A 571.30? OF THE BACTERIAL FLORA 0:7 THE CR0? OF NORMAL CHICKENS Thus“: {at the Dagmar at M. 5. #fifiCH‘sGAN STATE COLLEGE Mme! Dims: I945 THESIS This is to certify that the thesis entitled A Study of the Bacterial Flora of the Crap of Normal Chickens presented by Mabel Djang has been accepted towards fulfilment of the requirements for M.S. degree in Bacteriology 8: Public Health ““Ywd‘ deRJW~n£%rVVflV‘ Major pr fessor Date December 14, 1945 MW“I"*" ’ ' Fri- —--—.v . - . 21?? “viii. " .91_‘5:’..¥fi‘, t". ‘1 .1‘('& .1. i’-’£’X‘l . . '.' I '."' LIP-"'- f l ‘ i'kfitfi."f' “L‘A‘cJflplfii'fi, I} ' ' " ' .- "' "n.4l‘e":.",~ "a“, 5? 2'3“ 3.'.w“‘i "rhq: ' . 5 . I. . f. '- ' 1 31¢. "" M’fikfllywl ($1411?! '1‘ “ _, .‘ ' ' . 9f" 3' .3, ‘- ~9le 1511“!” - . ‘ ." ~ -' 1‘ . \ 3‘...‘ ' ,.- ..".. ”’5? ‘1). , v ’ M f 1» “ " C‘" I '4 l 7 i "3"". ' .H " ' ~l' ‘ , ‘2 5'1» ..r'l,5,'-’3,:1ll I. "3 l 1.3. I‘I -: ‘ » 'i .1 ‘l- T ‘ .. 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Pr- 1" - ‘tt‘. I1 I“II ‘ . .- £133.; m . u -. X' "r .. ”If ’. 2h , . x‘JI“ ~ ha . .‘;.3' ~' . J -' l ' 11%|". 181% . 'I I a . '. “l." .I 3. . O . . '1 .' - A V 3‘: “fi;§fi$1d iglffiéi ‘ ' '«6° s Xe -’,. ix 7* g v v ., 'c‘ - H ' "I. ~\ ,‘JJ' 4" ‘ I I .J. ‘ I ‘ .. ‘ 4244ij J, ;»,.:J?;v. 11:”; =9 l 1.9§LIIKQLA.3¢¥-?2 7 lily . A STUDY OF THE BACTERIAL mom OF THE CROP OF NORMAL cmcxms A STUDY OF THE BACTERIAL FLORA 01‘ m CROP OF NORMAL CHICKENS A MSIS SJBMI'I‘TED TO m FACULTY OI MICHIGAN STATE COLLEGE IN PARTIAL FULFILLMENT OF THE REWIREMENTS FOR DEERE 0! MASTER OF SCIENCE MABEL WANG 19’45 II. III. IV. V. VI. VII. TABLE OF CONTENTS Title Introduction Review of Literature Experimental A. Procedure for Obtaining Crop Samole B. Examination of Sample C. Results D. Tables of Biochemical Tests Discussion Summary Literature Cited Appendix AcknOWIedgement INTRODUCTION - A preliminary study relating to the value of a certain agent, to which the manufacturer attributes intestinal antisep- tic prOperties, raised the question as to what is the normal bacterial flora of the crop of healthy chickens. In the first study, feed and water were not removed previous to obtaining the crop contents for bacteriological examination so, naturally, some of the organisms isolated from those chickens could have been traced directly to such sources. In this study feed and water were removed, eighteen to twenty-four hours prior to the removal of the crop contents, for the purpose of preventing the introduction of organisms which could not very well be considered a part of the normal flora. However, one cannot disregard the initial effect of feed, water, etc., in the establishment of a bacterial flora of the digestive tract. The digestive tract of young vertebrates is sterile at birth but becomes contaminated in a few hours. Tanner (1) gives an extensive review of the work done by numerous investigators of the intestinal bacterial flora of various vertebrates and of the factors which influence its establishment. The chickens on which this study was made. had been on a standard Michigan Farm Bureau laying mash. Each bird was placed in an individual clean wire cage,but no precautions were taken to prevent it from picking at the fragments of fecal matter which adhered to the wire flooring of its own cage. 1 REVIEW OF LITERATURE There have been no reports on the bacterial flora of the crop.but that of the intestinal tract has been the subject of much discussion. Several authors, also, have given some information concerning the bacterial flora of the respiratory tract of fowls ill with respiratory diseases, and one author described the bacterial flora of the respiratory tract of normal healthy fowls. Gibbs (2) found Staphylococcis‘albus, Snirocheta, Micrococcus, Escherichia communior, Alcaligenes bronchisepticus and Sarcina lutea in the respiratory tract of normal healthy fowls. He also found a considerable variety of non-pathogenic organisms (probably secondary invaders) in the respiratory tract in conjunction with various diseases. Organisms encoun- tered in cases of infectious tracheitis were: Bsch. communior, Escherichia coli, Escherichia gastrica, Escherichia alkalescens, Escherichia pseudocoloides, Micrococci, Alcaligenes bronchisepti- cus, Pasteurella avicida, Eberthella septicemia, Torula and a yeast. Chickens ill with pullorem disease had Esch. communior, Esch. coli and Sarcina in their respiratory tracts. Staph. albus was found in the respiratory tract of chickens ill with chronic laryngitis and avian paralysis. Kernoham (3) found, in association with laryngotracheitis of fowls, Pasteurella avicida—like organisms, diplococci, Pseudomonas 2 pyocaneus and other unidentified bacteria.none of which produced laryngotracheitis by intratracheal instillation. Graham, Thorp and James (3) found a.pleomorphic, gram— positive, non-spore—forming.hemolytic organism in laryngeal and tracheal exudates of fowls suffering from acute infectious laryngotracheitis and subacute or chronic avian laryngotracheitis. Beach (5) also found gram-negative rods of the Pasteurella type, gram-positive cocci and diphtheroids in association with laryngotracheitis lesions. The results of Eliot and Lewis (6) were essentially the same as those reported by other investigators of the nasal and tracheal secretions of chickens in health and disease. They found certain staphylococci, streptococci and diphtheroids com— monly occurring on mucous membranes of man and animals. These were also a number of ovoid bacteria, gram-positive and gram- negative and several species of micrococci. For a time parti- cular interest was centered on the strains of Eggtgurglla isolated. These were present in a number of chickens suffering with infectious coryza although fowl cholera did not appear in the flock during the laboratory investigation. A number of authors have described organisms of the in— testinal tract and feces of fowls. Emmel (7) gives the most com- plete and comprehensive list of bacteria found in the feces of healthy adult birds: Escherichia coli, Escherichia communior, Escherichia neapolitana, Escherichia acidi lacti, Escherichia alcalescens, Micrococcus aurantiacus, Micrococcus subflavus, Micro- coccus percitreus, taphylococcus albus, Staphylococcus citreus, Bacillus mycoides, Bacillus subtilis, Bacillus cereus, Bacillus gytaceus, Bacillus tritus, Bacillus circulans, Bacillus megatherium, Bacillus petastites, Bacillus ramosus, Bacillus cohaerens, Bacillus vulgatus, Aerobacter a rogenes, Bacteroides bifidus, Clostridium sporogenes, Achromobacter liquefaciens, Salmonella icteroides, and Actinamyces microflavus. EXPERIMENTAL Procedure for Obtaining Crop_Sample To remove the crop contents, the technique devised by Stafseth (8) was employed. Ten ml. pipettes were cut off near the distal end and flamed so that the glass wall around the opening became somewhat rounded and smooth. Short pieces of rubber tubing (about three inches in length) were attached to the proximal end. The free ends of the rubber tubes were then plugged with cotton. The pipettes fitted with plugged rubber tubes were then placed in metal cylinders and were sterilized by autoclaving. After completion of the sterili- zation process, the pipettes were allowed to cool and the distal ends were dipped in sterile five per cent agar so as to form an agar plug about one cm. long in the end of the pipette. When the agar plug had become cool and firm, the pipettes were returned to the sterile cylinder in which they had been autoclaved. A sample of crop contents was taken by inserting a pipette into the crop, forcing out the agar plug by injecting 20 ml. of sterile saline by means of a 20 ml. Luer glass syringe attached to the rubber tube after removal of the cotton plug. The syringe and the rubber tube were then de- tached from the pipette, the crop was gently massaged for a few moments and by holding the bird head down, while the pipette was still in the crop, a sample of crop contents could easily be obtained, collecting it directly into sterile test tubes. Examinatignlgf Sample The samples were examined microscopically for organisms that might not grow on culture media and were then cultured. The microscopic examinations failed to show any protozoa or spirochetes; however, numerous gram-positive and gram—negative rods and cocci were present. In the preliminary study, by cultural methods the following genera were found: Alcaligenes, Cellumonas, Corynebacterium, Escherichia, Lactobacillus, Micro- coccus, Shigella, and Streptococcus. Four healthy white Leghorn cockerels were used for further study. The material obtained from the crop was shaken for fifteen minutes. Streak plates on five per cent blood agar (see Appendix for list of media and reagents used) were made directly from the undiluted sample. Further dilution was found unnecessary as discrete colony formation was obtained. The plates were incubated at 37° C. aerobically and under 10 per cent carbon dioxide. All the organisms grew well aerobically at 37° C. and grew even better under 10 per cent carbon dioxide. The sample from the first chicken was also streaked on anaerobic media, covered with Brewer’s anaerobic Petri dish cover and incubated at 37° 0. A.streptococcus was isolated,but no bio- chemical tests were employed and it was not identified. A blood 6 plate from the third chicken was also incubated at room tempera- ture. One organism (No. 35) grew well at room temperature, crowding out other organisms. It was identified as Escherichia gg_i var. communior. Blood plates from the fourth chicken were incubated at “5° C. aerobically and at 37° C. under anaerobic condition produced by replacing air in a pressure cooker with illuminating gas. It was thought that illuminating gas might furnish anaerobic conditions under which organisms, which had not been isolated aerobically, might be obtained. However, no organisms were secured which had not grown under aerobic conditions. Only one organism (N0. M1) grew well at M50 C. and it was identified as Escherichia coli var. acidilacti. After the organisms were obtained in pure culture, smears were made and stained by Burke's (9) modification of Gram's stain. Acid fast, methylene blue and Anjeszky's spore stains were also made of the organisms obtained from the second chicken. Since none were acid fast or revealed meta- chromatic granules or spores, all further cultures were stained by Gram's stain only. Bergey's Manual, Fifth Edition, was followed in so far as possible in the identifica tion of the organisms al- though frequently it was necessary to resort to other sources for a more detailed description of specific organisms. The numbering of the cultures was done in sequence corresponding to the number of the chicken from which they were isolated. The cultures numbering from one through nine were isolated from the first chicken. Those numbering 20 through 29 were from the second chicken. Those numbering 30 through 39 were from the third chicken. Those numbering “0 through 50 were from the fourth chicken. The results of the biochemical test employed are recorded in Tables I, II and III. Results On all blood plates incubated at 37° C. a beta- hemolytic organism was the predominating one. Subcultures from these were numbered 1, 21a, 30 and MO. The colony in all instances was circular in form, convex, with smooth surface, translucent to transmitted light, grayish in color and had an entire edge. The agar streak culture was similar on all subcultures: the growth was moderate, the form of growth was filiform, glistening, butyrous in consistency and the color was a gray- ish-cream. The tryptose broth cultures showed moderate clouding with considerable clearing in five days. There was a moderate amount of grayish viscid sediment. All these organisms were gram-negative, pleomorphic rods which occurred singly, in pairs and in filaments. The average size of the rods was 0.6 x lp and of the coccoid forms O.M x 0.6u. Some of the filaments were as long as 50m. Smears from the colonies on the isolation plates showed bipolar stain- ing organisms. Biochemically these four cultures showed some variation. Culture No. 1 gave an acid reaction on litmus milk in one day, had reduced litmus in one day and had formed a curd by the fourth day. No. 21a did not produce any change in litmus milk. No. 30 did not change the pH but did reduce the litmus of litmus milk. No. HO gave a slightly acid reaction in litmus milk in seven days. The morphological and biochemical reactions of culture No. 1, especially the hemolytic property, the coagulation of milk and the failure to produce indol, in- dicate that it was closely related to Pasteurella hemolytica described by Rosenbusch and Merchant (10) and Merchant (ll). Culture lo. 21a showed a similarity to Pasteurella avicida (Gamaleia) Trevisan except that it was hemolytic, failed to form indol and produced no H28. Patton (12) gives the results of studies on strains of E, avicida by nine investigators, and several found rare hemolytic strains, also strains that failed to form indol within a week. Cultures No. 30 and No. #0 showed characteristics resembling those of E: hemolytica and E, avicida. Because a beta—hemolytic organism, which showed a morphological,cultural and biochemical relationship to the Pasteurellae, was consistently isolated from the crop of the chickens studied, it was decided that the pathogenicity of the latest culture isolated (No. #0) should be tested. A young Rhode Island Red was given an intravenous injection of 0.25 ml. of a heavy suspension of an eighteen-hour—old culture grown on a tryptose agar slant. The suspension was made by adding, aseptically, several ml. of sterile saline. The tube was gently rotated, thereby creating a suspension 10 of organisms free from clumps large enough to produce embolism upon intravenous injection. After two days, blood was withdrawn aseptically and cultured by mdcing a pour plate (one ml. blood to fifteen ml. sterile nutrient agar at h5° G.) and broth culture(two ml. blood to fifty ml. sterile broth.) A hemolytic organism was recovered in almost pure culture. Morphologically it was like Culture #0 and exhibited a definite capsule. It produced strong acid reaction in dextrose, mannitol and sucrose, weak acid reaction in lactose and maltose and no acid in salicin. In lactose broth with a peptone base there was no reaction after a week. Blood smears were made five days after ino- culation but no organisms were found. The bird was still alive at the end of three weeks and apparently in good health. It is of course obvious that a test on one bird is misleading since it is known that individual birds vary greatly as to their susceptibility. Time was too limited for further study of the pathogenicity of this organism. The second most predominant colony on the blood plates was a small non—hemolytic one. Subcultures from this type of colony were numbered 29c, 36 and H2. Cn agar slants a scant grayish film was formed along the needle tract with discrete colony formation along the 11 edge of the film. Tryptose broth cultures were clear with scant, slightly flaky grayish sediment. Litmus milk remained unchanged in each instance. Microscopic examination of smears revealed small, pleomorphic gram-positive rods. There were straight and curved rods, some were club-shaped and others were coccoid. They occurred singly, in pairs and short chains. Some short chains showed branching-like arrangement. The size ranged from .5 x lu to .5 x 2.5». They were non-spore-forming, did not have capsules and were nonmotile. These appear to be diphtheroids and may possibly be related to Bacillus maculatus described by Graham-Smith (13). Culture No. 6, an alpha-hemolytic, gram-positive pleo- morphic rod, closely resembled Corynebacterium enzymicum (Mellon) Bergey at al. and the pleomorphic organism of Graham, Thorp and James (M). There was no perceptible growth in tryptone broth after one week and the test for indol formation was negative. According to Bergey Q. enzymicum produces slight indol forma- tion. Loeffler's blood serum, potato, dextrin and glycerol media were not employed. Culture No. 37 was a small alpha-hemolytic colony on blood agar. The cells were gram-positive, non-spore-forming. 12 motile rods of irregular shape. Some cells were straight, some coccoid, others were dumb-bell-shaped and some were club-shaped. The range of size was from 1 x 1.5u to .8 x lgu. On an agar slant the growth was a scant, grayish film with discrete colonies along the edge. The tryptose broth culture was slightly turbid, clearing in five days, with a moderate amount of grayish granular sediment. It was not identified for lack of information in available literature. Culture No. 27c was a puntiform, convex, alpha-hemo- lytic colony on blood agar. The growth on an agar slant was scant, with discrete small colonies. Tryptose broth did not show any visible growth. Iorphologically the culture consisted of short, plump, gram-positive pleomorphic rods which occurred singly and in pairs. Identification was not possible due to lack of sufficient information in available literature. Culturally and morphologically Culture No. 39 was related to Lactobacillus brevis (Orla—Jensen) Bergey et al. There was an especially close relationship with regards to the fermentation of xylose, levulose, galactose and the vigorous fermentation of arabinose. There was only slight fermentation of dextrose which was also in agreement with the preference of 13 some strains of E! brevis for levulose over dextrose. Io tests were made to determine the products of fermentation of the hexoses and the pentoses. No acid was produced in litmus milk or maltose. Calcium lactate, dextrin, glycerol and starch fermentation media were not employed. Cultures No. 5. 25, 38 and 50 were similar to Lacto- bacillus fermenti Beijerinck morphologically, culturally and biochemically in so far as the tests employed corresponded to those enumerated by Bergey. Yeast extract-dextrose gelatin, dextrin and starch fermentation media were not used; nor were tests made to determine the products of fermentation of the hexoses and the pentoses. There was no reduction of litmus in litmus milk. Cultures No. 9, 22a, 31, and N9 were identified as Streptococcus lactis (Lister) Lohnis from Sherman‘s (1h) description. Potato, glycerol, sodium hippurate and esculin media were not used. Chemical tolerance tests were not employed. Temperature tolerance was not determined. Neither antigenic analysis nor serological identification was attempted. Cultures No. 27. 32 and M6 were identified as Strepto- coccus equinus var. iggavus Holman as described by Sherman (1h). Sodium hippurate, esculin and glycerol media were not employed. in Temperature relations and chemical tolerance were not determined. Culture No. 33 corresponded closely to Streptococcus liquefaciens Sternberg emend. OrlaoJensen. It varied from the description in that the curd which formed in litmus milk was not peptonized, however caseolysis fails in variants which do not liquefy gelatin. Sodium hippurate, esculin and glycerol were not determined. According to the tests employed cultures No. 3. 23¢, 3N and H5 were similar to Neisseria catarrhalis (Frosch and Kolle) Holland. The biochemical, cultural and morphological characteristics of Culture No. 2Hc were similar to Flavobacterium pgoteus Shimwell and Crimes. Culture No. 35 was identified as Escherichia coli var. communior (TOpley and Wilson). Culture No. Ml was identified as Escherichia coli var. acidilacti (Topley and Wilson). Culture N0. M2 was identified as Escherichia coli var. neanoli- tana (TOpley and Wilson). Two micrococci were isolated: Culture N0. M3 was 15 identified as Micrococcus percitreus Bergey, et al and Culture No. h? as Micrococcus epidermidis (Kligler) Hucker. Culture No. uh corresponded closely to Shigella minu- tissima (Migula) Bergey, et al. 16 I T " E NO. Biochemical Characteristics and Motility .Hoadhm museum 0w OHOdrw 4. +— * - 4... Hepwsame * omOpHmE _++ swepoma + «r + + ‘b + 4- + @9-(+)-(+)- omospwmd + -+ + i-‘r - + sans maspaa ARC slA+++ ARC i- + + RAG RAG N sl.A -P + -t mmm opmauao Hosea don .nuoe .xmoam womob mamampmo mmz mo.voam mosses“: .mofiwaa .How O Iago.“ Cg +hau 08 m m masses .-t+»- sopasz massage 21a 22a 23c 2h 25 27¢ 29c A - Acid light "N - Neutral (+)- Acid and slight amount of gas R - Reduction C - Curd 4.. ~ — s Blank space - test not run -‘I 17 nouonpeu - H PJnO ’ O 39? pan pIUB‘-GD an; qou qseq - seeds quetg notices; pros qqfirts — v [s tedqnefi - N PI°V ’ V WSIIS - 1' 1E ni 2i IQ of La Culture Number mot. a a O 4. (D L. ferm f" ‘* gel. liquef. nitrites prOd. Of NH3 + is a catalase Voges Prosk. + meth. red + indol citrate H28 V I3 DEV litmus milk 4-‘v Is dextrose + 4 + '3 N lactose 4 ‘? maltose It + mannitol '1' sucro 86 ++++++OHY salicin + + ‘t i- + ‘6 + l-xylose 'I‘ 4 dulcitol d-sorbitol i-inositol l-arabinose l-rhamnose d-xylose d—galactose + + + +- e d-mannose 4' + d-levulose trehalose + + raffinose inulin + - -@@®®@Q®-@©®@-®®@@Hov - starch hydr. 18 LLITITON CH? SDILSIHELOVHVHO TVOIWEHOOIE II 'OH ETEVE sefi pus p199 — G) unfitts - I 9109 Ktzqfitts - v 18 GUIIVHIB - XIV notqezxuoqdag - a and son 4331 - seeds xuetg notionpeg - a pJno - o; tezznah - fl 09 6h 9h LN 1m in 8n In Culture Number mot. a a o L. ferm. 9' ‘* + gel. liouef. nitrites prod. of N33 4 + + catalase Voges Prosk. 4. *- meth. red q. indol 0V litmus milk dextrose ¢+QEV lactose + +3+ maltose mannitol a. SllCI‘O 86 ®®®®9® salicin l-xylose dulcitol d-sorbitol i-inositol l-arabinose IILrhamnose d—xylose d-galactose d-mannose * d—levulose trehalose + ‘4 raffinose inulin 19 aritzon pa? SOTQSIJGQOBJBUO leotmaqaolfl K 'ON ETEVL III DISCUSSION The flora of the crop of healthy chickens appears to be subject to some variation, however some organisms seem to be constant inhabitants. The Pasteurella-like organism was consistently isolated as was the Neisseria sp., Lactobacillus fermenti and Streptococcus lactis. Streptococcus eouinus var. ignavus and Corynebacterhmxsp. were isolated from the crop of three of the chickens. Escherichia coli was iden— tified from only two chickens. The Micrococoh, Shigella, Flavobacterium, Lactobacillus brevis, Corynebacterium enzymi- ppm and Streptococcus liquefaciens were each isolated from only one bird. This apparent variation might be due to in- adequacies of bacteriological procedures. It is quite pos- sible that these organisms might not actually have been absent. The colonies that appeared to be alike were picked in duplicate. But this did not guarantee that other colonies, apparently identical, might not represent other organisms. In subculturing colonies from the blood plates from the crOp contents of the third and fourth chickens this was taken into consideration and a greater number, but not all the alpha- hemolytic and non-hemolytic punctiform colonies were subcul- tured in semi-solid agar. This was effective in securing more species and more abundant growth. In regards to possible sources of contamination, the 20 following should be considered: 1. The possible contamination of the agar When plugging the distal ends of the pipettes. The pipettes had to be used soon after they were plugged since the agar dried in a few hours and broke the seal. Hence there was no opportunity for detection of contamination. However, if the agar was practically solid when the pipettes were plugged, the time interval was reduced to only a few minutes before the pipettes were returned to the sterile container. Since the table had been wiped-down well and the windows and the doors were closed to prevent cross currents of air, the amount of contamination was practically nil. The important area of the pipette, the inside, still remained completely sterile. 2. In inserting the pipette into the crop of the chicken, it is obviously possible that organisms may be carried down from the mouth and esophagus. However, this is not serious because of the fact that the bacterial flora of the crop is constantly conditioned by organisms from the upper respiratory tract and oral cavities. A considerable amount of difficulty was encountered in attempting to identify organisms which were isolated because of incompleteness of the available literature dealing with the genera concerned with respect to description of various characteri- stics of the organisms and also with respect to the exact nature of the media used. 21 In this study no antigenic analyses or serological identifications were attempted, the identifications were based solely upon cultural, biochemical and morphological features. Most of the organisms isolated were closely related to previously described organisms but might nevertheless be different species. 22 SUMMARY 1. A study of the flora of the crop of healthy chickens was made. 2. Organisms which were either identical with or similar to the following were isolated: Pasteurella sp. Neisseria catarrhalis Lactobacillus fermenti Lactobacillus brevis Corynebacterium enzymicum Corynebacterium sp. Streptococcus lactis Streptococcus eguinus var. ignavus Streptococcus liquefaciens Flavobacterium pgoteus Escherichia coli var. communior Escherichia coli var. neapolitana Escherichia coli var. acidilacti Micrococcus percitreus Micrococcus epidermidis Shigella minutissima 3. As indicated in the discussion only morphological, cultural and biochemical procedures were employed in the identi- 23 fication, therefore it is quite possible that some of the isolated species are not identical with the ones named above. h. A.Pasteurellarlike organism was tested for pathogenicity with negative results. 2h 2. LITERATURE CITED Tanner, F. W., 193%, "Microbiology of Foods," Textbook, Garrard Press. Gibbs, Charles 8., 1931, "Saprophytic and Secondary Microorganisms Occurring in the Respiratory Tracts of Domestic Fowls and Chickens in Health and in Disease." Jour. Bact. 21: 97 Kernohan, George, 1931, "Infectious Laryngotracheitis of Fowls," Jour. Am. Vet. Med. Asso” 78:196 Graham, R., Thorp, F. Jr., James, W. H., 1930, "PleomOrphic Micro-organism Associated with Acute Infectious Avian Laryn- gotracheitis," and "Subacute or Chronic Infectious Avian Laryngotracheitis," Jour. Inf. Dis., M7: 33 and 87 Beach, J. R., 1931, "A Bacteriological Study of Infectious Laryngotracheitis of Chickens," Jour. Exp. Med., 5k: 801. Eliot, C. and Lewis, M. R., 193”, "A Hemophilic Bacterium as a Cause of Infectious Coryza in the Fowl," Jour. Am. Vet. Med. Asso., 8h (N. S. 37): 878. 25 10. ll. 12. 13. Emmel, M. W., 1930, "A Study of the Bacterial Flora of the Feces and Intestinal Contents of the Fowl.“ Thesis for M. S. Degree, on file in library, Michigan State College. Stafseth, H. J. - Personal communication, unnublished work. Burke, 1922, "Burke's Modification of Gram Stain," Jour. Bact., 7: 178. Rosenbusch, C. T. and Merchant, I. A., 1939, " A Study of the Hemorrhagic Septicemia Pasteurellae," Jour. Bact., 37: 69. Merchant, I. A., 19H2, "Veterinary Bacteriology" - Text- book - Collegiate Press., p. 360. Patton, J. W.. 1925-26, “Avian Hemorrhagic Septicemia (Fowl Cholera), “ J. Am. Vet. Med. Asso., 68: 581. Graham-Smith, G. 5., 190%, “A Study of the Virulence of the Diphtheria Bacillus from 113 Persons and of 11 Species of Diphtheria-like Organisms, Together with Measures to Check Outbreak of Diphtheria at Cambridge, 1903,“ Jour. of Hygiene, h: 258. 26 1h. Sherman, J. M., Dec. 1937, "The Streptococci," Bact. Reviews, pp. 63 and 53. 27 APPEEDIX The Media Used Were Made Ag Follows: Gelatin liquefaction test medium: 120 gm. gelatin 5 gm. sodium chloride 3 gm. beef extract 1000 ml. distilled water Nitrate-peptone solution: 1 gm. Bacto peptone 1 gm. nitrite-free ENC, 5 gm. sodium chloride ’ 1000 ml. distilled water Proteose-peptone medium ( V~P ): 5 gm. Bacto proteose-oeptone 5 gm. dipotassium phosphate 5 gm. dextrose 1000 ml. distilled water Starch agar: 15 gm. agar 2 gm. peptone 3 gm. beef extradt 1 gm. dipotassium phosphate 1 gm. sodium chloride 5 gm.'soluble starch 1000 ml. distilled water Semi-solid agar: 3? g. Bacto brain heart infusion dehydrated 1.5 gm. agar 1000 ml. distilled water Nutrient broth: 20 gm. Bacto tryptose 5 gm. sodium chloride 3.5 mi. N. sodium hydroxide 10 gm. dextrose 1000 ml. distilled water 28 Blood plates were made by adding 15 ml. sterile defibrinated sheep's blood aseptically to 300 ml. sterile chicken infusion agar base at #80 C. The motility medium was a modification of Bacto' iotility Test Medium to which 1 per cent lactose, l per cent Andrade's indicator solution and .05 per cent dipotassium phosphate were added. Litmus milk was prepared from skimmed milk with sufficient litmus solution added to give good color. For fermentation studies a 1 per cent sugar solution was made with a tryptose broth base. Only 0.5 per cent of the rare sugars was used. Agar slants were prepared from Bacto tryptose dextrose agar. Citrate medium was Bacto Simmons citrate agar. Iron agar was prepared from Bacto Kligler‘s iron agar. Difco tryp- tone broth was used to detect indol formation. Anaerobic agar prepared by Baltimore Biological Laboratories for use with Brewer's Anaerobic Petri dish cover was used for anaerobic culture. *See Difco Manual, 7th Edition for all Bacto products. 29 Test Reagents Employed: Litmus solution: 1+0 gm. granular litmus 300 ml. 110% alcohol Indol Reagent: 75 m1. Isa-ml alcohol 25 ml. cone. hydrochloric acid 5 gm. p-dimethylaminobenzaldehyde Nitrite test solutions: ‘ 8 gm. sulfanilic acid Number 1. 250 ml. glacial acetic acid 750 ml. distilled water 5 gm. apnaphthylamine Number 2. 250 ml. glacial acetic acid 750 ml. distilled water Acetyl-methyl-carbinol test (Voges—Proskauer): 1 gm. c0pper sulphate in 10 ml. water 140 ml. conc. ammonium hydroxide 950 ml. (10% aqueous) sodium hydroxide For production of R'Hs Nessler's reagent was used. Andrade‘s Indicator: 100 m1.0.2$ aqueous solution acid fuchsia 16 m1. N/l NaOH . For catalase promotion: 1 ml. of Parke—Davis Hydrogen Peroxide (3%) to broth culture several days old. 30 ACKNOWLEDGMENT The writer wishes to express appreciation to those who have aided in this work: to Dr. H. J. Stafseth under whose supervision these studies were planned and executed,“ Miss Lisa Hen and to others of the Depart? ment of Bacteriologsr who assisted with pre- paration of media and handling of animals. 31 . 1 7... . . .. .. . . .- . {IIIJIIV : - I: . a . . o . V, . 1‘1 In}. . it . E V! «K . .ti' . . .‘I. n .. 3‘ ‘0 .‘ «IL \I' fix .un... 5 \ - Am . nu 4 - u ahkk a ‘ .0 .n, I . a n .. .2 nu . .m .. In Ar] - 1. . r e . .7 - - I , I \l. .. 1 I I n \I ’ s A > I \ m .1 1‘},..I Ills-Ilv.v I: lit. '1 \. l ‘ ’1“! a 6! I In J p s I n . \ b. . ol .J - III I (a .5 k (A u . 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