.’ f \l vi H l y 1 m I“ \ “I ‘ ‘ IV‘I‘ ‘ 1 0 l I ‘ll \ 1 1 it! t" H MI ,1 I! 1M AH E‘w’ALUATE‘GE‘J O? MESEA FOR 7&2 :‘aSCiLAflON 6F SAtMONELLA §ROM FECES Thesis $0: fhe Degree of M. S. é‘MCE‘iEGAN STATE COLLEGE Syivia $1.953 Laine i948 THESIS This is to certify that the thesis entitled An Evaluation of Media for the Isolation of Salmonella from races presented by Sylvia Lela Laine ' has been accepted towards fulfillment of the requirements for mm;_degree inJecieniolOgy ”UL jor professor DateJfiWfiS _ “-795 1.! EVALUATION 0! non 108 m I$IATIOI 01' W mom 11018 by lylvie Lela nine L THESIS hbnitted to the School of Graduate Studies of Itichigen State College of Agriculture and Applied Science in pertiel fulfilment of the requiremte for the degree of “ST!!! 01' 861W] Department of Becterioloa and Public Health 191*! .‘I’Hh‘agg ”“2532 lug? ‘3' é/ Ll wmmmr It is a pleasure to acknowledge the assistance and advice of Dr. I. L. Mall-nun in the work and the help of Prof. J. A. Davidson of the Michigan State College Poultry Departnent and nsnhers of the Regional Poultry Laboratory. 0.8.13.1" without whose cooperation in collection of specimens. the practical studies could not have been node. The writer also wishes to acknowledge the courtesy of the iiichigan State Health Labora- tories for the urological identification of cultures. 21?}01- mm 01' COMES mmOTIOIOOooooeeeoeeeeeeeeeeeeoeeeeeeeeeeeeeoeeeoeooooeeeooooo 1 mmmcn....00000 ..... O...00....OOOOOOOOQOOOOOCOO00.00.00.000... 2 ‘0 ”11d uwmoooee00.009000...0000000000000...eeeeeeoeoeeeeee 2 BO hdchent '“1‘0000000eeeeeeeoeeeeeoeeeeeeee00000000000000. 6 mmmOOOOOOOOOOOOOOOeeoooeeoeeeeeeeeeeeeeeeeeeeeeooeeoooooo 8 I. h" mm." Stud1°.OOOOOeeeeeeeeeeeeeoeeeeeeeoe00000000000. 8 ‘0 “11d non-dianOIS‘lO mediate...eooeeeoeeeeeeeeeeeeeeeooo 8 lo selective $116. “diacooeeeeeeeoeooeeeeeeeeeeeeeeeeeee0010 0. brichut [Mines-noon...”.c......o..............“.15 D. combined enrichnent and selective media.................17 11' liked. Games “6.06. m rec“ Haters-a1...eeeeeeeeeeeeeeeeeeols ‘0 Hon-diagoetlc ledlaeooonc.o.....u.....o.......o......18 3. hflcmelt media...eeeoeeeeeeeeeeeeeeeeeeeeeeeOOeeeeeecom O. Oonhined enrich-ant and selective nedia.................22 III. ’mtical Imfl'mmooecoooooeooooeeeeeoeeeoooeeeeeeeeeeocfi A. Selection of nedia (diagnostic and enrich-ant)..........26 3. 30‘1”! ”d dowrlptlon 0f fecal BQBplOQooooo...o...o...030 c. Emmation Of focal MIQBOOooeeteoeoee0.000000000000032 Do hm‘. f”. practical O”.h3t10fl.oeeeeeeeeeeeeeeoeee0.33 DIMSSIO'oeeeeeeeeeeeeeeeeoeeeeeoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeefi OMLUSIOIOeeeoooeeoeeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeee00.000.000.039 WIIOOOOOOOOOOooe000000000000eeeeeoeeoeeeoeoeoooeeeeeoeeeeooeoho MMIBOOOOOOOOOOOOOOCOOOOO0.000...0.00.00.00.00...0000.00...OQhS W The isolation of pathogenic organisms from mixed bacteriologi- cal cultures has always been a difficult problen. primarily because the pathogenic bacteria are generally a small minority of the organ- isms present. 1urthermore. the pathogens. as a rule. are fastidious organisms that demand special media. whereas. the non-pathogens fre- quently grow vigorously. particularly on the special media employed for the pathogens. To hold back the growth of the non-pathogenic bac- teria. it has been necessary to employ inhibitory agents which are in general. more selective for the non-pathogenic group than they are for the desired pathogen. an. isolation of the Salmonella group from feces is a good example of this problem. Numerous media and kinds and types of selective agents have been tried and recommended. Some have proved to be very useful and others which have been highly recommended have failed completely in practical use. A review of the literature revealed considerable confusion as to what constituted the best method of isolating enteric organisms. It appeared advisable to males a detailed study of the various recon- nended media both with pure cultures and in practical application. this thesis presents such a study. -1-- Historical Review lie attempt will be made intentionally of citing the various re- searchers and their contributions. but rather the different media will be reviewed. to show the author's development of a media and his rea. sons for recommending the medium. 10.143.119.912 The Wilson bimth sulfite agar (l) and Wilson and Blair (2) modi- fication of this medium have been used as selective medium for enteric pathogens by almost all investigators. he principle upon which this medium is based includes the fact that the Salmonella are able to re- duce sulfi tee to sulfides in medium containing a fementable carbohy- drate (glucose) and metallic salts (ferrous nulf'ate) and as a result of the reduction. the colonies of Salmonella and the medium surrounding the colonies are blackened. 'ilson and Blair (2) found the glucose an indi- spensible source of energy and found further. that the acid produced from this sugar facilitated the characteristic changes by bringing the metallic salts into solution. A combination of bismuth and sodium sul- fite causes a suppression of Escherichia 921i without reparably suppress- ing the development of the Salmonella colonies. Sodium phosphate was used as a buffer to absorb emcess acids produced by the fermentation of glucose. thus insuring the blackening of the Salmonella colonies. Bril- liant green (3) aided in the inhibition of the colon group as well as promoted the blackening of the Salmonella colonies. Schmidt (’4) in a -2. practical study of bismuth sulfite glucose medium confirmed the find- ings of 'ilson and Blair. Gunther and hft (5) obtained many more pos- itive results from bimsuth sulfite agar medium in a comparison of the results from Endo's. eosin methylene blue agar. desonycholate citrate agar and bismuth sulfite agar medium. Buy (6) found that bismuth sul- fite agar gave rather poor results for the isolation of Salmonellas. Hynes (7) concluded from his research that bismuth sulfite agar medium inhibited gram positive organisms completely and all _1_._ 921‘; and sup- ported a good growth of 3. mm. and the Selmonellas in general. Gibbons and floors (8) found bi-uth sulfite medium too selective in their experiments with egg powder. Iacconlsey's agar (9) is probably the most co-sonly used selec- tive medium for the isolation of the typhoid-dysentery group. lost state health departments employ Hacconlrey's agar in combination with other solid media for the isolation of enteric pathogens. This medium is based upon the general principles that organisms of the intestinal flora may be isolated by utilising lactose. bile salts and dyes. Bile inhibits the non.colon types and enhances the growth of intestinal bac- teria. Lactose indicates the presence of colon forms by the production of acid and gas. Neutral red is used as the indicator of this oxida- tion and reduction phenomenon. Crystal violet inhibits the growth of gr. positive organisms by its selective bacteriostatic action. Hynes (7) suggested a variation of the usual formula: peptone. sodium chloride. sodium taurocholate. neutral red and 1 percent lactose. In the place of the lactose. he used 0.5 percent sucrose and 0.5 per cemt lactose. Typical red colonies developed and sucrose fermenters as well as lactose fermenters were easily recognised. than. Sell and l’ollack (10) found that though Macconlney's med- ium was usually reliable and supported a good growth of pathogens it appeared to favor the lactose fementers at the expense of the non-lac- tose fermenters. Gibbons and floors (8) found MacOonhay's agar medium not selective enough for isolation of the Salmonellas from eg powder. Leifson (11) presented a medium based on the principles that if a solution of neutral red or a similar dye and sodium desoxycholate are acidified. the neutral red goes out of the solution with the precipi- tate of the desonclmlic acid. The bile reduces the toxicity of the dye to bacteria. and iron salts (ferrous citrate) increase the inhibi- tory effect of the medium upon contaminants such as 1; 32;}. and other lactose fermenters. while they lessen the inhibitory effect on the path- ogens. There are several modifications of Leifson's medium. Difco's modification S S agar was studied in this investigation. Difco adds brilliant green to the medium to inhibit gram positive and non-intesti- nal bacteria. Sodium thiosulfate indicates the sulfite reducers by pro- duction of ludrogen sulfide. Gibbons and Moore (8) obtained good results with desorycholate citrate medium. Hynes (7) modification of this medium gave good re- sults but was difficult to prepare. Gmither and luft (5) found bimth sulfite agar superior to desoxycholate citrate medium. Derby (12) race. mmemded S S agar medium for the isolation of g: puller“ from chicks. -10- Harrie-Bolt and league (1}) raconendad the use of aosin-math- ylene blue agar which utilised the two dyes in combination with lac- tose and sucrose. for the isolation by differentiation and inhibition. of enteric organisms. league and Olurman (1h) devised an eosin-brill- iant green medium which inhibited lactose farmenters and gram positive organise and differentiated between those that grew and the pathogenic non-lactose fermenters. lhox. Gall and Pollack (10) suggested a median utilising the threa dyes. eosin. methylene blue and brilliant green. Il'hay found that the gram positive organisms and lactose farmenters were supporessed by the brilliant green. the lactose fermentere that grew could be readily differentiated by the oxidation-reduction action of the methylene blue. his Proteus group was also inhibited. Brilliant green liver-infusion agar medium was reconended as a selective medium for g. m by liailmann. i'hmrp and Sumes (15). Liver-infusion agar was suggested by Huddlason (15) as a base medium for fastidious organisms. he effectiveness of brilliant green as a selective inhibitory agent has been proved by Torrey (l7) and confirmed by Rakieten and Rettgar (18) and others. Oruickshank (19) recommended Boyle's brilliant green acid fuchsia agar which n. round to be inhibitory to Pmteus. rm. medium which is comparable to ecsin-brilliant green agar (ll-t) in formula. was simples-“to prepare and more consistent results were obtained. The acid fuchsia did not have to be titrated for each lot of medium. as did the eosin. Better .6- colonial differentiation was obtained. his medim contains besides the base medium and the dyes. sodium taurocholate which is inhibitory to non-intestinal organisms and seems to stimulate the growth of en- teric pathogens when present. lnrichmant Media ‘l'he literature revealed that many enriclnent media have been used. Conradi (1h) demonstrated the inhibitiva action of brilliant green on 2:. 22;; at dilutions which allowed the typhoid organics to grow. Surrey (17) tested the efficany of brilliant green broth as a specific enrichment for the paratyphoid-dysantmry group. browning. Gilmour and Iackie (20) utilised brilliant green peptone water. They demonstrated the bacteriostatic action of the dye and the stimulation of the enteric pathogens. Rahatan and Rattger's methods were found to entail too much work by Orsechowslri (21). Buy (6) suggested a med- ium for enrichment which she found useful. Hynas (7) recouended Buy's medium. Be fotmd that it suppressed Proteus and inhibited _l__._ 22;; and gran positive organisms. This nedims utilises brilliant green dye which is inhibitory to the gran positive mid lactose fermenters and picric acid which is inhibitory to gran positive organisms. Sodium tetrathionate broth was first discovered to be useful as an enrichment broth by Mueller (21). The principles upon which this med- inn is based involved the regulation and inhibition of the physiological activities of contaminating organisms by sodium tetrathionate which is -6- formed by the reaction of sodium thiosulfate and potassium iodide. Iodine is inhibitory to gram positive organisms and the bile salts in. hibit the non-intestinal types of organisms. Protoose-peptone is used in the Difco foimula as a ready source of energy for bacteria and aids as a buffer against too great a change in the reaction of the medium. Khalil (23) found a high incidence of the Salmonella organisms in rats and mice and ascribed his success of isolation to the use of totrathio- nate broth and oosin-brilliant green agar media. Hoaden (214) obtained twice as many positive results from feces and obtained pure cultures of the pathogens directly after enrichment with tatrathionate broth by plating on Endo's medium. lggrinental 3tudies 1. Studies with Pure Cultures A. and fledia - Fon-digegstic \ Oonparative studies are useful and necessary in any scientific inquiry. '!o initiate this research. a series of tests was set up to denonstrate and compare typical characteristics and reactions of var- ious bacteria of the intestinal flora on non-diagnostic media. 31% [lococcus aureus. geeudononas aeruginosa. groteus vulflis. lgcherichia coli. Bacillus subtilis. Aerobacter aerogges. Shigglla disentariae. gonella m, Salmonella tn himuriun. Salmella Elgrun. and is]! m}. nella paratmhi were selected-as representative organics that night be isolated fron fecal samples. Plain nutrient agar and tryptose agar were conpared. Darby and hallnann (25) compared Bacto-tryptose. a pep- tone’used extensively for cultivation of fastidious nicroorganisns. one ' Beets-peptone and showed that nuch more rapid growth occurred with the former peptone. The following procedure was set up. A standard seeding of a 2’4 hour culture of each of the control organisms was suspended in sterile test tubes containing lO“=cc of ster- ile saline. and thoroudily nixed. lash of these suspensions was streaked on plain nutrient agar and tryptose agar. The plates were incubated for '21; hours at 37° centrigrade. At the end of this tine the plates were examined. Table 1 shows the appearance of the colonies of the various organisms on plain and tryptose agar plates. Table l APPEARANCE OF BACTERIAL COLONIES ON TRYPTOSE AND PLAIN NUTRIVNT AGAE AND A COMPARISON OF TH? RELATIVE SIZE AND AMOUNT OF GROWTH ON THE MEDIA AFTER 2h HOURS INCUBATION AT 370 C."‘ Bacteria Description of Colonies end Appearance of Medium Plain Nutrient Agar Tryptose Agar Staph. eureus PS, eerugingga, ,§L_Vulgaris §;_coli 3. subtilis §L_eerogenes 3h..d1ienhnnnm1 ia‘rnflmL §;_typhimurium §‘_gullorum Colonieq ere round, smooth, glistening butyrous, en- tire, raised, yellowish to orange in color. Media not changed by growth, No individual colonies, confluent growth surface of agar covered by moist grayish to yellowish glisten. ing even layer, Medium bright green fluorescent, No individual colonies; very spreading growth, flat Opaque, smooth, grayish, Distinct fishy odor. Media not changed. Colonies white to grayish opaque, smooth, moist, Some colonies slightly brownish, entire to undulate media not changed. Colonies Smooth, raised to convex grayish white, round to emebeio crenete, margins, butyrous adhern ent media Slightly grayed. n See 3L_coli; growth and colonies Colonies small, discrete, slightly raised; grayish in color, smooth entire, slightly convex media not changed, Colonies small, entire, smooth, grayish, convex trenSlucent, Media not changed, Seme as SL tyghi Colonies very small — same As §*_tg3hi. Size 2-5 mm, Good growth Not measurable. Very luxur- ious growth. Variable colonial size. Not measurable as to Spreading, Very abundant growth. Size 1—10 mm. Good growth. Size ”.6 mm. Growth abundant, Similar to E. coli Size 2-3 mm, Growth scant. size 2-5 mm, Growth Scent. Size 2-5 mm, Moderate growth 1-2.5 mm. Fair growth Size ZaS mm. Good growth Not measurable. Very luxurious growth, Size of colony not mes. surable. Very abundant growth. 1-12 mm, Size. Abundant growth, Size u-10 mm, Growth abundant. Similar to E‘_coli Size 2-h mm, Moderate growth. Size 2-5 mm, Good growth Size 2-5 mm. Good growth 2-3 mm. Moderate growth *Appearence similar in both media, However a variation of size and amount of growth on plain nutrient agar and tryptose agar plates was noted, The colonies of M. m. 2,5; asaginoss. and g: subtilis were quite characteristic. 2; vulgggie because of its spreading cher- ecteristic, could be readily identified. _l_._ gall. .L_._ eegggee. 932' gsenteriee end the Salmonella were nore difficult to identify be- cause of nsrlned colonial einilsrity. The need for selective nedie for the isolation of pathogens was quite obvious tree this series of tests. be tryptose plates duonstreted better growth and larger colonies then did the plain nutrient agar plates. particularly of the pathogenic or. genisns. The fonulee for plein nutrient and tryptose egnr nedin will be found in the appendix. 3. Selective Solid Medic Ehe following solid selective media were studied in the next series of tests: Mecconhey's eger. 'ilson end Blair's bi-uth sulfite eger. 8 8 eger. e. nodificetion of descsycbolete citrate eger. eosine. brilliant greenpnethylene blue eger suggested by then. Gel]. and rolleck. brilliant green liver-infusion eger recommended by Hansen. harp and Senses. end Hoyle'e brilliant green-acid fuchsia agar nediun. Pure cul- m. studies of the control organises were ends on each of the six eel. ective nedie. employing the saline suspensions of 2’4 hour cultures as in the first series of tests. The colonies were studied at the end of 2“ hour and M hours incubation at 37° 6. Pure culture studies of Vilson end Blair's bismuth sulfite agar indicated that this nediun supported e good growth of the Selnonellee. ’lhe gran positive organisms were inhibited. Proteus grew. but bed no -10- tendency to spread and was readily distinguished fro: the patlngens since it did not blaclnen on the nediun as did the pathogens. 1'. 22;; survived on bismuth sulfite agar. appearing as droplets of noisture and after ’48 hours very pale yellowish green. indicating that care had to be exercised in picking colonies fron nixed cultures to avoid con. tanination. Experiments with lacOonlney‘s agar showed that this sodiu- sup- ported a good growth of the migellas and Salmonellas. Gran positive organisms were inhibited. Proteus grew well and spread over the In- tire plate by the end of he hours incubation. giving off a distinct and characteristic odor. Lactose fenenters grew with characteristic red to pinkish color and a precipitation of bile salts surrounding some of the colonies. Brilliant green liver-infusion agar. when tested in pure culture studies. inhibited a. gal; and the gran positive organi-s completely. l’mteus was not inhibited and was difficult to differentiate fron the pathogens. l'he Ialnonellas grew well. !he preparation of the base nod- inn was tedious and difficult. The media studied up to this point. demonstrated more or less satisfactory results in elisinating or differentiating gre- positive bacteria and the coliforn and other lactose fermenters. However the Proteus group was the nost obnoxious non-pathogen that grew on culture plates. for it not only spread over nedia. asking isolation of individ- ual colonies impossible. but was difficult to differentiate from the pathogenic organisms. -ll- Knox. doll and Pollack (10) found eosine-brillient green-neth- ylene blue agar medium inhibitory to the Proteus. In this investiga- tion. tryptoee agar was ueed as the basal nediun instead of tryptic digest agar was suggested by the authors. The preparation of the nediun was difficult as fresh dyes had to be prepared often and the techniques were tine consuming. Pure culture studies showed eneell. ent results. !he lactose fernsnters that grew eere few'in nunber and these readily differentiated by colonies which were large. mucoid and with deep indigo centers. ‘Proteus was inhibited completely. the Sal- monellas grew luxuriously. 'L'he §_._ Ellerun colonies which are usually quite tiny on most media. grew to a.much.1anger size. as did the colonies of all the Salmonella tested. Gram positive organisms were inhibited completely. Oruickmhankr(l9) recommended Hoyle's brilliant green-acid fuchsin agar which had been shown to be inhibitory to Proteus. Iith pure cul- I tures. experimentally. the medium inhibited Proteus and grem positive organisms completely. Salmonellas grow well on the medium. Bhigellas were inhibited. This medium was sheen to be highly selective. was not difficult to prepare and kept well. !he comparative amounts of growth.on each of the media studied are presented in iable 2. In Table 3 are presented the typical colon. ial characteristics of the control organisms on the various selective media. l'ormlae for the nedia are given in the appendix. -12.. Table 2 i'he Growth of Organics Representative of Intestinal flora Compared on Selective Media B. G. 1.3.G. 3. G. Bacteria Inc. 0. Bi 8. S S L. I. NJ. A. I. @2212: - - 3 .- - - Easel; «H t 3- - 1' t B; subtilis - «- - - - - L: vulgris +++ +++ 4-H» +4- : - A; 22:25:92.1 H - 35 - - - 5.: m2! *4" ‘H" *H' 4-H- «H-H +4.4. 1. mm +4-0- +4~+ +++ +++ ++++ +++ g; m +++ +++ 4+4- 'H-‘I- ++++ M.- E: W +++ - +++ +++ 4'“ - Key: Io Growth - Very scant growth 1 loderate Growth 'H- Good growth *H' Very mod growth ++++ APPEARANCE Table 3 OF COLONIFS OF VARIOUS BACTERIA ON SELECTIVE AND DIFFERENTIAL MEDIA Bacteria lBismuth Sulfite Agar S.S Agar Brilliant Green- Aeid Fuchsin Agar MacConkey'S Agar Brilliant Green Liver infusion Eosin-Brilliant Green-Methylene Blue Staph. aureus 1.3:. P A is 3. S i. . coli subtilis ar 8 aerogenes typhimurium Pullorum dysentariae No growth No significant growth,those present pale, Opaque,yellow- 'ish to green without pitting medium, sometimes brownish. No growth Colorless to pale bluiSh or yellow green. No pitting in medium. 1-2 mm. No tendency to spread. USuslly inhibited. Similar to §‘_coli Round, about 1-3 mm. Jet black surrounded by black- ish zone, with intense me- tallic Sheen, young colon- ies, green with black centers. Soft with blaCk pit in medium. Colorless to bluish gray, convex, 1-5 mm. Smooth but- yrous, tranSparent. Clear area surrounding colony. Same aS above (Sal) though much smaller No significant growth No significant growth; very tiny pale pinkish brown. Growth inhibited. If present large Opaque, pink to red colored; mu. coid; umbilicate. Some whitish edges. No significant growth Res embl e Sal monell a though usually larger cloudier. Fishy odor. 1-5 mm. Colorless trans- lucent to yellowish or tan; May have ten center Smooth butyrous done Shaped sometimes umb 11 i- cate. Colorless to gray green slightly raised, labate clear 1-5 mm. Size. No change in medium. Same as g; tyohimurium' except much smaller and dark center Colonies same as §£ typhi No growth Mostly inhibited. If present, large mucoid umbilicate, opaque greenish blue with vi- olet zone. 1-5 mm. No growth No growth (No growth F Size 1-5 mm. Smooth transparent, color- less to slightly yel- low. Flat to slight convex. Older colonies develop central peak. Uniform pinkish pur- ple. translucent with lacerate edgesyumbil- icate. Size 3-8 mm. Grape 1 eaf 1 ike . Same as S. tflhimur- ium thou-gh- somewhat Smaller. No growth No growth Colonies 1-9 mm. opaque brick red to pinkish occas- ionally white or gray cream usually surrounded by zone of ppted. bile salts. Smooth glistening. No growth Colorless, transparent, spreading colonies. 2-8 mm. in size. Flat to convex. Similar to Salmonellas but larger. See E _,_ coli Colonies colorless to blu- ish gray, convex. Size 1-3 mm. Glistening smooth but- yrous. Clear area around colony. Transparent. Dark green to dark'brown lusterous, Size 1-5 mm. Flat to raised cohonies pit- ting of medium. Black cen- ters with transmitted light. Colorless to bluish gray. tranSparent, glistening. Smooth butyrous clear area surround colony tiny 0.5-2mm Colorless to whitish small 1-6 mm. transparent to opaque, uneven edges. Flat to slightly convex. No growth No growth No growth Dirty gray mucoid or somewhat dry. Spread. ing flat to convex. Opaque distinct odor. Grayish green, shiny raised centers, buty- rous, opaque. Medium greened. ' Lobate, dull grape-leaf like. Colorless, size . 1-5 mm. Medium some- times yellowed. Colonies similar to §L_typhi, larger. Dark brown to black center. 1-7 mm. Tiny colorless to pale+ bluish green. Clear 1 moist labate dull gran; ular. Size 1-2 mm. ‘ Similar to '§_._ tmhi No growth Scant growth, or none. Deep indigo centers. Large mucoid, umbili- cate or convex. No growth No growth usually. Sim- ilar to Salmonella if they do not grow. No growth Uniform.pinkish purple large translucent la- cerate edges. Umbili- , cate. Size 3-8 mm. Grape leaf like. Similar to §‘,typhi. Same as S; typhimurium or S. typhi. Colony size 1-6 mm. Similar to §‘,typhi and Salmonella -1M. A recapituletion of the findings showed that bis-nth sulfite agar was quite selective. and the pathogens could be readily identi- fied. MecOonlney's eger medium wes not very selective end seemed to fever the growth of the lectose fernontsrs more then the non-lectose fermenters. 3 8 eger. though selective. did not inhibit Proteus end these colonies were difficult to differentiate from the pethogens. Resins-brilliant green-methylene blue eger wes highly selective and very favorable to the Salmonellss. 1; 2.9.1}. could be reedily distin- guished free the non-lectose fernenters when they grew. and 2; Lu;- geris wes inhibited conpletely. l'he sodium was difficult to propere end did not keep well. Brilliant green liver-infusion eger inhibited 1. coli and gran positive organises completely but allowed the Proteus to grow. Brilliant green-ecid fuchsin eger nediun inhibited gren positive organises end 2:. vulgris completely. Ehe lelnonelles grew well. The nediun was not difficult to prepare end kept well. 0. Enrddlnnent Media. has nixt series of tests with pure cultures of the test organics wee node with the snriclnent broths. Ray’s nedim end sodium tetrethio- nets broth (Difco). A standerd seeding of eech test orgenisn wes planted into Roy's nedim end sodiun tetrethionete broth. the cultures nixed thoroughly end then incubeted for 18 hours et 37° 0. Iron eech tubs wes streelned e tryptose eger plete end the pletes incubated for an end ‘18 hours. At .the end of sub incubetion period. the plates were emined for growth. The results showed that Buy's nedimn wee very highly selective end was toxic to the pathogens as well as to the non-pathogens. -15.. Table h The Effects of Enrichnent on the Representative Bacteria" ‘Plated on tryptoee agar after 18 hours incubation in enriotnent nedin. fotrnthionato My‘ 3 Organic: Saline Broth Rodin. 3223; mm. H- - .. 19; aeggénosa 'H- - - £3; Qaentnrlne “- + - divulged: ++++ 4+» .. LE: 0211 4" - - 2:. mbtilie ++ - - L.- aeggenee ++ - - 2:. 5222.1. 'H' 'H' r is mum ++ ++ in Key: N Good Growth ++++ Very Good Growth 4» Scent Growth - 30 Growth (1) Streamed directly on tryptoee agar plates (control) -1 C- lodiun tetrethionate broth inhibited the lactose fer-enters and the gran positive organisns conpletely. ‘l'he pathogens grew well. Bow- ever. Proteus grew even lore luxuriously than the pathogens. In fable 1‘ are shown the results of enrichment by Bay's nediun and sodiu- tetrathionate broth of pure cultures of the test organisne. when plated on non-diagnostic nediun. D. lnrichnent with Sodium retrathionate Broth and Solid ledia The final series of tests to be run with pure cultures. utilised enrichment with eodiun tetrathionate broth followed by plating on six selective nedia. A plate of each of the selective nedia, MacOonkey's agar. bisnuth sulfite agar, 8 8 agar. brilliant green liver-infusion agar. eosine-brilliant green-nethylene blue agar and brilliant green- acid fuchsin agar was strealned directly hpn saline suspensions as con- trols of growth. A standard seeding of each of the test organisns was planted into tubes of tetrathionate broth and incubated for 18 hours at 37° 0.. and theme plate of each of the selective nedia was streaked from the cultures. i'hese plates were in turn incubated for at and h! hours. At the end of the incubation period the plates were examined. The lactose fementers were suppressed on all plates after en- richent with sodiun tetrathionate broth. Gram positive organisms were completely inhibited. ’i‘he pathogens grew well but denonstrated little variation tron the direct plating nethods. he Shigellas which did not 61" on bis-nth sulfite agar or brilliant green acid fuchsin on direct plating did not grow on these nedia after enrichment. Proteus see-ed -17. Ink-J A .“."‘.‘—~;“ 4- to be favored by the enrichment with sodium tetrathionate broth and grew even better on all the media except brilliant green-acid fuchsin agar which inhibited its growth with the direct plating :1er also. he compared results of this series of experiments are presented in table 5. II. nixed Known cultures added t9_l‘ecalwuateria;_l_ As a prelininary procedure to the study of nixed cultures. five unknown chicken fecal samples were strealned on tryptose agar and lacconkey's agar plates. Colonies were picked from both sets of plates after incuba- tion at 37° 0. the organisms which predoninated were from the genera Escherichia and Proteus. Practical application proved this to be true of nest poultry fecal samples. Proteus appeared to predominate and to be the lost obnoxious and confusing group of organisms found on the culture plates for the isolation of enteric pathogens fro- chickens in this in- vestigation. A.-N9n-Digggstic Solid Media Methods similar to those used by Hellman. Ryff and Matthews (26) were enployed. using pure cultures of g: vulgaris. 1: gal; and g; 3.129%." 52.1.92. to represent the genera of most sigificance to this investigation. Into each of five large sterile test tubes containing 10 cc of sterile sa- line was placed approximately 1 gram of sterile fecal material. Into the first three tubes, a standard seeding of 2% hour cultures of 2; vulgaris. 'Lgpli- and g: tnhiluriun was introduced respectively. To the fourth tube. a standard seeding of a mixture of 1.. 2.9.3. and _§_._ tzphimuriun was added. '!o the fifth tube a mixture of 2; vulgarig and g; tzghimuriun was introduced. The mixtures were carefully suspended and then allowed Eable 5 comparative Growth of Bacteria on Selective Media Iron Direct Plating and After hriclnent in Bodiun i'etrathienate Broth Solid Medial-4 s s 301...: )IM pew mine} 9 II: n In I e namj - - - - - - maeggggnosa «- - - - - g - gnéggentariae 4' + 4' + + - amt-.1; 1‘ * + - us- - hm; - - - .3. - .. Lashtilis - - - - - - Assamese: - - - - -~ - hm + + + «H» «H- + hm g+ + + ++ «- + I"Enrichment in sodiun tetrathionate broth for 18 hours at Key: - BGL- I EBB-NIB as.” 370 0. prior to plating. Illflllfllllt“ Good Growth lo Growth slight growth Hoderate growth Direct Plating Inrichnent IiacOonlney's agar H 2 Very good growth Bismuth Sulfite agar (Difco) 8 8 Agar (Difco) Brilliant green liven infusion agar losine brilliant green nethylene blue agar Brilliant green acid fuchsia agar tc settle. Bach tube was stashed on plain nutrient agar and tryptose agar plates. his plates were incubated for an hours at 37° 0., and then emined for growth. has first three plates of tryptose agar showed luxurious growth of the individual organisms with which each had been streaked. The fourth plate. which had been inoculated with the mixture of 2:. 293.}. and _S_._ tmhinuriun also demonstrated abundant growth. However. identifica- tion of ten colonies piched at random. since there was no way of distin- guishing the pathogen from the non-pathogen. revealed all ten colonies to be 5; 311;. The spreading growth of the _13_._ vulgaris on the fifth plate obscured any individual colonies and nade picking of isolated col- onies impossible. The five plates of plain nutrient agar showed similar results. though the growth of the pathogen was not as abundant as that on the tryptose agar plates. It was again obvious that a selective med- iun was essential for isolation of pathogenic organisms. Table 6 shows the comparative results obtained from this series of tests. 3 - grictment gedia and nixed Cultures To conpare the growth of nixed cultures in feces and with en- richment broth. into each of five tubes of sterile saline. Ray's medium (though it had proved too inhibitory in pure culture studies) and sodium tetrathionate broth was placed approxinately 1 gram of sterile fecal ma.- terial. and into the first three tubes of each medium, a standard seed- ing of 3.; tmhimurim. 1:. 92;; and g; vulggi . Into the fourth tube of each was placed a mixture of §_._ ggl_._i_ and E. typhimurium, and the fifth. a mixture of P. vulgaris and §_._ mhinuriun. The suspensions were care- .eoeafisouconoo on u om .. 393cc: .3an .89 ecuncaoc 9.1mm .. seaccaoo guarded“ .. doom 3an .39 seasons 0: here i souncaoo 33):...— ... 253554 canon» mcaussunm .. concedes A»: gauche ocean I canvass be» ”how .36? case 68% p.239 concedes o." e New». nude AM: o." gnaw?» d.» OH .3333? and .3238. v3.30: managed.» sequence H3332: ten—5s nouncaoo :3 .n was n39 8 .3339.” 3.2.34 sea 3 .32...an 2538“ on g .m m :8 am 3. .33 4a 3 £55 £885 .23 4a 2 5:26 savanna g «u. : Auoaacnoo £352: och as m as an #3 3 engrave causes weaves.” I. .a 9» .a 3 338% b.» «finale; 4» 2 pieces... to» 3..me .m n .fiqm «m 2 £3.83 :8 am 2 afie§p4 38 «m m ages. in 2 use a an S 38.8: 3% a... a ceasaosn souccuco conga c.9393: souncaco Apnea .838ng henna— 3.0.3 .8333 so 8338“ son .3... 33°38 .8 32.3.: .5 e8 .8330 can: 5 cadences eds .3599; .sBouquoan spouse scum 2-3593 no 5:95 23 3.803889 ca m 0.3!“ -21.. fully mixed. The saline suspensions were streaked immediately on tryp— toss agar plates. Into the tetrathionate broth tubes. a sterile piece of cotton was pressed. to force the coarser particles of fecal material to the bottom of the tubes. The enrichment broth cultures and the tryptose agar plates were incubated for 21% hours at 37° C. At the end of this period. tryptose agar plates were streaked from each of the enrichmnt broths. and incubated for 2‘4 and ’48 hours. Colonies were examined and identified from the direct plating at this time. The results were similar to those obtained in the previous mixed culture experiments. 2: _c_9_l_i_ and 2; vularis far outgrew the pathogens. The examination of the tryptose agar plates made from the enrichment medium showed that lny's medium inhibited 1.. 92;; and g; _V_‘l_l_1_- $9.21.! but was also toxic to the _S_._ typhimurium. The sodium tetrathionate broth. though apparently favoring the}..xnlga:1.s_ allowed the g. tzphimur- __i_m_ to grow. IThis medium inhibited 3. cell completely in this series of tests. Table 7 (a and b) show the evaluation of enrichment media with mixed cultures. To conclude the series of tests ”with mixed cultures. the three representative organisms were compared on the solid selective media with enrichment in sodium tetrathionate broth and from direct plating. The procedures used in the foregoing experiment were followed. except for plating on the six selective media instead of non-diagostic media. - 22.. Table 7.4 An Evaluation of lnrichnent Media! Plate Direct my's Sodium Ho . Organi an s Pl ating Medium ‘Te trathionate l g; typhimurium +++ + +++ a L coli. +++ .. + 3 2.2. 19.1939. *‘H'l’ 3 ++++ 11 Mixture of E. coli +++ : +4- and §_._ tv'o, hfiurfi'fi 5 Mixture of g; vulgaris 44-H- 1' ++++ end _S_=_ itvghimuriun Key - - = No Growth 3 I! Scent growth -1ess~than 10 colonies per plate. 1|! 2 Fair growth -apnroximate1y 10-20 colonies per plate. ++ =- Moderate growth +++ = Good growth 4-H”.- 2 Very abundant spreading growth *After enrichment in broth plated on tryptose agar medium. 23- Table 7-3 Identification of Colonies Picked from Plates ’4 and 5 which were Streaked with Mixtures or Microorganisms"l F Direct M's Sodium Plate Mixture 1’13.th Itedium Tetmthi%_ ’4 _3_:_ txphimurium 0% 100% 100% + __._ coli 100% 0% o;- .. - - .. .......... L .................. 5 §__._ tzggmurium 0% 70$ 140% + h vulgaris 100$ 30% J 60% trementages based on 10 colonies picked. -2h— After enrichment with tetrathionate broth. 1:22.13. grew poorly on MacConkey's agar. _P_._ vulgaris grew well as did the _S_._ typhimurium. Di- rect plating allowed all three organisms to grow well. On the plate inoculated with the mixture of E; 993;; and g: typhimurium, after enrich— ment. E; ggli grew lightly and could be readily identified. 0n the direct plate the growth of §_._ 393:; was much heavier though it could be identified as such. The plate containing the mixture of _S__._ typhimurium; and g; vulggris indicated that this combination of media was not of much value for elimin- ating the obnoxious Proteus group. On bumuth sulfite agar. after enrichment with sodium tetrathionate broth E. coli did not grow. 2; vulgaris grew better after enrichment but could be recognized. _§_._ tynhimurijlm grew well with enrichment and on the directly plated medium. The mixtures of 1; 29;; and g; typhimurium streaked on busmuth sulfite agar directly and after sodium tetrathionate enrichment showed no recognisable £1 93);}. colonies. The plates streaked with the mixture of g; vulgaris and S.ut1phimurium. directly and after enrichnent both allowed the 2:. vulggfis to grow. The colonies could be identified as such. and had no tendency to spread. On 3 S agar _I__._ gg_2_|._i_ was inhibited with and without enrichment. g; vulgaris showed little variation on direct plating and enrichment prior to plating. In the mixtures. g; c_9_l_i was inhibited and _S__._ typhimurium grew well. 2; vulgaris seemed to be favored slightly when enrichment with sodium tetrathionate broth was used prior to plating. On cosine-brilliant green-methylene blue agar. with direct plating. g; typhimurium grew luxuriously. E: 93;; grew scantily. and g; vulgar“ grew poorly. After enrichment with sodium tetrathionate broth E; coli -25. did not grow. 2; vulgaris showed a slight growth. and _S__._ typhimurium good growth. On the plate containing the mixture of the pathogen and E; coli. §_._ typhimurijgm could be readily identified. 2:. vulgaris grew better after enrichment with sodium tetrathionate broth on this medium and the colonies were difficult to identify from §_._ typhimurium col- onies in the mixed cultures. Brilliant green liver infusion agar suppressed E; 39}; com- pletely both with direct plating and sodium tetrathionate enrichment. 2; vulggris grow well with both methods. Brilliant green-acid fuchsin agar was not affected by the utilisation of sodium tetrathionate enrichment. the pathogens grew well on direct plating and after enrichment. The non-pathogens were suppressed with direct plating and enrichment methods. The results of these tests are presented in Table ’8 (a and b). 111- Practical Applications A. Selection of Media to use for Isolatiopl In selecting the media to use for practical application. the isolation of Salmonella organisms from feces, the following qualifica— tions were utilized: (1) the medium should allow the Salmonella group to grow well. (2) it should be inhibitory to the non-pathogenic organ- isms without being toxic to the pathogens (3) the medium should show adequate differentiation of various bacteria. (1;) it should be relap tively stable and give uniform results and (5) it should be relatively easy to prepare. -26.. .35»an 3 serum 595 opsccaflcupoa 538 an ascension s .ooqsaccaac hp mucosa assoc—54 has» n +++... .3353 0» vacate £5 museum venom u + soucoaco Mo engage 5.8.5 coco u +++ moan confines 53.30.? 5.5.5 venom u ..... £38m spandex a .1. 5:96 on. n I I I I I +++ +++ acme sauces» can 59% pasufiaam +++ +++ I I +++ +++ anus gigging: macaw unmadzan + + I H .11.... .III. 055 one Humvee—Icons» assuaauamdsusofi .I. .I. I I . +++ +++ .32 m m .1 u .. .. +++ i4 .53 3 a?» £53 +++ +++ M +1. .1... .I... need Bunkhouse: falcon-.3.» one.» use 0 some as u gonna ages: . .33 . . 8232 1 .Im afifioasmu not... Ea mummy“ pooawm £1 «3.: 3?» ocean: cc snaking» .m was Grumman» .m .300 .M no macaw 05 no confinemaoo a. 4 u 0.309 50333353 new 3ch none scan 339 noncouoo 0.7 $3.323. 2.2.3.. a. 8...... 3.28.8.2. 8 2822. .3828 E .393 no abaacancmsamba cacsmcnasnIccc sandman—woos.» oz Amy 35:35.3 hips... and 33.233an 123.30 wanes. coco .033 so commence oncomfiusadcn use csmfipsm as ”how A3 A3 “3 8V .3». 53.8 .2382.» S .2222.» S .2382.» S .2..er 2 3......8. 2.22.” R. 5 .288... m .2382.» 2 AS A3 a... 328.. 3398 m .2398 m .2822.» S .2883.» S 2.8 896482223 3 .5398 m 3393 .— Amv Amy news can; 28255.: .2322... m .238... m .2383 3 .2383 02 no...» «5222.-....8 5 3 .238... : .Epfim m A3 A3 338.— m .2882... m .238... S .2882... S a... m m 3 3 as g .2322.» S .2282.» S .2322.» S .2382... 2 a... .32.. 58.8 5.8. 32...... 33a .8»... scam I mounoaoo Amy “.5 .33. 25.8. 2.25.." .m .2822... 2 .2832... S a... ..a§..o..a_ Sascha 5.2.3 .028 .833. owe—2.2a .0... .52.:— 2.222.» Ilia .H + n c .3 . jag save: ovuuosnom user...» so .5383 on. uneven.” £33.83.” sheave on» no madcap—o mfiacpceucaaom no conga: scam noaccaoo no couamounuanouu min 0.369 CC With these specifications in mind, a compari son was made of the various media studied. 0f the enrichment media. Ruy's medium was disqualified because it was too toxic to the Salmonellas. Sodium tetra.- thionate broth was inhibitory to the gram positive organisms and the lactose fermenters and was easy to prepare. The medium was made in small lots for each group of specimens and was found to give better results when'freshly prepared. - A review of the solid selective media indicated that in pure culture studies. MacConkey's agar. while it allowed the pathogens to grow, it was not selective enough and seemed to favor the lactose fer- menters and the Proteus group to the desired pathogens. Bismuth sulfite agar medium was highly selective and differ- entiated those non-pathogens that grew. It allowed the Salmonellas to grow well in pure culture studies. Difco's prepared medium was used. 3 s Agar (Difco) showed good growth of the Salmonellas. It was inhibitory to the gram positive organisms for the most part. al- though during actual testing of specimens a few strains of Streptococci were recovered. Lactose fementere were inhibited and if they grew were readily identified. The proteus was not inhibited in pure culture studies and were difficult to differentiate from the Salmonellae in most instances. Brilliant green liver infusion agar was inhibitory to lactose fementers and gram positive organisms. It allowed the Salmonellas to grow well. The preparation of the base medium, liver infusion agar was difficult and time censoring. -29- Basins-brilliant green-methylene blue agar showed good results: It inhibited lactose fementers. gram positive organisms and the Proteus in pure culture studies. It stimulated the growth of the Salmonella group. However. the medium was exceedingly difficult to prepare. be- cause of the number of sterile techniques employed with the addition of each dye and the sugar. The dyes had to be freshly prepared often. as they were found to be relatively unstable. Brilliant green acid fuchsin was found to be highly selective and allowed a good growth of the Salmonellas. This medium was easily prepared and kept well. A comparison of these findings. led to the selection of the following media for the practical experimental phase of this investigap tion: bismuth sulfite agar, S S Agar. and brilliant green acid fuchsia agar medium in combination with direct plating methods and sodium tetra- thionate broth enrichment. Later in the investigation direct plating was found to be of little value for the isolation of Salmonellas from chicken feces and was discontinued. B. Euros end description of fecal samples. P. R. Edwards (27) stated in 1939 that "fowle are the greatest reservoir of paratyphoid in the United States". Since it was difficult to obtain an adequate number of specimens from chickens known to be suffering from Salmonella infections. the suggestion made by Mallmann, Byff and Matthews (26) of the possibility of the normal chicken as a source of infection (Salmonella) to man and animals. led to the choice of normal chickens for investigation. With the cooperation of the -10.. Michigan State College Poultry Department and the Regional Laboratory. U.S.D.A.. fecal samples were readily available. recal samples from 376 chickens were examined. Of these. 298 specimens were from drappings of live birds. These specimens were col- lected into sterile petri plates from the cages of the isolated chickens. numbered. dated and histories obtained for each chicken. Seventy-six specimens were from the intestines of killed birds. The intestines re- moved at autopsy were brought directly to the laboratory for examina- tion. or were refrigerated until they could be examined. All the birds studied had normal life histories and gave no clinical manifestation of any disease. The chickens had had no known contact with diseased birds of any kind. Of particular interest was the fact that the chickens studied were from groups of birds which had been segregated from all other flocks throughout their lives. The preparation of the fecal samples for examination varied only in the preliminary procedures because of physical differences. The intestines of the killed birds were divided into three parts. the large and small intestine and the caecum. each section split open and the con- tents removed to a separate sterile petri dish. The saxoples were labeled as to source. large. small intestine or caecum as well as the bird number. From this procedure. they were handled as individual specimens. Speci- men. of feces from the live chickens were obtained by collecting approx- imately 10 grams or more of the droppings from the tray under the cage of each bird. The trays had been lined with clean paper the night be- -31.. fore the collection was made. Portions of each sample of feces were taken from various parts of the tray. and care taken to collect any suspicions material. such as that which contained any unusual amount of none. was especially liquid. or contained gross blood. 0. lamination gf l'ecal Me; The following procedures were employed in examining each fecal specimen. Iron each specimen. approximately one gran of feces was trans- ferred to a test tube containing 10 cc of sterile saline. and one con- taining 10 cc of freshly prepared sodium tetrathionate broth. The tubes were mixed thoroughly. Into the tetrathionate broth tube was pressed a sterile piece of cotton to force the coarse material to the bottom of the tube. he saline suspension was allowed to settle during this pro- cedure and then a plate of biasuth sulfite agar. s s agar and brilliant green acid fuchsia agar sodium streahed from it. The sodium tetrathio- nate broth cultures and the directly plated agar plates were incubated at 37° 0. After 18 hours incubation. 8 B agar. bismuth sulfite agar and brilliant green acid fuchsin agar plates were streaked with a fairly heavy amount of inocula free the sodium tetrathionate broth en- richment tube. These plates were incubated for 2‘! and ‘3 hours at 37° 0. At the end of 2’4 hours. all the plates were examined for typical colonies. and when possible at least five colonies picbd for further identification. If at the end of 2h hours there was no growth or very light growth. the plates were reincubated for another 2’4 hours and reex- amined. The suspicious colonies were transferred into lactose broth. From the lactose broth. non-lactose fenenters were transferred into tubes of semi-solid agar for stock cultures and motility tests and into tryptone broth for indole production test. !he tryptone broth culture was tested for indole at the end of “8 hours. and if indole was produced. a transfer was made from the stock culture into gelatin and nigler's iron agar slant. A typical reaction in the nigler's agar slant indicated the inoculation of the following carbohydrates: glucose. lactose. sucrose and salicin. Pro.- dnction of acid or acid and gas in glucose and negative reactions in the other three sugars led to further indentification in maltose. Ian- nitol and edonitol. Yoges-Proskauer's test and a Gras's stain were run as supplementary examinations. If the 7-? test was negative and the area's stain demonstrated a typical gram negative rod. fresh stock cul- tures were made and incubated for 2’4 hours at 37° 0. These cultures were then sent to the Michigan State Health Department for antigenic identification. I The flow sheet which follows shows the general methods of pro- cedure. D. Results from practical emriments The findings from the practical applications were twofold: the normal chicken was proved to be a carrier of Salmonellas and a prac- tical evaluation of S S agar. bismuth sulfite agar and brilliant green acid fuchsin agar medium was made. An emination of 376 normal chickens showed an incidence of 1.6 percent of carriers of Salmonellas which were identified as 1.. oranienberg by antigenic identification. A study of the findings in relation to the media. showed that 21 colonies were isolated and tested serologically. rourteen of these colonies proved to be Salmonellas. five were identified as numbers of the paracolon group. and twa were rough colonies which could not be typed- J“- -gi- GENERAL METHODS OF PROCEUJRE Incubated for 18 hours ' at 37° C. Saline Suspension and Tetrathionate Broth Direct «i incubated at Bi S BGAF and N8 hours At least 5 suspicious colonies picked from each plate. X Incubated No acid or gas' Motility and for 2h Heurs produced Indole Negative Stock:Culture Lactose Broth Tryntone Broth Semi-Solid Agar L—T Checked daily for at least 30 days. // Gelatin Kligler's if a typical reaction in 2h hours Single sugars if typical reactions X:] I] X:] X:‘ \ I glucose lactose sucrose salicin maltose mann tol adenitol / Vegas-Proskauer G- e . negative ram negative rods Gram's stain ‘ 2h hour culture Tryptose agar slant State Health Department for Antigenic Identification All of the positively identified Salmonellas had been subcul- tured in sodim tetrathionate broth prior to plating. Of the fourteen positive colonies. 9 were made from brilliant green acid fuchsia agar asdiua. and 5 from 8 s agar plates. roar of the colonies identified as paracolons were from 8 3 agar and 1 from brilliant green acid fuch- sin. the 2 rough colonies were isolated from s 8 agar. lo isolations were aade from biasuth sulfite agar nediun. 'A compendium of the practical findings showed that approxi- mately 614 percent of the Selaonellas isolated were made from brilliant green aoif mchsin agar and approximately fi percent from 8 S agar. Ito isolations were nade from bimth sulfite agar. |l'he results of this experiment are presented in i'able 9. 'l'able 9 Results from Practical Experiluts ’in Relation to Iledia Isolati nsfuade Prom ”Ti—s ’r 5610' '31s. Bird Number No. of Isolations 8 P R S P R S P 505 u 1 1 - 2 - - - - D7126 - - - 2 - - - - Dash: 7 3 - 1 3 - - - - 2802 1 1 - - - .. - - - 5&1 1 - - - 1 - - - - 5503 2 - .. 1 1 - - .. - 8270 2 - 1 - - 1 - - - 1:951:32 2 - 2 - - - - - .. Totals 21 5 l4 2 9 1 o o o 13!: Total Salmonella - 114 Total Paracolons motel Bough 8 - Salmonella identified -5 -2 P - Paracolon group B - Rough colonies could not be typed. 8 3 - 8 3 agar plates BM - Brilliant green acid fuchsia agar plates. Bis. s. - Bismuth sulfite agar plates. DI 8608810] humorous selective and enrichment media have been used and recommended for the isolation of the Salmonella group from fecal spec- inens. A detailed study was made of the various selective and enrich- nent media both with pure cultures and in practical applications. he necessity of a selective medium for the isolation of Salmonellas was substantiated with pure culture studies. and in practical experiment. Studies of a number of fecal sample cultures revealed that organisms from the genera Escherichia and Proteus were the contamin- ants nest frequently found on media used for the isolation of intes- tinal pathogens fron chickens. On all the aedia studied, gran posi- tive organisms and lactose fermenters were in man inhibited or readily identified. The Proteus group proved to be the most prove.- lent and obnoxious organism to be dealt with. being difficult to inhibit or differentiate on nest of the media. in evaluation of Macconltey's agar. bismuth sulfite agar. 8 S agar brilliant green liver-infusion agar. cosine-brilliant green-neth- ylene blue agar and brilliant green acid fuchsia agar medium was made using pure cultures. HacOonloey's agar aediun though it allowed the enteric patho- gens to grow. appeared to favor the lactose fermenters and was not at all inhibitory to Proteus in pure culture studies. Bianuth Sulfite agar aedius in pure culture studies supported a good growth of the Salmonellas and though it did not inhibit Proteus the colonies could be identified as such on the medium Gran positive organisms and lactose fermenters were inhibited. -36.»- On 8 8 agar medium the Salmonellas grew well. and gram posi- tive and lactose fermenters were for the most part. inhibited and if not inhibited were readily differentiated. Proteus was not inhibited and the colonies appeared similar to the colonies of the Salmonellas. Brilliant green liver infusion agar medium was found to be completely inhibitory to the lactose fermenters and gran positive or- ganisms and supported a good growth of the Salmonellas. It was not of much value in eliminating the Proteus group. louse-brilliant green-methylene blue agar medium gave good results: inhibiting non-pathogenic organimss including the Proteus and at the same time stimulating the growth of the pathogens. Brilliant green acid fuchsin agar medium was found to be so.- perior to the other selective media studied with pure cultures. It was highly selective and supported a good growth of the Salmonellas. It was found to be inhibitory to the Shigellas. Two enrichment media were tested. Buy's medium and sodium tetrathionate broth (Difco). Ray's medium was found to be toxic to the enteric pathogens as well as to contaminants. Sodium tetrathio- nate broth did not appear to affect the growth of Salmonellas apprec- iably in pure culture studies. It was found to be inhibitory to lac- tose fermenters and gram positive organisms. but seemed to favor the Proteus group. Sodium tetrathionate broth combined with the selective media studied. showed somewhat superior results to direct platring though it had no inhibitory effect on the Proteus group and. in fact. seemed to stimulate its growth. - 37.. Bismuth sulfite agar. 8 S agar and brilliant green acid fuch- sin agar mediua were employed in combination with sodium tetrathionate enrichment broth and direct plating for examining fecal samples. Di- rect plating was found to be of little value in the isolation of Sal- mnellas from chicken fecal samples. An examination of 376 chickens specimens was made. All the specimens were from birds having normal case histories and had at no time shown any manifestations of any kind of disease. Size of these birds. or an incidence of 1.6 percent. were shown to be carriers of . Salmonella organisms. he organisms were identified serologically as _3_._ oranienberg. Iwenty-one suspicious colonies were isolated and tested sero- logically. Fourteen of these proved to be Salmonellas. 5 were identi- fied as members of the paracolon group. and 2 were rough colonies and could not be typed. line of the colonies that were identified as Sel- nonellas were isolated from brilliant green acid fuchsia agar plates and five from 3 8 agar plates. four of the colonies identified as paracolons were picloed from 8 8 agar. and 1 from brilliant green acid fuchsia agar. he 2 rough colonies were isolated from 8 8 agar. All the isolations were made from specimens which had been subcultured in sodium tetrathionate enrichment broth prior to plating. A compendium of the findings showed that 6h percent of the 1..- lations were made from brilliant green acid fuchsia agar medium. and fi percent from S 8 agar mediun. Io isolations were made from bismuth sulfite agar medium. CONCLUSIONS A detailed study of six selective solid media. MacOonlcey'e agar. bismuth sulfite agar. brilliant green liver-infusion agar. Siaeon, cosine.brilliant green-methylene blue agar and brilliant green acid fuchsin agar medium. and two enrichment broth media. My's brilliant green broth and sodium tetrathionate broth. both with pure cultures and in practical applications is presented. Ihe necessity of a selective medium for isolation of Salmon- ellas from feces of chickens is substantiated by results from pure culture studies and practical applications. Organisms from the genera Escherichia and Proteus were found to be the contaminants most fre- quently encountered on media used for the isolation of enteric patho- gens. Gram positive organisms and lactose fermenters were. in. general. inhibited or readily identified on the media studied in this investiga- tion. Ehe Proteus group was the most difficult to inhibit or identify on selective media. lethods utilizing combinations of sodium tetrathionate enrich- ment broth and three solid media. binuth sulfite agar. 8 8 agar. and brilliant green acid fuchsin agar medium for practical isolation of Salmonellas from feces of chickens is presented. in examination of 376 fecal specimens from none]. chickens. re- vealed an incidence of 1.6 percent carriers of _§_._ oranienbegg. The specimens from which isolations were made had all ban cul- tured in sodium tetrathionate broth prior to plating on solid media. Sixty-four percent of the isolations of Salmonella were made from bril- liant green acid fuchsin agar medium. Thirty six percent of the iso- lations were from S S agar medium. No isolations were made from bismuth sulfite agar medium. l. 2. 3. mm: i‘ryptose Agar mm.0000000eeeeeoeeee0.000000000000000009000000000000 20.0 m. &d1m Chloride......................................... ggou.................................................. Aproeeeeeeeeoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 5.0 grams h.0 m0 1e5 ”I 20.0 grns m.n11°d 'atoreeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeelmoc cc. Dissolve ingredients in steamer. adjust pH to 6.8 or 7.0. Autoclave at 15 pounds pressure for 20 minutes. Pour plates. Liver Infusion (For liver infusion agar plates) 1:1701‘(fl301y Md. ffllh)eeo........................o 1.0 pound bur ‘tap or d1.t111.d)0000000eoeeeeeeeeeeeeeeeeeeeeeeowo cc. No the ground liver in an agateware pail add the 500 cc water. mix thoroughly and allow to stand in a cool place (refrigerator) for not more than 16 to an hours. Strain meat infusion through clean cheese- cloth in a large funnel. thoroughly pressing out all the Juice. 500 as should be rsceovered. Sterilise in autoclave for 30 minutes at 15 pounds pressure. Ready for use in medium. Liver Infusion Agar w.-eeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeeeeeeeeooeeoeoeeeee Liver InmdonOOOOOeoooeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeoo Peptorm................................................. balm morideeeeeeeeeeeeeeeeeeese‘eeeeeseeeesseeseeeeee Distilled 'ator......................................... 20.0 grams 50000 OOe 10e0 grams 5.0 ‘1'“. 500.0 OOe Adjust pH to 7.0. Sterilise at 15 pound pressure for 30 minutes. Brilliant Green Liver Infusion Agar Liver InflIhn ‘8”00000000000000000000000000000emcee... Bflllimt maeeeoseeoeeeeseeeeoeeeeeeeeeeeeeeeeeeeeeee 1000.0 cc. 00.013 @e Add Brilliant green dye to the melted agar base aseptically and pour plates aseptically. Use about 20 cc in each petri dish. -110. 5. 6. hy's lnrichment Broth (6) PeptoneoeeOOOOOOOeOOOOOOOeeeoooeeeeeeeoneeeoeeeeeeoeee 200° STQIS abdin. Chlorideeeeeeeeeeeeeseeeeeeeeeeeeeeoeeeeeseeeee 50° gralfl m3t111°d 'atoroeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeeee ImOeO cc. Bfllllmt green ”lution (1310“,)eeeeeeeoeeeeeeeOOOoee 1000 CO l‘bflBh'. reagentoeeeeeeeeeeeeeeeeeoeeeeeooeeeeeeeeoeeo 20.0 00 (lsbach's reagent is prepared by dissolving 1.0 gra icric acid and 2 grams of citric acid in 100 cc. of distilled water. Dissolve all ingredients by stem and adjust pH to 7.2. Dispense in 10 cc amounts in test tubes. Sterilise at 15 pounds pressure for 20 minutes. ‘ Ietrathionate Broth (Bacto) Broth base . PrOtQOIODPOPtonO no. 2 OD1ICO)Oeeeeeeeeeoeeeeeeeeeeeeeee 500 ‘r‘.’ B‘OtO-bilfl .81‘.OeeeeeeeeeeooeeeeeeeeeeeeeeeOeeeeeeeeoee 100 gr... 03.131“ carbonate"..................................... 10e0 81‘“. goat“. thiOflnlfatheeeeseeeeeeeeeeeeeeeeeeeeeeeeeeeesees 30cc gran: nifltillfid 'atareeeeeeeeeeeeeeos-seeeeeeeeeeeeeseoe.eeeeeIOOOeo 00. ~ Iodine Solution 7. Indlno c:’.t81.00000000eeeoeeoeeeeeoee000009000000eeeeeo 600 gr... POtQ'Ciu- iodideeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeseeeeeeee 500 8r... DlfltIIIOG 'atorOmeeOO000000000000000000000000.00.009000. 200° 00. !o prepare 1000 cc of medium. to 1000 cc. of broth base which has been boiled and then cooled to below h5° 0.. add 20 cc. of the iodine solu- tion. Shaina well to mix and dispense in 10 cc. quantities in test tubes. taking care to obtain an even distribution of the insoluble ma- terial. She medium was found to give better results when freshly pre- pared and used on the sme day. Bismuth Sulfite Agar - (Bacto) (Dime-dehydrated) . Peptonfieeeeeeeoeeeeeeseeeseeeesoeoeeeeeeeeeeeeeeseeseee 1050 £78.. 300’ attractooeeeoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 5.0 6"”. n.2tnOCQOOeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeseeeeeeeeeeee .0 6r... DiCOdium Phosphate..............3...................... .0 ‘13-. Ibrfonj [alfatfieeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 003 gr'fl. Blcfluth Sulfite indicatoreeeeeeeeeeeeeeeeeeeeeeeeeeeeee 800 gr.fl' ‘garOOmeeeeeeeeeeeeeoeeeeeeeeoeeeeeeeeeeeeeeeeeeoeeeeee 20.0 gral. Brilliant groonoo...................................... 00025 gran. Difltilled 'atoreeeeeeeeeeeeeeeeeeeeeeeeeOeeeeeeeeeeeeee 100000 cc. uni. .1 9. 100 Dispend all dry ingredients in the distilled water and heat to boil as rapidly as possible: then allow to simmer for a minute. Into sterile petri plates pour 15 to 20 cc of the medium. i'his medium should not be autoclgved as prolonged heating destroys its selecti- vity. Final pH 7.6 In lchonhey' s Agar PaphnCOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 2.0 gm. wu- OmorldCOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeee 005 m. $6.1“ taurocholateoo.................................. 005 ‘1'... Distilled water........................................ lweo Gee lgar-agar.............................................. Zoo 31". LwtOIOeeoeesooooeeeoeeeeee.eeeeee.oeeeeeeeeeeeeeeeeeee 100 gr. 'fltrfll radoeeeeeoeeeeeeoeeeeeoooeeeeeeseeeeeeeeeeeeeee 0.001 ‘1'. he first four ingredients are steamed for an hour and filtered while hot. Agar. lactose and neutral red are added. the medium thoroughly mixed and then autoclaved for 20 minutes at 15 pounds pressure. Ap— proximately 12 to 15 cc. of the medium is poured into sterile petri dishes- !acOonhey' s Agar-Bee to (Dehydrated - Difco) Bwb-pcphnfieeeeeeeeseeeeeeeeeoeeeseeeeeeseeeeeeseeoeo 17.0 gm. Protease—peptone."...u............................... 300 gm. hate—lactose.......................................... 10.0 81'”. kc“ 3110 [alts no. Beeeeeeeeeeeeeeeeeeeeeeeseeeeeeeee 105 gm. “d1“ chlorideoo...................................... 5.0 grams Bwto-mreoeeeseeeeeeeeeeeeeeeeeeose.eeeeeeeeeoeeeeeee 17.0 m. Bach—nmtral red...................................... 0e03 grams MMfyjtal Violet (DeGn2)eseseeeeeeeeeeeeeeeeeseeee 0.001 ‘1". Diltlllod “taroeeeeeeeeeeeeeoeoeeeeeeeeeseeeeeeeeeeeeelmeo cc. Do prepare the medium 53.5 grams of the dehydrated medium are sus- pended in 100') cc. distilled water. Boil for one or two minutes to dissolve the medium. Sterilise for 20 minutes at 15 pounds pressure. Desoxycholete - citrate medium erOoeeeeeeeeeeooeeeeeeecooeeeeeeeeeeeeeeeeeeeeeeeee. 22.5 81‘”. Lab Imeoeeeeeeseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 5.0 m. 31:60 Pmt”.O-p.pton°eeeeeeeeoeeeeeeeeeeeeeeeeeeeeeee. 5.0 m. LutOSOOOeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeee 10.0 gm. ha!“ cltmtae........................................ 805 gm. “dim tnomra‘OOOOOOOeee000.0000c.0000...0000.000... 805 gm. Ionic cltrata......................................... 100 m. 8001‘ demmlatoOeeeeeeoeeeeeOOooeeeeeeeeeeeeeeeeeo 500 gm. Neutral ”do...eeeeeeoeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeOeO 00025 ‘1'“. "torOeeeeooeeeeeeeeeeeeeeoeeeeecceeeoeoeeoooeeeeeeeoto IMoO GO. -142. ll. Salmonella and Shigelle Hedium (Difco - dehydrated) Beef .xtr‘ctoeeeeeeeeOeeeeeeeeeeemeemeeeeeeeeeeeeeeeeoeo 50° Sta-fl PIOtOO'O—pflptonQeeeeoeeeeosoeeooseeeeeeeeoeeeeeeeeeeeeee 500 grams LactOQQQmeeeeeeeeeeeeeeeeoeeeeeeoeeeeoeooooo000000000000 10cc gran. 3116 Saltfleeeeeeoeeoeoseoeeeoeeeeeeeeeeeeeeeeeeeseeeseee 805 gram. Sodium citrate....,..................................... 805 graflfl Sodium thiogulfatooeoooooeeseoeoeeeeoeeeeeeeeeooeeeeeeee 8e5 gran. lorric citratOQeeeeeeeeeeeeeeeeeees.eeeeeeeeeeseeeeeeeee 1.0 grams ‘GarOQeeeeeeeeeeeeeeeeeeeeeeeeeeemeeeeeeeeoeeOOeeeeooeee 13.5 grams Brilliant groaneeeeeOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeo 0033 gran. Rhntral rOdOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 00025 at... Distilled 'fltereeeoooeoeeeeeeeeeeeeeeeseeeeeeeeeseeeeee*o 1000 cc. Dissolve ingredients in distilled water. Stem for 15 to 20 minutes or bring to the boiling point. Do not allow to boil or do not ster- ilise. Pour about 20 cc. medium in each sterile petri plate. Allow to dry with covers partially removed. 12. Rosina-Brilliant green - Methylene blue agar (10) Base Medium - ‘l‘ryptose agar (See Page 1 - Appendix) ITyPtOQQctgar‘bale I.d1“.eeeoeeeeeeeeeeeeeeeeeeeeeeoeose 8900 cc. Iogino 1 eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoeeeeeoeeeeeeeeee 3.5 cc. Brilliant 51..“ Oel$ooeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 2.5 00. nbthylane b1“. 0.1%..................................... 2.5 cc. IQBQOIO eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 205 cc. he base medium is melted by steaming and each dye and the lactose added using sterile techniques. Stock solutions of the dyes are made and diluted 1-10 immediately before use. Stock solutions for l—B—G-M B Medium. Lactocoo........o......................................e 1000 grail Blatillod‘wateroeeeeeeeeeeeeeeeoeoeeeeeeeeeeeeeeeeeeeeee 100.0 cc. Sterilise at 12 pounds for 15 minutes Store in dark bottle. Basins (10 percent) 308130 101.000.000.000.0.000.000.0000.eoeeeeeeeeeeeeeee. 10.0 8?... 'Bth'difltilledeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 100.0 cc. Sterilise at 12 pounds for 15 minutes. Store in dark place. -143. i3. Brilliant Green (1 percent) Brilliant guano........................................ 100 m. niIttllod watmroo....................................... 100.0 00. "ethylene blue (1 percent) ubthV1flne b1“...eeeeeoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeo 1.0 gran. Dietillod 'atOroeeeeeeeeeeeeemeeeeeeeeeeeeeeeeeeeeeeeeee 10°00 BC. Store in dark. 7 Brilliant Green Acid fuchsin Agar (19) | Base medium Agarbmmoooeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 20.0 gran. Peptonoo................................................ 2000 gran. abdiun tanrochOIQtheeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeo 5.0 ‘13-. sodiul chlorld9......................................... 50° 8T3“. nlfltilled mater......................................... IOOOoO‘CCe Autoclave at 10 pounds pressure for 20 minutes. adjust pH to 7.1: and filter. Add 10 grams lactose. mix and distribute in 200 cc. quantities in flasks and sterilize at 12 pounds pressure for 15 minutes. Io prepare plates. to 200 cc. of base medium. melted and cooled to 50° 00 add 2 cc. of Andrade's indicator and mix. Add 0.8 cc. of freshly prepared brilliant green (I percent) solution. Mix and pour plates. ' l. 2. 6. 7. 8. 9. 10. 11. 12. 13. MEMO]! Iilson. I. Janos. Reduction of ailfites by certain bacteria in media containing a femantable carbohydrate and metallic salts. igurl El" _Z_1_: 392- 192249230 '11.». v. James. and n. a. 1M Blair. Use of a glucose bismuth sul- fite iron nedius for the isolation of B. typhosus and B. proteus. Jour. £28. 26: 2714. 1927. Ibid. Further experiment of bimth sulfite nedia in the isolation of bacillus typhosus and B. paraptyphosue from feces. sewage and water. J__o___ur.gzg .1. 138.1931. Scimidt. Rudolf. Uber die Brauchbarbeit des elektiven lahrbodens fur i'yphusbasillen nach Wilson. Zentralbl. Baht. Abt. I Ori. 11:15. 1937. An abstract 31.1. up“? 131'7". "6'9"". 1533': Gunther. Cora B. and Louis Tuft. A conparative study of media employed in isolation of typhoid bacilli fun feces and urines. Jour. Lab. and 011“. "9d. a: 1.61. 19390 Buy. Charlotte A. The isolation of typhoid-pantyphoid and dysentery bacteria from feces and urine. Brit. iled. Jour. J: 6(5 19%. mes. lartin .7. he isolationn of intestinal pathogens by selective media. {cub Pat th. and Bact. 1:193. 19142. Gibbons. 1i. 1.. and R. 1.- floors. m. examination of dried egg powder for Salmonella. National Research Council rt. (20-6413) Division of Applied Biology. l. 1%}. MacDonlney. Alfred. Lactose fermenting bacteria in feces. {cum m. box. 3.. P.G.H. Gall and M. B. Pollack. Selective Media for organises of the Sal-ensue group. gour. Eat . and Bact. 55: N69. l9ll2. Leifson. linar. Rev culture media based on sodim desoxycholate for the isolation of intestinal pathogens and for the enumeration of colon bacilli in milk and water. Jour. Ea ath. and Bact. ‘10: 581. 1935. Darby. O. I. A practical bacteriological test for the diagnosis of pullorun disease in chicks. "Weterinarian no: 121. 19112. Harrie-Holt. J. E. and Oscar league. A new culu1re nediun for the iso- 119.1%” of Bacillus tflgsus fro: stools- Jour. Infect. Dis. 18: 5%. l . -us. 1h. 15. 16. 17. 18. 19. 20. 21. 22. 23. 2h. 25. 27. Teague. Oscar and A. I. Olunan. An improved brilliant green culture mediun for the isolation of typhoid bacilli from stools. Jour. Infec. m'. g: 6&7. 19160 uallnann. W. L.. Frank Tharp. Jr.. and Margaret Semmes. A medium for for the isolation of Salmonella pullorum and other members of the parap typhoid group fmm avian tissues. Jour. Amer. Vet. Med. Assoc. 13: 8250 19280 Huddleson. I. 1., DJ. Halsey and J. P. Torrey. Further studies on isolation and cultivation of bacterium abortus (Bang). Jour. Infect. Mac 39: 352s 1937’ Dorrey. J. G. Brilliant green as a specific enricinent nedium for gsmtyphoid - dysentery group of bacteria. Jour. Infect. Dig; l3: 3. 1313. Batista. I. L. and L. 1'. Rettgsr. Brilliant green and its use in an enrichsent medium in the isolation of typhoid and paratyphoid organisms. flour. Infect. Dis. 13;: 93. 1927. Oruickshank. J. c. A brilliant green acid fuchsin medium for the isolation of Salmonella. Bull. H25. 18: 26. 19h}. Browning, O. 3., W. Gilnour and I. J. Racine. an. isolation of _t_z- floid bacilli from faeces by means of brilliant green fluid medium. £01115 Else 1.}: 3142. 1913-191)“ Orsechowski. Gerhard. Versuche mit einem Verfahren xur Anreicherung von Typhusbacillen. (A Method of enrichment for typhoid bacilli) Zentralbl. Bakt. I. Abram-1E. _1__1_1_, 357.361. 1929. An abstract 3101. Ibse 5. 530. 1330. 513 a Manual of Muted Culture Media and Regents. Difco Lab. Inc.. Detroit. Michigan. etrathionate broth base. 138. 1939. Khalil. A. 11., Incidence of the Salmonella group in wild rats and mice in Liverpool. Jour. . x 77. 1938. Boeden. J’. van der. Der Tetrathionate nahrboder nach L. Muller in der i'ypimsdiagnostik. (Detrathionate medium of L. Muller in Typhoid diagnosis). Gantral'bl. Baht. I. Abt. Ori . 191. 1477-1482. 1927. An abstract 31.1. 1b.. _ 77. 51 o. 1930. Darby. O. I. and I. L. lallaann. Studies on media for colifora or- ganisms. gour. Amer. Water Works Assoc. 31; 693. 1939. Hallmann. I. L., J. I. hff and Evelyn Matthews. Studies on the sal- monella group onethods of isolation and pathogenicity of strains oc- cgring in the intestines of chickens. Jour. Infect. Dis. 12: 353: l 2. Edwards. P. B. and D. W. Brenner. The occurance and distribution of Salmonella types in the United States. Jour. Infect. Dis. 72: 58. 19h}. “" -hfi. as (”1 C) swim ‘I we »- |||H|||||||UIIUIHIIH 5 5484 .1: O 3 I! I III. III II II I || |