Q) U .k-JU I “7:“ mm." ”x... m. .. .. a, .. 3... ~ - o .3. .9” 8 “a... (Win 50.. . o. 3 We“ a... ‘a‘h (‘4‘ ”\l P v. f \ “ 438‘s” 3 hi .4 r ¢ 3 ’1 K! r." 0'. .C—t"s\5 ulna w... ‘- RWJ wmdmu .... arm.“ .17.. :5. "N I T” ‘4'. Io a” It. . ouun. J. .‘o {.5 ”A" . X r g r & Ex .3... 5.... 9“ . .KL 0:.... . . tun“. I .0...“ ‘0‘ - .T u g g... l... t- .. ,w. .‘I luv... 0.3. . c... ”“4 .;. TM. , A? «U (K , (J. .5'1 awn (Fw- . c. r \ yr .33. r... .1. O. I; I.» - v.9“; '1 ‘ a 0‘ u . L ‘oe Ohm IMOJ nu “ t I.» an... is M1 ism This is to certify that the ’ thesis entitled A Study of the Activities of Rumen Bacteria presented bg Clyde Konrad Smith has been accepted towards fulfillment of the requirements for M.S. degree in Bacteriology V. Maj professor Date May 22, 1953 l _ 0-169 A STUDY OF THE ACTIVITIES OF RUI'fEIN BACTERIA By CLYDE KONRAD TEMITH 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 mm OF SCIENCE Department of Bacteriology and Public Health 1953 THESIS CfiQ/53 ‘8; DEDICATION To Mary, whose sacrifices and encouragement made possible the completion of this work. ACKNOWLEDGEMENTS The author expresses his sincere appreciation to Dr. W. L. Mallmann and Dr. C. F. Huffman fOr their interest, aid and encouragement in the various phases of this work and for their critical reading of this manuscript. Thanks are extended to Professors C. W. Duncan, E. J. Benne and R. W. Luecke and their associates in the Department of Agricultural Chemistry for their timely suggestions and for providing facilities for the chemical determinations. Special thanks are extended to Miss Sarah T. Wade for her expert technological advice in regard to the amino acid assays. The author wishes to thank Mrs. C. L. Frank, Mr. R. L. Salsbury, Nb. R. S. Emery and Mr. H. Q. Tucker for their aid in various phases of the investigation. Thanks are also extended to many others that assumed responsibilities in the Dairy Bacteriology'laboratory, therehy'alloving time for these investigations. Grateful appreciation is expressed tOIhr. A. L. Bortree, who stim- ulated the author to begin investigations in the field of rumen micro- biology. TABLE OF CONTENTS _ Page Rm ODUCT ION '- O O O O O O 0 O O O O O O O O O O O O O O O O O O O O 1 REVIm 0F LImTURE O O O I O C O O O O O O O O O O 0 O O O O O O O l Function of the Rusten and Rumenf Microbiology . . . . . . . . . . blethOds Of Stlldfing Rmen lficrObiOIOgy 0 Q Q o o o o o o o o o o \JIWN Cultural Studies . . . . . . . . . . . . . . . . . . . . . . . . gmstudies........................12 PROCEDURE . . . . . . . . . . . . ; . . . . . . . . . . . . . . . . 16 AnimalsusedintheStudy...................16 Method of Feeding . . . . . . . . . . . . . . . . . . . . . . . 16 SamplingProcedm'e.......................1'7 MethodofAnelysis.......................18 REULTS..............................24 Composition of Human Ingesta of Cattle Fed Natural Rations . . . 2.4 Rate of Removal of Dry Matter and Crude Pi'otein from the Rumen . 24 ’8 Methionine and'Lysine Prasent' in Rumen Ingesta . . . . . . . . . The pH and Surface Tension of Ruman Liquors . . . . . . . . . . 29 Number of Organisms as Determined by Limiting Dilutions . . . . 29 PureCultureWork.......................29 SymbioticCultureS.......................31 DISCUSSION.............................38 Passage of Dry Matter and Crude Protein from the Rumen . . . . . 38 The buounts of Methionine and Lysine Present in the Runen . . . 1.1 The pH and Surface Tension of Rumen Liquors . . . . . . . . . . 1.2 rhmberofViableBacteriaCultured............... 43 Pure ointm-e Studies . . . Symbiotic Cultural Studies WWY O O O O O O O O O O O LITERRTURECITED....... atria? 22332121112333 Ruminant digestion and microbiology have been studied by various means and devices fer nearly a century. The goal of higher production per animal with low cost rations has increased the interest in rumen microbiology during the past decade. Numerous methods have been dove— loped fer studying the microbial digestion that occurs in the rumen, yet the fundamentals that many of the investigators seek still elude them because of the paucity of knowledge concerning the individual functions and requirements of the rumen microorganism. The purpose of this work was to use the best of the available methods of studying rumen bacteria with the desire to establish a method of routinely culturing rumen organisms that indicate the efficiency of digestion. ‘While this was not completely accomplished the results of this investigation will aid further research. LITERATURE REVIEW A number of review articles on rumen microbiology, physiology and ruminant digestion have appeared in the literature over the years; of particular interest may be those by Marston (1939), Goss (1943), Mcknally and Phillipson (1944), Hastings (1941.), Cole 91; _a_1_. (1945), Hungate (1950), Baker (1947—49), Owen (1947), Flsden (1945), Thorton 93 pl. (1952), and Doetch and Robinson (1953). The reader is referred to these articles for a more complete review-r of the field. Below appears a review of the literature and statements that appear pertinent to this study. Function of the Rumen and Human Microbiology The ruminant occupies a rather unique place in the animal kingdom by virtue of its polygastric type of digestive system as compared to that of the more common monogastric animals. This characteristic has allowed it to survive by utilizing a foodstuff that is unfit for the monogastric digestive tract. (The ruminant has achieved a high degree of symbiosis with the bacteria and protozoa in its digestive tract , allowing it to consume large quantities of cellulosic material. After mastication, the microorganisms of the rumen break down these materials, converting them to byproducts that may be absorbed directly from the rumen or pass on further along the alimentary canal to the abomasum and from there to be acted on as in the normal monogastric digestive tract. According to Hungate (1946) and Sijpesteijn (1948) the first record- ed study of a controlled feeding eXperiment was carried on with an ex by Haubner in 1855. This work showed that part of the cellulose disappeared in its passage through the digestive tract of ruminants and that this material had a nutritional value equivalent to that of starch. Later work by Hermeberg and Stohman in 1864, Homeister in 1861. and 1869 and Stohman in 1870 confirmed this early investigation and they extended these studies to sheep and goats. Tappeiner in 1884 showed that the site of celIulnytic activity was in the region of the rumen, reticule and cecum. This work was the first case of _i_n y_i__t_r_g incubation of rumen contents.. The following years brought similar experiments and in 1:119 studies such as the one by Hofmeister in 1881 when a capsule contain- ing grass was placed in the rumen and allowed to remain there for three days. Analysis at the termination of this period showed that 78.4 per- cent of the crude fiber of the grass had disappeared. Methods of Studying Rumen Microbiology Following the establishment of the site of cellulolytic action in the ruminant, various methods of study were applied to the microorganisms found in the rumen. Henneberg (1922) was one of the first to employ direct microscopy to study the disintegration of plant material. He used cthrozinciodine to stain the organisms and observed areas of eros- ion around the bacteria that were attached to this material. These cavities enlarged as the organism was allowed to act over a period of time.. Later, Baker (1931) studied the role of coccoid microorganisms in the disintegration of cell wall substances in material collected from the cecum and natural decaying vegetation. Baker (1939), Baker and Martin (1937, 1938), Baker and Nasr (1947) and Baker and Harries (1047— 48) used various microchemical tests, phase contrast microscopy and polarized light to observe the breakdown of the plant material in the digestive tract of ruminants and other animals. A large portion of their work deals with the iodophilic population of the rumen and com. Baker (1939) stressed the importance of these organisms in relation to cellu- lose digestion. Gall, Burroughs, Cerlaugh and Edgington (1949) published a tech; nique using a gram stain and a negative stain, water soluble nigrosine, to determine the morphological types and enumerate the number of bac- teria ‘found in the rumen. Smears of the 1-10 and 1-1000 dilutions of the rumen contents were made. The smear of the 1-10 dilution was used for the gram staining procedure. Using a Breed pipette, 0.01 ml. of the 1-1000 dilution was placed on the slide, and mixed with a loopful of nigrosine. This was spread over an area of 4 sq. an. and dried rapidly on a hot plate. Moir (1941) and Williams and Meir (1951) also used the negative staining technique to determine the total numbers of rumen microorganisms. They varied the procedure slightly by using formalized samples and using a circle with an area of 4 sq. cm. instead of the square as described by Gall gt _a_l_.. (1949). These techniques, though satisfactory for comparative work have difficulties that seriously re- flect on the accuracy of the results. These criticisms are deemed suf- ficently important to quote, Gall gt a1. (1949) who Stated‘ "Ebccess fluid often causes uneven drying. Too much or too concentrated dye causes cracking, while too slow drying causes a large area of shrinkage. Ln uneven plate causes ridges of dye, and any foreign particle causes an unstained area which tends to be round." "The slide was counted only after a gram stain of the original material .had been examined. This acquaints the examiner with the types of organisms which are present and during counting emf ques- tionable-looking spots on the nigrosine slide were examined care- fully unless they appeared to have the morphology of a bacterium seen on the gram stain. For example, a perfectly spherical white Spot would probably be an artifact, if no such perfectly round organism were seen on the gram stain. A spherical area with cracks around it is usually caused by an air bubble." William and Meir (1951) stated: "Some comment on the accuracy of the counts for total free bacteria and of the extent of diurnal and day to day variations in individual animals on a fixed dietary regime is appropriate at this point, . . . ." ". . . . It is exceedingly difficult to distinguish with certainty between artifacts and bacteria when their size is less than 0.05 u even though the presence of more minute organisms can be demonstrated in stained preparations. . . . The value of phase contrast microscopy in overcoming this difficulty is being investigated at the present time." Van der Hath (1948) and Bortree, Smith, Sarkar and Huffman (1948) used a Petroff—Hauser counting chamber to determine the total numbers of‘rumen microorganisms. The staining solutions employed were nile blue sulfate and crystal violet respectively. Hhile this technique involves positive staining of the organisms, it has the disadvantages of brownian movement and a delicate technique. Thus it does not lend itself to routine use in a laboratory. Pbunden and Hibbs (1948) used the gram stain to determine the mor— phological types, but no counts were given. Hungate (1946) employed a dilute solution of Ziehl-Nielsens carbol fuchsin to count rumen organisms, however the counts given are lower than those achieved by using either negative stains or the Petroff—Hauser counting chamber. Cultural Studies Margolin (1930) devised feur media which utilized agar, peptone, Lemco meat extract and Locke's solution as a base. These media were altered by adding various amounts of hay infusion, rice starch, pulped filter paper, magnesium oxide and saline citrate. Although he was pri- marily interested in the cultivation of ciliates from the rumen, the bacteria that were present in the inoculum.grew very readily. It was also noteé that ciliate subculture was more successt1 after the filter paper had been partially digested by cellulose digesting organisms intro- duced from earlier cultures. Kohler (1940) used two cultural procedures to determine the numbers of’rumen bacteria. The plate counts were carried out with a weakly alkaline bouillon agar, incubated aerobically at 37 C from one to five days. The average of the counts after one day of incubation was 20,000 per gm. while after five days of incubation the number had increas- ed to 137,000 per gm. He cited Ankersmits, who in 1905 and 1906 found a 70,000 - 900,000 count on gelatin plates and 2,160,000 -15,000,000 on dextrose afar. Tube dilutions made by Kohler, using dilutions up to 10'12 showed growth up to 10‘6. An average count of 550,000 was obtained by this method. Colonies picked from plates showed only Sporeforming rods on the surface while cocci were obtained from the depths. Dextrose bouillon agar was needed to grow cocci and these cultures lived for only three transfers. He was unable to culture cellulose digesting bacteria. Elsden (1945) used Stephenson‘s inorganic medium pH 7.4 and sup— plemented it with 0.4% Difco yeast extract w/v and 1% w/v sodium lactate. This was inoculated with one drop of rumen liquor and incubated for ID days under an.atmosphere of N2 containing 5% 002. After repeated sub- cultures on liquid and solid media a pure culture of Propionibacterium was obtained. These organisms, small gram negative cocci, developed colonies 152 mm..diameter dome shaped and cream colored. They were strictly anaerobic, with good growth in the bottom of a yeast extract lactate agar stab, and fermented glucose and lactic acid with the pro- duction of propionic and acetic acids and 002. Van der Nath (1948) isolated an iodophilic streptococcus and studied it in detail. The technique he employed was to place a small silk bag filled with crushed and shelled sterilized maize kernels into the rumen. This was suspended there fer 48 hours by a string through a rumen fistula. After this period of digestion the bag was removed to a sterile petri dish, and a few of the partially digested kernelstmueadropped into sterile saline. From this suSpension surface cultures were made on dextrose agar or starch agar slants. The small dewbdrop type colony usually' proved to be the streptococcus associated with the maize kernels. This organism.fermented glucose, lactose, sucrose, maltose, levulose, raffinose, starch and inulin to produce acid but no gas. It did not ferment mannitol, galactose, salicin, sorbitol, rhammose, inositol and dulcitol. It grew poorly at room.temperature but grew well at 37 C. Plain agar supported only feeble growth while dextrose or starch agar gave more profuse growth. Serum also supported growth but there was no liquefaction of the gelatin. Using the same technique Van der Hath suSpended chemically pure cellulose and casein in separate silk bags, and studied stained prepare, tions of the organisms. The cellulose encouraged the growth of various types of gram negative bacteria while the casein encouraged predominantely gram positive organisms. He felt that the organism cultured played a significant part in the breakdotm. of the starch while the other organisms studied assisted in the complete metabolism.of the ration. Gall (1946) and Call, Stark and 1.00811 (1947) outlined a procedure for isolation and studying some of the predominanting flora of the rumen in cattle and sheep, Theywievised a rich organic broth to culture the organisms. The rumen contents were obtained by use of a pipette or a dipper and care was taken so as not to aerate the sample. Dilutions were prepared in a 1% glucose solution, planted into tubes containing'hroth that had been recently heated to remove the oxygen and then sealed with a vaspar seal. These tubes were incubated at 38 C. Growth was shown by turbidity or "clumping" of the cellulose. In most cases growth occurred in 10'9 dilution in 48 hours but sometimes it was as long as 60 days be- fbre growth appeared in the higher dilutions. The organisms cultured were all capable of anaerobic growth and some of the cultures produced cellulose digestion ranging from 10 to 20 percent. These organisms appeared to be the types commonly found in the higher dil- utions. In a later study, Gall and Huhtanen (1951) described some of the organisms that were isolated from the rumens of about 350 cattle and sheep in three states. They also established five criteria fer Judging true rumen organisms, they were as follows; "(a) Anaerobiosis; (b) presence in the numbers of one million or more per gram of fresh rumen contents; (c) isolation of a similar type bacterium at least ten times from.at least two animals; (d) isolation from.animals in at least two geographical lo— cations; and (a) production by the organism of end products found in the rumen from substrates found in the rumen." The organisms described are alI carbohydrolytic and produce gas and lactic acid. The authors stated that though these organisms fit the criteria,they should not be considered "typical rumen organisms in general" because they are found in rations high in carbohydrate or in immature ruminants. Huhtanen, Rodgers and Gall (1952) altered the original medium of Gall 23 gl. (1947) and outlined in detail a procedure for the isolation and purification of cultures of rumen organisms. Cysteine was added to the media as well as to the dilution blanks of buffered distilled water in order to maintain the proper Eh.. The data reported showed that these "new techniques" were more satisfactory and gave growth in higher dilutions than did the techniques used previously. Hungate, in a series of papers (1944, 1946b, 1947 and 1950) discussed and described various types of cellulolytic bacteria, some of which were isolated from the rumen of cattle. The most recent publication described in detail the complicated isolation procedure used to isolate and purify these cultures. The medium.was composed of a complex inorganic salt sol- ution, finely ground cellulose, cysteine, resazurin, agar and rumen fluid. This was sterilized and diapensed in sterile 002 washed tubes. The tubes were then cooled and inoculated, flushing the tubes with 002 while they were being inoculated. After inoculation the tube is stoppered, inverted, then returned to a horizontal position and rotated to seal the junction between the glass and the stopper. This forms a thin layer of agar through- out the tube that 1££E1 allow the cellulose digesting colonies to be readily seen.. The colonies were subcultured with a Pasteur pipette and the same precautions regarding the maintenance of anaerobiosis were employed. The culture was considered pure after two consecutive subcultures produced single types of colonies macroscopicalIy and similar types of organisms microscOpically. USing these techniques Hungate (1943) isolated nine cellulose—decom- posing organisms and determined that they occurred in the rumen in numbers ranging from.I8,500‘to 1,200,000,000 per ml. By'calculation he estimated that the original rumen content contained 40,000,000 cellulose-decomposing bacteria per ml. In (1950) Hungate described two other types of cellulolya tic organisms that fermented cellulose to volatile and non-volatile fatty acids. - -10.. Sijpesteijn (1943) described in detail her work with cellulose- decomposing cultures from the rumen of cattle. The work was carried on over a period of five years, but only three years actual time was involved in the research. She obtained one pure culture of a cellulose- decomposing organism from the rumen and named it RMgoccus ggvefaciens. Another type that was obtained in "nearly pure culture" was named aiming- _c_g_9__c_:_u_§ m. She found that maintenance of anaerobic conditions was important. The decomposition products of the cellulose digestion were largely volatile and non-volatile fatty acids. Unemzu, Tuda, Katuno, Omori, and Minagski (1951a, 1951b) studied the biological characteristics of the aerobic bacteria of the rumen. They were unable to culture celluloly'tic organisms aerobically, but sug- gested that the aerobic bacteria may play a part in the cellulose digestion indirectly. The symbiosis postulated was one in which the reducing sub- stances were produced by the aerobes, therefore promoting the growth of the anaerobic organisms. Bryant (1952), and Bryant and Burkey (1953a, 1953b) isolated and described some rumen microorganisms. He used the technique outlined by Hungate (I950) to obtain in pure culture a Spirochete reported by Hungate (1947) be, which Hungate was unable to obtain in pure culture. This or- ganism is not a cellulose digesting organism, but lives on the breakdown products of the celluldse digesting organisms. Thus it is usually found in close association with the cellulose decomposing bacteria. The more recent publications deal with the numbers and more mimbrous groups of bacteria in the rumen. They grouped the organism cultured into thirteen groups according to morphology and biochemical characteristics. Only two _ 11 - of the 13 groups reported are cellulolytic in pure culture, but eight groups-attacked cellobiose. Heald (1952) used xylose in a.basal medium to study anaerobic xylose fermenting organisms of the rumen. He isolated 17 strains of gram-negative rods belonging to the colifonm intermediate group. These organisms showed marked variations in the end products of fermentation when the substrate was changed from.xylose or glucouronic acid to glucose or cellobiose. Like the other workers, the main end—products of fermentation found were vol- stile and nonpvolatile fatty acids. - many ip yitgg studies have been made of rumen bacteria using mixed cultures. 'Wenger, Booth, Bohstedt and Hart (1940) incubated rumen ingesta in 602 to maintain anaerobiosis. Louw, Williams and Mhynard (1949) imp proved upon this method by placing the rumen ingesta and the substrate in a visking cellulose casing to allow for dialysis of the byproducts of fer- ‘mentation.. These techniques have become known as the "artifical rumen" and has been used by Burroughs, Arias, Gerlaugh andBethke (1950), Oyaert, Chin, and Clark (1951),‘MbNaught (l95la),‘wasserman, Duncan, GhurchiII and Huffman.(l952) and Gall and Glows (1951) and Huhtanen and Gall (1953). Some investigators have used "washed cell" suspensions to study the metabolism of'rumen.bacteria. The rumen liquor is fractionated using various Speeds of a centrifuge and the fraction containing the majority of the organisms is resuSpended and used as inocqum on various substrates. This technique has been.usedrby'Lewis (1950, 1951) to study nitrate re- duction, Elsden and Sijpestijn (1950) to study the decarboxylation of succinic acid and Doetsch, Robinson and Shaw (1952) to investigate ketO' acid production. -12.. Quinn (1943), McAnally (1943), Walter (1952) and McBee (1950) employed a manometric technique to evaluate the activity of the rumen micro- flora. This consisted of adding the ruminal liquor to a buffered substrate and measuring the respiration of the cells using a Warburg apparatus. in 15:22 Studies Monroe and Perkins (1939) determined the pH of rumen contents at 2 hour intervals. They found‘ that the pH varied greatly depending upon the time of feeding and the type of ration. Generally Speaking, the pH before feeding was near neutrality and decreased to 6.0 within three hours after feeding, then rising slowly to neutrality again. Rations of hay and grain gave the highest readings, pH 7.01, and blue grass pasture gave the lowest reading, pH 6.47. Smith (1941) placed the electrodes of the pH meter in the rumen and found that the pH of the rumen ingesta in gig; was 0.6 of a pH unit lower than when the sample was withdrawn and checked. He also found that closing the cover of the fistula lowered the reading 0.3 of a pH unit. When feeding an alfalfa ration the pH of the rumen ingesta was 6.3.. Monroe and Perkins (1939) have shown that the pH of rumen ingesta 12-18 hrs. after feeding is about 7.34.. This alkaline condition is brought about by the constant flow of saliva that has a pH of about 8.5 to 8.71 (Reid and Huffman 1949). Patel (1952) investigated chemical and nutritional properties of rumen contents of cattle and found the surface tension of rumen liquor to vary from 54.86 to 69.87 dynes per sq. cm. This is somewhat higher than was found for saliva by Reid and Huffman (1949) when they recorded a range of from 45.54 to 49.20‘ dynes per sq. cm. Armsby (1911) stated that non—protein nitrogen could be a partial substitute for protein'in the ration of ruminants. Although true protein - 13 - .was needed,the noneprotein nitrogen could be used for maintenance, milk production and growth. Johnson, Hamilton, Mitchell and Robinson (1942) stated the protein of a ruminants ration is digested in the abomasum in the same manner as in any monogastric animal. The protein produced in the rumen by the bac- teria is then available to the host animal and the biological value of the protein would be relatively constant. They found that the biological value of the nitrogen in the ration was about 60 when the rations contain- ed from.10-12% protein. Loosli and Harris (1945) fed lambs rations containing urea and urea plus methionine. The added methionine caused increased gains and nitrogen retention. They considered urea formed bacterial protein mildly deficient in methionine.— Reid, Hair and Underwood (1949) investigated the value of rumen bacterial protein. They separated the cells by fractionation using various Speeds of a centrifuge and concluded that they were low in digest- ability, high in biological value and mildly'deficient in methionine. Bacterial protein formed from green and dry rations had about the same cystine content but the protein from the green feed contained a higher level of methionine. Later, Meir and Williams (1950) feund that about 50 percent of the ingested protein was converted to bacterial protein, as the protein in the ra+ion increased, the biological value of the bacterial protein increased. Block and Stekol (1950) and Block, Stekol and Loosli (1951) fed radioactive sodium.su1fate (835) to ruminants and found that the cystine and methionine formed contained radioactive sulfur in appreciable amounts. In case of the goat, the radioactivity was the same for the methionine and qystine in the milk, serum albumin, and rumen. These results indicate that methionine and cystine were synthesized at approximately the same rate as were used by the tissues to make protein in the quanities needed. The synthesis of lysine by bacteria during incubation of rumen contents in _v.i_tr_g has been shown by McNaught (1951) , McDonald (1948) fed a lysine free ration to sheep that had both rumen.and duodenal fistulas. The organisms recovered contained 7 percent lysine in their mixed proteins. They estimated that about 40 percent of the zein had been converted to microbial protein. Agrawala, Duncan and Huffman (1953) and Duncan, Agrawala, Huffman and Luecke (1953) studied rumen synthesis and passage in steers fed natural and purified rations. They concluded that about 60 percent of the dry matter was removed from the rumen in six hours irreSpective of the ration fed and that "true" protein synthesized varied from 33 to 109 gm. depend- ing upon the ration fed. Considerable amino acid synthesis was demon- strated, and with the exception of histidine, the amino acid pattern found in the mixed protein of the purified diet was essentially the same as found in the natural ration. The synthesis of lysine was prOportionally greater than that of methionine. Chance (1952) studied rumen synthesis and passage in steers on natural and purified diets supplemented with aureomycin. The passage noted.here was somewhat slower than.reported by'lgrawala gt‘al. (1953‘ and could be attributed to the feeding of the aureomycin. The majority of the passage had occurred by 12 hours. The amount of amino‘acids present in the rumen ingesta was less when aureomycin.was fed as compared to the control period. -15.. He noted that the rate of removal of amino acids was increased when 0.5 gm, of aureomycin was added but reduced again when the amount of aureomycin was increased to 1.0 gm per day. . . Many'methods have been.used to study the activities of the rumen microorganisms. This study was undertaken to determine if a combination offseveral.of these techniques could be employed to determine the activity of the rumen bacteria on a particular ration. PROCEDURE Animals used in the Study The animals used in this study were two three-year old steers 707, a Guernsey and 714, a Holstein which were fitted with lucite fistula plugs. These plugs were fitted with caps that could be easily removed when a sample was desired, or could be disassembled and removed to empty the rumen contents. I’I‘evious to this study the animals were maintained on a ration of hay alone for 30 days. A‘ ration of hay and corn was fed during the first two week period and a. ration of hay and distillers solubles was fed during the second two week period of the exp rirzent. I-ic‘hod of Feeding The steers were fed the total ration of 1. pounds of corn and 13 pounds of hay once daily. The same was true during the second period when the corn was replaced with 4 pounds of distillers solubles. The corn was placed in the manger and usually consumed in 30' minutes at which time the hay was fed. The hay was eaten readily, usually being consumed in less than three hours. The distillers solubles (containing 20.5% rye solubles) were refused periodically. In these cases the solubles were emptied directly into the rumen through the fistula for two or three days after which time the animals would again eat the ration. Chemical analysis of ingredients in the ration are given in Table I. Water was fed §_d_ libitum. -17.. TABLE I Chemical Analyses of Corn, Hay and Distillers Solubles Used in the Natural Ration Dry Crude Matter Protein Methionine Lysine Ration % % 75* gas Hay 91.9 10.00 0.026 0.080 Distillers solubles 86.2 30.25 0.500 0.711 * Based on dry matter Sampling Procedure Chemicg gelysis. The rumen contents were removed through the fistula opening after disassembling and removing the plastic plus. The ingesta was thoroughly mixed, weighed and composite samples were taken for chemical analysis. Approximately alOOOgm sample was taken for amino acid, total nitrogen and dry matter determinations. These Operations were done quickly so as to prevent excessive aeration and cooling of the ingestahthat was returned to the rumen. The sample taken for chemical analysis was placed in a brown glass bottle and one liter of 95% ethyl alcohol was added to stop bacterial action and facilitate later handling and drying. These samples were taken on the 14th day of the experimental period. ct iol i . I ic c c sis. These samples were taken four hours after feeding at four or five day intervals throughout the experimental periods and three times daily on the thirteenth day (the day preceding the sampling for chenical analysis) .. These samples taken on the thirteenth day were taken before feeding, four hours and -13.. nine hours after feeding.. Rumen organisms can likely withstand the treatment accorded them when the.ingesta is weighed and then returned to its natural enviroment, but attempting to culture these organisms on an artifical medium at that time may be more difficult. These samples were taken with a sterile 10 ml dipper so that a representative sample of the ingesta could be obtained. The ingesta was placed in a screw cap sample bottle and tranSported to the laboratory. Care was taken to fill the bottle to capacity to prevent excessive aeration. Mbthod of Analysis Ch c as. The samples of rumen contents and feeds taken for chemical analyses were dried in a ferced-hot air oven at 60 G. The dried samples were ground in a‘Wiley'mill to pass a 20 mesh screen and stored in air tight glass containers until analyzed. An extended period of time passed between drying and the actual analyzing, so the samples were redried and all determinations were carried out on oven.dried samples. The total nitrogen determinations were carried out using the Kjeldahl method as outlined by the now (1950) . Hydrolyzates for the amino acid determinations were prepared accord- ing to the procedure of Stokes, Gunness, Dwyer and Caswell (1945). One gram of the material was diapersed in 25 m1 of 65 H01 and autoclaved for eight hours at 121 C. The hydrolyzates were cooled, neutralized with NaOH, made up to 100 m1 volume, filtered, covered with a few’drops of toluene and stored in the refrigerator until analyzed. The concentration of this solution was 10 milligrams per ml. The microbiological method was used to determine the amino acids. The composition of the media used for determining the amino acids is -19.. TABLE II COMPOSITION OF THE MEDIA USED IN AMINO ACID ASSAY (Per 500 milliliters of double-strength medium) vi Composition I II H202 treated peptone (gm) ~— 7.5 DL (-)-Alanine (mg) 200 ---- L (A-Arginine-Hm (mg) 100 _ L-ASparagine (mg) 200 -—- L(-)-Cystine (mg) 200 100 L(/)-Glutamic Acid (mg) 400 .— Glycine (mg) 100 -- L(/)-Histidine-HC10H20 (mg) 100 _._. DL-Isoleucine (mg) 200 -- DL-Leucine (mg) 200 ~— DID-Methionine (mg) 200 .— DLPhenylalanine (mg) 100 .— L(-)-Proline (mg) 5O .— DL—Serine (mg) 200 --- DL—Threonine (mg) 200 ___ DL-Tryptophan . (mg) 100 100 L(-)-i‘yrosine (mg) - 100 1'00 DL-Valine (mg) 200 .. Glucose (gm) '20 20 La acetate (anhyd.) (m) 20 12 131401 (gm) —-- 6 KH2P04 (mg) 500 500 TABLE II (concluded) Composition I II KQHP04 (mg) 500 500 MgSO4'7H20 (mg) 200 200 Fe804°7H20 (mg) 10 10 Noam-41120 (mg) 10 10 NaCl (mg) 10 10 Adenine 304.2320 (mg) 10 10 Guanine 1101-21120 (mg) 10 10 Uracil ' (mg) 10 10 zenttine. (ms) 10 .. Thiamine-H01 (mg) 0.5 1.0 PyridoxineoHCi (mg) 1.0 2.0 DL-Ca pantothenate (mg) 0.5 2.0 Riboflavin (mg) 0.5 2.0 Niootinio acid (mg) 1.0 2.0 grdminobenzoic acid (mg) 0.1 0.01 Biotin (ug) 1.0 5.0 Folic" acid (mg) 0.01 0.0015 6.8 7.0 pH before autoclaving given in Table II. Medium I, McMahan and Snell (1941:.) was used for ngcgnostoc mesentroides P-60 (801.2) to determine lysine.. Medium II, Lyman, Mosely, Butler, Wood and Hale (1946) was used for Lactobacglug mesent_r:o_ides for the determination of methionine. The assays were carried out according to the procedure of preparing a standard in triplicate Of the amino acid being assayed. Diplicate 1.0 ml, 2.0 ml and 3.0 ml portions of the diluted hydrolyzate were used to determine the amount of amino acids in the sample. The tubes were incubated for 72 hours at 37 C to permit development of the lactic acid. This acid was adjusted electrometrically with 0.1 21 NaOH to pH 7.0. Bacteriological w. The methods of Huhtanen, Rodgers and Call (1952) were followed to culture the rumen bacteria. The rumen in- gesta was weighed directly into a wide mouthed glass steppered bottle containing 90 ml‘ of sterile buffered distilled water. This dilution blank had been saturated by bubbling with 002 for two minutes and lime-- diately before use 0.1 ml of sterile 10% cysteine solution in 2.2% NaH003 was, added. All dilution blanks used were treated in this manner. Dilup tions of 10-1, 10‘"3 and 10"5 were prepared and a 1.0 m1 portion was inoculated into a tube containing 9ml' of broth, outlined in Table III. Dilutions of 10’6, 10'7, 10’8, 10-9, 10'10 and 10-11 were prepared by planting 1.0 ml from the preceding tube to the tube containing the dil- ution being made. Vaspar seals were used in the first part of this work and later it was found that a neutral oil‘ would be as satisfactory (Mall- mann, 1953). Any tube showing turbidity within a. 12 day period was con- sidered positive. One loopful of this culture was transferred to an agar deep (Table III) and maintained there by subsequent transfers until it could be purified. TABLE III COI’LPOSITION OF MEDIA USED FOR CULTURAL STUDIES Carbohydrate fermen- Broth Agar tation Base 3 Bacto Beef Extract 10 gml 10 gm .. Bacto Peptone 10 gm 10 gm 10 gm Bacto Tryptone 10 gm 10 gm 10 gm Bacto Yeast Extract 10 gm. 10 gm. 10 gm Glucose 1 gm . 1 gm -- KZHPO4 1 gm 1 gm 1 gm E21304 1 {rm 1 gm 1 gm Agar 15 gm. - --- Cysteine (10% solutign 0.1 ml 0.1 ml 0.05 ml in 2.3% NaHCOB) . Alfalfa.meal 2 trace trace -——- Carbohydrate being -- -- 10 gm tested . I. Ingredients sufficient to make 1000 ml. 2. Added directly to the tubes after dispensing. 3.. Usually dispensed in 5 ml amounts. Nmflia.were sterilized at 121 C fbr 10 minutes. _ 23 _ The purification of the cultures was carried out by using a transfer from.the agar deep to a broth to establish a 48.hour culture. One loopful bf'the'mixed culture was placed “in an agar shake tube, this was mixed well, a transfer was made from this tUbe to a second shake tube and so on until three tubes had been inoculated. These were poured into petri dishes and incubated in Brewer anaerobic jars. The cultures that were pure microscOpically and gave only one colony type upon subsequent trans- fer as well as maintained their purity microscopically were transferred to carbohydrate fermentation broths (Table III).. The cultures that were not pure were again replated using the shake tube method in attempts to purify them. ' (The bacteria-free filtrates were Obtained by filtering the materials through an.ultra fine fritted glass filter. ’ Egypiéal Chemical Analyses, The rumen liquor was expressed from the remainder of the sample and used far these determinations. The pH determinations were carried out using a Cenco electrometric titration pH meter. The surface tension was measured using a Cenco DuNouy'teng siometer. The method followed was that described by Central Scientific ‘ Company, Bulletin 101. Triplicate readings were carried out on each sample. 3 RESULTS Composition of Rumen Ingesta of Cattle Fed Natural Rations The weights and a partial analysis of the constituents of the rumen ingesta obtained at O-, 4:- and 9- hour collections are presented in Table IV. These data are arranged by hour of collection and ration fed so that the data pretaining to the two rations fed a given animal maybe compared. It was felt that there was considerable individual variation in the animals used in this experiment. The consistency of the rumen ingesta of steer 707 was consistently lower in dry matter and more finely mascerated than that of steer 741. This increased breakdown of the fibers would allow for greater bacterial action. Rate of Removal of Dry Matter and Crude Protein from the. Human The amount of dry matter “and crude protein present in the rumen at O-, 4— and 9- hours, as well as the dry matter and crude protein fed were calculated. From the amount present in the rumen and the amount fed, calculations were made to determine the rate of removal of dry matter and crude protein during the 0—4 hour and 0-9 hour periods (Tables V and VI). The rate of removal was based on the intake of the component. ‘ calculations were also made to determine the amount and rate of removal of dry matter and crude protein from the rumen during the 4-9 hour periods. The AP hour collection was used as a- base, and the rate of removal was based upon the intake of the constituent. - 25.. monedm A¢dbe¢z Duh mqaa¢o ho H mdmda oo.sa ommH om.mH woes mom.oo m . ham use moans oo.ea mama os.mH NmNHH www.me : are -Hom osoaadpoan Hm.mH mafia oe.m~ memm amm.oo o am.mH mmm oe.m Hsmm omm.om m ; mom was moans me.mH mmca 0H.0H Heme www.mm a non uHom oeoflaapodn oo.sH was om.c ream oms.mm o om.ma mmoa HH.aH moms ooe.mn a Hm.NH HNOH mm.m~ mmmm mon.rn a are sex ass ssoo om.NH mma He.HH some mnm.mm o Hm.ra mmm em.oa ammo www.mo m .on.mH sum mm.e cane serene a . new mom can ssoo me.me one mm.m amen- mmm.sm o m aw m am am .mnm .02 com :oaudm :aopopm ousnu soppmeHnn mpcopcoo cofimm oaaa Headed no pawns: TABLE V REMOVAL OF DR! MATTER FROM THE RUMEN OF CATTLE FED NATURAL RATIONS Animal Sampling Dr Matter Ration Fed No, Time In Eugen Ted Tota;_ Removed* gm 8m gm gm 0 5152 7050 12202 0 0 Corn and Hay 707 4 6510 0 6510 5692 81 9 6655 o 6655'55u7 78 4-9 hr -1u5 ‘ 0 6307 7050 13357 0 0 Corn and Hay 741 4 8296 O 8296 5061 72 9 7563 0 7563 5794 82 4-9 hr 733 4 0 5374 6988 12362 0 O Distillers 801- 707 4 6571 0 6571 5791 83 ubles and Hay 9 5541 o 5541 6821 98 4-9 hr 1030 15 o 8268 6988 15256 0 o Distillers Sol- 741 4 11282 0 11282 3974 57 ubles and Bay 9 7766 0 7766 7518 98 4-9 hr 3544 51 * Based on dry matter intake. TABLE VI THE REMOVAL OF CRUDE PROTEIN FROM THE RUMEN OF CATTLE FED NATURAL RATIONS Animal Sampling Crude Protein w p cf .4- O :3 rs; (D p. No. Time ‘In Rumen gm 8m gm 0 676 721 1397 Corn and Hay 707 4 879 O 879~ 9 952 O _952 4-9 hr 5 0 788 ~721 1509 Corn and Hay 741 4 1021 0 1021 .9 1025 0 1025' 4-9 hr 0 775 1015 1770 Distillers Sol- 707 4 1035 0 1035 ubles and Hay 9 883 0 883 4-9 hr 0 1142 1015 2157 Distillers 301- 741 4 1812 0 1812 ubles and Hay 9 1320 o 1320 4-9 hr gm 0 518 _ nus _ 735 947 212 345 837 492 Fed Total Removed‘ 72 62 68 67 72 93 21 34 82 48 * Based on crude protein intake. .90pde and co somem * sex one means Iaom mamaaapman hem was mean: IHom mamaadpman hem was ago 0 new use apoo aa.Hm o :H.Hm Hoa.o ma.m o ma.m moa.o m em.om o no.8m mom.o ~8.HH o mo.aa moe.o n Ham mm.am ma.ma mH.om ema.o rowed mm.m em.e mmo.o 0 ~28 o «23 emmd 84. o 84. 320 e Nn.mm o Ne.mm Hmm.o om.e o ow.e HHH.o a son no.8m mr.mH NN.HN mom.o so.oH mm.m mm.o ama.o o oe.em o or.em mom.o em.m o em.o mmo.o m rs.om o 8 .mm can 0 me.“ o mm.m moo.o a are mm.mm mH.o o .eH mmm.o mr.e sm.m ea.: 880.0 0 mo.nm o me.mm mmm.o mm.e o mm.e mmo.o m oa.mH . o os.ma emm.o no.0 . o 30.8 mmo.o : nos mm.rH nH.o os.m moa.o 55.0 sm.m om.m moo.o 0 am am am am am new m AS A com - cossm :H as» a com sossm GH seas .oz ..|..I.. .i I . 0. m4 mug. . _ .. _ __ __ ._ .. .. ....ol5..i:lloanlplozllllll madamsem Headed. 0mm coaudm m20H9 mnm