STUDIES ON THE SPORULATION OF CLOSTRIDWM BOTUHNUM, TYPE A Thai: for the Domino of M. S. MICHIGAN STATE uwvmsm Lawrence E. Day 1960 (“453:3 LIBRARY Michigan State University MSU LIBRARIES “ RETURNING MATERIALS: Place in book dr65_to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. STUDIES ON nus sponuu’rxms or 0ngQO Bommmz, rm: A By Lawrence E . Day A THESIs Submitted to Michigan State University in partial fulfillment of the requirononto for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1960 ABSTRACT 3TUDIE3 ON THE SPORULATION 0F CLOSTRIDQUR BOTULINUH, TYRE A by Leta-once E. Dey i etudy wee nede of the eporogenic proceee of Cloetridium_ggtulinun. ATCC 62.1. in order to determine whether or'not thie orgeniem would undergo endotrophic eporuletion. end eleo to determine the nutritive require- nente for eporuletion. It was denonetreted tbet 9;. bgtglinum would not eporulete erter being repleced in weter. phoepbete buffer. eeverel eelutione of verioue e-ino ecide end vitenine. e eolution of ecid hydrolysed ceeein. end e eolution of eneymetic hydrolyeed ceeein. A four percent eolution of tryptieeee. e pencreetie digeet of ceeein. wee found to be highly eporogenic both ee e culture medium end ee e replacement medium. when the individual enino ecide known to be present in trypticeee were combined ee e eynthetio medium end ueed ee e replece~ nent eolution, eignificent eporuletion occurred. A etudy of the effeote of penicillin. chlorenphenicol. end vencomyoin on the eporuletion proceee wee eede. Penic- illin end venconycin were {bund to inhibit eporuletion if edded to the culture before or shortly efter the eporuletion proceee bed begun. however, e leg was observed before vanoo~ eycin demonstreted eny effect. Chlorenpbenicol erreeted eporuletion when edded to the culture et eny etege of eporuletion. with no leg being demonetreted. ACKNOWLEDCHELEN T3 The euthor wiehee to extend hie eincere epprecietion to Dr. R. N. Coetilow of the Depertment of’nicrobiology end Public Heelth for hie kind eeeietence and guidance, end for hie technicel edvice. The many helpful euggcetione of Dr. H. L. Sadoff of the Depertnent of’Microbiology end Public Health ere ecknowledged with much gratitude. ii TABLE OF COIE‘I‘EI‘ITS mmODUCTIONOOOOOO0.00000COOOOCOOOOOOOOOOOOO0. 1 REVIEW OF LITERA'I‘URE.......................... The replecenent technique end endo- trophic Spatulttionooe............... 3 Nutritionel end environmentel require- HCB‘. far .p0r030n031.eeeeeeeeeeeeeee k The role of dipicolinic eoid in Iporulation.......................... 9 The node of ection of penicillin. chlor- .mph.nic°1 .nd 'Incomy01neeeeeeeeeeee 10 IXPERIEENT‘L METHODSeeeeeeeeeeeeeeeeeeeeeeeeee 1h RESULTEeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 20 DI’CUSSIONeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee h, smmYUOI...OOCDOOOOOOOOOOO0.0.0.000...COO... so BIBLIOGRAPHIOOOOOIOOODOOO0.00.0.0...0.0.00.00.0 52 iii LIST 0? 1181.138 TABLE I. summery of the reeulte of the endotrophic IPUfU1I31°D .f.£l° botulinun................ II. Smery of the reeulte of the e tion of gl. petulinum in chenicelly def ned replete- meat «an? oelle oultiveted in rap medium... III. Sumnery of the reeulte of the eporuletion of §_,l_. botulinum in chenicelly defined replete- ment mad as celle cultivated in 1. percent tfyptt..l.eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee iv PAGE 29 32 3b LIST OF FIGURES FIGURE 1. 3. b. 5. 6. 7. 8.m 9. Comparison of growth end eporuletion of g_. botw Lnum in TSP medium end in b percent cryptlceee broth conteining 1 ug per ml of mLmneOOOOOOOOOOOOO0.0......COOOOOOOOOOOOOOOOOO Comparison of growth end eporuletion of Cl. botulinum in 5 percent trypticeee end in" 5 percent trypticeee conteining 1 us per ml of tmmiDOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Mperieon of totel refreetile epore counte hoet reeietent epore counte end the level of dipicolinic ecid preeent during .Wtion of Le botulinmgnn................. The effect of replecenent of the vegetetive celle of . botuling! in the pretence of .msphe 0 oxygen............................... The effect of replecenent of the vegetetive eelle of‘£_.w tggim min the preeence of thgen......................................... The replecenent of the vegetetive celle of Cl. botulinun in e eynthetie mediul eonteining efiIno end the enino eeide of trypticeee....... The effect of penicillin on the eporuletion Of 9... botulinme................................. Meffect of chloreepheniccl on the .pmtim 0: Re bOtwwmeeeeeeeeeeeeeeeeeeeee The effect of vencomycin on the eporuletion Of 9;. botulinum................................. 21 23 26 28 37 39 £0 LIST OF APPENDICES “PEBJDIX I.OOIOOIOOOOOOOIOO000......OOCOOIOOOO P‘@ 57 1. INTRODUCTION Practically ell etudiee concerning the nature of the eporogenic proceee of glggtridium botulinum have been carried out in complex. artificial.nedia. and no real dietinction has been made between conditione which favor growth of the vegetative cell end those which favor eporul- ation. However. 31.1:- (1950) demonetrated limited spore formation of 9;. botulinum in variatione of the eynthetic medium of Roeeeler end Brewer (19th). In order to etudy the proceeeee of eporulation of the anaerobic bacteria more directly. the need exiete fer the development of a technique and/or a medium which allowe eporulation to proceed to com- pletion in an environment of known compoeition. The technique of replacement offere a method of partially eegregating the growth phase from the aporogenic proceee. Hardwick and teeter (1952) demonstrated the practicability of the replace- ment procedure for aerobic spore-forming bacteria by tranc- ferring vegetative cells that were committed to eporulation to dietilled water. A high percentage of eporulation wee ehown to occur in thie non-nutritive environment. They have deeignated thie phenomenon ae endotrophic eporulation. There are no reporte that thie procedure can be carried out with‘gl. botulinum. although Collier (1956) reported that the vegetative celle of Cloetgidium £25233 will eporulate in an environment devoid of nutrients. Thie work wee undertaken to inveetigate endotrOphic 2. eporulation of 2;. Qotulinum ae a poeeible approach to a more detailed etudy of the eporogenic proceee of thie organiell; and. failing thie, to determine the nutritive requiremente neceeeery for celle to epornlete. Some of the more general aepecte of eporulation of thie epeciee were eleo etudied in order to gain more ineight into the nature of eporulation of the anaerobic bacteria. 3. REVIEW 0? LITERATURE geplgcement and onentrophgg epggglgtion. than the vegetative cells of aerobic epore-formere are harveeted and reeuepended in dietilled water. eporulation occure (Buchner. 13?O: Schreiber. 1896; and Knayei. l9t5). There wee no experimental utilization of thie observation until 1952. when hardwick and Foeter (1952) noted the value of thie technique ae an experimental approach to eporogeneeie. Using Eggillgg,ngoidgg and gacillgg tico . they feund that eporulation occurred over a two hour period after re- placement in dietilled water. and a final level of 70 - 90 percent eporee wee attained. The heat reeietance of the spores thue produced wee the cane ae the heat reeietence of sporee produced in a growth medium. However. Black gtwgl. (1968b) obeerved that eporee of ggcillue gggggg are heat susceptible when produced endotropically. It ie the hypotheeie of hardwick and Foeter (1952) that eporulation in dietilled water ie accompliehed by the degrad- ation of'materiale already in the cell for the utilieation in the eporogenic proceee. The abeence of’exogenoue nutrition would eupport thie conclueion. while it ie fielt by some inveetigatore (Powell and Hunter. 1953! Black 22H£;e. 1960b) that the replacement eolution ie actually a dilute nutrient medium due to the lyeie of vegetative celle after the tranefer to water. Perry and Footer (1955) were unable 1.. to detect lyeie during endotrophic eporulation. Hardwick and teeter (1953) harther obeerved that the enaymee of glycolyeie are degraded and utilised ee cubetrate by amino acid enzyme in the eporogenic proceee. Ihen glucoee ie preeent in the environment. eporulation ie inhibited. indic- ating that the glucoee metabolism takea precedence over the letabolian eeeential for eporulation. Collier (19») removed aub-eemplee from a culture of 9;. m under anaerobic conditione when the culture wee in the proceee of eporuletion. The celle were washed. and incubated at 30%: in aterile dietilled water under anaerobic conditione. me culture eporulated eeapletely under theee cceditione. However. Lund (1956) obeerved that a mutant of the Putrefactive Anaerobe (PA 3679-11) would not eporulete in a replacement unatrum of buffered thioglycollate or in thicglycollate and gloom after being cultivated in a "fortified epent aediun of 2 percent trypticaee broth. then the vegetative calla of thie eaeerorzani- were re- placed in a non-eporogenic uedim euch aa 2 percent trypticaae. eporulatien took place in the replacement media before eporulatien ia the control neural. him. W m W new has: W Much inveetigatien hae been done in thie particular area. eepecially with the aerobic eporecfer-ere. Ordal (1956) hee eteted three require-ante for eporogeneeie: vie.. (a) the celle auet be of the eporogenoue type. (b) the 5. cello must be in the proper phyeiological condition. and (e) the cello aunt be in the proper environment. The firet of theoe requiremente ie. of course. primary. The eecond of the requirement” proper phyeiologieal condition. ie much acre difficult to aaeeee. The right phyeielogical condition for eporogeneeie apparently requires minerale. nutriente. growth factore. end enviromeental conditiene which differ from thoee required for growth. The aanganeee ion ie of prime importance to aporulation in the genua Mil-E! (Charney 23 g... 1951; Powell. 1956). Curran and Evane (195“ noted that the reguiment for it!” could be replaced by large addition- ef Fefi‘”. but Heinberg (1955) denenetrated that thie quantity of For” H contained minute amou- of Mn“ ae an impurity and the enmmt of manganeee that ie required for eporulation ie only one part in twenty million. The function of thie ion nay be coneidered fr:- two aepecta; it may inactivate eoue epore inhibiting compounde. or it lay .be required for the action of enaynea of eporegeneeie Weinberg. 1956). Other metallic iene have been found to be of importance in eporogemeie. Ia oddition to manganeee. inaha £3. Il- (1956) ebeenec that to“ and m“ enhanced aporulation in the genua w. Footer and Heilipan (1959) found that x" lo deficient in complex organic media in the fluid etate for operulatien of g. m; while Brewer gt 5;. (191.6) have reported that Mg” would enhance eporulation of gaoillue gthraeie in a eynthetie medium. It wee also noted 6. by these inveetizatora that Ca++ increaeed eporulation b.5 timee in a eynthetic medium compared to the same medium containing no CI++e The effect of the ealt concentration or the ionic strength of the medium on aporogeneoie has not been studied extensively. The percent eporulation of a culture of'gl. botglinum decreaeed at the concentration of ”325050 haCl and K01 increased in the culture medium. and, when the concentr- ation of NaCl nae increased to 2 percent in brain-heart- infueion medium, eporuletion was inhibited completely (Wynne. 1958). Leifeon (1931) also noted the inhibitory effect of haCl. and reported that eporulation took place in a basal medium of 1 percent peptone only when hhk*'and P0“a were added. The addition of eulfate atinuleted eporulation eomeuhat. The role of glucose in eporulation ie controvereial. Leifeon (1931) reported that a 0.2 percent concentration of thie carbohydrate is inhibitory to eporulation of 9;. ‘bggglipgg,due to acid formation, and Kaplan and Hilliame (19k1) noted the eame effect with Qloetgidium eporoggnee. Blair (1950) noted inhibition of eporulation cf‘§_. botglinum in synthetic medium, but Wynne (l9k8) reported that 0.8 per- cent glucose in brain-heart-infueion.medium did not adveraely effect eporulation of thie organiem. The significance of growth fectore in the eporogenic proceee has not been atudied extensively. Ordal (1956) reported that the amount of folic acid required for 7. aporulation no greater than that required for growth of manna $3.2M!) and Williams and harper (1951) noted that when p-aminobenaoie acid wao deleted from a medium. the oporuletion of a. some one reduced. Lund (1956) observed that the medium in which Pi 3679 had been cultivated demon- otrated oporogenic activity. He believed that thie could be dm to exhaustion of acne eeeential nutrient for growth of the vegetative cello; or to the utilization of come oporulation inhibitor; or to the eyntheeio of some factor which io required for oporulation. The nedim need in thie inveotigation no 2 percent trypticaoe with 0.1 percent oodim thinglycollate. b'hen thie aediua wao fortified with 10 pg per ml of thiamine. all cello were in the swollen etate at the end of 2!. heuro of growth. However. the cello never completed the eporulation proeeoo. and at the end of 72 , hour-o only vegetative cello were preoent in the nedim. If the cello were removed frat thie aediua after 18-20 houro of growth. and the nedim rofortified with the initial quantity of dry ingrodiento. oporulation took place to a level of about 90 percent. 1 control of I. percent trypticaoe. which correoponded to the concentration of trypticeoe in the refortified opent medium. did not demonotrato ao great a degree of eporulation. Thio oeened to eliminate the theory of the removal of a oporulation inhibitor or the exhauotion of nutriento ae the factoro initiating oporulation. 0n the booio of thie oboervation. it wao theorized that oomething 8. is eyntheeioed and elaborated into the medium by the agenism which is required for sporogeneeio. Blair (1950) working with the synthetic medium of Roeosler and Brewer (191.6) demonstrated that the deletion of methionine and phenylalanine from this medium ouppressed the eponiletion process or 91. yptxggmt . The percent spatula- tion has increased by addition of ec-alanine. glutamic acid. glycine. ornithine, oodiun butyrate. eodim valerate, arzinino and proline. The stimulatory effect of glucose towards omgeneeie in aerobic bacilli in a synthetic glucose-glutamate nediue wae reversed by the addition of Ill-alanine (Foster and Heilimen. 191.9). The effect of oxygen tension on eporulation of the onaerebee ie not clear. Leifoon (1931) reported that veriouo anaerobic organism exhibit different responses under increasing oxygen tension. Samar (1930) working with g}... hetlulinmn noted that a broth culture will spend-- Ite more quickly when exposed to the air than when the culture is sealed with veoeline. and Eety and Feyer (1922) reported that small amounts of anger; in the culture meditm were beneficial to oporogeneeio. However. it has been oboorved that the level or oporulation in broth cultures is identical in either oxygen or in natural gas atmospheres (Wynne, 191.8). All of these observations were node with g_l_. Melina. The optimum pH for the oporulation of £1. haggling hao been established to be 7.0-7.2. Thio wee noted by Kohrke 9. (1926) and by Collier (1956). Leifson (1931) stated that the pH at the time of sporulaticn is of greater importance than the initial pH, but he did not recommend what this pH should be. 1e. £2.13 2:. W 1c n is...“ n W. mum-2. 6-dicarboxylic acid or dipicolinie acid was first isolated from the spores of 219.133 W by mu (1953). Perry and Foster (1955) isolated DPL froa endotrephicelly produced spores ore-mmww- In rapidly sporulating culture of 9;. means, DPA synthesis as found to correlate directly with the oporogenic process (collier and Krishnamurty. 1937): but in other work with this same organism. it was noted that the sexism on was formed an hour before heat resistant spores could be detect- ed (Halvoroon. 1957). This was also observed with oporul- eting g. w (Black 93 31.. l960e). Young (1959) ascribes the thermostebility of spores to a complex of DPA and amino acids in the presence of caleim: however. this cmplox was only demonstrated on the filter paper of e chrmtog-as. This combination could be the means whereby dipicelinic acid renders stability to proteins and nucleic acids of the spore. The work of Black gt 3;. (196%) would indicate that on does render heat stability to the spore. These investigators demonstrated that endotrophically produced spores of E0 m were heat susceptible and DH deficient. If Ga“ were added to the replacement suspension shortly 10. after transfer of the vegetative cell s, tlze EPA dc ficiency was relieved; however. tre Spores were still heat sen sitive. These spores were as resistant to gamma radiation or to phenol as normal spores. Church and Ealvorson (1§59) re- ported that if phecyinlnrire were [resent d1::irg Sporcgencsis of P. so , the EPA level of the spores was reduced with a ccncommitant lowering of the heat resistance. to other amino acid 3 demonstrated this phenomenon. The most recent report mold indicate that the D? A content of the Spores cf‘gl. rescue is not necessarily correlated to the heat resistance. Eyrne £§_g;. (1960) reported that spores produced in the presence of L-alanino had a lower level of dipicolinic acid than spores produced in a medium containing no Laalanine; however. the spores with the lesser amount of EPA were more Lost resistant. Obviously. much work needs to be accomplished on the role of DPA in the spore and on its connection with heat resistance. it one time it was surmised that DPA gave ultraviolet (U.V.) resistance to the mature spore because it absorbs stroaagly in the U.V. region of the spect— nrn. but Rania and byes (19 57) noted that resistance to U.V. appears at the time of the fermntion of the foreopore of g, gggggg while the DPA dose not appear until mature spores are present. This work was done with cultures sporulating in distilled water. The node of fiction“ of penicillin chlcrw picnicol, an vcn o n. nest recent reports attribute the action of 11. penicillin to the inhibition of division of the bacterial «11 by the combination with and inactivation of m sssential constituent of the coll which is :13un for division to occur (Lederberg, 1957). It does not prevent * aspiration or synthesis within the cell (Pratt, 1953). The canpomd inactivated by penicillin which is acquired for cell wall synthesis is N-acetyl mursmic acid {Park and Straningor. 1957). Most osrlior reports, how-over. attrib- utsd the mechanism of action of this antibiotic to the inhibition or am synthesis (Mtchell and Hoyls. 1951] Gale. 1949) or a disturbance in tho nucleotide balcncs (Mitchell. 191.93 firmpits and Worms. 191.7). This disturb- nss of nucleic acid synthesis mks by blocking sssimilstory processes. thus. argsnisns winch are dependent upon assimilation rather than synthesis for growth on unsitivs to this drug (Gals. 19W). Gram positive orgmicms loss thsir chuactsrisuc staining reactions. This as sham by “Alysia to be due to the altsration by penicillin of tbs rstio of ribonuolsio phosphorous to dosoxyribcnuclsis phosphorousin the gran positive sensitive coll toward the ratio in the gram negative organism (Oslo. 191.8). Gals snd Folkes (1953) “ported that chlomlphenicol acts by the inhibition of protein synthesis, but they addsd that tho am soncsntration of the antibiotic stimulated the synthesis of RNA. With the use of labeled amino scids. it an rm that protein synthesis was inhibited by the 12. provontion of amino acid utilisation (Galo and Folk”. 1953; Smith. 1953). Tho inhibition of notaboliam of fato and ootoro by tho action or chloranphonicol hao aloo boon domonotratod to bo of aignii‘ioant importanoo in tho inhib- ition of growth of tho bactorial coll (Smith. 1953). Viaoimn at Ll. (1952) raportod that tho inhibition of tho aaainilation of NH, and tho concm-ront oxidation of glycorol vao a nochanion of thio 4mg. whilo Ga1o and Paint (1950) found that chloromphoniool had 1itt1o offoct on glutamato aoaimilation ovon at high concontrationa but inhibitod tho oynthoaia of protoin conplotoly. Primarily. than. tho nodo or action of chloramphonicol would appoar to bo tho inhibi- tion of tho oynthooio of protoin motorialo in tho con. A minim amomt or invootigation hao boon roportod on tho mochanim of action of mcomycin. Of tho information avaihblo. it in known that this antibiotic ia bactericidal. Thio io accompliahod by tho inhibition of tho aynthooio of ribonucleic acid with tho rooulting inhibition or collular divioion. Doath ia duo to tho unbalancod growth brought about by tho inability of tho organics to oynthoaizo mu (Ionian. 1959). Co1lior (1956) romvod mono of a culturo of 9;. m at varioua intomlo or time and placod tho mp1“ in aeration tuboa with penicillin at a final concentration or 100 units per uni. It as found that. furthor oporulation vac amatod by tho ponicillin. but no offoct no obaonod 13. on tho oporoo olroady in tho oomplo at tho tino of tranofor. Uoinborg (1955) invootigatod tho offoot of nino antimicro- bial drugo on tho oporulation of g. gubtig o culturod in a nodimi containing in“. i 1on1 of ponicillin which did not provont growth had no offoct on oporulationg higher 1on1o of thia antibiotic and ito offoot on oporulation taro not diocuoood. Chloronphonicol inhibitod oporogonooio at a lover concontration than no roquirod to inhibit myth. Thia offoct wao not rourood by addod Mn“. lit. EIP’ERII .342». 211.. i-‘ETZECDS g logtrigiun botulinm, €2-i. was the organism used in all aspect: of thia investigation. The culture was originally obtained from tho American typo Culture Collection and was maintained in oporo ouszsonsicns produced in either the trfiaticoso-ealto-peptone medimn (TSP) of Zoha and Sadofi‘ (1:63) or in a medium of 1. percent trypticaoo broth (BBL). men oponco woro prooont in the culturo at o level of 107 or 103 per m1. tho spores were harvested from the growth median by contrifugetion, washed twice in sterile distilled water and roouoponded in 0.067 It phosphate buffer. Thooo suspension won then otorcd at 5'0 until needed. Subsequent inoculation woro made from these suspensions. Griginolly. TSP medium: was used for the cultivation of vegetative cello and spores; however. subsequent work demonstratod that a medium of 1. percent trypticase contain- in; 1 115 per m1 of thiamine resulted in more rapid and complete aporulation. Yeast-voxtract-oterch-bicarbonate agar (mom) or Wynne g3, 51. (1955) was used for making colony counto with ovel tubes. The composition of these media are given in tho appendix. ‘I‘ablo 1. Preliminary work indicated that an atmosphere devoid of oxygen would be moosnery to carry out the transfer of the vegetative collo of 9;. botglinum from the growth sodium to the replacement monotmun. Thio was achieved by the use of a ploxigloso chamber containing an atmosphere 15. of nitrogen. Ihie chamber was fitted with.erm~longth rubber gloves attached through Openings in the front of the chamber to allow handling of equipment inside the chamber. The chamber was filled with nitrogen from a cylindrical tank of the comproeecd gas after passage over hot copper shavings to remove oxygen present in the nitrogen tanks. The gas entered the bottom of the chamber through gaa Jets fitted to the lower part of the chamber. and the atmoophore in the chanbor'oocaped through a zoo Jot fitted to the top of the chamber. The chamber wuo equipped with an airuinter- lock in the roar which allowed cultureo or'oquipment to be removed without the necessity of refilling the entire chamber with nitrogen. in electrical outlet wee fitted to the inaido of the chamber. end a Sorvall anglo~hoad contri- fuge wee utilized for centrifuging the cello from the growth medium. The replacement technique woo achieved by first cultivating the organiwm in TSP medium or k percent trypticaoo at 37“C for tho doairod length of time. Then. using the nitrogen chamber. a 10 n1 aample of the culture was tranc- ferrcd to a atorile tube. and the cello removed from the growth medium by centrifugation for 10 minutoo. The cello were washed three timoa in oterilo distilled water. and reounponded in the appropriate replacement menctnnun. Anaerobiooio woe attained by one of two methods. Sodium thicglycollate in a concentration of 0.1 percent was used in conjunction with YESBA medium need for the lo. enumeration of cells and Spores. Sodium thinglycollate was also used in some cases with replacement solutions. This method was the most convenient to handle of those employed but was of doubtful value if the incubation times extended much beyond 72 hours. The sodium thioglycollate apparently loses its ability to maintain anaerobic conditions after thie time interval. The other method of attaining anaerobioeis was by the use of the Brewer anaerobic Jar. This method was utilised exclusively with replacement cultures. The cultures were placed in the Jar. the Jar exhausted by vacuun, and then filled with nitrogen from which the oxygen had been removed. The Jar‘vas exhausted and refilled three times to insure anaerobioeis. Total counts and spore counts were usually made by the use of the Petroff~fiaueecr counting chamber. This is a higlly reproducible method of enumerating cells and spores, however the method is of no value in determining viable or non-viable organisms. Also, it is of little use in differentiating heat resistant and non-heat resistant. or mature and immature spores. In those experiments concerned with the effect of antibiotice on the eporogenic process. YESBA and oval tubes were utilised to determine viable cells and viable spores. This again offered some difficulty in determining total counts since not all Spores germinate when placed in germination medium without a heat shock. Sauples for spore counts were heat shocked for 10 minutes 17. at BO'C. Four tubee were poured per dilution in running viable cell and/or epore counte. In order to determine the DH content of eporee at varioue etagee of development a large quantity of celle eae required. Therefore. a 2L Erlenmeyer flack containing 1500 ml of 1; percent trypticeoo medim and fitted with appropriate inlet and exhauet tubee to allow the paeeage of eterile natural gee through the medium nae inoculated with ' e 10 percent inoculuu of vegetative oelle of 9;. botflgm. At various time intervale 200 ml euplee of the growth ‘ euepeneion eere moved and total counte and epore counte made both by plating and by direct aicroeoopic counte. Celle were then reacted from the media by centrifugation m reeuepended in 5 .1 or eterile phoephate buffer, 0.06m. The dry weight of the celle or each eaaple wee determined by placing 1 ml in a crucible and drying at llO'C for 2!. houre. The eamplee were then diluted to contain 5-20 mg of celle per 5 ml of eample. i dipicolinic acid. aeeay wee then run .on theee eemplee by the method of Janeeen 33 L13 (1958). The percent of DPA of the dry weight of the «He wee calculated. Penicillin at a concentration of 100 unite per ml. chloramphenicol at 100.»; per ml, and vancoeycin at 10 ug ‘ per al were utilieed to determine the effect of theee antibiotice on eporulation. For each of then determination tour fluke containing 100 ml of 1. percent trypticeoe aediua were need, each {leek being inoculated with a 5 percent epore inoculum. To the first culture. the microbial 18. inhibitor being utilized was added during the logarithrdc grmfih phase, or eonetime dwing; the interval from 10 to 16 houro of growth. During this time no eporulation was apparent and no forceporce could be observed. it the end of the logari'tlwic growth or at about the 20th hour when almoet ell cells mm in the cradled state but only about 1—5 per- cent Symon could be observed, the microbial imfibi tor was added to the seemd culture. At the point when there mre from 10-50 percent spores (between the 2640le hour). the witagorxiot was added to the third flask. The fourth flask served no a control. At various time intervals. samples were removed from each culture and total counts and epore counte made both by plating and by direct microocmdc count. Zine preparation of trypticaoe ash for use in replacement solution wee accomplished by aching; 4 3; of try- pticaoo no SZS’C in a porcelain crucible in a muffle furnace. Thie was then dieeolved in 96 ml of water. It was found that it was necessary to loeor the p}! to about 3.0 in order to dieeolvo the aeh. When the pii was Again raieed. precipit~ ation of some constituent of the ash began at a pH of about 5.0. "moi-cram the pH wee held Just below thie value. A dielyoetc of trypticnoe one prepared by placing 100 ml of a 1. percent trypticeoe solution in dialyeio taking and suspending in a chromatography Jar containing distilled nter. "The dialyeie procedure was carried out for 72 hours at 5°C. The distilled water wee stirred constantly and changed every 2% hours. The dialyzo ryptucaao solution was than atorillzod 1nt solution. d I: a ,0 § .- p0 _' “I" . 5. L236 19. 20. RESULTS The initial phass of this investigation was concerned \with the sslsction of a medium which would support growth and good sporulation of Clostridium botulinum. Originally. 13? medium was used for the cultivation of vagatativa calls and sporas of this organism bacauss it had been demonstrated by Zoha and Sadoff (1958) that this medium would support the sporulation of a similar organism. They used Putrs- {active Anaeroba 3679 (PA 3679) and achiavsd sporulation at a laval of 109 par ml in it hours. However. these results could not be reproduced using 9;. botulinum in this work. lnvariably the number of spores in the culture did not roach 80 - 90 psrcant of the total count until after 5 - 7 days of incubation. Therefore. a broth of A percent trypticass was tasted as a culture medium. and it was obscrvad to be of a such grsatar sporogsnic nature than that or TSP medium. A comparison of the two media was made and it was found that sporulation in the TSP medium reached maximum sporulation after about 150 hours incubation, while sporulation in the 5 percent trypticass attainad a lsval or 90 percent in about to hours. This comparison may be scan ianigura l. Trypticasa is a pancreatic digest of casein marketed by the Baltimore Biological Laboratory. Inc. of Baltimora. Maryb land. in approximate assay of the product shows it to be deficient in thiamine (Vera. 1900). and Lund (1956) reported that the addition of thiamine to trypticasa stimulated spore 21. .8115 no Ashen soc meets 833.53 assets.— can '«welasuld Hosouaenshodeeseguesoeg influx.— mmoo: 2. m2; oom om. ow. 03 ON. 00. on ow oe ON 0 q u d q q u a w u q q u 4 1 q 4 u 1 as u i _ bzaou 4:0» \ _ wm¢._, o\o¢ - o M mumodm $40.22... oxoe . . o\ mmmoam mm? o . P2300 45.0.. amp v o o o\ o[ 0 ol\\ d 4 1., c I 'ON 50 90'] 22. formation of Pa 3679 and C . botglinum. This investigation also demonstrated that the addition of l‘pg of thiamine per ml of A percent trypticase medium stimulated sporulation o£.§}. hotulinue (Figure 2). Sporulation in the medium con- taining the thiamine reaches a level of 106 per ml about 9 hours earlier than in the medium containing no thiamine. and the final spore population was increased about ten told by the addition of thiamine. Gas evolution was apparent in the thiamine containing medium. but none was observed in the control. No methods of counting spores was used in this work. The first was a direct microscopic count by use of the PetrofIAfisusser counting chamber, and in this method only those spores which were highly refractile were enumerated as nature spores. This was compared wdthcolony counts made in heat shocked samples by the oval tube method. A comparison of these two methods in conjunction with the dipicolinic acid content of the spores during various stages of the sporogenic process is presented in Figure 3. From these results it should be noted that the direct microscopic count wee consistently higher, usually by about 70 percent. This difference in the number of spores can be attributed to a number of factors; vis.. (a) it is highly unlikely that all spores germinate and produce colonies. (b) due to clump- ing. a single colony may arise from more than one spore. and (c) not all retractile bodies observcd microscopically have heat resistance. 1 measurable concentration of DPA was LOG OF NO. 23. g - xz-X\ // X I, \ X A ‘\ / ‘ . /"' C 1X. a 4%, TRYPTICASE TOTAL COUNT ' o o 496 TRYPNCASE spoaes \ \ 4% TRYPTICASE s THIAMINE I l TOTAL COUNT ‘- ‘ O 4% TRYPTICASE a THIAMINE SPORES I) 1 L l 1 a 1 I I0 20 3!”: 4O ' 5O 60 7O 80 TIME IN HOURS Figureil. (Reminisce Cflpfilflflllfl‘ epmnmunuemned'fil. *itgyuuggin i;punumm.trywuuuulnana percent trypticase containing 1 .nslmu-sd.et1nneldne. LOG OF NO. D \o 3 t6 P l : o I O 4 I 0 -OJ / e REFRACTILE SPORES . I _ I . HEAT RESISTANT I : scones ll “.m l 1 o mmcoumc ACID b l x I I J L I 1 IO 20 30 4O 50 60 TIME IN HOURS Figure 3. comparison or total refractile spore ecumulwdflhlwmtxnudsuunsspam! counts and the level ct’dipiccl- isic acid present during sperul- atien 01' El- W. 25. observed only after an appreciable number of spores were present. When the heat resistant spores were present in a concentration of 103 per ml, the amount of EPA present was not detectable by the method being utilised for the assay. it this point the spores accounted for only 0.03 percent of the total count. is sporulation proceeded. the amount of DPA present also increased. When the spores reached a level or 105 per ml, the on accounted for 0.10 percent of the total dry weight of the spores and vegetative cells. although it was assumed that the DPA.was in the spore. This trend continued until spores were present at . level of 107 per ml and the DPA reached 3.hl percent of the dry weight. gggotggphig sporulgtion. Initial experiments were de- signed to determine the effect of exposure of the vegetative cells of 9;, botalinum to the atmosphere during harvest and transfer (replacement) to a fresh medium. Cells were culti- vated in a k percent trypticese medium and were removed from the growth medium at the 25th hr of growth and replaced in fresh sterile 5 percent trypticase in the presence of atmospheric oxygen. Figure A illustrates the results. it the time of replacement. there were 107 spores per ml and this level remained constant in the replacement medium while the total number of cells declined. Sporulaticn proceeded as expected in the control culture and reached almost 100 percent in 70 hours. with 35 percent spores at ‘5 hm.e LOG OF NO. 36. Q 1 CONTROL TOTAL COUNT ° CONTROL SPORES —‘-_ o D x REPLACEMENT TOTAL COUNT s REPLACEMENT SPORES I 1 L I a J to 20 so ~ 40 so 60 70' Figure 5. The signs? oliN re; ‘33... o! the vegetative cells or . e i in the presence or 36';th oxygen. 27. The replacement technique use then carried out using a chamber containing an atmosphere of nitrogen. This time TSP medium was used both an the cultivation modiun and the replacement medium. The replacement spores in this instance reached a higher level than the control spores (Figure 5). indicating that no cell damage use incurred by the procedure of centrifugation and replacement. The vegetative cells of‘glt hotgligum were cultivated in T8? medium for use in the first attempts to Sporulste this organism.in an environment containing no exogenous nutrients. At various stages of growth, the cells were har- vested by centrifugetion under nitrogen and fractions suspended in (e) sterile distilled water. (h) .067 M phosphate buffer containing 0.1 percent sodium thioglycollate. and (c) .067 M.ph05phate buffer. All three suspensions woro incub- ated at 37'C3 the water suspension being incubated under - nitrogen in a Brewer anaerobic Jar. The results of these three experiments are given in consolidated form in Table I, and detailed results are found in the appendix. Tables I-a. II-A. and III-A. The spore counts indicate that replacement was carried out about 1 day hetero the onset of sporulation. again Just prior to or shortly after the onset of sporulation. and lastly after sporulation had taken place in 10 percent or“more of the total cells present. The total populations in both the control and replacement menstrua were found to decrease at all incubation times after the initial harvest LOG OF NO. gs: X x/_‘ . cou'rem. TOTAL coum O o CONTROL spans /— D o e REPLACEMENT TOTAL I l / COUNT i REPLACEMENT SPORES // // a J_ h l l I j IO 20 30 4O 50 60‘ 70 80 TIME IN HOURS Figure 5. he effect of replace-est or the vegetative sells «fibwminthepresesee er recon. eeézbegegoosgagaelflelg hope saves noses» e5 aw assuage led—subs as» as 0.333 s I defiance... N... o o o O o a o o guano: vacancies 2.... 8+ 8+ 3+ 8+ 8+ .8... 8+ 3+ «Houucoo no.“ use.» oust 3 ounces 38.3.. 45 mm: mm... mm 3.. mm... on: 3.. 3| Essen agmom a en- 3.. on an- an man 3.. 3.. 3.838 .5 £500 33 5 ewnsno oneohem V V V V #3 Oh on N H .n A av ,5" A .n «ovowuwagoe ed» a on ad end on 3 2. «a 3 an." 79:: aged“: teams '3 53% Q. .3 an and. g on «a 3 an Tenn. val-813: ae suede «o 84 as .3 Setup e- a1oiflo£wommsi “we? 813-23 2 So. tea?» Riflesm 2 So. he»! 3383 gnaw—ex uni-3&8 313 no 83.98... suites...- 1» «a Seat no i H135 30. time. However. while the spore counts of the control cultures increased by 80 percent or more beyond the papu- lations at replacement time, no increase in the number of spores occurred in any of the replacement ssnstrua irrespec- tive of the harvest time. Similar results were observed in five subsequent experiments using water or phosphate buffer as the replacement menstruum. Therefore, it is concluded that the culture of El. botulinum 62-1 tested will not undergo endotrophic sporulation. . The observed decrease in total pepulations, which un- doubtcdly resulted from cell lysis, uss greater in the phos- phate buffer suspensions than in the water. This may have resulted from incomplete anaerobiosis in the former. since they were incubated under atmospheric conditions. wnmumumnw dgginsd replacement Eggig, Since the foregoing results lead to the conclusion that sndotrophic sporulatiOn was not possible with this organism. a search was initiated for a chemically defined or simplified sedgmnwhich would support final sporulation. The cells were again cultivated in TSP medium and transferred at various stages of develop- ment into media of known composition. The three replace- ment menstrua tested had the following composition: No. I biotin 0.01 percent methionine 0.03 percent pH 7.1 MhSO“ 0.01 percent 31. No. II biotin 0.0001 percent methionine 0.0600 percent nicotinic acid 0.0001 percent folic acid 0.0001 percent pH 7.1 pantothenic acid 0.0001 percent nnso 0.1000 percent nobensoic acid 0.0001 percent No. III DI. -ac- alanine 0.2000 percent L - glutamine 0.2000 percent L - ornithine 0.2000 percent glucose 0.2000 percent pH 7.1 glycine 0.0500 percent biotin 0.0005 percent p-aninobensoic acid 0.0002 percent thiamine 0.0002 percent nicotinic acid 0.0002 percent KHzPOk-KzHPO‘. 0.034 The results of sporulation.studies in these replacement menstrua.are given in Table II. and the detailed results are found in the appendix in Tables IV-A. V-d, and VI-A. None of these three replacement solutions containing vitamins and amino acids would support further eporulation of the crgsnism. A control culture exhibited normal eporulation during the same intervals ot'time. shen spores eere present at the thus of transfer to menstrua II and III. a decrease was noted in the number of spores during the incubation period. This decrease use attributed to gerlinstion of the spores. The decrease in the total counts in both the controls and the replacement aedia undoubtedly resulted from lysis of the vegetative cells. Since eporulation proceeded quite slowly in the TSP medium. a percent trypticase broth containing lung per’nl of thiamine las utilised in subsequent experiments as the 32. «do on: an «n- 4u+ man was 0 05+ an: an: .eeeedaeh he veneeeuen use one Heeeeeca sea» ease and been sauces mesons as» ea veneeuanne_eeuseune and no o at 00+ one encodes e s_donenoos o om+ but no: NV «.44N “one H.oz o ennnuecefl pnneeoedmem 00+ eaonucoo “an assoc shone nu smudge unsound on: asshuecen p343 on be: edouanou and peace Hence nu «wanna unsound . v in... ”equate... so“ acnaeoeflmenanunww sea» cede-nuenu we Agata. ace-eoeansn as surname no see HH sauna {434... eaan_uo sanseen an» «canteens» 33. initial culture medium. This medium gave highly reproducible results as to the time of the swelling of the cells and the onset of eporulation. Therefore. all succeeding replace- ments were carried out between the 13th and the 20th hour of incubation. it this time almost 100 percent or the vegetat- ive cells were in the swollen state. and the center of spores in a culture accounted for about 1-10 percent of the total count. It was believed that a large percentage of the cells should be committed to eporulation at this stage of develop- ment. A culture of 9;. botglipum cultivated for 19.5 hours in a.medium or 5 percent trypticaee with 1 pg per ml of thiamine was replaced in: (a) sterile distilled water. (b) a solution of calcium acetate at a concentration or 200 ppm. and (c) a solution of thiamine at a concentration of 1 PP“. The tiret three columns of Table III show’the results of this investigation. The total counts decreased in both the control and in the replacement media. although the per cent decrease see much less in the replacement solution of calcium acetate and the solution of thiamine than in the control. He increase in the number of spores use observed in any of the replacement menstrua. while during the same time interval the spores in the trypticase control increased over 99 percent. Detailed results of these experi- ments are given in the appendix, Table VII-i. In an attempt to characterise the type or compound(s) .veeeansn so concerned cos.ann «unheard slaw ales and hero suave-ennsonu as» ea eeoesuunno sequence as» no encode» e |.Houucooe 3.. 3.. «a... 3. o o o Sane—nun unmannednem nn+ nc+ um+ nae one one am+ eacnacoo a canoe enons as o: 0 ocean.» mm... on: an on nu on R genes pooeeoeanem man an: we: we: on: con 001 eaonucoo an“ ounce Hence an omnsgo ocean-s 4H J." HV V V t 3 on a a nouomu.§ a «h «h on ma n.«« n.«« n.«« ~.sn£. anessceanen necks and» couounrucn 8 8 3 3 “.2 “.2 . «.2 tan: cg... oe.ennunnc «s and - 1 3 1 .- - 3.355 3:333 383 AF... 3 an. n :2 8... one cums-sane “was Idaho “4 eeeuueac ends-«no ++eo ocean -.i¢o‘.-.H€.-«£Mn L.- .131. . .... “; 1391- z- i - M . _nsseoenneu can . . accuser ninuaumaamumqmmmmmmmuwwm.ue new» on» as sources as» us annea_hnessoa nun eases 35. Vital to the final sporulction process. a dialpucd L percent trypticnse solution and a solution containing he sch of i grams of trypticase per 100 ml.were used as replacement media. the vegetative cells were cultured for 13 hours in s t percent trypticase broth with added thiamine. The results (columns 3 and t of Table III) chow'that the spores in the control increased 93 percent while in the replace- ment menstrua a net decrease was observed. Table VIII-A. appendix, gives the detailed results or this experiment. It must be noted that the pH of the solution of the ssh of trypticnoe use 5.0 in order to solublise the ssh. Evidence cited previously (hohrke. lpzb; Collier. 1956) indicated that the optimum pH for spcrulaticn or this organism is about 7.0 - 7.2. Products similar to trypticsee were also used as re- placement:nedis. a 5 percent solution of-s vitamin free enzymatic hydrolysnte of casein (General Biochemicals. Inc.) and a t percent solution or Bactoacssamino acids (Difco) were both utilised in this manner. The replacement was per- formed after 20 hours of growth in 5 percent trypticase fortified 81th thiamine. At this time sporulation had taken place in about 10 percent of the cells. do further sporulntion was observed beyond this level in the replacement menstruai instead a large decrease was observed (inhle III. columns 6 and 7. and appendix Tables II-i). It was noted that after replacement in the ensynstic hydrolyontc or 36. casein. there was clmping of the cells. After the 10th hour it was impossible to enunerate the number of cells present by use of the direct microscopic count. and no spores could be observed at all. In the acid hydrolysed casein replacement medium, there was a loss of 97 percent of the Spores present at the time of transfer. No doubt, the decrease in spore counts resulted from germination. in assay data table (Appendix. Table II) obtained from the Baltimore Biological Laboratory containing the approximate composition of 8-8-1. peptones was used as a reference for the preparation of a solution of amino acids of the same composition and concentration as found in a solution of k percent trypticsse. Only the seine acids were used. along with 1 fig, panel of thiamine. This solution was then used as the replacement solution fer cells cultivated for 2!. hours in 1. percent trypticsse. At the time of replacement thorelss about 1.2 percent sporulstion in the culture. Sporulation in the replacement medium increased to a level of about 33 percent over the first 16 hours after replace- ment (Fi‘wo 6). The spores in the control medias reached almost 99 percent of the total count in the same time inter- val. Although sporulstion in the replacement median did not reach the level of that in the control. it was the first time that a significant increase was noted in a replace- mont solution of known composition. m effect 91 antibiotics on the sporulation 2g LOG OF NO, Nb A CONTROL TOTAL COUNT o CONTROL SPORES * REPLACEMENT TOTAL COUNT 0 REPLACEMENT SPORES l 1 I i 1 K) 20 thmnrb. 30 40 so so 70 TIME IN HOURS nulnduuu-wtottunvupunwul«n10 tmt imlnonnunmc-unnn cmggi uflnuamltMIuflno IfluulOf‘HTFNHMIOO 38. ‘Qigggzggggg,§2§g;igggp Throo antibiotico woro utiliaod in thia phaao of tho inroatigation; vil.. poniciliin, chloram- phonicoi. and vanconycin. Tho firat of thooo. ponicillin. loo addod at variouo otazoa of growth of tho culture to giro a final concentration of 100 unito por mi of culture Iodiun. If ponicillin was addod to tho culturo during tho log growth phaoo or at tho ibth hour, further oporulotion appoarod to bo inhibitod. and tho oporoo alroady orooont docroaood. Thio population docroaoo night havo boon duo to tho gornination or aporoo (Figuro 7). If addod at tho 20th hour. uhon thoro woro about 10 porcont aporoo in culturo. oporulation continuod ovor tho noxt four houra and thon ooaaod. lhon tho antibiotic woo addod to tho cuituro at tho zith hour. when oporuiation uaa about 30 poroont comploto. tho antibiotic appoarod to havo no offoct and oporogenoaia continuod. microooopic oounta of rofractiiooporoo undo in conjunction with tho oounta of hoot rooiatant aporoa ohouod ooaontially tho aalo rooulto. Chloramphonicol uaa firot uood in a concontration of 10 us por al. but at thia lovol no offioct uaa oboorwod on oporulation. Houovor. uhon tho concontration uao incroaaod to 100‘s; por mi. oporulation wao arroatod inmodiatoly on addition. Thia provod to bo true uhothor tho drug uaa addod oarly (10th and 20th hour) or lator (30th hour) in tho oporulation procoao of tho culturo (Figuro 8). LOG OF NO. 39- o/._fi. 24'h HR A ‘ X X A 20'h HR A/ 7 " \ / x \x I I CONTROL ° PENICILLIN- 16’" HR :6” HRHA x PENICILLIN- 20'“ HR 0 PENICILLIN- 24'“ HR L I I 1 . o IO 20 so 40 so TIME IN HOURS , m t or oimn on “I “n" " T'Jmhu .721. m- LOG OF NO. M3. TIME IN HOURS nm'o. nu offoot or ohloraaphoniooi on tho aporuiatioa of Q. m. 8 I / ‘ I Lo— “—. 7 n 30Ih HR \ /: 20’h HR A 6 .. \ / Ih a IO HR I < _____.< *-——x x x _x 5 . O\ O—O— -———— O o O 4_ , CONTROL 0 CHLORAMPHENICOL“ no") HR 3. x CHLORAMPHENICOL - 20'“ HR 0 CHLORAMPHENICOL - 250'h HR 1 l l 1 l l A 0 IO 20 so 40 so so 70 1.1. Vonconycin. at a concontration of 10 pg por ll. woo obaorvod to arroot aporulation. tho tino intorvalo of growth boi'oro tho addition of tho antibiotic woro again 10. 20 and 30 ham. With tho antibiotic thoro m an intonal of obout lOOhouro ai‘tor tho addition of tho anti- biotic boron any ofroct could bo oboortod. Aftor thio ti- intorval no i‘urthor oporulation woo obaorvod (Figuro 9). NO. LOG OF 1.2. CD ./—I74 30'“ HR / - \A: , . fl / , 20 HR\/o A 6- éofio O o *0 I II ,, . CONTROL // I, h o VANCOMYCIN — 10' HR 5 u. x VANCOMYCIN - zo'h HR 1h 0 VANCOMYCIN - 30 HR 1 1 l l J 1 '0 20 30 4o 50 60 70 TIME IN HOURS- riguro 9 Tho otfoot or Vance-rein on tho . oporulation of 2;. bowling, :- \I.’ o DISCUSSION Thoro aro oovorol pointo of importanco in tho dioin- ilarity of tho oporogonic preportioo of TSP modium and of L porcont trypticaoo broth. Firot. it ahould bo notod that trypticaoo. ao proparod by tho Baltimoro Biological Labor- atory, io a highly variablo product. Ito oxnct compooition io unknown. and ito conpooition and charactoriatico-vory from batch to batch; thio depending on tho caooin boing utiliood in tho proparation of tho trypticaoo (Voro. 1960). Thio variability. howovor. oppoara to bo ovorcono by tho addition of thiamino to tho Iodiuu in o concontration of lwpg por mi (Lund. 1956)., Tho concontration of trypticaoo in TSP ia 1.5 porcont, whilo in tho trypticooo Iodiun tho concontration io i porcont. Horoin.oay lio tho ditforonco in tho oporulation cupporting proportioa of tho two Iodia. ipparontly. caooin containo oomo thctor which io nocoooary ftr tho oporulation of thio organion. and it would appoar thot concontrationa of trypticaoo undor i poroont may not contain enough of thia fhctor to aupport oporulation. Sinoo tho tryptioaoo io a varioblo product ao to compoai- tion. tho batch of trypticaoo uood by Zoho and Sadoff (1958) noy’havo boon particulorly high in tho oporogonic tactorto). and tho oubooquont loto uood in thio work woro not of tho oowo charactoriotico. Whilo tho original work with 13? waa with Pi 3679 and tho prooont otudy'wao with .Q;. bgtgligun. nany othor trialo conductod in this 1.1.. laboratory with PA 3679 (Coatilow. 1960) rooultod in failuro to obtain a high poroontago or aporulation. ‘Ihio no truo ovon whon tho nodiun wao aupplolontod with thia-— ino. ' Anothor aapoct of m oodiun to bo conoidorod ia tho poaonco or glucooo in thin aodiu at a conoontration of o.) pomnt. It hoo boon notod by .‘....-.1 inuotigotm (Blair. 1950) that thio carbohydrnto ia inhibitory to . oporulation or 9;. 29% ot o oonoontration or 0.5 pomnt. A 0.} poroont lonl nay vory all. thou. dolay and partially inhibit conploto oporulation of thio organ- ion. Tho work or Wok and Footor (.1952) condoning ' tho odour. or on. inhibition or oporulation by on. prolonoo or glucooo would aloo indicoto thia. o nodiu of 0. porcont trypticooo would not contain gluoooo oinoo trypticooo. no oooayod by tho Baltinoro Biologiool Loborotory. ia troo of tormentablo carbohydratoa. Tho ocototo oontont or tryptioaoo io quito high. oinco acotio acid ia uood for purpoaoo of pa adjuotnont in ordor to ovoid a high chlorido contont or on. product (Vara.l960). It in known tron concurront work in thio loborotory. (Simono. 1960) tbot 9;. W 629i; containo o my aotivo acotokinoao. tbuo tho ocotato contont of trypticaoo no'y oonohow bo involud in aporozonooio ao a oouroo or onorgy. Futuro intootigationo will otudy thio oopoot of tho oporogonic naturo of trypticooo. Tho rolo of thiamino in oporulation io not woll ootobliohod. Howovor. tho rooulto would indicato thot it io not oooontiol for growth and oporulation of thio organ- ioo. but that it dooo onhohco both. Again tho oonpooition or trypticaoo canoa into play. Thianino nay bo prooont in trypticooo in ninuto. catalytic quontitioo but not in mot onough concontrationo to ho conducivo to comploto oporulation. Thoroi‘oro. tho conploto aboonco or thia factor may rooult in tho conploto abaonco of oporulntion. Thin can only bo ootabliohod by uoo of a oynthotio oporul- ation nodiun. oono thingo noy bo doduood tron tho rooulto of thio work about tho conpoundo of trypticaoo which do load to oporulation of 9;. m. In onprobability. thooo tactoro aro dialyoablo. Trypticooo io conpoood of amino ocido and mil poptidoo and it would not ooon unrooaonablo thot moat of tho conpononto or trypticoao aro dialyoablo. iloo. tho ooh of tryptioaoo alono io not oporogonic in a roploconont nonotrum olthougb it ouot bo ronombomd that tho pH of thio oolution no 5.0. 01‘ couroo. tho poooihility oxioto that oithor on. rooted-- in aah or in on. dialyaod portion of tryptioaoo in combination with othor foctoro or with oach othor night to oporogonic in tho roplaconont nodim. Thio poooihility no not ominod oxporimntally. ill oi’ tho variouo toctoro tootod. awino acido. ninoralo. voriouo inorgonic iona. digooto of oaooin othor to. than trypticooe. end vitamino proved to be non-oporo- sonic when uood oingly or in combination in replacement eolutiono. m thia baoio. tho oporogonic nature of theoo compoundo end factoro cannot be precluded. It can only be etotod that they have no oporogonic value by thenoolvoa. or in combinetiono uood in thio work. no replecement nonotmo. Ihile it ia obviouo that thia organion cennot under- go tho phenomenon of ondotmphie oporulotion. tho orgw, ion may very well becone omitted to oporulation. but not to tho extent that it can complete oporogonooio in the absence of exogenouo nutriento. Ihilo it io poooible thot exieting protoino and nucleic acido ere utilised in opore eynthooio by thia orgenion. eono oxogonouo nutrient or factor in alao required. Perhepo the moot important clue ao to the rocuirononta for oporulntion of thia organioo io given by tho work which demenatratod that a combinetion of tho amino acide of trypticeoe and thinnine io at leaot partially opera- genie in nature. Although oporulation did not opproach tho level of oporulation obtained in o normal oporogenic environment. a definite inoroaoe in oporulation could be oboorvod. In thie oolution of andno acido. the ionic otrength would be conaidorably higher than in tho trypticaao nodium. oinco all amino acido were added on their eolto. end thie condition may here a limiting effect on the b7. eporogenic proceoo. Leifcon (1931) and Wynne (192.3) pointed out that oporulation decreased aa the eelt Concentration increeoed in n culture medium. It may he that the oynthetic amino acid oolution in not continue enough in iteelf to warrant complete eporulation. If the latter io the cone. the next step in the work is to odd to thie mine ecid nonotrumn the rerioue metellic ione. inorganic iono. vitamins. and other factors of known importance to the eporulation of this end similar organismo. The high level of acetate in trypticeso. and the ooetokineoe activity of thie organism indicates thot a combinetion of the anino acide end acetote ohould to inventigeted for oporogenic propertioo. The effect of penicillin on eporulotion in difficult to explain in light of the hypothetio thot thie antibiotic prevents the oynthoeio of cell well material (Prett. 19533 Lederberg. 19573 Pork and Strominger. 1957). It would be expected that if the cell were omitted to eporulation. that thie drug, would hove no effect on the (proceoe. end oporulntion would proceed to completion. The possibility doea exist. however. that the eyntheoio of the epore cont ley proceed .by a pathway similar to that of the cell well. and then the notion of penicillin might be explained. If thie antibiotic note by the inhibition of ERA oyntheeis. ao reported in earlier inventigationo (Mitchell and Hoyle. 1951; me. 19.9). it would offect eporuletion in the some k8. leaner ao uncomyoin. which ia known to inhibit mu oyntheeio (lnnioo. 1959). However. the reoulto do not eloerly ahow thet penicillin doeo act on a oporuleting culture in the come manner on nnconycin. Before on explanation con be lode ae to how penicillin interferoo with the oporulotion procooo. the node of action of thie antibiotic must be known with oerteinty. when unconycin in added to a culture of Q. We the inhibition of eporulotion did not com inoodietely. i lag of ebout lo houro wao oboerred before the oporulation oeeoed. If RM ie eooontial to protein eynthooio. oo io hypothooioed in on. sou-pm. theory. then on. inhibition of REA oynthooio rooulta in the inhibition of protein oynthooio. and thie antibiotic ultimately note in the oano manner oo chloranphenicol. The leg which ie obeervod with the am inhibitor would be explained by the foot thot oono cello. ot the tine when the entibiotic io edded. already hove the RNA which io neceoeary to oerve ao ten- plateo for the oynthooio of woteina which are oaeentiol for oporulation. Theoo cello would then. continue to eyntheoiee opore protoine. and oporogenoeio would go to completion. Proonably thie would take plece over the lag period. eccounting for the incroooe in the anchor of oporeo. Thooe cello which had not yet formed the RNA tenplotoo required for epore protein ot the time of the oddition of the vancomycin would be prevented frou oynthooioing #9.- theee particular Rhi noleculea. and the eporulation would be etopped at that point. It ie felt by the author that the use or antibiotice and other antimicrobial inhibitore may offer a practical tool in the elucidation of’the eporogenic proceee. If the node or action of the inhibitor ie known.with certain- ty. it would be poeeible to determine which reactions are being_inhibited and thue which pathwayie) are essential to eporogenooie. Some work hat been carried out by the author with the trivalent areenic ion and ita effect on eporulation. it thie tine the reaulta are not clear. but it ia felt that thia inhibitor of cellular metabolism may preeent oluoe ea to the uechanicma of epcrulation. Other compounde of thie nature ahould be inveetigated ae to their effect on eporulation. i great deal of work romaine to be accompliehcd in the area of eporulation of the anaerobic bacteria. The development of a good baeal medium which can be rendered free of metallic contamination ie required for the .deteruination of the mineral requirements or eporulation. lerhape the moat valuable technique already develOpod for the etudy of eporcgeneeia ia the technique of replacement becauee it offere a method of practically eoparating the eporulaticn proceee from growth. Future work will utilise thie technique in order to further elucidate the nutritional requiremente for eporuletion of‘gl. bogglinum. SWARY Studice on the nature of eporulation of glgctzigium hotulimm. iTCc 62A. revealed thia organise will not under- go endotroPhic eporulation. Mines-ale, amino acids. inorganic ione. and vitamins need by thonaelvee and in ., various combination would not pranote aporulation in replacement construe with one exception. i aynthetic median of the amino acida reported to be in tryptieaae. fortified with thiamine. would eupport aporulation to a aignii‘icant extent. The nature or the eporogenic quality of thia solution no not inventigated further. It wee alao noted that trypticaae with added thiaeine ie an excellent cporogenic medium for thie organien, however trypticaee which hae been dialyled doea not exhibit the eporogcnio property. It Ina concluded that aporea of Q. W cannot be turned aolely tron the degradation of cellular material and the recombination of the producta of thie degradation into compounda neceaaary for eporulation. Three antibiotica. penicillin. chloranphenicol. and vancomycin. were etudied to aeoertain their effect on the eporulation moon of thie organiee. Penicillin inhibited aporulation it added before the oneet oi' aporulation or during the early etagea of aporulation. Added at a late etage of opera development. thie antibiotic had no apparent effect on eporulation. Vanccnycin affected aper- ulation in much the lane manner. however, a lag of about ' 51. 10 hours was observed after the addition of this anti- biotic before eporulation was arrested. Chloramphenicol inhibited ferther eporuletion regardleee of the stage of growth when the drug was added to the culture. From these results and the hypothesized mode of action or these anti— biotics, it was concluded that the synthesis of protein material is an essential mechanism in the final aporogenic process of this organism. fl 5;. BIBLIOGRAPHY MU. MeZe. Ordal. Jo and T011“. ‘e 1956 Sgorulation roquircmenta of §:§é%;33| e var ermo cid- urane in.complex. a. . ctoriol.. . -- . Black S.H.. Haohinoto. T. and Gerhardt. P. 1960a cvolo ment or fine otructure. thereootability. and dipico nato during oporogeneoio in a bacillue. Can. J. Microbiol... 9 (1). 203-212. Bth 8.H.. ”3011133030. To and 63th. ’e 1960b énlcium reveroal of the heat aunceptibility and dipicolinic deficiency of oporeo termed 'endotro- ggically' in.water. Can. J. Microbiol.. 9,12). 213-» 0 Blair. 8.5. 1950 Observationa on bacterial opore torm- etion in oynthetic media. M.L. Iboaio. university or fine. Iblto in m RCPtle 8101. “do. g. ’61. Brewer. 0.3.. McCullough. w.a.. Milla. n.c. Roeoaler. w.e.. Herbot. E.J. and Howe. i.r. 1945 Studies on the nutritional requirementa of fiagillgg anthragig. Archiv. of Biochen.. L9. 65-75. Buchner . 1890‘ Uber die Ureache der Sporenbildung m nuanmuuuu.2mu.mnuun “mama" Orin. 3.3.. L6. (c.1‘. xnayei. 191.8). Byrne. a.F. Burton. T.H.. and Koch. 3.8. 1960 Relation of'di colinio acid content of anaerobic bacterial endoeporeo to their heat reoiotance. J. Bacteriol.. Q. 139’qu Chaney. J.. Fioher. w.r'.. and Hogarty. c.r. i951 B'anganeoe ao an eeoontial element for oporuletion in the germ. We Jo BIcuflOJue. 23. “5’1‘0’80 Church. 8.0.. and Halvorocn. 3.0. 1959 Dependence of the heat rooiotance ot’becterial endoeporeo on their dipicolinie acid content. Nature. lg}. 12h. Cootilow. R.N. 1960 Pereonal communication. Collier. 3.6. 1956 in ap ch to eynchronoue growth for epore production in g oetridium eeum. In ”Sporee'. Edited by h. 0. Halvoraon. rican Inetitute of 81010510“ “1.110... Waflhlngton. DQCO. 1957. PP 10.“. It”? Jo“. Collier. 8.2. and Kriohnanurty. 0.0. 1957 The correla- tion of DPA ayntheoio with oporulation of gloatridiun £056Me BlCtOdOlo ”We. £222. 320 Curran. H.R. and Evano. F.R. l95t The influence of iron or mangenece upon the formation of eporoo by meno- ghilic nerobee in fluid organic media. J. Bacteriol.. _13 #89-t97. Eety. J. and Meyer. K.!. 1922 The heat rooiotanco of botulinun and allied anaerobeo. J. Infect. Dio.. . 005. Footefafg.w. and Heili 1. Pi 19h9 giochemical tactoro uenc ng opo t on n a otra n o gagillgg gagggg. J o B‘C “T101" . 11. 639“6wo Footer. 1.3: and Perry. J.g. 135$ Ehe nonoinzolrefent of o ur ng oporulat on 0 21° iii: a co a n .tim “tor. Jo 3.63.910 o. W Cele. E.F. 19t8 The nitrogen netabolioe of’gree-pooitive bacteria. Bull. Johno Hopkine Hoop.. 52. 119-175. Gale. E.F. 19h9 The action of penicillin on the aeeinilao tion and utiliaation ot’anino acido by gran tire bacteria. Sympoaia of the oociety foruExpe mental Biology. 111. Selective Toxicity and Antibiotico. Academic Preoo Inc.. Publiohero. Rewrlork. Now'York. 6.1., EJ. ”1d FOR... Jo'o 1953 Th. aooililation 0: amino acido by bacteria. 15. Nucleic acid and protein oyntheoio in W m. Biochoa. Jo. a. 1.33-5.92. Gale, 8.?. and Polkoo. J.P. 1953 The aooiwilation of amino acids by bacteria. 20. The incorporation of labelled amino acido by diaru ted otaphylococcal ”ll-o Biochen. Jo. :2. 661 75o 5.1.. EJ‘. and Paine. 'r.r. 1950 The effect or inhibitoro and antibiotico on glutnmic acid accumulation and on tein oyntheoia in.fitgpgxlggggggg,gggguo. Biochen. e. AZ. mi. ' Halvoreon. H.O. 1957 Rapid and ainultaneoue oporulation. Jo Appl. Bacteriol.. 29. 305-3“. Hardwick. W.i. and Footer. J.V. 1952 On the nature of eporogoneoio in acne aerobic bacteria. J. Gen. Phye.. 22. 907-927. (I (*9. fiardwick w.i. and Foster J.h. 1953 Eneyeatic changes during a re eneeio in some aerobic bacteria. J. Lecterio .. ' 355-360. . - Ionics. 3.3. 1959 Studico on poaoiblo nodes or action or a new antibiotic (vanoomycin) on etraina of §§cthvloco ue‘ggzgug. h.3. Thoaia. Univcroity of JIBBMZI F.1d" Lund. ‘oJo lad. AndeaOfl LeEo 13:33 . Cc crimetric acoay for dipicoloinic acid in bacteria oporeo.w Science. 131. 26-27. up? -. I. and Williams. MI. 191.1 Spore formation more the anaerobic bacteria. 1. The formation of spores by file tridium e orccenc in nutrient agar. J. £301.53? 0 o. g I " 533 e , inayoi. G. 19b5 A etudy of acne environmental factore which control endoopore formation b a otrain of 35'!!th ngoédgig J. EECMI'iOlo. a “73"4930 hnayei. G. 19k3 The endooporc of bacteria. Bacteriol. ROYo. l2. 19'77o Kranpito 1.0. and workman. 0.1. 19h? 0n the mode of action of penicillin. Arch. Biochem. g. 57. Lederberg. J. 1957 mechanism of action of penicillin. Jo EficudOIo. 21. Mo . lei fsggi E. 1931 ”€3.31“ epcrea. Jo Bacteriol.. a. Luna. A.J. 1956 Diocuaoion in “Sporeo'. Edited by H.0. Kalvoreon. American Inatitute of Biological Sciencoo. waehington. 0.0. 1957. pp 26-30. Mitchell. P. l9t9 Nature. 15'. 259. (cited by: inti- biotico: a ouroey or thEI§ propertiea and uses. London: Pharmaceutical Prone. 7 Bloomsbury Square. weCo 1o Mitchell P. and Eoylo. J. 191] Relationahi a between on growth ourrace propertiea and one oic acid prodnction in normal and penicillin-treated %é$rcccccgglgzg;gggg. J. Gen. Microbiol.. 5, h21~ s. Kohrhc, r. _l926 Ein,naueo Ycrfehron our.Eineporcnkultur anaerober Bakterien. octet homerkungen uber one VorsporungeOPtimun dor anaerobior. Zent. Baht. Parasitonk. Abt. I. Orig. 23:3,. 533-5h7. (c.1'. Inaysi, l9bo). Crdal, Z.J. 1956 Tho affect of nutritional and environ- mental conditions of aporulation. In “Spores". Edited by H. 0. Halvoroon. American Institute of MOlOLfiCIl 80‘0110... Washington. D.C. 1957. pp 18-25. Park. J.T. and Stromingor J.L. 1957 lfiode of action of penicillin. Biochemicalbaaia for the mechanism of action of nicillin and for ite eclectivo toxicity. Science. 99-101. Perry, J.J. and Footer. J.W. 1955 Studiee on the bio» ayntheoie of di ioolinic acid in aporae of §g§f%;gg coz‘o‘g VII mzco QOQe Jo Bacterial" £2. 33 " a Powell; J.F. 1953 Isolation of dipicolinic acid (p - no 2.21312: ~2, 6-dicarboaylic acid) from a rea of the i . Biochcn. J.. 25, 210- 11. Powell J.F. 1956 Informal discussion in “Sports”. Baitod by H. O. Halvoraon. Anarieaa Inetitute of Biological Sciencoa. Washington, 0.6. 1957 pp 9. Powell J.F and Hunter J.R. 1953 8 rulation in .1 ’ 3 £3 atilled tutor. e “no Phne. . 01-6069 Pratt R. 1953 Sympoaiun on the node of action of anti- biotics. IV. Enchaniama of penicillin action in V113”. Bactcriol. ROVo. HO 1.1-1.5. Roeoelor W.O. and Bro-or. 0.3. l9k6 flutritional etudiee uitfi C oatr dium botulinum.-toxin types A and B. J. BQCter 0 e. . 57I“5720 Ronig.‘V.R. and Uyae. O. 1957 Bone affecte of ultra- violet radiation on oporulatir cultures or‘gggillgg 32533;. J. Bacterial..‘25, 38 -391. Schroiber, 0. 1896 Uber dio physiologiachon Endingungon dor ondoganon aporon bildung die Bacillua anthracia. subtilie, and tumeeccna. Zontr. Bakt. Parasitank. Abba 1e. £2. 353-371” (c.f. KMYS1. 191:3). 31mmons, R.J. 1960 Poroonal communication. SmithfiiG.N. 1953 ‘8 poaium on the mode of action of anti- otico. I. 1 o asibla modes of action of chloro- mycotin. Bactorio . Rev..‘;2. 19-29. 56. ~ Somnor E.W. 1930 Boat rooiotance of tho aporoe of C Cfitr‘d in W. J. meta Di.e. E. $5- ‘ v.r.. H.D. 1960 Poroonal communication. weinbcrg. 5.9. 1955 Tho etfbct of En++ and antimicrobial drugs on aporulation of F cillu anytilig in nutrient bmtlia Jo E‘cuflOIO. " vi)" o - wcinborg. 5.9. 1956 Discussion in "Sporee‘. Edited by n. G. Halvoroon. Amorican Inotitute of Biological Sciences. walkington. 0.6. 1957. PP 6.8. Williams. 0.3. and Harper. 0.7.. Jr. 1951 Studies on heat resistance. IV. Sporulation of figci§lno serous in synthetic modia and tho boat roe otanco . ¢ £19 8130". producod. Jo bactarioln a. 551-556. ‘ “.813333. 0.1... Jre. “y. “aloe Jr.. Ind H‘hn. LE. 1952 its action of chloram ho col on.aicroorsanions. Lactoriol. Proc.. 122 . 92. “WM. 3.3. 191.3 Ph iological atudiu on spore ion-tra- f‘ , tion in gloatzid gg_§gtglinya. J. Infect. Dis.. «£22.. 2&3" e Wynno E s Schmieding 3.8. and Daye"0 T Jr. 1955 cimélifiod mothoa for counting giogtriéigg spores. FOG-d italic. 29.. 9"“. Young. E. 1959 A rolationehif botwocn tho froo anino acid pool. dipicolinic ac d. and calcium from root- ing spores of {ac line . r.th . Can. J. iicrobiolu 20%. 5 Zoha. 8.x.3. and Sadoff. H.L. 1953 Production of oporco ggéazputrofactivo anaorobc. J. Eacterio1.. fig. 0%. 57. APPENDIX 1. TABLE. I”Ao Tho Pfigllsfiflont of the vogotativo colic of o trioinn bctq;intx in otoriio. ii tilled water. Inc*bntion at a H ' a "‘ . rel 3 ‘: (“o under ultra on. and Growth tixo in Time in.ronancc- Total count in Sports in T“? niliqv w ' a “7‘1 "“ r , 25. 213*}? a 72 its. is hrs. 9.0xio; 1.txlC 72 hPSo 5o9X10 1.9x10 TSP CCYECCL crayon tire Total count Spores 1.2 17.13 a 7a 2311'..- g . < 1‘36 210‘ 3:1'3 e G o #1.“) ' < bib 3 V‘r‘ '} at hrs. ,.tli.‘ <1gb .n iro. 2.U21£8 <1og €33 lira. lofixlu,‘ ‘ he LIKES, ?L £33. .5x100 1.2xlfl so hrs. l.§x1£3 5.txlU7 no flay". "o- C l”: m .453. OHM 1" EFQ‘HXlV 2. .rw'r- v, . Tu t1 "rrfiJ *u'qug‘fl The n ;“ wortont of the vcdc Him cells of Clo-Mi r13. rm otulin on in 0.067 M phoonhato buffer. pH 7. U with o’ .I fill. 3...; ”i H... 1.11:1 01110., 1; 'CO...:.-'3. cue 4 C1... ‘fl .ed a” ?~:;e .9 -i In: .31 M ,. .. Hr. Grant?) " “.5? in .irno in re......::cr-- .-o‘:..l count in. res in TS? :._:;:T.‘*:'z mganng";‘.m; m. olgcoment 0 hrs. 3.1.1.103 10 . "CS-o 1.1.22.3.” 22 m. 8:9110’ 53 1:2. 32 hrs. 7.5leg 1.6 hrs. < 1C 70 33's o < 106 0 hrs. 1.3::1033 < log 10 3".."30 9 063.113.! < 1‘36 72 '1... 3. 2!; hrs. < 10: < 10/ I... bra. < 100 < 10° 0 hrs. b.3310 1.01.10: :3 3'12"".3o 20 11.3.. 5.6.-L106 13:32.10 50 hrs. 2.0110 1.03106 TSP 60::51‘ .a...‘ LR; Grcrr'gfi t-f;+21 Tot-31.1 Grit; é’.’-.‘-{‘-?*i‘3 1:} 1:1‘3e 60 1..-“C10 Vi; ‘1‘36 23. . “a. . 13:39:; <10? 5:" mo. 3.73m <10? oo-nrs. 3.1x1C5 <1x5 72 113959 2eCXlLa < [cl-""8 3:33 hrs. “'13- < 13? 36 hrs. 1.1x13“ 1.0. ‘L9 12...) hips. i. as; 1.15.? .2;-..;°~§1t) 13.13 hrs. 6.3319,. 6.151.180 lflg 1"3. 6.23519, 1.325.537 J. TAB“ III-A. Tho ”phenom. of the '0 000110 00110 of W n 0001110 0.06 :2 pm 00 but!» . . m at. 37% undo:- tho 0&1 OWN. mun-u 11-010"qu- ‘lbmomuuapmntn WWW o m. 6.72107 <1 20 m. 9.1a <1 9 m. 74:1 <10‘5 2:. m. 7 m. < < 06 139 m. < 06 < 0 hrs. 0.33108 2.03105 19 hrs. 7.2210 1.03106 U. hrn. ‘7 .17.. 8.211 30081 119 m. 2.031 2.031 0 m. 1.5.00; .6110 10 an. 9.mo .mo.’ m. .7110; 3.5210 73 m. m. .gfio game 90 m. . 07 .5210 m common W W m 10 m. 0.1::107 <10‘ m. m. 9.1.103 <106 . 1.2110 3.03106 In. 1.0:10‘ 7.6:}.05 9 m. 3.0007 8.9110? 115 m. . 07 1. 01, 9 m. a. o7 2. ‘ L. TLBLE I‘V-Ag m replacement; or the "genitive «11- ot WEE}: canny-m 1n n lolution o biotin. “5510““. am “‘in gm (.1 Incubanod at 37°C under nitrogen. Grazanh time in Time in uphce~ Total count. in Spores 1n '1‘??? ”mafia _ mags mnetruwa nglacgmeng mvlacefigfi o m. 2.6mm; <1£2 '06 hrs. 2.0110 (106 '53 hrs. 118 ”.0 2.0110; ‘106 2610 hrs. 103110 ‘15 o m. Luzog < 10-? 26.5 m. 1.01107 < 1 1‘) 6705 I’LX‘CQ 980’ hr.- 803110 <16! 21.5.5 m. 6.03107 <10” 0 hrs. 8.03107 3.6::102 m. zaxm" Leno 9m: 1m. bro. .6x107 3.1mm; 7 13". 5.2x10 3011.109 21 m. 34:10 3.321;: is? ccrzmm. 12‘: tim 321:1; £2“ 53 acres hrs. 9.82.2107 <15? ‘v 31373. 602.3103 ‘10; 705 RPS. 1.6310 <10“, 91. hrs. 8.3:;107 bclxluir? 113 hrflo 6.3110 5.6x1G" hrs. 5.9x10 2.22267 163 hm. b.9210 rescue 312 b.1331 5018.10 .1210 5. TABLE 740 no ”placenta: or ch- '- unit-u an. or . ermamzhm “figmgfi. ° 3 a pan 0 ‘o n . man an u’wflgg 7.1. demand it we: an» 5.3: manhunt: ulnar-plun- woman-1n [min WWW. 0 m. - .8310: ‘ 106 a m. $063108 < 1 It In 72 5'3" J'mo‘ 3106 ' no m: 933337 < 0 ha. 1.2.103 2.5::102 3 :3... hum 1:31:85 2"“ 2m 22223 $222? 120 m2 iono‘ 1.61106 C I 2 fig; no ha. 3% Sgt-:3: gigg gong; no m. 5.1210 .ou 'm comm mm mm In 2* ".0 5.2:“): < 1.: in. {72103 < 97: h": 1'33 ! 9x1 120 hrs. 7:63107 . 1 3' Z'fig} 2‘ ' C O 7 . 222:: 2:”? 1:54:33 * :2 2a. . 7 5.73107 6. TABLE ‘31-}... Tie replacement of tho vegetauvo 2.11: or (12223222222 2- 2: 17.022222122222223 in a solution of DL- can-alanine, L-J.........., 1..-703733123 2. lycino glucoaa. biotin p-aalnobanzoic 1:16. thia? n nico tfn1c acid in 0.065 a phonphato buffbr. p22 7.1. incubaud .2. 372a under mmgen. may? :12 2.23 in an. in roplaco- Total count. in 3312:. in .. e (105 200110 2.62:103 <105 3.2x10 :06 21 hrs. 2.112103 1.3310 :10 0 2:“. 1.62103 23.2.2210? 21. 3222:. 1.1.1108 2. 022106 125 brie ’08 l“. 1.1.11037wx105 76 m. 1.31.1228 5.222106 0 hrs. 9.6210; 3.29:107 21. hrs. 8.1::10 2.2.x107 69 hrs. 52 hrs. 8.2.140; 1.3x10 120 hrs. 7.9x10 2.72210 2:39 comm. mmmmg m count; 82222:: 22238 :1 hrs. 3.1210: < 10? 31 1.22222. 3.6mm. _< 1223 LS hm}. 2.2flué 8.53:10 6.2;: hrs. 1.21.12.“ 5.2222107 93 hrs. 9.22.1.6; 5.22.3.0; 1.21 2.22. 9.22.20? 3.722157 1:53 31:80 3.5110 .9110 7. TABLE VII-A. 1h. 1231:2th12. of tho ”return «11. or 61233231212112... Malaya in n Ioluuon 0? admin in a lo ut. cm 0 «1.1—... acetate. and in sterile. 1132111“ Inter. Incubated :1 37%: in com-up Till. ”muons 4:011an 0.1 pomnu ”dim thiaglyco :0. Growth time Rophcmnt am: 111 ram counts Span. in in I. want mutt-um replacement in "plan- replan- Ezg 1.1.2.131 I w W... M . Ez‘b ’ O 111‘. 1502110: <102 sterile. 30’ 3111. 1.41.10 23 <106 1905 £22.25. distilled 6.5 hr“ 051103 (106 “a? 1205 m. 002103 < 06 2205 hrs. .8310 < 0 3 6 a nu. bones. <106 .5 hrs. 3.38108 < 10 1905 hrs. Gilchn 05 m. ’OmC‘8 < 6 «at». 12.5 m. 3.22108 <10!) (ZOOPPB, 22.5 bro. 1.2110 ‘10 o m. 1.3310 <10g’ 3.3 MI. lqulOa (106 60.; ’lr.l g0m08 < 06 1905 m8. think! 1205 m. 031108