“mm WmSA m QELLA (1‘? SALMC’M \‘ARIQUS WEGRATQRY MEI-BIA ' rr‘LDIJ .1. It... THISII 0-169 , s l ___.__... x .—.C ‘._L .. fu__ ‘ ..L-..—.- w -Aot A—-—- -h— L- This is to certify that the thesis entitled Persistence of the VI antigen of Salmonella tgghosa in Various Labo ratory Media presented by Milton Bernard Dobkin has been accepted towards fulfillment of the requirements for AS;— degree in Mgr—19.10 55' M, Major professor I . . -‘- o— _ ' w—.. u.' '-.'.._.. ‘fi . . an. - on -— o 0". .- PERSISTENCE OF THE VI ANTIGEN OF SALMONELLA TYPHOSA IN VARIOUS LABORATORY MEDIA BY MILTON BERNARD QQBKIN A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology 1950 TH 6815 ACKNOWLEDGMENTS The author wishes to acknowledge his gratitude to Dr. Walter N. Mack, Associate Professor of Bacteriology and Public Health for his guidance and criticisms and to Dr. H. J. Stafseth, Head of the Department of Bacteri- ology and Public Health for his corrections of this manuscript. He also wishes to express his appreciation to Mr. H. W. Schoenlein, Director of the Biological Laboratory, Difco Laboratories, Inc., Detroit, Michigan for his helpful suggestions. 244570 TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . MATERIALS AND METHODS . . . . . . . Typing Broth Medium . . . . . Typing Agar Medium . . . . . . Phage Dilution Broth Medium . Stock Cultures . . . . . . . . Selection of Stock Culture Media Typing Procedure . . . . . . . RESULTS . . . . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . REFERENCES CITED . . . . . . . . . Page 11 11 ll 12 l2 l3 15 26 28 3O 32 Table I. II- III. IV. VI. VII- VIII. XI- LIST OF TABLES Characteristics of the V1 Bacteriophages Reactions of the Vi-type Strains of S. typhosa to Routine Test Concentra- tions of Typing Phage Preparations . . Grouping of the Strains of S. typhosa with Regard to Sensitivity to V1 Phage II 0 O 0 O O O O O O O O O O Persistence of the Vi Antigen of g. typhosa at the End of 30 Days . . . Persistence of the Vi Antigen of S. typhosa at the End of 60 Days . . . Persistence of the V1 Antigen of §. typhosa at the End of 90 Days . . . Persistence of the V1 Antigen of §, typhosa at the End of 120 Days . . The Persistence of the V1 Antigen of fiié‘iifléu-fi-SSP—"Sf‘? YaE‘i‘f“? . . . . The Persistence of the Vi Antigen of Strain DAHP56 S. typhosa in Various Media (SummaryT' T'T’T‘T'. . . . . . . The Persistence of the V1 Antigen of §Z§?:“(§&m%;r-§-¥Pl‘—i’—‘°’—? f“."‘i“‘f°‘fs. . . . The Persistence of the Vi Antigen of fiZSi‘iWEfiaW 297?“??? . . . Page 18 19 2O 21 22 23 24 25 INTRODUCTION The development of the technique, by Craigie and Yen (1938), of identifying strains of Salmonella typhosa by bacteriophage typing has assumed a role of major im— portance to the epidemiologist. With this method, out- breaks of typhoid fever can be rapidly traced to the carrier source and prevented from further spreading. Craigie and Felix (1947) tried to standardize the technique of Vi-phage typing in order to validate the results coming from any laboratory which might set up a program utilizing this method. In devising a standard method, the primary regard must be for simplicity, from the standpoint of time, materials used, and actual tech- nique of operation. This is necessary in order to per— mit any laboratory, regardless of size, to use the standard methods. The authors of this proposed standardized technique suggested the use of Dorset egg medium as the best medium to be used in preserving the stock cultures of the V1- type strains of S. typhosa. However, they implied that this medium was suggested only because no work had been done up to that time to prove that any other media would be more suitable. Since lysis of the bacterial cells is dependent upon the presence of the Vi antigen, cultures must be trans- ferred frequently to prevent the organisms from disso- ciating to the W form. Ordinarily, stock cultures should be transferred at least every three months in order to retain a significant number of V form organisms. The purpose of this work is to determine which com- mon laboratory media will preserve the V1 antigen. REVIEW OF THE LITERATURE Prior to the work of Felix and Pitt (1934), the virulence of g. typhosa was attributed to the somatic antigen. These investigators reported, however, that another antigenic component was present on the organ— ism which enhanced the virulence of the typhoid bacil- lus, at least for mice. This Vi, or virulence antigen, as it was called, was found to be contained in all freshly isolated cultures; and it was this particular point which provided the impetus to future investiga- tions. The V1 antigen was found to possess certain pe- culiar properties. Felix and Pitt (1934) were the first to demonstrate that there may be an inhibition of O agglutination in the presence of this antigen. An ag- glutination test has been described by these two inves— tigators whereby the presence or absence of the Vi anti— gen can be detected by using 0 agglutinin. This test is more sensitive than bacteriophage typing, but is not as simple to carry out. According to Almon (1943), other properties ascribed to the V1 antigen are: It is heat labile, and unstable in the presence of certain chemicals. Kauffmann (1934) developed the terminology of V form to describe those typhoid organisms which will not agglutinate with 0 antibody and, therefore, contain the Vi antigen. The term W form is used to describe the degraded state of the organisms which have lost the Vi antigen and can no longer inhibit O agglutination. The VFW form is the intermediate stage. With the one exception found by Kauffmann (1934), this reaction is irreversible. The original contention that the V1 antigen alone is respon- sible for the virulence of the bacteria has been dis- carded, since rough organisms containing the Vi antigen are not virulent. Almon states that it is now believed that both the Vi and O antigens are reaponsible for the virulence of S. typhosa. The discovery of the Vi antigen paved the way for further investigations, including the work of Craigie and Brandon (1936), who reported that there were bac- teriophages whose specificity for S, typhosa was based on the presence or absence of the Vi antigen. Later on, Craigie and Yen (1938) described four distinct bacterio- phages specific for the V1 antigen. Type II was unique in that it could be propagated in such a manner as to show irreversible specificity for the substrate strain. While experimenting further with this preperty of Vi phage II, Craigie and Yen (1938) found that S, typhosa consisted of a number of strains. TABLE I CHARACTERISTICS OF THE VI BACTERIOPHAGES Neutral- Vi Relative Thermal ifiizigg 2y Lytic Activity Pha e Particle Death Segumg for V forms of ‘ g Size Point §, typhosa II III Iv Type Large 0 _ _ Lyses all V I phage 67'70 C ' forms Develops a high selective 1ytic Type Medium 0 _ _ activity for the II phage 69’72 C + type of §. typhosa on which II Is propagated Type Small 0 _ Lyses majority III phage 61—64 C + ' of‘V forms Lyses majority T796 Medium 59-620 C _ _ + of V forms with IV phage exceptions of type F and D1 The claims for the existence of these strains was based on the assumption that Vi phage II occurred as several mutants, and the strains of S, typhosa differed only in their Vi antigen. These mutant phages showed specificity for these various strains of bacteria, and by selectively growing a mutant phage specific for a substrate strain of typhoid bacilli, the other mutants could be eliminated, with the final occurrence of homologous phage and bac- terial types. At present, there are 25 strains of S, typhosa and their homologous phages. Craigie and Felix (1947) have tabulated the reactions between homologous and heterolo- gous phages and bacteria, as indicated in Table II. The validity of bacteriophage typing is based on the fact that these types are stable. Craigie (1942) has also found that the organisms sensitive to the action of type II Vi phage may be placed into three main groups. Those organisms belonging to the alpha group are completely sensitive to lysis by type II Vi phage, and when the phage, specific for one alpha type, is prepagated with another alpha type, it becomes fully active for the new organism. Reversibility of this re— action has not been demonstrated. The beta group comprises those organisms which, when grown with a phage specific for another type, do not produce a phage having irreversible specificity for the new type. Finally, in the gamma group there are those organ- isms which are considered to be untypable since they are resistant to lysis by Vi phage II. The group into which each type falls is shown in Table III. In describing the procedure for bacteriophage typing, Craigie and Felix (1947) recommended that the stock cultures of bacteria be maintained on Dorset egg medium. Originally, this medium was developed for the growth of Mycobacterium tuberculosis, but it also has been found useful in the cultivation of S. typhosa. Transfer to fresh medium must be made every 3 months in order to retain all antigenic characters. Siler, gE_§S, (1941) suggested the use of lyophilized cultures for preserving and storing cultures. This is a good method, but one that requires the use of special equipment not available in all laboratories. BacteriOphage typing has proved itself to be an invaluable aid to epidemiologists as a means of tracing outbreaks of typhoid fever to their carrier source. In Canada, Desranleau (1942) was the first to apply bacter- iophage typing on a practical scale, while Vener and Stevens (1942) are given credit for introducing this REACTIONS OF V PING PHAGE PREPARATIONS V1- type Strains A A CL 2+s M .— N .. CL +s 3+s +n CL +s 3+s +n CL CL CL CL ll Confluent lysis. Normal plaques. x r+*$ 10). Increasingly numerous plaques. A few plaques usually present. TABLE III GROUPING OF THE STRAINS OF S, TYPHOSA WITH REGARD TO SENSITIVITY TO VI PHAGE II Type 2.1.233 13513311. 3:23? A + - - 131 + - - B2 - + - 3 ' + ’ C + - _ Dl - + - D2 + — .. D3 — + _ D4 + - - E1 + — _ E2 + - - Fl + - - L. +++++++ I I lO. technique into the United States. Its use in laborator- ies in this and other countries is becoming more and more commonplace. It is doubtful whether the basic procedure can be improved upon, but certain aspects of this method are still questionable. Perhaps, in clarifying these small points, the over-all picture regarding the nature and action of the V1 antigen will also be clarified. MATERIALS AND METHODS It is important to remember that the fastidiousness of bacteriophages is such that precise duplication of conditions must be adhered to in order successfully to demonstrate lytic activity repeatedly and with accurate results. Deviation from the prescribed conditions, no matter how slight, is likely to cause erratic results. Craigie (1947) recommended the following media to be used in the typing procedure: Typing Broth Medium This medium is diapensed in 2.0 ml quantities into 13 x 100 mm tubes and is used to invigorate a tranSplant of the stock culture prior to typing. The components of this medium are: "Bacto" nutrient broth (dehydrated) . . 15.0 grams Sodium chloride . . . . . . . . . . . . 7.0 grams Distilled water to . . . . . . . . . . 000.0 ml Typing Agar Medium The medium on which the typing is actually carried out consists of: "Bacto" nutrient broth (dehydrated) . . 20.0 grams Sodium chloride . . . . . . . . . . . . 7.5 grams "Bacto" agar . . . . . . . . . . . . . . 20.0 grams Distilled water to . . . . . . . . . . 1000.0 ml 12. The agar is dispensed into plates in 25-30 ml quantities. Incubation for two or three days, or left at room tem- perature for four to five days will remove excess mois- ture. It is important that the surface of the agar be fairly dry. Phage Dilution Broth Medium It has been found that bacteriophages survive best in an environment and under the same conditions that are optimal for their specific hosts. The concentrated phage is diluted to the critical test concentration using a medium having a composition of: "Bacto" nutrient broth (dehydrated) . . 8.0 grams Distilled water to . . . . . . . . . . 1000.0 ml Stock Cultures Four strains of S. typhosa, types A, D4HP56’ El’ and F1’ and their concentrated homologous Vi-phages, obtained from the Connaught Medical Research Laborator- ies of the University of Toronto, through the Michigan Department of Health, were used in the experiments. The concentrated Vi-phages were prepared in the fol- lowing manner: Rapidly growing cultures of each of the four strains of S. typhosa were transplanted into tubes containing 2.0 ml of broth and incubated at 37° c for two hours. Each tube was then seeded with one loopful of the homologous 13. strain of concentrated phage and again incubated at 370 C for four hours. The bacteria were killed by heating the tubes at 560 C for 45 minutes, immediately centri- fuged, and the supernatant fluid poured into sterile vials. Each vial of concentrated phage was now available as a stock culture from which the critical test concen- tration (C.T.C.) could be prepared. Critical test con- centration is defined as the highest dilution of bac- teriophage which will produce confluent lysis on the homologous strainof S. typhosa. In this case, 4.95 ml of broth and 0.05 ml of concentrated phage were combined to form the C.T.C. for these experiments. The diluted phages were kept at a temperature of 4° C for the dura- tion of the experimental work. Selection of Stock Culture Media Some of the media most commonly used for the culti- vation of S. typhosa, and other for comparative purposes were chosen for this work. The selection was not limited to solid or liquid media, or to particular compositions, in order to cover as wide a variety as possible. The following media were prepared: 1. Dorset egg medium 2. Petragnani medium 2. Brain heart infusion medium . Veal infusion medium 5. Beef heart infusion agar 6. Bismuth sulfite agar 7. S S agar l4. 8. MacConkey agar 9. Kligler iron agar 10. Tetrathionate broth 11. Selenite broth 12. S I M medium 12. Desoxycholate medium 1 "Bacto" nutrient broth "Bacto" nutrient agar All except the first two media were prepared from Difco dehydrated products. Because media 1, 2 and 4 become significant later on in this work, their formulae will be mentioned here. The formulae for the remaining media may be found in the Difco manual (1948). Dorset egg medium was prepared by combining three parts of whole egg and one part of 5% glycerol water, tubing, slanting, and inSpissating. Petragnani medium was prepared according to the formula described by Kol- mer and Boerner (1945). Potato . . . . . . . . . . . . . . . 75.0 grams Milk (cream removed) . . . . . . . . . 150.0 grams Potato flour . . . . . . . . . . . . . 6.0 grams Peptone . . . . . . . . . . . . . . . 10.0 grams Eggs (whole) . . . . . . . . . . . . . 4 Egg yOJ-k e e o o o e o o o e o o o c o 1 Glycerol . . . . . . . . . . . . . 12.0 ml Malachite green (2.% aqueous) . . . . 10.0 ml The formula for the following medium was taken from the Difco manual (1948): Veal Infusion Medium- Veal, infusion from . . . . . . . . . 500.0 grams Proteose peptone, Difco . . . . . . . 10.0 grams "Bacto" agar . . . . . . . . . . . . . 1.0 gram With the exception of Petragnani medium, which was dispensed into screw-cap tubes, the other media were dispensed into cotton-plugged tubes. 15. Duplicate tubes of each medium were inoculated with strains A, D4HP56’ El’ and F1 of S. typhosa, and incubated at 370 C for 48 hours in order to obtain a fairly heavy growth of organisms. Each tube was then tested for the presence of the Vi antigen, using homologous Vi-phages. Each week thereafter, for a total period of 12 weeks, those organisms which remained viable were typed and the results recorded. All media were stored at a tempera- ture of 4° c after the initial incubation period. Typing Procedure The typing procedure used was that described by Craigie and Yen (1938). A heavy loopful of the organisms was removed from each of the stock cultures and seeded into tubes containing 2 ml of typing broth medium. The tubes were incubated for two hours at 37° c. At this point the organisms had grown sufficiently so that the tubes appeared cloudy. If the tubes were not cloudy, then a heavier inoculum was used. A fairly large growth is desirable in order to differentiate more clearly be- tween the lysed and non—lysed areas. The remainder of the procedure will be described as it was written by Craigie and Yen (1938) in order to avoid any possible misinterpretations that might occur through paraphrasing, or accidental omissions. 16. Two standard platinum loops, 2.75 mm in internal diameter, are used alternately to apply the culture and phage to the plate. These are mounted on long handles, the balance of which may be improved with advantage by weighing. While one loop is actually in use, the other is cooling after sterilization. In transferring a loopful of broth culture to the plate, the loop is used merely to direct the natural spread- ing of the broth and should not be rubbed over the agar. The shank of the loop is angled so that the loop will lie parallel with the agar surface during inoculation, the loopholder being graSped between the thumb and first two fingers like a pen. A full loopful of broth culture is obtained by sharply mov- ing the culture tube so that the loop quickly breaks the surface of the fluid. Then, with the holder held in the way described and with the forearm and side of the hand resting on the bench, the loop is lowered until the drop held in it touches the surface of the agar. At this point, the loop is given a slight horizontal circular movement by moving the fingers, guiding the spreading of the drop over an area ap— proximately 15 mm in diameter. A number of areas, corresponding to the number of phage preparations used in the test, are inoculated with each strain. When the agar has absorbed the fluid which has been applied (Optimum 5 to 10 minutes), the phage is ap- plied from the standard loop to the center of the inoculated area. In this case the loopholder is best held horizontally between the thumb and fingers, these pointing downwards with the side of the arm resting on the edge of the bench. The loopful of phage is lowered until it almost touches the surface of the agar when slight rotation of the loopholder will move the loop into a vertical plane, thus a1- 1owing its contents to flow onto the center of the inoculated area. It is to be noted that the drop of phage must be allowed to spread naturally on the plate, and the actual contact of the loop and inocu- lated area is to be avoided as far as possible. When the fluid which has been applied has dried sufficiently to permit the plates being safely moved, they are incubated for two hours at 37 C and trans- ferred to the cold room overnight. They are returned to the incubator and are ready for reading in four to six hours. It should be noted that the plates must not be stacked, since at temperatures significantly below 37° C S. typhosa will grow without exhibiting lysis with VI-pfiage. The method of interrupted 17. incubation has two advantages: It permits the phage reactions to be observed at their optimum and before they are obscured by late growth, in the case of some strains. Further, the intermediate period in the cold room permits diffusion of the phage into the surrounding normal culture, thus enhancing the clar- ity of the reactions. The reactions are best ob- served by oblique artificial illumination and against a dark background. Bacteriophage typing is a rough qualitative test, and so the results of the reactions were read as +, -, and 0. The positive sign indicates that lysis, visible to the naked eye, had occurred. In routine laboratory work transplants of the test cultures are streaked out on plates, and the smooth, Opaque V form colonies are selected prior to typing. For this reason, inoculated areas hav- ing any visible plaques were recorded as positive. A negative sign indicates no lysis, while a zero sign means the organisms were no longer viable, or grew so slowly that there was no differentiation between the phage inoc- ulated area and the outer area containing organisms only. 18. .Emacmwpo pmop no npzonw mo xomfl mo omsmoon oosaahopoocs mamhq u o .Uopmnpmcoaoc no: mfimhq n u .Uopmnpmcoeoo mamhq u + .opmoaflasv CH vooaooon muHSmom * o o o + + + + + name psoHApsc sopomme .ma + + + + + + + + shown escapes: souooms .aa 0 o o + o + o + opmaonozxomom .MH + + + + + + o + enacts 2 H m .NH o + o o o o o o npoan opflcoaom .HH 0 o o + + + o + anode oossoasoohooe .oH + + + + + + + + news coed adamaam .m + o o o + + o + ammo hoxcooomz .m o o o o o o o 0 some m m .s + + + + + + + + some opHMHSn spssnam .w + + + + + + + + some soansmcH pane: doom .m + + + + + + + + scansmsfi Hmo> .: + + + + + + + + Coamsmsfi Damon sawhm .m + + + + + + + + Hcscmsapom .m + + + + + + + + wmo ponnoq .H as Hm mmimsn a IIIIIII._I asfivoz smonahp .m mo cdmnpm *mwdm om mo mzm mmB B< «mommwa .m mo ZMUHBZ¢ H> mmB mo wozmemHmmmm >H mqm .: + + + + + + + + scansmsa ammo: eaohm .m + + + + + + + + asscwoapom .m + + + + + + + + wmo pomaon .H Hm Hm amazed a :IIIIII. I. suave: wmonazp .m mo Cashew *mM mus mo MoszmHmmmm > mqm¢8 20. .snficswno poop mo saxonm mo Essa mo omsmoon confisaopocc: manna u o .Uopwppmcoson no: mamhq u I .vmpmapmcosoc human u + .omeHHQSU cw uocnoooa mpazmom * I I o o + + I I some escapes: sopomme .ma 0 o + + + + + + noose escapes: sopooms .aa 0 o I I I I I I oumaonohxomom .mH I I I I I I I I ESHUoE SHm .NH 0 o o o o o o o snows obadoaom .HH I I o o I I I I Sponn opwcoazpmnpoa .OH 0 o o o + + + + some Coma ponHHx .m o o o o o o o 0 news hoxnooomz .w o o o o o o o 0 some n m .e I I o o I I o I hows opdmadm Sufismfim .m o o o o I I I I ammo scamsmcfi undo: moom .m + + + + I I + + scamsmsfi Hwo> .: + + + + o I + + coamsmcH ammo: cfimam .m + + + + I I + + acmcwmapom .m + + I I o o + + mwo pompom .H as Hm mmmmam a ESHUmE mmonahp mm Mo cfimapm *WV¢Q om mo sz mmB B< mma mo mozmamHmmmm H> mqmga 21. .Emacmwno poop no npzonw mo Roma mo omsmoon cocasaopovcs mammq u o .oonohensosoo eon wanna I I .oooohuncosoo named u + .opmoHHQSc SH coonooon mpadmom * o o o o o o o 0 some answers: eonosme .ma 0 O O O + + O 0 $9093 QCQHHPSC :Opodm= o#H o o o o o o o o oeoaosoaxonoe .ma 0 o o o o o o o ssaoos 2 H n .NH O o o o o o o 0 shown opacoaom .HH 0 o o o o o o 0 spots newcoaeeohboa .OH 0 o o o I I + + some coma soawfiax .m o o o o o o o o ammo aoxcooooz .m o o o o o o o 0 some m m .s o o o o o o o 0 some obaeasn enssnam .m o o o o o o o 0 some seaweeds swoon doom .m + + I I I I + + soamsmca Hmo> .: o o I I I I + + COHmSMSH ammo: madam .m + + I I I I + + acmcwmnpom .N + + o o I I + + mwo pomson .H as Hm mmmman a IIIIIII. I. enacts mm029hp .m no Sachem *mM mma mo mOZMBmHmmmm HH> mqm<8 22. .snasmwno pump mo cpsoaw mo good we omsmoon confisnopocc: mamhq u o .Uopmnpmcosov poc mamzq n I .oopmapmcoEoc mdmkq u + .oumoaaasv CH woomooon mpadmom * o o I I + + + + name escapes: sonosme .ma 0 O + + + + + + SQOHQ PCTHQQSC =Opomm= .#H o o I I + + o + onwaonohxomom .ma 0 o I I + + o + Esfiooa E H m .NH 0 o o o o o o o snows opasoaom .HH 0 o I I + + o + snows ooneoaeeossoa .oa + + + + + + + + some coma nonHHx .m o o o o o o o + ammo moxcooom: .m o o o o o o o 0 some m m .s o o o I + + + + home opHMHSm cpssnam .w o o I I + + + + some cOHmSHCH peso: «mom .m + + + + + + + + eoHnSHCA Hso> .: + + + + + + + + soamswcfi pesos Camam .m + + + + + + + + Hescmmnpom .m + + + + + + + + wmo ponhom .H name one news om mass on mass om assoc: Ashosesnv *aHnmz mBOHma> zH awomdwa .m.a zHamem mo zmeHaza H> use so wozmemHmmmd mas HHH> mamas 23. .EmHsmwuo poop no npzomw Ho xomH Ho omsmoon vocaahopowcs mHmHH n o .oopmnpmcosoc poc mHmHH u I .copmameoEov mHth n + .opmoHHasv GH cocsoooa mpHdmom * o o + + + + + + ammo pcoansc 2090mm: .mH + + + + + + + + SPOHD uSmthSC =Opowm: .#H o o I I + + o + opmHocohxonon .MH 0 o I I + + + + 85H605 2 H m .NH 0 o o o o o o o sponn ouHcoHom .HH 0 o I I + + + + snows obscoasooheoe .OH I I I I + + + + some soaH nonHHm .m o o o o o o + + Home Hoxcooomz .m o o o o o o o 0 some m m .e o o I I + + + + some mpHHHSn cpsanm .w o o I I + + + + Home COHmdmcH Damon poem .m I I I I + + + + COHmsmcH Hmo> .: I I o I + + + + COHmsmcH peso: sHmsm .m I I I I + + + + Hcmcwmnpom .m I I o o + + + + mmo nonnom .H name omH name om ease om name om asaooz Ashoessmv saHmmz mBOHma> 2H «mosses am omamsa zHamam so zmeHeza H> mas so wozmamHmmmd was xH mamas 24. .schsmno pmmp mo npzonw Ho xosH Ho omsmoon UoCHEHopooss nHmHH u o .Uopmapncosoo po: mHmHH n I .copMHpmcoEoo mHmHH u + .omeHHaso cH vooaooon mpHSmom * o o o o o o o + ammo pcoHapsc sepomms .mH o o + + + + + + :ponn pcoHapss =0pomm: .pH 0 o I I o + o + opmHonohxomoa .MH 0 o I I + + + + achoE : H m .NH O o o o o o o o sponp opHcoHom .HH 0 o o o + + o + :popp opHCOanmapoe .OH 0 o o o o + + + ammo conH HonHHM .m o o o o o o o 0 some Hoxcooomz .m o o o o o o o 0 some m m .N o o o o + + + + news opHHHsm spasmHm .0 o o o o + + + + Home COHmsmcH pnmon Hoom .m I I + + + + + + scansmcH Hno> .2 I I + + + + + + COHmsmcH peso: :Hmnm .m I I + + + + + + Hcmcwmapom .N o o I I + + + + wmo pomnom .H name omH name om mace om mass om aspen: AhhmsEsmv *aHQMS mDOHmd> 2H «mommwa mm Hm ZH mmB mo MozmamHmmmm mmB x mqmde RESULTS The amount of growth of the organisms varied in the different media, the most extensive growth appearing on Petragnani medium. The Sparsest cultures occurred on bismuth sulfite agar, SS agar, and MacConkey agar. At the end of the 48-hour incubation period, all strains were tested by means of bacteriophage typing and appeared to be in the V form, as was evidenced by the confluent lysis. Tables IV through VII show the results of phage typing of these cultures at the end of 30, 60, 90, and 120 days. From the results obtained and indicated in Table IV, SS agar, although a good selective medium for the initial isolation of S. typhosa, did not support growth of the organisms for longer than 30 days. The V1 anti- gen persisted only as long as the organisms survived. The organisms grown in desoxycholate medium and selenite broth responded in the Same manner, with the exception of strain Fl’ which was still lysed by its homologous bacteriophage at the end of 30 days. During the second 30-day period the organisms stored on MacConkey agar failed to grow on tranSplant. At this time, the rest of the strains on their different 27. media, when showing growth also were lysed by their ho- mologous bacteriophages. At the end of 90 days some of the strains on the different media showed growth on transplants but did not show lysis by the phage. The V1 antigen was no longer present on some of the organisms. Finally, at the end of 120 days, strains A and F1 on Dorset egg, Petragnani, and veal infusion media showed growth and phage lysis. Strain A stored in brain heart infusion medium and on Kligler iron agar produced growth on transplants and was lysed by its specific phage. The D4HP56 strain stored in nutrient broth also survived storage and retained its Vi antigen. It is of interest in comparing the results obtained from growing organisms in nutrient broth and nutrient agar that the V1 antigen persisted for a greater length of time in the broth. Tables VIII through XI represent composite results of the bacteriophage typing of each strain, at 30—day intervals, for the 120 days of storage. Petragnani medium was least affected by dehydration since it was kept in screw-cap tubes. Loss of water was quite noticeable in the other media, and it is likely that the increased concentration of the constituents, due to dehydration, had a toxic effect on the organisms. DISCUSSION Many of the cultures became non-viable in a rela- tively short time, with the resultant loss of the Vi antigen. It is likely that dehydration of the media was a contributing factor in the loss of the V1 antigen. Ex- cessive drying of the media can be minimized through the use of screw-cap tubes. Choice of a single medium for the storage of the typhoid organism in its V form is a difficult one. The common media like Kligler iron agar and brain heart in- fusion medium cannot be used since only one strain (A) retained its Vi antigen over the experimental period of 120 days. Of the remaining media, examination of their formu- lae presents no correlative information to indicate the reason for the persistence of the Vi antigen of strains A and F1. Further investigation, using a greater vari- ety of media, would be necessary in order to permit any speculation with regard to the answer to this question. On the basis of the results of this work, Petragnani medium would be the most satisfactory. The greatest growth of the organisms appeared on this medium, and the presence of malachite green, which is an inhibitory agent, 29. would be an aid in keeping contamination to a minimum. The fact that this medium is not easy to prepare as com- pared with dehydrated products is, admittedly, a disad- vantage. However, it would be more difficult to work with the infusion media since several loopfuls would be required for inoculation of typing broth in order to produce the desired cloudiness. On the other hand, only one loopful of organisms taken from a slant would suffice in order to produce the same degree of cloudiness. SUMMARY A study was conducted on 15 laboratory media in order to determine which ones might be more suitable than Dorset egg medium for preserving and storing the V1 antigen 0f.§° typhosa cultures. The method of bacteriophage typing was used to indi- cate the ability of the Vi antigen to persist in these media. At the end of 120 days of storage strains A and F1 on Dorset egg, Petragnani, and veal infusion media showed growth and phage lysis. Strain A stored in brain heart infusion medium and on Kligler iron agar produced growth on transplants and was lysed by its homologous‘ phage. The D4HP56 strain stored in nutrient broth also survived storage and retained its Vi antigen. Petragnani medium is perhaps the most suitable of all the media used in these experiments for other reasons aside from the fact that the V1 antigen of 2 strains of S. typhosa persisted for 120 days. The greatest growth of organisms appeared on this medium after the initial incubation period at 370 C. The presence of an inhibi- tory agent to reduce contamination to a minimum, and the fact that a solid medium is preferable to a liquid me- dium, because it is easier to remove large numbers of 31. organisms, overrule the disadvantage of its being dif- ficult to prepare. It is not known at this time what factors contribute to the persistence of the Vi antigen. REFERENCES CITED Almon, Lois 1943 The significance of the Vi antigen. Bact. Rev., 7, 43-53. Craigie, J. 1942 The present status of phage typing of Bact. typhosum. Can. Pub. Health J':.§§: 41. Craigie, J., and Brandon, K. F. 1936 Identification of tge V form of B. typhosus. Can. Pub. Health J., 27, l 5- 170. "' Craigie, J., and Yen, C. H. 1938 The determination of types of B. typhosus by means of preparations of type II VI -pfiage. I. Principles and technique. Can. Pub. Health J., 29, 448- 463. Craigie, J., and Yen, C. H. 1938 The demonstration of types of B. typhosus by means of preparations of type II VI—pfiage. I . The stability and epidemi- ological significance of V form types of B. typhosus. Can. Pub. Health J., 29, 484- 496. Craigie, J., and Felix, A. 1947 Typing of typhoid bacilli with Vi bacteriophage. The lancet, 252, 823- 836. Desranleau, J-M. 1942 Typing of B. typhosus with bac- teriophage. Can. Pub. Health’ J., 33, 122- 126. Difco Manual of Dehydrated Culture Media and Reagents for Microbiological and Clinical Laboratory Pro- cedures. 1948 Difco Laboratories, Inc., Detroit. Felix, A., and Pitt, R. M. 1934a Virulence of Bacterium typhosus and resistance to 0 antibody. J. ofFPath. and Bact., 3§, 409-420. Felix, A., and Pitt, R. M. 1934b A new antigen of Bacterium typhosus: its relation to virulence and tdractive and passive immunization. The Lancet, 2, 186. Henderson, N. D., and Ferguson, W. W. 1949 Bacterio- phage typing of Salmonella typhosa. J. of Lab. and Clin. Med., 33, 739-743. 33. Kauffmann, F. 1934 Uber einen neuen serologischen formenwechsel der typhus-bacillen. Zeit. fur Hyg. und InfeCto, 116, 617‘6510 Kolmer, A., and Boerner, F. 1945 Approved Laboratory Technic. D. Appleton-Century Co., New York. Siler, et a1. 1941 Immunication to Typhoid Fever. The JofifisIHopkins Press, Baltimore. Vener, H. I., and Stevens, G. M. 1940 Typhoid outbreak in the city of Los Angeles. Epid. invest. report. Los8Angeles City Bd. of Health Comm. Bull., 65, 1'2 0 —- Milton B. Dobkin ABSTRACT OF THESIS PERSISTENCE OF THE VI ANTIGEN OF SALMONELLA TYPHOSA IN VARIOUS LABORATORY MEDIA. The discovery of a new antigenic component oqu. typhosa gave new impetus to further experimental work with this organism. It was found that the lytic action of certain bacteriophages on the typhoid bacillus was dependent upon the presence of this antigen. It was called the V1 antigen because it was believed that this antigen was responsible for the virulence of the organ- ism. On the basis of selectively propagating these bacteriophages, it was determined that there exist 25 strains of S. typhosa. A technique of bacteriophage typing was described, which became the forerunner of a proposed standard method, to be used in tracing out- breaks of typhoid fever to their source. In the standard method it is recommended that all stock cultures of S. typhosa be stored on Dorset egg medium. This suggestion is made only because no previous investigations had been conducted to determine whether other media exist which would be more suitable. In this study, 15 laboratory media were tested for the persistence of the V1 antigen of S. typhosa, using strains A, D4HP56’ E1, and F1, and their homologous Milton B. Dobkin bacteriophages. At the end of 120 days of storage, strains A and F1 on Dorset egg, Petragnani, and veal infusion media showed growth and phage lysis. Strain A stored in brain heart infusion medium and on Kligler iron agar produced growth on transplants and was lysed by its homologous bacteriOphage. The D4HP56 strain, stored in nutrient broth, also survived storage and retained its Vi antigen. 0f the media used in these experiments Petragnani medium appears to be the most satisfactory, although not the least expensive nor the simplest to prepare. This statement is based on the observation that the organisms grew most abundantly and the Vi antigen persisted on this medium. Also, the presence of an inhibitory agent will minimize contamination. I .1 ' V. .l. . '1’ E I’ IN ‘I’I't U R . 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