HI IIIIH H 4“ ‘ Ll IHHIII WW llll‘ 117 361 THS ENFLUENCE OF MILK AM} STOCK DIETS ON THE ENTESTINAL FLQRA OP CECEC?OMIZEB AND NORMAL RATS Thesis far iha Qagree of M. S MICHIGAN STATE COLLEGE 3hirf0ybe|l McClure 1949 J 9’)? (/7; Jig/L, \ THEbIS ' ‘ , *' ii‘_ _.: 3 WWIIllll|I||lllll|llllllllllllllllllllfllllllllflllll i 1293W00776 0063 a »s‘ -._'p .. 'I‘. \a q A 9.9:: 2:. «is. E; INFLUENCE OF inlLK 1:111 STOCK DIJTS C'EJ Till ILI'I‘ZSHL’II'éAL FLOEA OF CiCLCTUEILJD th fiUthL HuTj b! SHIRLEYBELL IQQCLURE A THEQIS Submitted to the School of Graduate ptudies of hichigan State College of Agriculture and Applied science in partial fulfillment of the requirements for the degree of MnSTaR 0F SCIEECE Department of Bacteriology 1949 THESIS AC KN 011113 BC Eiaifli T The author wishes to express her sincere appreciation to Dr. H. J. Starseth for his advice and assistance; and to Dr. Dane C. Cederquist and Dr. Margaret.A. Ohlson for their.000peration and counsel in connection with this study; and to Dr. Pauline 0. Paul and Dr. Walter R. Mack for their assistance in the photography. Tl TABLE OF corsrmrs Introduction......................... 1 Review of Literature................. 1 Experimental hethOGS................. 13 Results and Discussion............... 22 Summary.............................. 3h References........................... 35 INTRODUCTION This study was undertaken in an effort to determine certain factors that might possibly prove of value in the investigation of the intestinal flora and its nutritional significance. Work in this direction has been in pregress for some time in the Department of Foods and Nutrition, School of Home Economics, Michigan State College. The bacteriological study was undertaken with the hOpe of determining possible differences between the intestinal microflora of cecectomized rats and normal controls; and to observe how these floral differences might be affected by certain stock and milk diets. Only the numerically important organisms of the microflora of the two groups were considered. REVIEW OF LITERATURE The importance of the bacterial flora of the intestines probably was recognized for the first time in 1885 when Louis Pasteur suggested that animal life would be impossible without the cosperation of the microorganisms found in the digestive tract. Soon, thereafter, Hirstler (1886) and Winternitz (1892) found that certain foods gave rise to putrefactive products in the intestines, and later the alterable characteristic of the intestinal flora was demonstrated by Eetchnikoff (1901). These studies suggested to subsequent investigators that the diet exerts a marked influence over the bacterial flora. tith this thought in mind, studies on this problem were started by Belonsky (1907), Tissier (1908), Herter and Kendall (1909) and Bull and Bettger (1917). These and other early studies demonstrated that a pre- dominance of proteolytic bacteria was induced when the diet consisted largely of meat or other protein food, and that an aciduric flora became dominant when the diet was high in carbohydrate (Torrey, 1919; Cannon, et a1., 1920; Porter and Rettger, 19AO; Winblad, 19h1; Mitchell and Isbell, 19h2). Nath et al., (1948) found contrasting results with dextrin, but the majority of workers have discovered that of the many carbohydrates investigated, dextrin (Herter and Kendall, 1909; Cannon et al., 1920; Porter and Rettger, l9t0) and lactose (Mitchell, 1927; Ruickshank, 1928) were most ef- fective in producing a flora primarily aciduric in nature. Since these are the carbohydrates most incompletely digested, and reach the lower part of the digestive tract they probably provide a suitable substrate for the development of an aciduric type of flora. Along with the diet studies, observations were made which brought attention to the possibility of bacterial synthesis of accessory food substances within the intestines. Among the early observations were those of Osborne and hendel (1911), who noted that rats, maintained for long periods of time on isolated food stuffs, became cOprOphagists, and that the addition of a small amount of feces from a normally fed rat stopped the decline in growth of the deficient animals. COOper (l9lh), however, was perhaps the first to suggest the -2- actual synthesis of a vitamin by the intestinal flora. about the same time Theiler, Green and Viljoen (1915) advanced a similar hypothesis. Steentock, Cell and Nelson (1923) and Dutcher and Francis (1923) called attention to the fact that the ingestion of excreta by rats, subsisting on a so-called vitamin B free diet led to the cessation of vitamin B defi- ciency symptoms. Heller, hosiery and Garlock (1925) confirmed these findings. These and similar studies, simulated a vast amount of research aimed at the determination of the factors involved in the probable synthesis of vitamins within the intestinal tract. The synthetic activities of certain of the intestinal organisms were very difficult to determine, until a satis- factory bacteriostatic agent for the organisms was discovered. The early use of drugs as modifying agents (Bouchard, 1887) was for the most part disappointing. In 19h0 Marshall et a1., reported the use of sulfonamides, and Black et a1., (1941) and flackenzie et a1., (l9h1) immediately demonstrated their satisfactory bacteriostatic effects. Certain of these drugs have but a slight toxicity, are slowly absorbed from the intestinal tract and modify the flora by inhibiting the deve10pment of certain groups of organisms. The feeding of the sulfonamides (9.3. sulfaguanldine, succinylsulfathiazole) was found by various workers to de- crease the coliform count in the feces (White, 1942; Gant et a1., 19h2; Miller, l9th). Evanson et a1., (19h5) found that succinysulfathiazole depressed the lactobacillus as well as -3- the coliform groups, and that the lactobaoilli showed a slower decrease in numbers, but a more permanent one. Several workers discovered that, deepitc the decrease in numbers of organisms within certain groups, the total numbers or organisms seemed to remain fairly constant (Light et el., l9t2; Cant et a1., 19h3; Miller, l9hh; and Evaneon et a1., l9h5). The reduction in numbers found in the lacto- bscilli and coliform groups in the studies of Evanson et 81., (19L5) was found, in part, to be compensated for by the in- crease in numbers of enterococci and yeastlike forms, and Gent et a1., (19h3) found that enterococci replaced the coliforms during the time of repressed Escherichia 22;; growth. The inhibition of the growth of certain orgunisms by the use of sulfonamides has been found to be temporary. It appears that the organisms become resistant to the drug and often resume their original numbers, (Gent, et a1., 1943; miller, 19A5) even though some strains may lose their ability to resynthesize vitamins. In addition to depression of bacteriological growth, there is a reduction in the rate of growth of rats on syn- thetic diets receiving certain sulfcnamides (Black et 81., l9hl; mackcnzie et a1., 1951; martin, 19t2; Tepiey at al., l9h6). The retarding of growth and the signs of nutritional deficiency seen in rats fed purified diets containing the sulfonamides (Black et a1., 19Al; Reich and :right, 1943: miller, l9hh) is thought to be due to the inhibitory action -h- of the drugs on the vitamin synthesizing bacteria of the intestines. Tepley et a1., (l9h6) indicate some variance in this connection depending on the bacteria, drugs, and vitamins involved. Their studies revealed that phthaly- sulfathiazole produced a marked decrease in the concentra- tion of niacin in the cecal contents, but the neon increased in size so that the total amounts remained about the same. The folic acid content, however, was greatly decreased even on a total basis. It appears that rats possess limited ability to syn- thesize members of the B-complcx by means of he intestinal microorganisms. Neilson et a1., l9t2; Ioolley, i9b2: Light et a1., 19t2; Kitchell and Isbsll, 19h2; Taylor et a1., 19L2; schweigcrt et a1., 19h5; Eivehfiem, 13n6 and Tepley et a1., 19h7. are among the many workers who have conducted experi- ments concerning this synthesis. Dam et a1., 19bl and Day et a1., 19h}, studied the synthesis of vitamin K in the in- testinai tract. In view of the fact that diet is highly influential in the deveiOpnent of a certain kind of flora, it is not unex- pected to find it of considerable i portance in the bacterial synthesis of certain vitamins. Recently Hitchell and Isbcll (19n2) and Taylor et a1., (l9t2) have emphasized the marked effect diet has not only on the quality but also on the quantity of the intestinal flora, and point out that these factors in turn are reflected in the vitamin s"nthesis. Etudies dealing with the effects -5- of the carbohydrates on the vitamin synthesis have been con- ducted extensively beginning with the early eork of Hull and hettger (l9lh) to the more recent investigations of Gall et a1., (l9t8). Investigators point out that irreSpective of the B vitamin being studied, the requirements for that vitamin are reduced when relatively insoluble carbohydrates like dex- trin or etarch are used in the diet; lactose greatly favors the production of some of the B vitamins, but glucose, sucrose, and other readily assimilated sugars are generally without appreciable effect in this respect (Fridericia et el., 1927; Cruickshank, 1928; Guerrant et el., 1935, a, b, 1935. 1937; horgan et al., l938; Schweigert et el.. l9h5: Sarma et 81-. l9t6; Tepiey et a1., l9h7; Elvehjem end Krehl, l9h7). The fat component of the diet has received less atten- tion than the carbohydrate portion, but considerable attention has been given to its effect on the intestinal flora. Among the recent studies on the role of fete, Boutwell (19L3) demon- strated that when lactose is the sole carbohydrate, butterfat is superior to corn oil in effecting the growth of rats; Tepley (l9h6) found that on "synthetic milk" diets butterfat resulted in greater vitamin synthesis than did corn oil, and hath et 31., (19L8) found the aerobic and anaerobic plate counts as well an the numbers of coliforms to be decreased in the cone of most of the rats fed sucrose diets containing a high level of corn oil. Eennering et el., (l9hh) demonstrated that riboflavin deficient rate survive for shorter periods of time when fed a high fat ration than when maintained on a high carbohydrate -5- diet. Elvehjen and Krohl (1947). however, have pointed out he effect 0! rot in moiifyin; tno ruiuironcnt for some of tho B Vi’ngius. In tho studios of vitigln synthesis by the integtinal flora, torkern hove endeavored to establish the exact location or such agnthczin in tufl into tlnol tract. The cecum nus been postulated by tony to he the main site of vitamin synthesis (Tissier, 938; Cannon at 51., 1326; Guerrant at 61., 193A; Griffith, 1335; liaison et a1., lfitfi; Taylor et ul., 19L2; Day et al., 19h3; flchteigert et 31., (l9t5). fiomever, Day et a1., (1943) state, that while the cecum is an important site or the :gntbsais or vitamin K, this vitamin can also be formed in otLer parts of the intestinal tr ct. Griffith (1935) falnd that copropnu;y i proved the condition or ceceotomizod nninnla and concluded that vitamin synthesis occurs in otnor pnrts of tno alimentary tract. ainoe the ocean hon been considered to be the site of much vitumin synthesis, several workers hive conducted nutri- tional atuniao on rats deprived of tneir ceoa (Jriffith, 1935: Taylor et a1., 1942; buy at al., 1943; :ohaeigert et a1., l9h5)o Provided the diet man I git, the oooum was founfl to make a contribution to the rat's supply or some of the B vitamins and it mus of intereet to observe, that as a whole, cacao- tomized anixals grew as well as the controls provided the diet was nde,u3te (Griffith, 1935; Taylor et a1., 19h2; _ Day et a1., 1943). However, Taylor at al., (19t2) found -7- that, on a diet such as the pyridorin-deficibnt ration II or (Donner and Elvobjem, the cocoa adds very littlc to the vitamin sources already contained in the diet, and Lohweigert at 31., (19L5) indicatod that rnto arc not dependent on the cecum to any'oxtent for the production or absorption or certain of the B complex vitamins then on a sucrose dict. With the knowledge that bacterial vitamin synthesil occurs within the inte tinnl tract, the availability or these vitamins to the aninal was to be determined. Among other “workers, Guerrant et a1., (193t) and Griffith (1935) indi- cated that althou h vitamin synthesis occurred in the intestinal tract, these vitnninn did not appear aVailablo for absorption until tho animals resorted to cOprOphagy. This seemed probnblo when the work of Abdfil - oalum et a1., (1938) showed that there in a nynthosia of thinnin by a mixed cecal flora in vitro, althouih no diffusion of the synthesized vitamin B from the bacterial cells into the broth medium was detected; indicating that the vitamins may be kept within the bacterial celln baking them unavailable for abnorption. Fitcholl at al., (19L2), discovered, how- ever, that in addition to thinning inositol, niootinic acid and riboflavin are found in considerable guantitien in bac- terial coils and that puntothonio acid and folio ucid diffuso to a greater extent, while biotin and pyridoxin apparently move freely from the cells into the surrounding medium. Kitchell et o1., (19t2) indicated that the vitamins that do escape into the medium are vit nina which can be absorbed by body tissues. Schweinert et sl., (l9h5) believed that intestinal absorption of some of the vitamins synthesized accounted for increased growth rates observed in some of their experimental enioals. Eitchell at 31., (1942) point out that the amount of vitamin absorption is a reflection of the amount of vitamin in the medium surrounding the bacteria and not the total Quantity synthesized; and that the problem of the total quantity of absorption is compile cated by the possible synthesis of vitamins by body tissues. It appears probable that at least a part of the vita- mins synthesized by the intestinal flora is absorbed by animal tissues and might thus partially or completely satisfy the reguirements of a rut for particular vitamins, while the rest are not readily available to the animal unless coprOphsgy is practiced. Along with the absorption, the anatomical segment where absorption occurs has been studied. Selye, (19k3) found that in the small intestine, riboflavin in both absorbed and ex- croted, while in the cecum and colon injected riboflavin is destroyed with little if any absorption. The studies of Hitchell at 31., (19L2) and Schweigert at 31., (l9h5) indicate the cecum to be the principal point of vitamin absorption. In all probability the kind of vitamins and the form in which the vitamins are hold is an important factor in this reapect. It is known that diet is influential in establishing the nature of the flora and certain flora are believed to be syn- thesizers of particular vitamins, but the exact role of bacterial vitamin synthesis is still questionable. It is possible that some of the organisms that occur in small numbers may directly or indirectly affect the floral products, and in their metabolic activities maintain or produce conditions helpful or harmful to bacterial vitamin synthesis and absorption. Considerable nork indicates thnt coliform end lectobecilli firoups are major influences, and theme organisms are reported by Torter and Eettger (l9h0) to be the most pre- volent in the intestinal tract. Eitchell et ul., (l9b2) demonstrated some interesting points concerning the above groupa of orgsniams es the re ult of feedint around lean beef to eupport e proteelytio flora and ground lean beef containing 255 lactose to enamort en eciduric flora. It W93 observed that the acidophilic flora allowed the pentothenic ecid preduced to diffuse more freely into the surrounding medium thn did the coliform flora. A siniler situation existed with the folio acid but to a lesser extent. on Opposite effect was demon- strated with pyridoxine end thiamin where the vitamin ' availability was favored by the coliform type of organisms. In studies of this kind it is well to keep in mind, an Day at 31., (19h3) brought out, that Variability is shown by individual organisms and the vitamins they produce. Attempts have been made to find additional information in connection with the intestinal flora by attempting to raise animals eeepticolly. (Glinstedt, 1936; Lobund Reports, 1946) Although this will not offer finel proof of that heppene under normal conditions it will undoubtedly revcel many facts of prime importance concerning the intestinal flora. Essentially the intestinal flora of men and animals are much alike. The white rat has been found to be the most suit- able experimental animal for studies of this character and has therefore been used by the majority of investigators in this field. Although this review has dealt primarily with investigations involving rate many contributions to the study of the bacterial flora of the intestines have been made using other animals: monkeys, Waisman et al.. (1943) McCall et al., (l9h6); mice, White (l9hl); dogs, Poth (l9h2); guinea pigs, Crelius and Rettger (l9h3): humans, Hajjar et el., (19hh): fowl, Luchey et a1., (19L6); Johansson et a1., (l9h7); Couch et 31., (l9h8). Ae work advances in determining the nutritional role of the intestintl flora it is clear that before too many assump- tions can be made, an overall picture is imperative, if indeed, the true picture can be seen at all under the multitude of complexities and variebilities produced by the bioloaicel pro- cesses that take place in the intestinal tract. This in evidenced in this particular review by the variety of results cited on the various problems discussed. In this connection, it is of interest to note that very recently Nath et el.. (l9h8) found that a lactose diet induced a high coliform.ee well as a high lectobecilli count in the cecwm of the rat. The effect of lactose in inducing a high aciduric flora has been known since early times, but its effect in maintaining a high coliform flora has not been reported in previous work. as formerly stated, many workers indicate that conditions supporting a lactcbacillus flora tend to discourage the growth of proteolytic flora and it is reported that this is due to the acid produced as a result of the metabolic activity of the lactcbscilli. Weinstein, Weiss and Gillispie (1938) found a connec- tion between an asidephilio flora and a low pH value, but' they were unable to ascertain any absolute correlation. hath et 81., (1958) state that since the proteolytic organ- isms are lactose fermenters there is no reason why they should be repressed by a lactose diet. In view of this statement, it is interesting to note the pH ranges for bac- terial growth given by Porter (1946). It is observed that the optimum pH for the growth of Laotobacillus ecidoghilus is 5.8 . 6.6 and for §&_ggli’6o7, while the minimum pH values for growth are h-h.6 for the 5:.ecidcphilus and k.h for the 3;,2gli. Perhaps then, if further work does support the ma- Jority of previous studies and e lactose diet is found to favor the lactobacillus groups it is possible that the acid produced, due to growth of these organisms, may not be the only factor of primary importance in the depression of proteolytic organisms in such a diet. It is more than likely that the environment, supplying Optimal conditions for the aciduric bacteria, causes population increases that depress ccliform groups by virtue of their numbers. -12- host of the recent work has pertained to those factors which would indicate the bacterial flora to be of nutritional value to the host. However, certain types or bacteria may be deleterious because they could possibly destroy or utilize vitamins and amino acids or produce toxic materials which may be abeorbed end retard normnl metabolism. vaiously studies on nutrition may have little meaning until more is knoen about the production and destruction of nutrients in the digestive tract. EXPELIQLNTnL HETBODS All experiments were conducted with weanling albino rats from the Spregue Dewley Colony, fiedieon, tieconein. The rat; were individually housed in raised, wire- meehed bottom cages to prevent ooorophegy. The milk diet used consisted of evaporated Carnation milk, diluted equally one to one with tap water, and supple- mented by a mixture of iron phoephate and magnesium and capper sulphate. The stock diet consisted of: yEIIOW corn meal............5,000 parts linseed oil meel............l,600 parts alfalfa MGfilooeoeoeooooeoooo 200 parts casein.........o............ 500 parts Whflat germ.................ol,000 parts yeast....................... 500 Part5 DOWGOPOd flilkooooooeoooooooo 500 part8 sodium chloride............. 50 parts calcium cerbonete........... 50 parts A: dispensed this meal was moisted with corn oil. -13- In the choice of media it was considered important to have a set of media in which determinations could be made' under the same conditions. Considering the lerze numbers of organisms to be estimated, a dilution count method was thought most practical, and was engloyed for all organisms so that, as nearly as possible, conparsble results could be obtained. It was considered necessary thst one medium give e total count of the viable bacteria, end that the selective media allow estimation of the relative numbers of each of the individual groups or bacteria. Evenson at 51., (19L6) reported a useful set or media which, for the most part, was found satisfactory in this study. Liver infusion broth was used for estimating the total numbers of viable bacteria because it offered an edeguute nitrogen end carbon nutrient complex for the diverse intes— tinal types, and has been found unentisfnctory only for the yeast-like orjsniens. Because of the difficulty in obtaining fresh dried liver when this experi.entcl study wee begun, dehydrated becto liver powder was used in making the liver extrect for this medium. Before dispensing the medium into the test tubes, approximately .25 am portions or the dehy- drated liver were placed in the bottom of the tubes in lieu of fresh dried liver chunks. This may have partially accounted for the fact thct t is medium was not found sutisfuctory for estinuting -11,- anaerobic growth, as it was reported to have been by Evuneon et a1., (l9t6). headings or bacterial growth were based on turbidity of the medium. Rinbled’e (19Ll) acetic acid medium, without the agar, nee used for the estimation of numbers of lectobecilli. Kulp'e tomato Juice medium.wue used eimulteneouely with the acetic acid medium at the beginning of this study. he wee noted by Eveneon et el., (l9t6) the growth results in the two media were comparable end the use or the tomato Juice medium wee cincontinued. einbled'e medium was also reported to eupport the growth or yeuete which could be detected by god trapped in Durham tubes. since considerable variance was exhibited by this method, and since the liver infusion medium used for total counts did not support the growth of yeasts, the determination of these organisms was not made in this particular study. headings were based on the tubes of tinbled‘e medium shoeing turbidity. The E 0 medium 0: H3333 and Perry (l9t3) was employed as a selective culture medium for the colirorm group. It has been reported the bile eelts mixture in this medium in- hibits the growth of fecal streptococci and spore tormere. Because enterococci have been reported tolerant to bile by other workers (heieeenbacn, 1918; Evans, 19t7) and because of what was considered as possibly low coliforu counts at the beginning or this study, the leuryl sulfate tryptoee broth of Mdllmenn and Derby one used in conjunction with the E C medium under the same set or conditions. The medium or -15. Eellmenn and Derby consists of tryptcse lactose broth plus the selective agent sodium leuryi sulfate in a dilution which has no toxic effect on the desired organisms. Both media were incubuted in two series; one at 37° C. to detect the coliform bacteria as a group; the other, at 45° C. was used for §&_ggli. determination. headings were made on the basis or gas produc- tion. For the enumeration of the enterococci the E F medium or Eujne and Perry (19L3) was employed. Ritn this medium a selective temperature of incubation (LSO CJ one used, and sodium azidc mus incorporated as an inhibitory agent. Tur- bidity and an acid reaction shown by the color change of the brom,creeoi purole were growth criteria. Lince the selective temperature of L50 C. was reported by EVuno (19b?) to separate the enterococci from the other streptococci and 37° C. was reported as the optimum temperature for their growth, a set of tubes was incubated at 37° C. us tell no at A50 C. All media employed in this study were checked periodi- cally by microecooic examination. Gram'o stain Wee ueed. Figures 1-3 illustrate smears typical of those obtained throughout this study, and show that the groups or bacteria being determined in this study predominated in the selective media employed for the estimation of their numbers. A group of A0 weanling rate were placed on the exocri- mentel diets. Twenty of these nnimale were fed the stock diet, and twenty others were fed the milk diet. Half of the animals in each diet group were cecectonized. couanauo «on .0 9R 5.3.: n m «cocoouoaam n Eva I6d¢5dac man .u a: .33.: o a Ilka-«dado u i: .floduaqnfihouou .305 “on 003 denudauv and .0 aka Ifldcflfl n.0ddnqun «AduoufioaOGH ' ;\\\\\\\~\~\~\~‘\. \‘ .— é: ducal ouuuoouoo 0:» ca wddaouw aauuazmuo wuuaqqaeoooua ona an ac coon one: aluouaaoo can .«aaaoanouond .«ooooououao on» naaooaaoououodu .nunu anodes nuuonno gnu uo maqouaoo naauuaoaau on» no aohauaso Aauuoaoon n¢hu oc¢l_nudllm .17. The eni ale in this eXpori;entel work were subjected to cecectony employing the method devised by Dr. Rode Brinker of the School of Veterinary Hedicine, Eichi;en Etete college. The erperirental rate were anesthetized with sodium pentoborbitnl. The etendurd solution contained 1 grain per ml., and for immediate use .5 ml. Woe diluted with 9.5 ml. of distilled water. Injectione of .1 ml. per 10 grams of body weight were made into the abdominal cavity. The Opera- tive area was clipped to remove the heir, scrubbed with eoep and meter, and ewebhed with 703 ethyl alcohol. This area was draped with a sterile shroud.- A 1/2 inch incision was mode through the skin, abdom- inal muscles and peritoneum. The incision wan made on the left side starting on a level with the umbilicue and extended posteriorly. The cecum nee loonted ,nd drewn throu h the incision (F1?. 1). The serious attachment between the cecum end the intestine one severed, and two Fficll straight mos- r'uito forceps were claced at the nose of the canon where it joins the inte tine (F12. 2). The cecum wge excised between the too forceps. The stump of cecum mes swebbod with pure phenol and 70$ ethyl alcohol. So. 100 cozton thread ens used as suture meteriul. The euturinq Was begun with a riiht unfle Cushin; etitch taken at the neecnteric border or t e cecun close to the forceps; it was not tied. The needle nus then carried over ("3 he forceps to the other side where another ushin; stitch was mode. The suturing was continued by alternnting from Cocootomy Figure ‘0 .9 00.n- Exponod Figaro 5 Forceps at bgso of ceoum Clamping off or the cecum. -10- one side of the forceps to the other (Fig. 3), covering the entire length of the ntunp. The stitchee were applied approx- imately 1/8 inch and 1/16 inch from the ed;e of the forceps. Tension was applied in Opposite oirectione to the loose ends of suture material and at the some tize the forceps were opened and slowly retracted. After the {creeps more withdrawn 3 short diet use they more dipped into the lumen. This pro- cnnure aided in the inversion of the bcel edges. A second row of Cunning stitching was inserted to reinforce the inner row (tin. L). The abdominal muecle, peritoneum and skin were closed with interrupted cotton euturee. Studies of the to expcri.entel rats were node by Lnnnda (19h?) previous to their autopey. the bacteriolcnicnl cork in the present study w e started by eutognying 1-2 afliguls at a time, and continued until the no experimental anionic hnd been killed. Before nutcpsy the animals were allowed to fast 12 - It hours. Bacterial total counts and colircrn determinations were mode on the intertinel contents following nutcpey (Study I). Approximately .1 gram samples of the contents from the large intestine, small intestine, and the cocum were obtained. Each sample was transferred to a separate, previously weighed sterile tent tube containing :lnea bonds. The weights of the samples were determined, Known dilutions (1:100) mode by the addition of eterilc distilled water, end a uniform auegeneion obtained after thorough shaking. The eunpeneicns or material 11 were further diluted in decimal series to 10' . -20.. CE»;CTGLY Fig”. 60 Exalted oooum; Cunning stitching Operation Completed -21- L, Inoculations or media were made in triplicate, in 1 ml. amounts for each éiiution (10 - 1011). All media were incubated for a period of 72 hours at a temperature of t50 C. or 37° 8., as the mcflium culled for. The counts were con- putcd by a reference to tho Dilution Count Tables of Buchanan and Fulmcr (1923), and averages were obtained by geometric mean. Approximately 9 ycnr later, n second group of uni cla of tho acne number his put under identical cxpcrimcntal con- ditions, and cnterococci and lactobacilli as well as coliform ani total counts more determined ( tuiy II). The fiilutiono were made as previously, except that the entire contents were obtained from the three intottincl sections for tnc first dilution (1:1C0) instant of the .1 gram amounts. This was cone with the possibility that a more representative picture of that section might be obtained. Tables 1 and 2 chow the counts of the bacteria found in the Various sections of the intestines in the to experi- mental rutt uncd in ”tudy II. The counts are expressad as the geometric noun of the individual bacterial counts ob- tained from L groups of animals. These 4 groups consisted of the 13 cacaotomized and 10 control uniruls fed a stock diet, and the same number of cecectomized and control rats fed a milk diet. Differences are seen in the bacterial mo.g; oduanaooo nu connouono nausea Houuououm» ooo.ooo.4~ ooo.oum ooo.ooo.u ooo.o4~ ooo.oo4.a ooo.ooo.o~n conga uooom coma-on a . tooooo o ooo.ooo.oo ooo.o~ ooo.oo~ ooo.o ooo.on ooo.ooo.ooo Hanan gooom oouuoow on .5000 ooo.ooo.do ooo.na ooo.no ooo.o ooo.- ooo.ooo.ooo noooo noooo Houoooo ou ooo.ooo.ona ooo.- ooo.mn~ ooo.o ooo.wm ooo.ooo.o~o canon nooum Houoooo o” ooo.ooo.an ooo.o ooo.n~ ooo.~ ooo. ooo.ooo.ooo ”Noam noouo Hoooooo oa ooo.ooo.oo ooo.oa~ ooo.oo4.o ooo.oo ooo.oo~ ooo.ooo.ooo.a gonna no“: oondaoo taco-u on ooo.ooo.oo ooo.on ooo.ooo ooo.o~ ooo.oo ooo.ooo.omn Hanan noon oouooo» .588 S ooo.ooo.«d ooo.oo ooo.oo~ ooo.u~ ooo.no ooo.ooo.ow~ , noooo noon Houuooo o” ooo.ooo.~n ooo.on ooo.owo ooo.an ooo.mo ooo.ooo.omui .maoa undo «cannon on ooo.ooo.o ooo.o ooo.¢4 ooo.o. ooo.o~ ooo.ooo.om "noon sad: donoooo on «aaoooo «oooo doooo «goo .n.uuooaaoo dooou oooooom undo “nooaoo .oz noaoog nouoouom‘aoooouom woo“ .ooR .83 .ooR 603 .ooR .ook .. 3 w din: , .oooooo ooouo on. Hoops ..HH hogan. .qooaooo Hoooooooou .oo oo noon oo undo ooooodouoo a no ooouom coo pogo o» ooooouuo.oooo nooam ooo noun o on» naam Houaaoo can voudnooooooo no nonuumouan can «a coach cannon «oeuoaodm one .H Odnofi doom cannoncou ca voonounxm ouuooo Houuouooma ooo.ooo.4~ ooo.onn ooo.ooo.m ooo.ooa ooo.ooa.~ .ooo.o~n ownon aw oouoaou a nooooo o ooo.ooo.oo ooo.o- ooo.oo4.o ooo.oo ooo.o4~ .ooo.ooo.~ cocoa noon oowowou on s o co coo.ooo.oo ooo.o~ ooo.oo~ ooo.o ooo.om ooo.ooo.ouo euooao nooon ooooaoo . cocoon 04 ooo.ooo.oo ooo.on ooo.ooa ooo.oa ooo.no .ooo.oon “noon soda voodoo» .638 3 ooo.ooo.oo ooo.na ooo.no ooo.o ooo.- .ooo.ooo cacao nooao Houoooo oa ooo.ooo.- ooo.ao ooo.om~ ooo.- ooo.no .ooo.oo~ noooo. noon Houoooo oa ooo.ooo.ooa . .~m coo.nmo coo.» ooo.~n .ooo.o~o among noon» «ouoooo on ooo.ooo.~m ooo.on ooo.oo4 ooo.An ooo.om .ooo.on~ condo noon Hoooooo oa ooo.ooo.nm .o ooo.n~ coo.“ ooo.~ .ooo.oo¢ Hogan noooo ”ouuooo om ooo.ooo.m .o ooo.4o coo.» ooo.o~ .ooo.on «doom loan Houoooo o” 2:23 «oooo «38 . :3 .u 3838 H38. 333m :8 3333". .oz nooooq ioaoouom -oooouom «noon .OONM .oan4 .OObn .oom4 .ooun .oohn ouoaou auauo< nuoooo ooouo one aoaoa .AHH hcduwu macaw anucmmukcnnm aauaoom HooaanouoH howsoauuom m 30m Iona doom ”chance can 9N Odpak on» oo,oomw no oouoouum on on oomooooo «can and: can goooo a com wonunouoooou uo moouamouaw adv a“ coach nuance «nauouoom Qua -25- counts obtained from the intestinal sections of the cecemtc- mized and control animals fed the some and different dicta, (Table l) and the effects of these diets on the counts in a particular section of the intestines. (Table 2). Tables 1 and 2 also show that, irreSpectivc or the diet, the coliforms were the least numerous and the loctobucilli the most numerous organisms found in the intestinal tract. Lactobucilli counts have been seen to exceed coliforn counts on other stock diets (Evenson et a1., 19t5). The high luctobacilli counts seen on animals fed a milk diet Was not unexpected. Milk, with its high lnctocc content has been known since the early studies of Hull and hettgcr (1917) and Cruiokchunx (1938) to favor the develOpment of an ociduric flora and depress coliform growth. The resultant ociduric flora noon in animals fed the stock diet was thought to be duo to the combined influences of a number of factors. Certain ingredients in the stock diet were considered perti- culorly important in this respect. among these uero: the milk incorporated into the stock feed, the mixture of grain: which have been reported by hull and hettgor (1917) to favor an aciduric flora, the help or certain inorganic elements in maintaining an nciduric growth (Bppricht at 31.. 1937), and the corn oil which has been seen to repress coliforms with little effect on luctcbucilli (Torrey, 1919; Roth ct ol., 19h8). Tables 1 end 2 show that the enterococci were lower in numbers than the lactobacilli, but numerically exceeded -25- the coliforme. ?he writer believes thct the enterococci found conditions favorable to their growth due to the en- vironment produced by the diets used, and the fact that his diet also caused the predominating growth of lacto- bacilli and depressed coliformo. Evenoon et a1., (19h5) found that depression of the coliform group and Gent ct 81., (l9t3) depression of 3; 3211 resulted in an increase of enterococci; Osterlenh and hunter (1946) observed that high total counts were more adverse to the growth of coliforms han to enterococci. Osterlenh and hunter (19L6) also observed in their studies that of 51 fecal samples examined, 37 per cent showed enterococci occurring in canal or in greater numbers than §;,ggli. Tables 1 and 2 further show that the coliforms, other than g, 231; dominated consistently. This has been seen previously by Evenson et a1., (19L5). iablo 3 shows the sum of the total and coliform counts obtained from the intestines of the orperinental animals used in Ftudy I and ftudy II. The counts, obtained a year apart, show similar results. Although the coliforne were found in greater numbers in ’tudy I there was u close correlation in their counts in each study. The ceoectomized animals showed higher numbers of coliforms then did the control sni.als; the differences were seen to be greater in the stock fed eni ale. In both studies the total counts also followed the same general trend. It was of particnlcr interest to note that the cecectonized ani ale on a milk diet showed udou oauuoaooa ad oommmpmmo nausea «auuoaoum» ooo.mo ooo.ooo.o-.~ HH nusam Mooun Houauoo ca ooo.o¢4.~ ooo.ooo.¢om HH have» xooam ccsaaopooooo oH ooo.osd ooo.ooo.~on HH hogan uaan. Heuauou ca ooo.uom ooo.ooo.oma.~ HH nugaw ”dun quuaaaao.ooo od coo.oaa ooo.ooo.aas.~ H macaw macaw “ouuuoo ca ooo.mmo.~ ooo.ooo.o4n H nusum nocaw ooNHeOpooooo on 000.005.“ coo.ooo.umm H unsum and: Houpnoo ca oao.ooo.o ooo.ooo.ooo.o~ H nusam um“: caudaoaooooo ca m§uom~flao aaqsoo Hanna .unnn noun noduaoaoo non-ya .0 ohm .0 can _ mamaaqd .AHH nan H ~aaun. aoan soapy can xaqa a a.» naam Houacoo cam eoudeoaooooo no moadpmoauu on» ad aqua» menace auoudaoo can Haauoaoum annoy one go saw one on canon. -27- showed highs total counts than the control animals on the some diet. Opposite results were seen in the animals fed the stock diet. The cecectomized aninuls on his diet showed lower total counts then the control animals. thnda 519;?) found similar results in fecal samples obtained from the anus or the animals used in Study I. The growth curves sheen in Figure 8 reveal that cecec- tomy had no untoward effect on the growth reten of the cccectomized animals in Ftudy I. Hormel growth was also seen in the ceccctomized animals in .tudy II. The animals were autoneied at approximately the same age and no growth curvee were node, but the neightn of all animals more found compar- able. In the cccectomized enimale, possibly the large numbers of bacteria, the increases of lectobecilli and coli- forme in the milk fed group and the decided increase in the colirorme or the stock fed group may have provided the necessary elements for growth in the form of synthesized accessory food rectors. Black et a1., (l9hl); Gent et a1., (1943); and miller, (19L5); among others believed §;.22ll. to be particularly active in vitamin 3 production. It is well Known that there are many importent componentsin the B complex, and among them thiemin chi niacin are i portant in carbohydrate metabolism, choline in fat metabolism, and riboflavin in biolo:icel ozidetions. The large intestines showed numbers of bacteria compar- able to and often exoccdin: t 036 in the cecu. This was noted most frequently in animals subsistinr on the stock diet. -23- AH hoopmv mBmHQ MAHS QZ£ Mooem zo mB4m QWNHEOBoMomo 924 H~.~m gauche coadpaoo ooo.ooo.aom annoy noopm conduouooooo oa ooo.amm.ama anyone gondaaoo ooo.ooo.oma.~ ”anon 3H“: usa«aoaooooo oH .ousoo wean nodawuaom nonamng .AHH hogan. .ucda uooam can aaaa a com oanflaqd.aouunou cum conaaouooooo no nouaumounm on» noun uoaauuno audaoo nacho Gnu no mafia voaanfloo on» can nulaoo H1009 Addkoaoam one no lam 0&8 .n Ganja Eveneon et el.. (l9h5). In this study it is attributed in pert to the necessity of ueing differential media, and the fact that many of the organisms believed present in the in- teetinel tract were not determined as groups, but may possibly have found a favorable environment in the liver infusion broth employed. The author believes that the organisms accounted for constitute the major portion of the organisms taken into consideration in this study. A great many verieblea are necessarily encountered in study of this kind. In this work it Wee 1 possible to immed- iately examine the samples obtained at collection. They generally remained in their diluent from four to six hour: before cultures could be made. hicroscOpic examinations of cultures were not made after each determination, however, the media were checked periodically throughout the study. In both study I and II considerable variation was ob- served in the bacterial counts from animals which were under the some experimental conditions. thite (l9h2), miller (l9h7). end hath et el., (19t3) found this to be true in their studies. In the cecectomized enimele undoubtedly the absence of the physiological functions of the cecum, e.g.. the absorption of water. influenced to e greet extent the resultant bacterial flora found in these studies. semen? A study was made of the intestinal flora found in cecectomized and control rats fed on stock and milk diets. The cecectomized animals on a milk diet showed higher total counts than the control animals on the some dict. Opposite results were seen in the rots fed the stock diet: the cccectomized animals on this diet showed lower total counts than the control animals. Total counts end lectobecillus counts corresponded in trend. Irrespective of the diet, the coliforme were the least numeroue end the lectobucilli the most numerous organisms found in the intestinal tract. The enterococci were lower in numbers than the lectobscilli, but numerically exceeded the coliforme. Within the coliforn group, organisms other then.§&wggli dominated consistently. Coliform counts were higher in cecec- tonized animals regardless of the diet. Considerable variation was seen in the counts from individual animals under the some experirentsl conditions. The large intestines showed counts comparable to and often larger then those found in the coca. high numbers of bacteria were present in the small intestine of cecectomized and control animals. Cecectomy did not retard the growth of the exocriwentel 83115313 0 “mg/1. & LU? :3 Lu.‘ U“ l. Abdel-Jelnn, 3., and Leong, ?. C., 1938, iyntheris of vithmin El by inte.tine1 b3ctexi3 of tie r t. Eioctem. do. 2:,‘JW"1330 2. Eeloneky, 5., 1907, Influence du ferwont 13ctiguc eur lo flora Gee excremente des sourie. An . Inst. rueteur, 3. Elect, 5., refibbin, .. 2., end 'lvchicm, C. 3.. 19Ll. Use of eulfhfiu.u dine in not: iti'on c. -ri.ont3. Lroc. 306. Exp. Biol. and 10d., £1:33.-3nu. A, Boucherfi, C. 3.,1337. Leccne nor lee eutoint03103tions Gene 133 fifllfidll- :arie. fitted by Lettjur, L. ?., end ChenliL, L. 3., 1321. Yale Univcreity Ercee, ice Eaten, Con . 5. Bouteell, K. 2., Sayer, R. F., Elvehgem, C. A., and hurt, E. 3.. 19L3. Turticr 333C133 on the cowo:3rative value of hatterfet, vegetable oils and c::or.: r; 3rinee. J. Kutrition,‘§§: 601-639. 6. Buchorh n, h. 2., and ul her, 3. 1., 1928. Ehy 1010 y and Bioche'iatry of hectarie, ”illi.-e end hileine 50., Zoltimore, 33.,‘1: 10-12. 7. CE -nnCn’ P. fig. Dr§55tfldt. Lo Lo. find FraflthEt. C. no. 1920.1nte:tin31 obetwiictios. n etufiy of the influo ence of tr -e bacteria 1 flora on the tozerh 3 of acute obstruction. J. Inf. 313.,.g1: 13?-1&h. 8. Cooyor, 1. 3., 191$. On the protective 3nd ceretive preportie 3 cf ccrte in foodstuffs 3;3inet polyneuritie in need by birds by a diet of poli:hed rice. Hart II. J. 13"}, 1.3:: 12-220 9. Couch, J. R., Crevene, I.. 3., Livehjen, C. 3., end Lelpin, J. 3., 19L8. R313tion of carbohydrate to intestinal tyntheeie of Liotin end h3tch3hility in mature foul. J. filtrition, 22; 57-72. 10. Creceliue, fi. 9., and Eettger, Leo F., 19L3.’ The intes- tinal flora of the gainee giz. J. Buct..gg: 1-13. 11. Cruickchank, 3., 1,23. The exporizeutul transfornotlcn of the intestinel flare. Erit. J. EEptl. Puth. 2; 313-325. 12. Dem, H., 81 vino, J., Crle-J eneen, i., and Orle-Jencen, D. l9h1. Eildun van tit .nin K in criibe ctcrien euf syn- thetischem eubetrot. 1M.tll 133, ?”: 23?. Cited by Evensen and nssocietee,1935, J. Loot. 2_; 513-521. 13. 1h. 15. 16. 17. 18. 19. 20. 21. h) h) 23. 24. Day, 3. 0., Fukim, K. 6., Krider, M. fl., and Q'Banlon, E. 3., 19h3, Effects of cccectony auccinyuulfuthlazole and p-amlnobenzoio acid on vitamin K syntheaia 1n the integtinul tract of rats. J. Lutritlan, fig: 585-600. Dhanda, Eulkh £33.. 19A7., Effect of cactuxy on the fecal flora of white rats. (5 130613. ulchigun {tats College). Eutchar, fl., Adams, ant F ancis, Emma, 1923, Vitamin etudiea. E. Feeding technigua in vitamin atuflies, 1923. Proc. boa. ixgtl. E101. fiad.,‘§;: 139-193. Elvehjen, ?. A., 19bé. hale of intagtinnl bacteria in nutrition. J. am. Dietet. 55300. 3;; 939-963. Elvahjem, C. 3., Exenl, $illard H.. 19h7. Imbalance and diettry inter-relaticnahips in nutrition. 3. Am. fled. ASBOGo. 122: k79~237. Eppright, E. 3., Valley, aeorgo, and :mlth, 3. 5., 1937. Influanoo of salts in the diet on the intettlnal flora of the albino rat. J. tact. 2&; 81-97. Evans, Alice 0.. China, Alice L.. l9h7. The entarococci with special referenca to their usaociution with human 6156359. 3. Fact. 25: h95¢512. Eveneon, h., mosey, Elizabeth, Geyor, E. h., Elvehjem C. h., 1946. The cecal flora of white rats on a puria fled diet and ita modification by succtnylaulfuthiazolo. J. 330‘. 2;: :13‘5210 Fridericia, L. 3., Fraudenthal, 9.. Gufljonnson, 8.. Johansen, 6., and Lchoubye, fi., 134?. Reflection, a transmlaslhla change in the inteatlnal content, enabling rats to grow and tirive without Vitamin B in the $006. g. Eyg.,‘§1: 70-102. gflllg Larralne. to, Fenton, P. ?o, and 0033111. G. R.. 1?;83. The nutrition of the mange. I. Effect of diet on the bacterial flora of the inteatine and the cecum. J. kutritlun,lgi: 13-25. 3311 Lorraine 3.. Illinflfiorth, 3. B. Cowgill. 3. at. an Fenton, r. F.. 19L80. The nutrition of the gouge. III. Ealutlon or aitt t3 the aynthetio activity or the predominating flora iaclated from the small intoatlno and ceoum. . Rutrltlcn. 2;: 27-38. Cunt, Ola 3.. Lunaone, fiavarly, EcCoy Elizabeth, leehjam, C. 3.. 19h}. Intentinal flora of rats on purified clots contaiging sulfonamides. Proo. :00. ixptl. H101. Had., ‘23: 276-279. -35- 25. 26. 27. ‘3 2:. 29. 30. 31- 32. 33. 34- 35. 35- Slimctodt, 6., 1935. Bakteriefreie Heerscheeinchen .flOta. i‘ath. at .ilCI'ObiOlo Scanilntzr, Li: 7012. Griffith, P. 3., 1935. Ltufiies on Grouth, III. B and G tvitueinoeie in ceCeotamizcd rete. J. nutrition l2: 607-074. Cuexrunt, fl. L., end Eltoher, h. 5., lgfiie. lffoct of type of ccrcohydrete on viteainc B and Q potency of feces voidei by rate. Proc. :00. sztl. £101. Hed., Guerzeut, d. 2., and Lutcher, L. A., 1934b. {one ef- fects of the conooeiti-c of the diet on the vitnmrn B and the vitamin 3 re uiremect of the growing rat. J. fictriticn g; 397-320. Guerrent, E. 5., Eutcher, L. 5., and Brown, h. h., 1937. Further stufiiec Concerning tLo formation of the B vitcaiee in the Cigectlve tract or the rat. J. nutri- tion, 3:2: 205-315. Guerront, K. 5., Lutcrer, L. A., and 70393, L. F. 1935. The effect of the type of c: botydretc on the eyn‘Lasis or the E vitamine in the fliiective tract of the rat. J. Biol. Chem.,.£;2: 233-243. Eejno, A. 3., uni ferry, C. 3., 19b). Comparative etudy of presumptive and conf rnctive media for boctcrie of the coliforn group and for fecal etrectococci. Am. J. Pub. fleeith,‘2}: 555-556. fieller, V. 3., icLlroy, C. H., and G rlock Hertha. 1925. lhc effect of the bacterial flora on the biolociCul test for vitamin B. J. Liol. Chem. £2: 255-26a. fierter, C. e. and gecdall, A. 1., 1109. the influence of dietary alternations on the t pee or inteetiuel flora. J. Biol. Chem.‘l: ENE-230. iull, T. G., en: Lettger, L. F. 1917. The influence of milk end carbohydrate feeding on the character or the intaiitififll flora. Jo Eticto 3’: 2047.71 Eutchingn, B. L., Echo og, 3., and Peterson, ., fi., l9hl. Growth factors for bacteria. LII. EurifLCution and properties or an eluete rector re:uircd by c ztein lactic acid bacteria. 3. Biol. Chem. lglz 521-523. Johensson, K. h., ohapiro, J. fl., and Series, S. B. 19h7. The flora of fecee of heme as infllcnccd by various carbohydrates in a biotin deficient ration. J. Bact., 21;: 35’360 -17- 37. \J an 0 LG. Al. #3- bk. #5. L6. L7. LS. Krchl, 1. n., {orno, P. f., Topley, L. J., eni Elvehjem C. n., sectors offectin: the dietaryr coin and tchtcn. -no re uixe ”(at of the firfimififi rut. J. Ku- trLt13fl 21} .)-l 00 ' Lifiri ’ T1. T”... CPL-'30 ””3, L. 0., U1C0tt, 3. TI. $11111 1:19;}, C. T., 1952. Inui,.ti n of ;»e oJ'aiotic synthesis of B. c~mole1 foctoxe bJ o; fcvam-d o. J. Lutrition _Ii: 1.27-2.35. I lobund, chorte. léto. ficrm free life studies. A Pub. from the ions. of Eect., oniv. of Lotte LLme. Lucxoy, T. 3., ioore, P. h., leehjem, C. n., and fort, E. B., 17L6. ffect of diet on the reeponse or oLicee to folic acid. Proc. Loo. Erotl. Liol. Led. 93: 3c7-JlZ. rockenzie, Julie 3., Lootenzie, C. 3., and LcCollum, L. V., lfitl. iffect of sulfuoilyl containe on the thyroid of the rat. ecicnce 9’: 513-519. 15911961111 5: ’."0 I10. 1.153120“, w... Ac, ifllvehjei“ a, U. «JO. 19h“. }.ol.tion of diet 1y th to rioofl- vin re: ulrcment of -:rouin g rate. .100 coo. .xptl. _E‘iol. zed., £2: 1&0-10ko ‘ Earsnell, 1. 2., Bretton, n. 6., thitc, B--’. 5., and Litchficlfi, J. T., Jr., 1340. uulfanilylzuonidine: n chemotherepeutlc o out for into tinel infections. Bull. Jonn Pogkine Hosp., 2]: 103-138. Eiortin, Sue tov J., 19L2. Folic acid in nctritionel zoLro . tric'i., :roc. L-oc., Lag. E101. one Led., 21; 353-3350 $012311,”., 'eieman, F. e., ilvehjom, C. 5., hfld Jones, 3,. 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