R O C O C C I E FFECT OF PENICILLIN ON FLORA — x PLATE COUNTS -28- is in agreement with the findings of Shapiro and Sarles who studied two chickens from "‘birth11 for thirty weeks. They assayed bacteria major classifications weekly or bi-weekly. of all four Variations in numbers from time to time deviated by at least ten-fold, frequently more than one hundred-fold. The majority of chicks were assayed at four weeks' age, a few at six v/eoks. These may be compared with no real differences in the amounts the others,since Shapiro and Sarles noted of bacteria (per gram) at any age, once the flora became established (in 37 hours). In an effort to relate quantities of bacteria to growth rate, the dietary groups were placed in five classes representing the greatest to the least growth responses without regard to diet. ains and others for total weights. Four weeks weight: Some lotswere averaged for weight The following categories were established: Class 5, under 100 g, class 4, under 110 g, class 3, under 120 g, class 2, under 130 g, class 1, over 130 g gain. At six weeks the corresponding weights were 360 g, 390 g, 420 g, 450 g, and 475 g. Loga­ rithmic averages for individual sampling are given Table VII. bacteria were most numerous in the poorest growing birds. Generally, the An appraisal of Table X reveals that the trend is for bacteria to be most numerous when chicks are growing rapidly or when growth rate is slowest. This suggests the possi­ bility that in the former cases the digestive processes of the chicks are so efficient that a supply of nutrients is available for bacterial proliferation, m the latter case the bacteria may be robbing the host of nutrients. Data presented in Table XI reveal no marksd differences in water content oetween ingesta from diets containing penicillin and those free from penicillin, -or this reason bacteria, were not calculated on a dry weight oasis. Effect of Penicillin on Surface gension and pH A news report in Chemical and Engineering Hews (2) cited nn.publish.ed work by Stern et al. Certain detergents (fatty acid derivatives and quaternary ammonium compounds) failed to stimulate chick growth consistently. These were fed at concentrations from ten to one hundred times that of eenicillin, which v/as far more stimulatory to growth. These workers found that penicillin does not lower the surface tension of feed in vitro. But intestinal contents of chicks fed oenicillin-supplernented diets had signi­ ficantly lower surface tensions than those receiving control diets. A number of detergents tested at Michigan State College failed to accelerate growth comparable to that of penicillin-fed chicks. One of these, 11'Tide11, is reported in Sable VIII. Ton-fold dilutions of intestinal contents of four-week chicks fed control nets, basal plus 10 ppm procain penicillin G, and basal plus 1000 ppm Ethomid ,J/l (Armour) we re prepared. ritn distilled water. Samples v/ere. weighed to the nearest mg and diluted After being thoroughly shaken the samples v/ere allowed vo settle and v/ere refrigerated until all samples v/ere prepared. Tubefuls oi the dilutions v/ere rapidly brought to room temperature and tested on Cenco -esearch-type Du-Eouy -rule .-jlI. -e:vo li. tensiometers model 70530. The results are given in Values found in Experiment I were slightly higher than in ExperiThis may be due to differences in the basal diets. oo the feed used in Experiment II is listed in the Appendix. dr.tp.nt portions of the diluted samples v/ere tested. The compostion Only the super- Acidity v/as measured clocir ometri cal ly. Penicillin showed a slight tendency to restrain acid production in the -uodena and small intestines out data on cecal mate.-lad are equivocal. The same may be said for cecal surface tension. Surface tensions of duodenal mucus v/as not consistently altered, "but that of the small intestines was depressed. Ethomid C/l5, which in water solution (0.1 percent) has a surface tension of 31 dynes per cm v/as less effective than penicillin in all areas tested. Cecal areas possessed higher surface tensions than those of control "birds. Further studies appear1 to "be required to establish statistically significant data. Miscellaneous Tests In vitro plate assays of the sera obtained from four mature chickens of which two v/ere fed penicillin diets demonstrated no antibiotic activity upon penicillin-sensitive organisms. Full strength sera v/ere placed in penicylinders for assay. Similar assays were made by soaking filter paper discs in undiluted intestinal contents. Ho antibiotic activity v/as observed. Discussion Johansson and Sarles (39) have stated that the ecological system of intestines is delicately balanced and very complex. itself poorly to analysis. They also state: It therefore lends "he know that there are numerous influences on the types, numbers and activities of intestinal microorganisms, e.g., diet .... pH, Eh, the animal species, the normal digestive activity, and possibly other factors (surface tension, mineral concentration, natural metabolic antagonises, synergisms, end age of the animal)11, (p. 40). To these may be added other uncontrollable factors v/nich apply to obtaining a sample. Peristalsis alters the amount of -31- material obtainable from any limited, portion of the intestine. Hecency of d vacation and habits of eating also affect the nature of the samples. Nevertheless, certain information has been obtained which warrants re-enaaination of some of the theories proposed to explain the mode of action of penicillin: 1. Suppression of Clostridium perfringens and related toxemia. Few workers have found perfringens to be present in chick intestines when birds were restricted to brooders or kept from extraneous sources of contamination. Ho anaerobes resem­ bling the Clostridia were isolated from 242 colonies picked at random from agar plates representing high dilutions of samples from chicks in each dietary group. (The majority iso­ lated resembled Escherichia coli var. intermedium and Lacto­ bacillus bifidus or cauce.sicus. were tentatively identified). diarrhea. A fevr micrococci end Proteus Ho chicks showed symptoms of If penicillin prevents enterotoxemia the result is indirect, since it is recognized that thrifty animals with normal flora usually resist pathogenic invasions effectively. 2. Antibiotics stimulate the appetite. A survey of pertinent literature previously reported reveals slight increase of feed intake per unit of time by antibiotic-fed birds. However, efficiency ratios, i.e. - pounds of weight gained per pound of ingested feed are greater when the customary antibiotic supplements are employed. The net result is a saving of feed. I In the case of crude preparations, it rear "be true that trace minerals are present and exert an influence on the "oacterial flora. However, pure crystalline penicillin and aureoraycin map he injected into the “blood strea . or added to feeds. t’-is form they promote growth. In Unless the various secretory membranes are especially permeable to trace minerals, there seems to 'oe no evidence of their effecting intestinal flora, of the chicks. Eke "low disease level theory" nay possibly apply to non­ experiment al groups of animals or fowl which are probably more frequently challenged by pathogens. Zxperimental animals and fowl are limited to specific diets and maintained under conditions unfavorable to infection by pathogens. It is, of course, recognised that thrifty animals (such as are favored by antibiotics) resist disease. But since levels used are far below therapeutic doses, the reduction of mor­ tality seems not to be due to antibiosis. The consistent suppression of the coliform group of bacteria has not been demonstrated. In fact, the coliforms have been demonstrated to produce riboflavin, thiamin, niacin, brotin, p-amino benzoic acid, folacin and vitamin K, some of these in excess of their own requirements. cited.) (See reviews previously The ecology of the gut appears to be so complex and obscure that simoie exclamations of this sort are unwarranted. -S3 - The absorption of nut-rients from the chick gut is relieved to take place in the cecum and small intestine. The duodenum, although its villi are longer then, elsewhere in the gut, considered to be chiefly secretory and digestive - a sort of extension of the gizzard. (14, IS). The write'r has never found material resembling chick feed within the duodenum. Probably the passage of ingesta is so rapid through this area, that the feed becomes located in the small intestine immed­ iately after the chick is sacrificed. It seems apparent that bacteria even in somewhat elevated quantities do not affect absorption in the upper level of the tract, v.'olterink et si. (98) noted a more rapid absorption of radio­ phosphorus (phosphate) and radiocobalt (cobaltic) in chicks fed penicillin supplemented diets than v/as absorbed by control chicks. One function of bile salts has been described as aiding the dispersal of lipides to effect improved digestion. This, at least in part, is a result of surface-tension activity. Reports of surface-tension activity by penicillin and growth stimulat­ ion by a few surfactants suggest the possibility of greatly altering concentrations of materials at the interfaces between enzymes and substrates, or the absorbing membrane and nutrients. The mechanism of surface tension depression must not be consider­ ed without reference to the physiological or biochemical effects of the chemicals utilized. The excellent review by Lawrence (47) lists numerous effects of surfactants upon microorganisms. Some I of these effects are related, to the chemical characteristics of the agents. Studies on the effects of growth-promoting detergents, includ­ ing certain antibiotics upon digestive juices may he required to evaluate the significance of surface tension depression. 7. Consistent or considerable alteration of flora by growth stimu­ lators has not been demonstrated in the absorptive areas of the intestines. It must he noted, however, that environmental conditions do affect the metabolic activities of bacteria while they maintain their identity when cultured on diagnostic media.. She possibility of alteration of vital processes with­ out affecting the ability to reproduce led the writer to per­ form exploratory experiments which follow -35- THE EFFECT OF DIETARY PENICILLIN G H PRODUCTION OF C3RTAIM B-VITAI-UHS Introduction Biely (8) suggested that the acceleration of chick growth hy feeding the chicks antibiotics may be due to alteration of the activities of the intestinal flora so that vitamins or other essential nutrients become more available than when no antibiotics are included in diets. It was believed desirable to perform exploratory tests of this hypothesis by analyzing chick intestinal contents for certain B vitamins and also to assay in vitro dialysis of B vitamins in a "synthetic gut" apparatus. In vitro studies on rumen bacteria have produced techniques for studying mired flora within dialysis sacs utilized as synthetic rumens (36, 49). ’/asserman (93) studied the effect of several bacteriostats and antibiotics upon cellose digestion by rumen microorganisms in vitro. This work suggested a method for evaluating the effect of penicillin on vitamin production by flora of hens' cecal feces added to a typical chick starter diet. EXPERIMENTAL Apparatus and Methods The equipment for vitamin analysis consisted of the usual glassware and electrometric titration instruments. All assays were made by measu­ ring acid production by bacteria sensitive to graded amounts of biotin, riboflavin, niacin, vitamin B 12 and folacin. ard procedures (60). Methods conformed to stand­ Details regarding bacterial species and media-, are -36- given in the Appendix. A series of four simulated intestines v/ere employed in each experimental run. Eight of each v/ere made so that one group might he cleaned and prepared while others v/ere in operation. Fig. 2 is a be- tailed drawing of one of these apparatuses. She feed-v/ater slurry v/as mixed with pancreatin, adjusted to approxi mately pH 8.2 and incuhated at 37 0 for four hoars. This v/as followed hy introduction of fresh cecal feces obtained from chickens which had no knov/n previous contact with penicillin. in a Haring Blender. The mass v/as thoroughly mixed The mixture v/as settled or centrifuged to remove air and divided into two equal portions: one being the control and the other mixed hy blender with appropriate amounts of procain penicillin G solution. The pH v/as adjusted with hydrochloric acid to approximately 6.3 to 7.0 before feces v/ere mixed with the feed. served as buffer. Bisodium phosphate The feed formula and also the details of feed-v/ater slurry preparation are given in the Appendix. Ilitrogen gas passed through pyrogallol v/as bubbled into the dialy­ sis sac contents to displace air and to keep the slurry agitated during the incu'bation-dialysis period. Two 50-ml volumes each of control and penicillin slurries v/ere funneled into four dialysis sacs each suspended in 800 ml distilled water Hide mouth jars into which the apparatuses were suspended v/ere painted black at the tops to exclude light. The lower portions v/ere kept below a black spacing board which excluded much light. The lower parts of the jars v/ere not painted in order to permit periodic visual inspection. PULLEY 1 PULLEY 2 GAS O U T L E T 3 K J E L D A H L BULB 4 WATER S E A T 9 5 F IL L IN G T U B E ?mm 6 GAS I N L E T 7 NO.6 STO P PE R 8 2 - 1 / 2 "C O R K Vs. 9 SPACING BOARD 10 F EE D M IX T U R E I I GAS D IS P E R S E R 12 C E L L O P H A N E SAC 15 13 D I S T I L L E D W A T E R 14 I 7 0 0 ML 15 STIRRER J AR S C H E M A T IC DRAWING OF APPARATUS FI G. 2 -A o NITROGEN TANK O VALVE (J) PYROGALLOL, 4 5 0 ML. O PYROGALLOL, 4 5 0 M L . (j) C I T R I C ACID, 3 0 0 M L . HEATER .41 C CL V. \ f PINCH COCKS 11 i>

-{••/''.'iuW >i! \ ■ f /■! siilliS -48- 87. Slinger, S. J., J. E. Bergey, Vi. F. Pepper and D. Arthur, 1951. effect of antibiotics on the protein requirements of broilers. Poultry Sci. 50: 936. The 88. Standard Methods for the Examination of Water and Sewage, 9th Ed., 1946. Amer. Pub. Health Assoc., New York, N. Y. (p. 188). 89. Stern, J. R., J. M. Sieburth and J. McGinnis, 1952. Lack of growth response of turkey poults to certain antibiotics and bacteriostatic agents. Poultry Sci. 31: 179-180. (Res. note.) 90. Stokstad, E. L. R, and T. H. Jukes, 1950. Further observations on the "Animal Protein Factor". Proc. Soc. Exp. Biol. Med. 73: 523. 91. Stokstad, E. L. R. and T. H. Jukes, 1950. Growth promoting effect of aureomycin on turkey poults. Poultry Sci. 29: 611-612. 92. Stokstad, E. L. R., T. E. Juices and H. P. Broquist, 1951. The effect of aureomycin in the requirements-of chicks for various water soluble vitamins. Poultry Sci. 30: 931. (Abst.) 93. Wasserman, R. H . , 1950. Thesis, Michigan State College. The effect of certain antibiotics, crystal violet and sodium azide on the "in vitro" cellulose digestion by ruminant microorganisms. 94. Whitehill, A. R . , J. J. Oleson and B. L. Hutchings, 1950. Stimu­ latory effect of aureomycin on the growth of chicks. Proc. Soc. Esq?. Biol. Med. 74: 11. 95. Whitlock, G. P., 1951. Mode of action of antibiotic feed supplements in poultry and animal nutrition. Feed Age 1: 37. 96. Williams, R. J., R. E. Eakin, E. Beerstecher, Jr., W. Shive, 1950, The Biochemistry of B Vitamins, New York, Reinhold Pugl. Corp. 97. Winter, C. E. and L. A. Sandholzer, 1946. Recommended procedure for detecting the presence of enterococci. U. S. Dept. Interior, Fish and Wildlife Service, Commercial Fisheries. T. L, 2. 98. Wolterink, L, F., R. Ogle, R. Kraay and A. C. Groschke, 1951. The effect of vitamin B 12 and penicillin on the absorption and distri­ bution of radiophosphorus and radiocobalt in chicks. Paper presented at 55th ann. meeting Mich. Acad. Science, Arts and Letters. March 23, 1951 (Mimeographed.) TABLE I - PRELIMINARY EXAMINATION OP CHICK INTESTINES Bacterial Numbers Per Gram (wet) A t . Wt. Gain Grams (1) 76.3 Diet Basal (2) Basal Basal plus 3 Y fa B 12 (2) Basal plus 3 Y $> B 12 102450 Birds 2018 Duodenum 105 105 200,000 Small Intestine (!) 105 105 1,500,000 Cecum 108 107 850.000.000 Duodenim 104 105 70,000 Small Intestine 105 10® 1,000,000 Cecum 107 107 27.200.000 Duodenum 106 104 310,000 Small Intestine 105 103 60,000 Cecum 107 10® 170.000.000 Duodenum 106 103 23,000,000 Small Intestine 10® 102 2,000,000 Cecum 107 104 128.000.000 2024 102450 Birds 2034 2036 Gp. 2 1. 2. Plate Counts Aerobes TGE Sample . Gp. 1 145.2 Most probable numbers Coliforms Enterococci Group In this and the following tables ’’small intestine” refers to the small intestine below duodenum. "Y” is gamma or micrograms. Yfo is micrograms per 100 g. Table I - Continued Av. Wt. Gain Grams Diet Group Sample Most Probable Numbers C o nf or m s Enterococci 156.1 Basal, 3 Y # B 12 and 0.01# Stretomyc in 102750 Birds 2090 Duodenum 104 105 — «» Small Intestine 105 105 100,000 Cecum 104 107 300.000 Duodenum 103 105 2,080,000 Small Intestine 103 ■7 10 7 4,775,000 Go. 7 Cecum 105 10? 89.000.000 102750 Birds 2098 Duodenum 103 106 575,000 S u b 11 Intestine 105 10 6 725,000 Cecum 105 10* 2.400.000 Duodenum 105 103 30,000 Small Intestine 103 103 20,000 Cecum 10? 106 6.000.000 Basal, 3 Y # B 12 and 0.01# Strep­ tomycin Basal, 4# whey, 0.01# Streptomyc in 152.1 Basal, 4# whey, 0.01# Streptomycin 2094 2099 Gp. 8 1. 2. Average of group at four weeks. Basal A plus vitamin mix* Plate Counts Aerobes TGE TABLE II - EFFECT OF STREPTOMYCIN ON BACTERIA IN CHICK: FECES Pooled Samples Expressed from Live Birds Counts given in millions per gram (wet) D i e t Gp. TGE Agar Plate Coliforms Enterococci . Cocci - Gram Stain (l) Rods / .. " 7 Short Rods - Basal (2) 1 104.00 14.00 1.40 23.4 18.0 16.9 12.8 12.8 7.7 Gp. 1 plus 3 Y # B 12 2 30.00 14.00 1.40 10.1 14.4 23.4 9.0 19.8 1.8 Gp. 2 plus vit. mix 3 29.00 15.00 0.45 46.6 27.3 3.6 9.0 18.0 1.8 Gp. 3 plus 4$> lactose 7 16.00 110.00 1.40 28.8 16.2 16.2 Gram-positive:: 57.25 Average: 6.27 14.00 1.10 282.6 12.6 23.4 Gp. 2 plus streptomycin 9 2.45 0.95 11.00 52.2 32.4 5.4 Gp. 3 plus streptomycin 10 5.44 2.50 11.00 277.2 0 Gp. 7 plus streptomycin 14 29.00 0.95 0.25 165.6 0 Gp. 1 plus 8 streptomycin Average: 1, 2. Average ten fields smears made from 1:100 dilution. Basal "A* without vitamins. 0 3.6 9.0 0 Negative: 36.70 7.2 7.2 0 54.0 12.6 9.0 0 18.0 0 23.4 0 9.0 0 Gram-positive: 231.75 Negative: 17.1 TABLE III - EFFECT OF STREPTOMYCIN ON BACTERIA IN oHICK FECES Log Averages of Numbers Per Gram (wet) Number of Birds Diet Most Probable Numbers Colifoms 114 (24) samples Various diets (whey, fish solubles, vitamin mix, etc.) with no antibiotic added. 138 (24) samples Same diets with streptomycin added. Enterococci Plate Counts Number of Birds Aerobic Gram Stain (Millions) Positive Negative 43,750,000 3,719,000 37,030,000 114 241 31 13,200,000 2,610,000 14,120,000 138 162 34 TABLE IV - BACTERIAL FLORA OF TURKEY FECES Experiment I Birds Diet Pooled Samples Basal diet plus 0.0071% arsanilic acid. Pooled Samples Basal only Most probable numbers Coliforms Enterococci Plate Counts Aerobic Anaerobic 400,000 9,500,000 34,000,000 80,000,000 65,000 4,500,000 26,000,000 27,000,000 Experiment II Pooled Samples Basal plus 0.0071% arsanilic acid. Pooled Samples Bas al only 45,000 25,000,000 103,000,000 132,500,000* 192,000,000 760,000,000* 450,000 4,500,000 33,500,000 53,000,000* 88,000,000 270,000,000* * Same medium enriched with B-conplex vitamins# TABLE V - ACTIVITY OF ARSANTLIC ACID ON BACTERIA IN VITRO Action on Bacillus subtills Concentration of Arsanilic Acid. 0.1$ 0.05$ Zone of Inhibition 16 mm. 16 m m 12 nm stimulation 0.01$ stimulation stimulation 0.05$ 0 .001$ none stimulation none none none none none none Action o n Escherichia coli Zone of Inhibition none none none none none none TABLE VI - BACTERIAL FLORA OF CHICK INTESTINES Group 10551 Bird 3651 Diet Basal "A" (l) Basal plus .002$ Peni­ cillin Gp. 3 10451 Bird 4242 Basal plus B 12 (3Y %) Gp. 5 Most probable numbers per gram Conforms Enterococci Aerobic Plate Counts Anaerobic Av. Gain (2) Grams 400 400 1,000,000 20,000,000 140,000,000 45.000.000 557,550,000 250,000 Cecum 11,000,000 110,000,000 5,386,500,000 1.449.000.000 Feces 110.000 14.000.000 1.100.000 2.550.000.000 Duodenum 400 400 100,000 10,000 Small Intestine 250 4,500 800,000 920,000 Cecum 140,000,000 250 1,100,000,000 371,700,000,000 Feces 2.500.000 75.000 2.000.000 3.000.000 450,000 4,500,000 3,969,000 3,230,000 Small Intestine 14,000,000 14,000,000 270,900,000 327,600,000 Cecum 14,000,000 14,000,000 2,205,630,000 4,725,000,000 Feces 14.000.000 14.000.000 1.606.500.000 3.748.500.000 Duodenum Small Intestine Gp. 1 10351 Bird 4364 Sample Duodenum 73.9 84.0 126.4 Table VI - Continued Page 2 Group 10451 Bird 4168 10551 Bird 3617 10551 Bird 3956 Diet Basal "A” plus B 12 (3Y fo) Penicillin .002 f> Gp. 7 Basal 3Y £ B 12, Peni­ cillin .002 % plus 3 % Lactose Gp. 10 Sample Most probable numbers per gram Coliforms Enterococci Aerobic Plate Counts Anaerobic Duo den urn 400 700 790,000 690,000 Small Intestine 900 45,000 6,460,000 7,000,000 Cecum 11,000,000 250,000 18,000,000 48,000,000 Feces 14.000.000 250.000 50.000.000 64.500.000 2 90 100,000 100,000 90 200 5,000 10,000 Cecum 300,000 9,500 101,500,000 185,000,000 Feces 4.500.000 1.500.000 156.870.000 46.400.000 2 250 2,520,000 2,250,000 250 250 1,590,000 2,400,000 Duodenum Small Intestine Basal plus 3Y £ B 12 Pencillin .002 1o. Small Intestine Gp. 9 Cecum 250,000,000 15,000 522,900,000 126,000,000 Feces 1,500.000 4.500.000 957.600.000 1,102,500.000 Duodenum Ave. Gain Grams 136.8 138.0 137.9 Table VI - Continued Page 3 Group 10651 Bird Diet Basal "A" plus 3Y £ Vit. B 12 Gp. 5 10651 Bird 4010 1. 2. Basal 3Y $, B 12 plus .002 £ Peni­ cillin plus 4 fo Raw po­ tato starch Gp. 11 Sample Most probable numbers per gram Coliforms Enterococci Aerobic Plate Counts Anaerobic Duodenum 450.000 450,000 2,712,150 2,529,450 Small Intestine 90,000 95.000.000 56,700,000 7,994,700 Cecum 250.000 20.000.000 4.819.500.000 2,494,800,000 Feces 11.000.000 4.500.000 1.474.200.000 10.615.500.000 2 40 100,000 450,000 750 4,500 350,000 100,000 Cecum 25,000 1,110,000,000 120,000,000 5,350,000,000 Feces 950.000 450.000 10.000.000 30,000.000 Duodenum Small Intestine No vitamins added. Weight gains at four weeks. Av. Gain Grams 126.4 131.4 Table VI - Continued Page 4 STMIAHF Log. Averages Per Gram (wet) Diet Without penicillin Sample With peni­ cillin Plate Counts Anaerobic 3,160 1,288,000 3,266,000 224,400 5,675,000 58,880,000 1,259,000 Cecum 3,112,000 8,185,000 776,300,000 557,200,000 Feces 2.564.000 9.594.000 137.400.000 4.656.000.000 10 145 275,000 233,900 328,100 2,150 427,600 268,500 Cecum 4,920,000 299,700 166,000,000 1,172,000,000 Feces 2.951.000 355.000 451.900 494.300 Duodenum Small Intestine 5 groups Aerobic 1,650 Duodenum Small Intestine 3 groups Most probable numbers Coliforms Enterococci TABLE VII - RELATION OE CHICK GROWTH TO BACTERIAL FLORA Log. Averages of Numbers Per Gram (wet) of Samples from Individual Chicks DUODENA Weight No. of Coliforms Enterococci Aerobes Class ______ Birds_________________________________________________________ Anaerobes • 1 2 3 4 5 4 2 5 6,714 158 17,740 354,800 1,465,000 514,000 1,321,000 2 100 100 316,200 4,477 351 8,995 1,782 2,065,000 366,400 19,140,000 197,200 2,858,000 187,100 449,800 21,135,000 479,700 445,600 2,238,000 103,500,000 294,500,000 278,000,000 345,900,000 SMALL INTESTINE 1 2 3 4 5 4 2+ 5 CECA 1 2 3 4 5 4 2^ 2 2,128,000 1,659,000 39,260,000 166,000 2,432,000,000 23,230,000,000 FECES 1 2 3 4 5 4 2+ 3,076,000 12,390,000 933,300 93,110,000 7,943,000 1,932,000,000 ________ 2______________ 523.600__________ 1.025.000 * No groups were in this class. - 99,770,000 6,310,000,000 1.483.000___________ 87.500.000 TABLE VIII - BACTERIAL FLORA OF CHICKEN INTESTINES Group Diet Sample 11751 Birds 4446 4449 4452 Basal "A" (l) only Duodenum 11951 Birds 4518 4524 4521 11951 Birds 4490 4493 4499 11751 Birds 4563 4564 4567 4572 1. 2. Small Intestine Most probable numbers per gram Colifonns Enterococci Plate iCounts Anaerobic Aerobic 250 25,000 10,000 110,000 25,000 2,500,000 100,000 740,000,000 1.400.000.000 1.100.000.000 8.190.000.000 18.900.000.000 Gp. 1 Cecum Basal "A" (l) and .002 fo Penicillin Duodenum 250 40 620,000 810,000 Small Intestine 250 40 850,000 583,000 25.000.000 25.000 320.000.000 286.650.000 450,000 4,220,000 53,233,500 Gp. 7 Cecum Basal "A" (l) plus 3.2Y jo B 12 Duodenum 95,000 Small Intestine 4,500 450,000 8,473,000 7,434,000 45.000.000 14.000.000 175.000.000 220.500.000 25,000 25,000 620,000 810,000 140,000 250 850,000 419,000 15.000.000 45.000 320.000.000 295.000.000 Gp. 5 Cecum Basal "A" (l) plus 3.2Y % B 12 plus .002 # Penicillin Duodenum ..PP.- 1 1 _________ Small Intestine Cecum Av. Wt. Gain Grama (2) 68.3 118.7 127.3 . No vitamin mix added. Birds depleted two weeks, then fed indicated diet two weeks. 139.8 Table VIII - Continued Page 2 Group Diet Sample 13151 Birds 4614 4616 4622 4623 Basal ’’A" plus 0.7Y ia B 12 Gp. 2 Cecum 13151 Birds 4687 4688 4691 4695 Basal "A" plus 0.7Y $ B 12 plus 0.002 % Peni­ cillin G Gp. 8 Duodenum 20251 Birds 4651 4653 4654 4655 Basal "A" plus 5.6Y £ B 12 20351 Birds 4723 4724 4725 4728 2. Most probable numbers per gram Coliforms Enterococci Aerobic Plate Counts Anaerobic Duodenum 400 400 10,000 10,000 Small Intestine 400 400 10,000 10,000 40.000.000 20.000.000 85.000.000 8.505,000,000 140,000 25,000 4,252,500 4,567,000 25,000 40 10,000 10,000 Cecum 250.000 250.000 1.000.000 146.160.000 Duodenum 450,000 25,000 270,000 285,000 75,000 14.000.000 11,340,000 23,600,000 Small Intestine Small Intestine Gp. 5 Cecum 2.500.000 95.000.000 215.000,000 5.827.500.000 Basal "A" 5 .6Y £ B 12 plus 0.002 ia Penicillin Duodenum 1,100,000 250 352,800 1,400,000 Small Intestine . 1,100,000 90 9,733,500 15,500,000 Gp. 11 Cecum 95.000.000 9.000 129.780.000 1.900.000.000 Birds depleted two weeks, tben fed indicated diets two weeks. Av. Gain (2) Grams 97.9 111.8 106.3 132.4 Table VIII - Continued Page 3 Group Sample Diet Basal"D" 33151 Birds 5337 5348 5354 5358 Duodenum Small Intestine 33151 Birds 5334 5315 5316 5319 40251 Birds 5107 5112 5113 5123 40351 Birds 5205 5214 5283 5225 3. Most probable number per gram Coliforms Enterococci Aerobic Counts Plate 1 Anaerobic 90 9,500,000 31,560,000 2,555,000 2,500 450,000 250,430,000 554,400,000 140.000.000 4.500.000 560.000.000 2.710.000.000 Gp. 10 Cecum Basal »D» plus Tide .05 % Duodenum 950 2,500,000 45,600,000 19,900,000 Small Intestine 950 450,000 103,320,000 131,670,000 40.000.000 250.000.000 1.060.000.000 2.140.000.000 950,000 2,500,000 23,000,000 1,000,000 2,000,000 1,400,000,000 1,127,000,000 459,900.000 950.000.000 950.000 540.000.000 1.452.000,000 25,000 4,500 720,000 1,200,000 Gp. 9 .... Cecum Basal "D" plus peni­ cillin 2 mg 1 lb. Duodenum Gp. 1 Cecum Basal "D" plus Baci­ tracin 2 mg 1 lb. Duodenum Small Intestine 11,000,000 250,000 3,900,000 8,900,000 GP* 5 Cecum 45,000.000 9.500.000 295.000.000 485.000.000 - Small Intestine Birds not depleted. Total weights at six weeks shown. Wt! (3) Grams 358.0 376.0 485.3 451.0 Table VTII - Continued Page 4 Diet Group Sample Most probable number per gram Coliforms Enterococci Aerobic Plate Counts Anaerobic 52451 Birds 6321 6325 6328 6331 Basal "A" plus 3Y $ P.P. B 12 plus .002 io penicillin(4) Gp. 11 Duodenum 95,000,000 25,000 6,450,000 11,900,000 Small Intestine 45,000,000 950 38,000,000 55,000,000 115.000.000 300.000 210.000.000 300.000.000 52451 Birds 6297 6300 6305 6308 Basal "A” 3Y $> P.P. B 12 Duodenum 4,500 2,500 195,000 1,575,000 110,000 45,000 4.500.000 450.000 20.480.000 30.870.000 950,000 2,500,000 27,090,000 31,185,000 11,000,000 2,500,000 80,955,000 64,890,000 140.000.000 15.000.000 318.150.000 1.187.550.000 52451 Birds 6334 6338 6343 6344 Small Intestine Gp. 9 Cecum Basal "A" 15Y $ P.P. B 12 Duodenum Gp. 12 4. 5. Cecum Small Intestine Cecum Av. Gain (5) Grams 127.0 114.8 4,000,000 P.P. • fermentation product, a whole dried culture of Streptomyces olivace3es. Gain at four weeJss. 128.9 Table VIII - Continued Page 5 Group Diet Sample Aerobic Plate Counts Anaerobic 250,000 450,000 4,000,000 990,000 95,000 9,500,000 29,900,000 29,900,000 30.000.000 25.000.000 1.435.000.000 1.580.000.000 Duodenum 4,500 250,000 724,000 596,000 Small Intestine 4,500 2,500,000 27,000,000 74,000,000 Cecum 95.000.000 2.500.000 172.000.000 404.000.000 Basal plus vitamin mix plus B 12 (3Y $) peni­ cillin .002$ Gp. 3 Duodenum 25,000,000 2,500 14,650,000 32,100,000 700,000 9,500 1,850,000 3,500,000 660.000.000 25.000 970.000.000 1.230.000.000 Basal plus vitamin m i x plus F.P. plus 3Y $ B 12 & Peni­ cillin .002$ Gp. 7 Duodenum 450,000 9,500 667,000 1,200,000 Small Intestine 950,000 150,000 1,540,000 2,550,000 250,000,000 250,000 525,000,000 1,640,000,000 6E151 Birds 6403 6406 6408 6412 Basal plus vitamin mix Gp. 1 Cecum 62551 Birds 6418 6421 64— 64— Basal plus vitamin m ix plus B 12 (3Y $) Gp. 2 62251 Birds 6425 6428 6429 6431 62251 Birds 6473 6474 6479 6483 6. Most probable number per gram Coliforms Enterococci Duodenum Small Intestine Small Intestine Cecum Cecum Gain at four weeks. Av. Wt. Gain (6) Grams 99.5 123.5 132.5 131.3 Table VIII - Continued Page 6 Group Sample Diet Most probable number per gram Conforms Entero cocci Aerobic Plate Counts Anaerobic 70951 Birds 6896 6898 6899 6900 Basal plus 3Y $ B 12 plus .00255 Penicillin G (7) Gp. 5 Duodenum. 1.500.000 4.500 3,252,000 2,000,000 Small Intestine 1.500.000 7.500 22,900,000 28,900,000 95.000.000 450.000 175.000.000 250.000.000 71051 Birds 6944 6945 6946 6948 Basal (7) plus 3Y % B 12 in P.P. content Duodenum 1,500 25,000 322,000 349,000 25,000 1,500,000 740,000 625,000 Gp. 9 Cecum 250.000.000 4.500.000 640.000.000 545.000.000 71051 Birds 6915 69 7 6 6983 6986 Basal (7) plus 3Y fo B 12 in P.P. .002$ Peni­ cillin G Gp. 12 9,500 950 103,500 124,000 450,000 2,500 3,020,000 5,190,000 1.100.000.000 15.000 410.000.000 690,000.000 7. 8. ...... Cecum Sinall Intestine Duodenum Small Intestine Cecum Basal "A" contained vitamin mix. Birds depleted two weeks then fed two weeks on diet indicated. Av. Wt. Gain Grama (8) 144,0 128.8 130.9 Table VIII - Continued Page 7 Group Diet Sample 80251 Birds 7038 7202 7232 7238 Basal (9) plus 3Y £ vitamin B 12 Small Intestine Gp. 1 Cecum 80251 Birds 7030 7051 7230 L.B. Basal(9) plus 3Y fo Vitamin B 12 plus .002fo Peni­ cillin Gp. 2 ....... Duodenum 80351 Birds 7031 7034 7102 7221 Basal (9) plus 10Y $ Indolacetic Acid plus 3Y % B 12 Gp. 3 80351 Birds 7090 7091 7099 7101 Basal (9) plus 1 0 Y # ethyl ester of Indolace­ tic Acid plus 3Y $ B 12 Gp . 4 9. 10. Most probable number per gram Coliforms Enterococci Plate Counts Anaerobic 40,000 4,000 1,045,000 1,980,000 450,000 2,500,000 85,000,000 100,800,000 25.000.000 25.000.000 179.000.000 780.000.000 450,000 45,000 650,000 1,480,000 7,500,000 2,500,000 38,430,000 48,510,000 45.000.000 950.000 15.000.000 570.000.000 4,500,000 4,500,000 19,600,000 21,300,000 140,000,000 ' 17,200,000 114,030,000 Duodenum Small Intestine Cecum Aerobic Duodenum Small Intestine 140,000,000 Cecum 450.000.000 20.000.000 400.000.000 6.340.000.000 11,000,000 14,000,000 20,000,000 23,600,000 150,000 950,000 88,200,000 168,840,000 250.000.000 250.000 640.000.000 970.000.000 Duodenum Small Intestine Cecum Basal diet "A” plus vitamin mix. Birds depleted two weeks, then fed diet indicated two weeks. Av. Wt. Gain Grams (10) 142.0 124.2 143.0 139.8 Table VIII - Continued Page 8 Group Diet Sample 80451 Birds 7017 7106 7107 7086 Basal (9) plus 40 ppm crude olivacein plus 3Y £ B 12 Gp. 5 Duodenum 80451 Birds 7024 7053 7071 7080 Basal (9) plus 100 ppm diphe­ nol indole plus 3Y fo B 12 Gp. 6 Duodenum 9. 10. Small Intestine Cecum Small Intestine Cecum Most probable number per gram Coliforms Enterococci Aerobic Plate Counts Anaerobic 14,000,000 250,000 14,200,000 15,800,000 4,500,000 45,000,000 29,900,000 32,400,000 450.000.000 95.000.000 510.000.000 670.000.000 350,000 2,500,000 3,200,000 2,950,000 25,000 25,000,000 23,800,000 32,200,000 25.000.000 9.500.000 106.500.000 204.000.000 Basal diet "A" plus vitamin mix. Birds fed depletion diet two weeks,then fed indicated diet two weeks. Av. Wt. Gain (10) Grams 137.8 122.3 Table VIII - Continued Page 9 Group 102951 Birds 8008 8014 8026 8066 103051 Birds 8003 8004 8009 8011 103151 Birds 8002 8005 8013 8047 11. Diet Basal rtC» Sample Duodenum Small Intestine Most probable number per gram Colifoims Enterococci Aerobic Plate Counts Anaerobic 740,000 250.000 66,500,000 82,220,000 1,180,000 920.000 210,000,000 239,000,000 22.000.000 9.200.000 1.420.000.000 Gp. 1 Cecum Basal "C" plus Peni ciUin 10 mg 1 lb. Duodenum 20 430 110,000 190,000 Small Intestine 73 7,400 6,000,000 11,800,000 Gp. 2 Cecum 9.000.000 147.000 19.000.000 3,500.000.000 250,000 2,500 375,000 295,000 250,000 250,000 6,200,000 1,750,000 118,000,000 43,000,000 111,000,000 3,960,000.000 Basal "C" 0.1$ Duodenum plus Ethomid C/15 (Armour) Small Intestine Gp. 3 Cecum Birds depleted one week, then fed three weeks on diet indicated. Weights are total, not gain. Av. Wt. (11) 180.7 228.9 194.0 Table VIII - Continued Page 10 Group Sample Diet 564,000 912,000 9,200 430 255,000 1,125,000 9.200.000 3.900 31.500.000 1.135.000.000 91 92,000 4,130,000 3,460,000 240 24,000 4,900,000 51,700,000 25.000.000 9.200.000 200.000.000 220.000.000 Duodenum 24 43,000 435,000 205,400 Efcnall Intestine 24 2.500.000 6,268,000 7,600,000 25.000.000 2.200.000 77.000.000 74.500.000 240 25,000 3,110,000 1,270,000 2,500 4,300 5,350,000 34,600,000 140.000.000 430.000 704.000.000 3.740.000.000 Duodenum 111251 Birds 8389 8442 8758 8832 Basal (12) plus 3 Y # Crystal B 12 Duodenum Gp. 2 Cecum 111251 Birds 8256 8354 8404 8483 Basal (12) plus 3Y % P.P. B 12 111351 Birds 8203 8403 8496 8861 Basal (12) plus 15Y % Crystal B 12 Small Intestine S E i .6 ...... Cecum 12. 13. Plate Counts Anaerobic 240 Basal (12) 3Y % B 12 (Crystal .002$ Peni­ cillin Gp. 1 ..... Aerobic 740 110951 Birds 8215 8412 8435 8620 Gp. 8 Most probable number per gram Colifoims Enterococci Small Intestine Cecum Small Intestine Cecum Duodenum Basal "A” feed with, vitamin mix. Birds depleted two weeks, then fed indicated diet two weeks. Av. Gain (13) Grams 137.8 132.9 128.6 124.0 Table VIII - Continued Page 11 Group Diet Sample 111351 Birds 8310 8319 8693 8829 Basal (12) plus 3T $ F.F. B 12 plus .002$ Peni­ cillin Gp. 7 Duodenum 111351 Birds 8216 8321 8650 8819 Basal (12) plus 15Y % P.P. B 12 Duodenum 12. 13. Gp. 12 Small Intestine Cecun Small Intestine Cecum Most probable number per gram Colifonns Enterococci Aerobic Plate Counts Anaerobic 36 9,200 189,000 7,310,000 430 2,500 283,500 5,103,000 45.000.000 25.000 21.500.000 2.813.000.000 9,200 2,500 2,561,000 1,745,000 240 2,500 55,000 9,450,000 920.000 2.500.000 37.500.000 5.720.000.000 Basal ”A" feed with vitamin mix. Gain at four weeks. Birds depleted two weeks, then fed indicated diet two weeks. Av. Wt. Gain (13) Grams 130.4 124.8 TABLE IZ - EFFECT OF PENICILLIN ON BACTERIAL FLORA. Log. Averages of Numbers Per Gram (wet) Pooled Samples Diet No. of Birds Enterococoi Colifonns Aerobes Anaerobes DUODENA. Basal A,D,C Same / Pen. 12 12 8,017 1,683 403,600 3,500 3.515.000 1,161,500 2.183.000 535,800 Basal / B 12 Same / Pen. 48 48 10,260 147,200 33,110 7,763 1.175.000 1.067.000 1.429.000 1.910.000 SMALL INTESTINES Basal A,D,C Same Pen- 12 12 51,530 165,200 1,770,000 152,400 19.900.000 26.300.000 232,300,000 97,950,000 Basal / B 12 Same / Pen. 48 48 7,430 291,100 185,800 2,723 3.565.000 1.968.000 8.974.000 3.243.000 CECA Basal A,D,C Same / Pen. 12 12 106,650,000 59.710.000 32,660,000 151,400 1,750,000,000 150.650.000 4,325,000,000 1,132,500,000 Basal / B 12 Same / Pen. 48 48 26.730.000 54.700.000 5,662,000 66,830 153.100.000 90,160,000 68,702,000 683,900,000 TABLE X - RELATION OF BACTERIAL NUMBERS TO CHECK GROWTH RATE Log. Averages of Numbers (wet) Pooled Samples Weight* Class No. of Birds No. of Samples Coliforms Enterococoi Aerobes Anaerobes U1 if- M M H DUODENA 48 40 20 24 20 12 10 5 6 5 209,400 27,420 29,510 9,890 10,940 37,900 56,890 14,000 10,300 150,700 3,048,000 1,938,000 1,315,000 516,400 1,600,000 2,917,000 3,564,500 1,503,000 763,000 1,614,000 183,200 127,400 12,160 82,410 712,900 9,931,000 5,875,000 641,200 6,339,000 13,430,000 17,700,000 18,493,000 1,014,000 19,187,000 114,300,000 UI^WMH 3IALL INTESTINE 48 40 20 24 20 12 10 5 6 5 388,200 41,700 9,480 54,570 65,460 U1 it* W » H CEGA 48 12 142,560,000 849,500 314,100,000 837,500,000 40 10 67,325,000 1,778,000 109,140,000 626,600,000 20 5 18,880,000 642,700 59,020,000 195,000,000 24 6 21,630,000 5,003,000 203,200,000 3,221,000,000 20____________5____________ 150.000.000______ 57.325.000_________ 920.450.000_______ 5.861.000.000 * Weight classes: 1 = over 130 g, 2 «= 120-130 g, 3 = 110-120 g, 4 = 100-110 g, 5 - under 100 g at four weeks. TABLE XI - WATER IN CHECK INTESTINE CONTENTS Basal Groups or Basal Plus Vitamin B 12 Date Bird Nos. Duodenum Small ___________ Intestine 62151 6403,6406 6408.6421 84.439 87.900 80.315 Basal plus vitamin mix 62551 6418.6421 83,787 81.400 79.789 Basal plus 3Y % B 12 71051 6944,6945 6946,6948 82.166 85.981 79.809 Basal plus 3Y $ P.P. B 12 80251 7038,7202 7232,7238 83.848 84.521 82.257 Basal, 3Y £ Cry. B 12 102951 8008,8014 8076,8066 91.433 84.082 81.105 Basal »C" 111251 8389,8442 8758,8832 82.264 84.202 80.721 Basal "A" plus 3Y fo B 12 111351 8256,8354 8404,8483 83.290 85.005 83.532 Basal "A" plus 3Y P.P. B 12 111351 8203,8403 8496,8861 85.154 86.355 82.325 Basal «A" plus 15Y f, B 12 11751 4446,4449 4452 - (3) 82.584 86.957 77.297 Basal 11951 4490,4493 4499 - (3) 81.158 84.652 82.172 Basal plus 3.2Y % B 12 13051 4614,4616 4622,4623 94.691 82.199 78.835 Basal plus 0.7Y % B 12 20251 4651,4653 4654,4655 81.736 82.991 79.325 Basal plus 5.6Y % B 12 33151 5337,5348 5354,5358 78.880 82.685 80.968 Basal "B" only 52451 6297,6300 6305,6308 86.417 86.641 82.627 Basal "A" plus 3Y f, P.P. B 12 52451 6334,6338 6343.6344 87.513 85.714 79.461 Basal "A" plus 15Y B 12 Total Average Cecum 1356.539 1357.231 1291.196 84.784 84.827 80.700 Diet Table U - Continued Page 2 Date Bird Nos. Duodenum 62251 6425,6428 6429,6431 85.551 86.409 81.787 Basal "A"plus Vit. Mix. (3y$) with B 12 62251 6473,6474 6479,6483 83.425 85.112 81.422 3Y $ F.P. B 12 3Y 70951 6846,6898 6899,6900 84.260 84.199 81.473 With 3Y $ B 12 vitamin m i x 71051 6975,6976 6983,6986 78.569 82.679 80.703 With 3Y $ B 12 vitamin m i x 80251 7030,7051 7230, L.B. 85.841 83.364 79.863 Basal "A" with vitamin m i x 103051 8003,8004 8009,8011 86.932 82.916 78.723 Penicillin 10 mg 1 lb. 110915 8215,8412 8435,8620 83.285 82.562 81.308 5Y $ B 12 111351 8310,8319 8693,8829 83.904 85.546 11751 4563,4564 4567,4572 81.493 85.609 11951 4518,4521 4524 80.803 82.400 13151 4687,4688 4691,4695 81.008 83.144 80.511 Basal, 0.78$ B 12 and Pen. 20351 4723,4724 4725,4728 81.657 79.437 78.916 Basal, 5.68$ B 12 and Pen. 52451 6321,6325 6328,6331 85.747 87.134 86.095 Basal "A", Yit. M i x & 31$ F.P. B 12 1082.475 83.264 1089.511 83.808 Total Average Small Intestine Cecum 80.969 82.183 Diet 3Y $ F.P. B 12 Basal, Pen., and 3.2Y $ B 12 Basal and Penicillin 973.953 81.163 Table XI - Continued Page 3 Date Bird Nos. Duodenum Eknall ______________________________________ Intestine Cecum 103151 8002,8005 8013,8047 82.135 83.094 80.437 Basal plus 0.1$ Ethomid C/15 80451 7017,7086 7106,7107 88.494 84.857 80.954 40 ppm olivacein 80451 7024,7053 7071,7080 83.600 80.284 100 ppm diphenol indol 33151 5314,5315 5316,5319 82.008 87.068 81.880 .05$ Tide and basal 40351 5205,5214 5223.5225 82.405 83.676 80.563 Bacitracin 335,042 83.760 422.295 84.459 404.118 80.823 Total Average STMMA.H3T Basal or Basal 8c Vit. B 12 84.784 84.827 80.700 Basal & Penicillin 83.264 83.808 81.163 Basal & Other Additives 83.760 84.459 80.823 Diet TABLE XII - EFFECT OE FEED ADDITIVES ON SUHFACE TENSION AND pH OF CHICK INTESTINE CONTENTS (Dilution 1:10. Surface tension in dynes per cm.) DDODENIM Feed______ Basal________________ Basal & 0.001$ Penicillin Bird Dynes pH Bird Dynes Basal & O.ltfo Bthomid C/15 pH Bird Dynes pH Experiment I 4148 34.39 6.10 4049 34.67 6.10 4179 33.34 5.90 4163 38.09 5.80 4196 35.55 6.20 4115 34.11 5.90 4028 36.10 6.00 4069 34.06 6.04 4010 33.67 6.10 4096 34.11 6.00 4171 34.94 6.10 4142 35.00 6.08 Av. 35.67 5.97 34.81 6.11 34.03 6.00 Experiment II 3891 27.79 6.08 3901 29.91 6.38 3950 32.43 6.08 3943 31.26 6.12 3976 29.00 6.17 3941 32.48 6.40 3958 31.31 6.10 3947 30.95 6.52 Av. 30.13 6.11 31.15 6.31 Av. I & Table XII - Continued Page 2 SMALL INTESTINE Feed_______ Basal Bird Dynes Basal & 0.001$ Penicillin pH Bird Dynes Basal & 0.1$ Ethomld 0/15 pH Bird Dynes pH Experiment I 4148 32.90 6.30 4049 33.73 6.40 4179 35.82 6.45 4163 38.97 6.50 4196 34.67 6.50 4115 36.76 5.43 4028 35.82 5.88 4069 32.24 6.70 4010 36.76 6.00 4096 48.36 6.18 4171 34.00 6.25 4142 37.20 5.40 Av. 39.01 6.21 33.36 6.46 36.64 5.82 Experiment II 3891 32.16 6.31 3901 30.95 6.29 3950 34.55 6.52 3943 31.67 6.20 3976 33.97 6.48 3941 30.90 6.10 3958 36.13 6.41 3947 33.83 6.77 Av. 34.20 6.43 31.84 6.34 Av. I & II 36.605 6.32 32.60 6.40 Table XII - Continued Page 3 CECTM Peed_______ Basal_____ ___________ Basal & 0.001$ Penicillin Bird Dynes pH Bird Dynes Basal & 0.1$ Ethomld C/15 pH Bird Dynes pH Experiment I 4148 45.21 6.20 4049 48.13 6.20 4179 51.28 6.64 4163 49.74 6.32 4196 48.30 6.69 4115 50.76 6.50 4028 50.18 5.64 4069 48.19 6.62 4010 52.99 6.50 409 6 49.29 5.28 4171 49.85 6.49 4142 52.33 5.69 Av. 48.61 5.86 48.62 6.50 51.85 6.33 Experiment II 3891 45.51 6.49 3901 43.31 5.96 3950 48.96 6.49 3943 45.90 5.96 3976 46.62 6.33 3941 44.46 5.88 3958 45.93 6.54 3947 46.25 6.00 Av. 46.76 6.41 44.98 5.95 Av. I & II 47.69 6.14 46.80 6.28 Table XEII - Effeet of Penicillin on Bacterial Populations SHAKER Code Description of Sample Niacin Biotin y/ml my/ml (pH 8. with pancreatin 0.4$) 0 Control "zero” hours 16.17 11.30 I Basal 8 hours 15.42 5.35 II Basal 8 hours 13.50 5. 65 III Penicillin 8 hours (.002$) 14.83 1.75 IV Penicillin 8 hours (.002$) 14.45 2.45 Same as above, pH 6.8, no pancreatin 0 Control "zero" hours 17.80 15.70 I Basal 8 hours 20.30 13.70 II Basal 8 hours 18.70 11.95 III Penicillin 8 hours (.002$) 20.38 12.70 IV Penicillin 8 hours (.002$) 20.42 13.10 and Vitamin Content of Chick Feed Suspension in Eight Hours TESTS Vitamin B 12 Folic Acid Ribo­ flavin my/ml my/ml y/ml 5.86 82.9 1.80 950 450 26 2.25 61.3 0.63 140,000 950 324 1.30 96.2 0.66 110,000 140,000 220 1.42 64.3 0.61 110,000 10,000 480 1.33 78.0 --- 25,000 10,000 220 3.00 137.5 0.954 25 250 3.30 174.3 0.835 220,000 25,000 220 2.58 147.8 0.717 92,000 25,000 215 3.22 171.8 0.756 250,000 3.6 50.7 2.17 158.3 1.074 92.000 3.4 50.7 Most Probable Numbers in Plate Counts Millions coliform aerobic cocci 2.4 Table XIV - Bacterial Flora and Vitamin (Pooled Samples from Date Group Description of Sample Av. wt. at 4 wks. Niacin Biotin y/mi my/ml 102951 1 Basal "C" 180.7 71.60 34.0 103051 2 Basal WC" and 10 mg pen. 1 lb. 228.9 57.25 34.0 113151 3 Basal MC" and ■ 0.1$ Ethomid C/15 194.0 63.60 32.0 110951 1 Basal "A" 3Y $ cry. B 12 and Pen. (1) 137.8 55.25 30.0 2 Basal "A" 3Y $ cry. B 12 132.9 60.50 73.0 6 Basal "A" 15Y $ cry. B 12 124.0 62.70 53.5 7 Basal "A” 3Y $ FP B 12 & Pen. 130.4 63.85 69.0 8 Basal "A" 3Y $ FP B 12 128.6 58.10 72.0 Basal "A” 15Y $ FP B 12 124.8 51.05 51.5 12 1. 2. Abbreviations: cry. - crystal line; FP - Fermentation Groups 1,2,3 on exp. diet 3 wks. Total wt. shown. Content of Chick - Duodena 4 Chicks Each Group) Vitamin B 12 Eolic Acid Ribo­ flavin my/ml my/ml y/ml Most Probable Numbers per ml. coliforms cocci Plate Counts Millions (in thousands) aerobic anaerobic 33.5 125 14.39 740,000 250,000 66,500 82,220 20.2 242 15.33 20 430 110 190 122 13.80 250,000 2,500 375 295 32.5 450 11.77 740 240 564 912 63.9 525 12.88 91 92,000 4,130 3,460 116.5 342 12.66 240 25,000 3,111 1,270 81.0 498 13.99 36 9,200 189 7,310 65.0 418 13.60 20 43,000 435 63.8 293 10.91 9,200 25,000 2,561 5.85 205.4 1,745 Products containing B 12, Pen. = Procaine Penicillin 0.002$. All other groups on basal 2 wks., exp. diet 2 wks. Total gains shown. Table XV - Bacterial Flora and Vitamin (Pooled Samples from Date Group Description of Sample Av. wt. at 4 wks. Niacin y/ml Biotin my/ml 102951 1 Basal »Ctt 180.7 20.45 39.5 103051 2 Basal "C» and 10 m g 1 lb. Pen. 228.9 30.25 27.5 103151 3 Basal "C" and 0.1$ Ethomid C/15 194.0 25.55 23.2 110951 1 Basal **A" 3Y $ cry. B 12 & .002$ Pen (l) 137.8 33.90 37.0 111251 2 Basal "A” 3Y $ cry. B 12 132.9 16.45 19.0 111351 6 Basal "A" 15Y $ cry. B 12 124.0 24.00 28.5 111351 7 Basal "A" 3Y $ FP B 12 & .002$ Pen. 130.4 24.00 27.0 111251 8 Basal "A" 3Y $ FP B 12 128.6 25.65 29.0 111551 12 Basal "A" 15Y $ FP B 12 124.8 23.40 28.0 1. 2. Abbreviations: cry. r crystal line; PP = Fermentation Groups 1,2,3 on exp. diet 3 wks. Total wt. shown. Content of Chick - Small Intestines 4 Chicks Each Group) Vitamin B 12 Folic Acid Ribo­ flavin my/ml my/ml y/ml Most Probable Numbers per ml. (in thousands) coliforms 35.00 133 6.63 34.2 242 9.40 31.1 166 8.08 82.0 590 6.25 9.2 40.4 441 4.40 0.24 63.8 375 4.34 0.24 79.5 479 8.15 0.43 63.4 492 4.75 0.02 102.0 478 6.62 0.24 1,180 0.073 250 cocci Plate Counts Millions (in millions) aerobic 920 anaerobic 210,000 239,100 6,000 11,800 6,200 1,750 255 1,125 24 4,900 51,700 25 3,110 1,270 7.4 250 0.43 2.5 2,500 2.5 283,.5 5,103 6,268 7,600 55 9,450 Products containing B 12, Pen. r Procaine Penicillin 0.002$. All other groups on basal 2 wks., exp. diet 2 wks. Total gains shown. Table XVI - Bacterial Flora and (Pooled Samples from Date Group Description of Samples Av. wt. at 4 wks. Niacin Biotin y/ml my/ml 102951 1 Basal "0" 180.7 71.55 305 103051 2 Basal "C" and 10 m g 1 lb. Pen. 228.9 High 363 103151 3 Basal "C" 0.1 % Ethomid C/15 194.0 56.60 234 110951 1 Basal "A" 3Y % cry. B 12 & .002% Pen.(l) 137.8 63.30 450 110951 2 Basal "A" 3Y % cry. B 12 .002% Pen. 132.9 59.70 670 110951 6 Basal "A" 15Y % cry. B 12 124.0 63.65 770 110951 7 Basal "A" 3Y % FP B 12 & .002% Pen. 130.4 93.75 395 110951 8 Basal "A" 3Y % FP B 12 128.6 77.50 960 110951 12 Basal "A" 15Y % FP B 12 124.8 59.40 550 1. 2. Abbreviations: cry. = crystal line; FP e Fermentation Groups 1,2,3 on exp. diet 3 wks. Total wt. shown. Vitamin Content of Chick - Ceca 4 Chicks Each Group) Eolic Acid Riboflavin my/ml y/ml Most Probable Numbers per ml. coliforms Plate Counts Millions cocci aerobic anaerobic --- 16.83 1,180 920 1,420 2300 15.00 9,200 147 19 3,500 2638 18.75 118,000 43,000 111 3,960 3334 15.36 9,200 3392 14.58 25,000 9,200 200 220 5400 13.50 140,000 430 704 3,740 6350 17.33 45,000 25 5650 12.75 25,000 2,200 77 2576 14.87 920,000 2,500 37.5 3.9 31.5 21.5 --- 1,135 2,813 74.5 5,270 Products containing B 12, Pen. s Procaine Penicillin 0.002$. All other groups on basal 2 wks, exp. diet 2 wks, Total gains shown. TABLE XVII - DIALYSIS OF CERTAIN B VITAMINS AS AFFECTED BY PENICILLIN Four to Six Hour Dialysates From 50 ml Feed-water Mi x into 800 ml Distilled Water. Feed mix seeded with, fresh cecal feces except where noted. Date 12952 13052 20152 Description of Sample Hrs. Dialysed Niacin Biotin Vitamin B 12 Folic Acid Riboflavin y/ml my/ml my/ml my/ml y/ml Basal feed 6 0.822 .230 .137 2.52 0.02104 Basal feed 6 0.540 .380 .123 3.67 0.02854 Basal feed & Pen..002$ 6 0.720 .310 .117 3.04 0.01633 Basal feed & Pen..002$ 6 0.777 .385 .129 1.17 0.02483 Basal feed 6 0.705 .610 .121 1.54 0.03500 Basal feed 6 0.565 .460 --- 6.48 Basal feed & Pen..002$ 6 0.535 .750 .123 0.98 0.02542 Basal feed & Pen. .002$ 6 0.500 .760 .123 2.15 0.02846 Basal feed 5 0.812 .760 .084 1.13 0.02266 Basal feed 5 0.880 .790 .118 1.05 0.02179 Basal feed & Pen..002$ 5 0.404 .730 .104 0.70 0.02233 Basal feed & Pen..002$ 5 0.417 .755 .096 0.70 0.02229 Table XVII - Continued Page 2 Date Description of Sample Hrs. Dialysed Niacin y/ml 20252 20552 20752 Biotin Vitamin B 12 Folic Acid Ribo­ flavin my/ml my/ml my/ml y/ml Basal feed 4 0.4L0 .590 .029 3.74 0.02158 Basal feed & Pen. .0004$ 4 0.460 .656 .029 1.05 0.02333 Basal feed & Pen. .0004$ 4 0.777 .550 .015 0.40 0.01978 Basal, not seeded 5 0.755 .450 .020 0.75 0.01896 Basal, seeded 5 0.805 .550 .127 1.17 0.02212 Basal & Pen. .0004$ 5 0.805 .518 .117 1.17 0.02142 Basal, not seeded 4 0.822 .620 .026 1.30 0.02315 Basal, not seeded & Pen. .0008$ 4 0.827 .660 .026 0.90 0.02283 Basal, seeded 4 0.868 .716 .061 1.38 0.02399 Basal, seeded & Pen. .0008$ 4 0.798 .610 .065 0.98 0.02042 TABLE XVIII - BACTERIAL FLORA AM) VITAMIN CONTENT OF CHECK FEED SAMPLES AS AFFECTED BY PENICILLIN Contents of Dialysis Sacs at 0 and 8 Hours (41°C) (Per ml) Experiment I Date Description of Sample Niacin y / m i .. 90651 90851 Biotin my/ml 0.60 4,500 2,500 77.83 0.16 140,000 950 1,418 8.10 --- 0.13 15,000 140,000 721 1.7 9.90 --- 0.21 45,000 110,000 134 6.5 9.20 ___ 0.20 140.000 750 154 --- --- 1,500 1,500 116 --- 0.26 45,000 250,000 108 0.31 25,000 250,000 299 ---- 8.1 Basal 8 hrs. 0.50 13.0 10.35 Basal 8 hrs. 0.60 4.5 Penicillin (.002$) 8 hrs. 1.50 Penicillin (.002$) 8 hrs. 1.00 16.38 13.7 Basal 8 hrs. 1,60 --- Basal 8 hrs. 1.47 --- Penicillin (.002$) 8 hrs. 3.00 --- Control ffzerow hrs. Plate Counts Millions aerobic --- --- Control "zero" hrs. Penicillin (.002$) 8 hrs. 2.96 92651 Vitamin Folic RiboMost Probable B 12 Acid flavin Numbers in _____________ ___________________ Thousands_________ coli f oiffl cocci my/ml my/ml y/mi --108 --- 11.6 108 --- 0.69 250,000 25 300 108 _____ 0.23 450.000 95 1.291 82.9 0.60 950 450 26 Control "zero" hrs. 16.17 11.30 Basal 8 hrs. 18.40 2.70 59 21.8 0.26 110,000 650 124 Basal 8 hrs. 19.65 1.30 35 29.7 0.27 7,500 700 135 Penicillin(,002$)8hrs. 17.60 0.22 42 50.5 0.41 25,000 4,000 37 Penicillin(,002$)8hrs. 19.20 41 44.7 0.20 25,000 6,500 35.5 1.34 Table XVIII - Continued. Exp. II. Date Hrs. Dial­ ysed Description of Sample pH ..... -... ........... 12952 13052 20252 205 52 20752 Contents Niacin y/ml Control (not dialysed) 0 ---- --- Basal 6 4.3 1.88 Basal 6 4.3 1.88 Basal & Penicillin .002$ 6 6.12 1.98 Basal & Penicillin .002$ 6 6.20 1.75 Control (not dialysed) 0 6.70 --- Basal 6 5.88 1.55 Basal 6 6.22 4.60 Basal & Penicillin .002$ 6 6.43 2.93 Control (not dialysed) 0 7.50 --- Basal 4 7.65 2.86 Basal & Penicillin .0004$ 4 7.63 2.60 Basal & Penicillin .0004$ 4 7.75 4.08 Control, seeded 0 7.56 --- Basal, not seeded 5 7.93 0.08 Basal, seeded 5 6.55 1.15 Basal, seeded & Penicillin .0004$ 5 6.55 1.07 Control, seeded 0 7.41 --- Basal, not seeded 4 7.81 1.45 Basal, not seeded & Pen. .0008$ 4 7.97 1.75 Basal, seeded 4 6.82 2.62 Basal, seeded & Pen. .0008$ 4 6.90 2.83 of Dialysis Sacs at 0 and 4-6 Hours (41 C) Biotin Folic Acid Ribo­ flavin my/ml my/ml y/mi --- --- Most Probable Numbers in coliforms Plate Counts cocci aerobic 4.50 45.00 0 ---- 4.28 .2399 140.00 140.00 2,200.00 0.640 5.12 .1937 140.00 140.00 1,920.00 4.80 4.46 .3771 140.00 0.40 470.00 5.04 . 4.25 140.00 2.50 . . .4016. _ ..._ . 0.025 11.50 _ . 270.00' 1“ 0 ---- 3.68 .4480 25.00 2,500.00 3,470.00 0.30 6.52 .4059 14.00 25,000.00 4,730.00 0.50 7.15 .4550 14,000.00 0.09 3,500.00 ---- --- --- 0.43 92.00 6.30 4.20 76.50 .0880 7.50 7.50 24.60 1.00 78.00 .0738 25.00 0.90 42.40 5.84 81.10 __ 45.00 1.50 25.00 --- 0.25 4.50 2.28 --1.26 66.60 .1666 1.95 0.95 12.90 4.10 67.00 .3610 450.00 15.00 2,060.00 0.74 68.00 .3333 1,100.00 0.75 2,050.00 ---- --- --- 0.43 0.24 0.65 1.03 12.30 .2100 2.50 2,500.00 0.70 1.16 10.30 .2158 2.50 0.03 1.24 2.20 60.00 .4083 43,000.00 39.00 1,880.00 6.60 69.00 .4292 43,000.00 0.03 1,635.00 i APPEMDIX STARSliTG HASH Used in Experiment II Surface Tension Studies lbs. Ground yellov/ corn Soybean oil meal Dehydrated alfalfa meal Oyster shell flour Iodized salt Pish oil * Fortafeed 249C Choline chloride Vitamin 3 12 crystalline Haganese sulfate Bone meal 58.4 35.0 2.5 2.5 0.25 0.20 0.15 17.0 g 0.3 g 10.0 g 2.5 100 2 * 500 Units A and 2250 Units D vitamins. BASAL CHICl FEED Used in in vitro experiments• Per Kilogram feed: Corn Soy bean oil meal Alfalfa Bone meal CaCOg 554 350 50 5 g g g g jiaOl Fermentation solubles B-4 (500 Riboflavin units per g) Choline chloride I-InS04 Hiacin D g HuO nig 50 mg 0.049 g ii COMPOSITION 01 BASAL Cl11CK BBBDS Basel "A11 (Us) Soybean Oil meal 50.0 59.4 Ground yellov; corn Dehydrated alfalfa meal 5.0 5.0 Steamed lonemeal Oyster shell flour 1.5 B-Y feed (500 micrograms riboflavin oer gram) 0.3 Pish oil (4-00D 2000A) 0.2 0.5 Salt (iodized) Choline Chloride 0.1 0.005 Nicotinic acid 0.022 manganese sulfate ilerck B 12 supplement (4) 0 Lederle Bortafeed (2-49c) r\ Basal "3" (1 1 s) Basal "C" (1 1 s) 35.0 55.3 5.0 3.0 0.5 32.0 61.4 2.5 2.5 0.3 --- 0.2 1.0 (1 ) 0.5 C.l 10.0 g 2.5 g (2) 1-25 g 0 Vitamin mixture (mg. per kg. feed) used in all feeds: Thiamin KOI 2 Calcium pantothenate 15 'vitamin B 6 4 Polic acid 1 Biotin 0.2 ,-,600 units D, 2250 units A ^grams per 1 0 0 1 1 . feed ^300 units 3D, 2000 units A "’12.5 meg vitamin B 12 11. 0.2 0.25 17.0 (2 ) ---1 0 . 0 g (2 ) 1.0 g 0.15 11. B a s a l "D" (Us) 35.0 5 5 .3 5.0 ■3.0 0.5 0.3 0.2 0.5 (3) 0.1 2.25 g 10.0 g 0.1 g O Ill COMPOSITION 0? MEDIA Dextrose Aside Medium. * Percent Tryptose (Difco) Beef extract Dextrose Sodium chloride Sodium azide Final pH - 7.2 1.5 0.45 0.75 0.75 0.02 * Modified to include three ml of 0.5 percent alcoholic Bromthymol blue indicator per liter of broth. Carrot-Liver Medium Carrot extract (l) Liver extract (2) Difco peptomized milk Difco neopeptone Washed a.gar Distilled water Adjust pH to 7.0 £ 0.1 pH (modified) 1 0 0 ml 1 00 ml 10 g 5 g 12 g 300 ml 1. One pound of carrots steamed one hour in one liter of distilled water, filtered through cheesecloth, bottled and sterilized in 100 ml of portions. Sediment removed by centrifugation prior to making medium. 2. Substituted Difco dehydrated liver in equivalent of one pound of fresh liver. Heated 70°C. for thirty minutes. Treatment same as for carrot extract. One of Squibbs B-complex Formula Tablets was crushed and ground by mortar and pestle and added to the medium. IV PSSPASAi‘1Oil OF F3HD SLUFHY FOF DIALYSIS Chicle feed (l) 50 g ilagKPO^ . 12HP0 15 g (or anhydrous Has HFO4 ) 5.95 g IfaOII, 10?i, to adjust pH to 8 .2-8.3 nil (phenolphthalein indicator) HpO 250 ml Cecal chicken feces (2) 4-8 g 1 See appendix, p. iii, for formula 2 Feces from chickens not fed antibiotics or antibiotic fe ed suoplemen ts. The first five items were thoroughly mixed and incubated 4 hours at 37 C. The slurry was then neutralised to pH 5.8-7.0 using conc. HC1. (half-strength, C.P.) mixed in a daring Blendor. Cecal feces were added and The slurry was equally divided, one half saved as "control11 feed, the other mixed \*ith penicillin to give final concentrations of 0.002 percent or 0.0004 percent or 0.0008 percent. Two 50-ml batches of each vere fumieled into cellophane dialysis sacs, the remainder (controls) for bacterial assay. V DETHRMIITAI11OH OF B VI1AMI1TS Hi ac in Biotin Range Assay organism Vitamins Lactobacillus arabinosus ii it ii it 0 - 0.5 jr 0 .- 1.0 y Vit. 3 12 Lactobacillus leiciiaanii 0 - 1.0 y Folic acid Streptococcus faecalis 0 - 5 . 0 my Riboflavin Lactobacillus casei 0 - 0.25 y Media, for carrying cultures: Tomato juice agar made with, freshly centrifugated canned tomato juice. Assay media conforms to Methods of Assay (60) stan­ dards. Incubation at 57 C for 72 hours followed by titration with ll/lO HC1. gave values comparable with standardised values by extrapulation from plotted curves. were provided for each assay. Standards