PRODUCTION AND ISOLATION OP THERMOVTRIDIN , AN ANTIBIOTIC PRODUCED BY THERMOA CTIN OIVEYCES Y IR ID IS N By David M. Schuurmans AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in p a r t i a l fu lfillm ent of the requirements for the degree of DOCTOR OP PHILOSOPHY Department of Bacteriology and Public Health 1954 Approved ProQuest Number: 10008422 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008422 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 481 0 6 -1 3 4 6 AN ABSTRACT A thermophilic actinomycete was iso lated from a com­ posted manure pile* Since no previous description of th is organism was found, i t was considered a new species of the genus Thermoactinomyces (Tsiklinsky). The name Thermoactino­ myces v i r i d i s was suggested for the organism, and the name thermoviridin for the a n tib io tic produced by i t . Thermoviridin was produced I n i t i a l l y In stationary b o t t l e c u ltu re s. Subsequent production was carried out on a Gump rotary shaker for the most p a rt, and also in a 30 l i t e r laboratory fermenter. The fermentation medium consisted of Bacto tryptone 4%, Bacto beef extract 0.5%, and had a pH of 6.9 - 7 .1 . addition of a number of n u trie n ts The to th is medium did not i n ­ crease a n tib io tic potency above the 32 units per ml obtained in the basal medium. Maximum a n tib io tic production was reached in 27 hours at 45°C. Recovery of thermoviridin was accomplished by acid p r e c ip ita tio n a t pH 3. The p re c ip ita te was washed with water and then extracted with a quantity of 80% acetone equal to one tenth the original volume of the a n t i b i o ti c beer. The acetone extract was evaporated, and the remaining water residue was dried from the frozen s t a t e . The solid material obtained by th is prodecure had 64 units per mg a c t iv i t y against M. pyo­ genes v a r . aureus 209P. D a v id M. Schu u rm an s Some p u rific a tio n of the active solid m aterial was accomplished by fra c tio n a l p re c ip ita tio n from methanol using ethyl ether as the p r e c ip ita tin g agent. The countercurrent d i s tr ib u tio n method of Craig was also an effectiv e method of p u r if ic a tio n . A preparation having 270 units per mg a c t iv i t y was obtained by th is method, using a butanol-water system buffered at pH 6. Thermoviridin was found to be stable at pH 2 fo r 21 hours a t 37°C, but at pH 10 for the same time period approxi­ mately 75% of the a n tib io tic a c tiv ity was l o s t . The s t a b i l i t y of thermoviridin to temperatures higher than 37°C was not i n ­ vestigated . Chemical t e s t s and s o lu b ility data have indicated th a t thermoviridin is an organic acid which contains no su lfu r or phenyl groups. The activ e material appeared to be non-protein and non-carbohydrate in natu re. Thermoviridin was dialyzable and could be p rec ip itate d with saturated ammonium s u l f a t e . The a n t i b i o ti c preparations tested showed maximum ab­ sorption in the range of 268~272mu. Thermoviridin was primarily active against the gram positive b a c te ria t e s te d . Preliminary to x ic ity t e s t s in mice, consisting of a single Intrap erito neal injection of 32 mg of material (800 u n i ts ) , did not cause death in any case. An autopsy of the mice showed no abnormalities in the gross appearance of the organs. D a v id M. S ch u u rm an s PRODUCTION AND ISOLATION OP THERMOVIRIDIN, AN ANTIBIOTIC PRODUCED BY THERMOACTINOMYCES V IR ID IS N. SP. By David M. Schuurmans A Thesis Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in p a r t i a l fu lfillm en t of the requirements fo r the degree of DOCTOR OP PHILOSOPHY Department of Bacteriology and Public Health 1954 ACKNOWLEDGMENTS The author wishes to express his gratitude to Dr. C. L. San Clemente, his major professor, and to Dr* B. H. Olson of the Michigan Department of Health for t h e i r help and encouragement during the course of th is work. Thanks are also extended to Dr. G -* D. Cummings, Direc­ to r of Laboratories, and to Dr. H. D. Anderson fo r allowing th is work to be carrie d on a t the Michigan Department of Health. The author is also g ra te fu l to Dr. H. J . Stafseth for his I n te r e s t In th is work. TABLE OP CONTENTS Page INTRODUCTION............................................................................................................... 1 HISTORICAL .................................................................................................................... 3 MATERIALS AND METHODS............................................ . ................................. S c r e e n in g ........................................................ ...................................... 12 • 14 ........................ ............................................................................ 15 U ltravio let Ir ra d ia tio n Assay 12 Fermentation...................................................... Recovery. . . P u rific a tio n . ....................................... . . . 17 . . . . . . . . . ................................. . . . . . . . . 20 21 Toxicity............................................................................ 2 RESULTS.............................................................................................................................. Cultural C h a ra c te ris tic s.....................................• • • • • • 25 25 U ltraviolet Ir ra d ia tio n 32 Fermentation......................................................................... 33 Recovery......................................................................... P u rific a tio n . • • • • • . . . . . . . . . . . . 39 • • • • • • 42 Biological and Chemical Properties. Toxicity............................................................................ 5 DISCUSSION................................................................................................................... 53 SUMMARY.............................................................................................................................. 56 REFERENCES................................................................................................................... 58 LIST OP TABLES T a b le Page I Agar media used in screening • • • • • • • 13 II Cultural c h a ra c te ristic s and biochemical reactions of Ther moa ctinomy ces v ir id is on various organic media. . . . . . • » 28 III The e ffe c t of u l tr a v io l e t Irra d ia tio n upon the viable spore count of T. v i r i d i s ................................................................................ 33 IV Tryptone-beef extract fermentation medium. V Nutrients te s te d in the tryptone-beef ex­ t r a c t medium for stimulation of a n t i ­ b io tic p r o d u c t i o n ..............................................................34 VI Metallic ions added to the tryptone-beef extract medium without effect ....................... 36 P u rifica tio n of thermoviridin by fra c tio n a l p re c ip ita tio n . . . . . . . . 40 P u rifica tio n of thermoviridin by countercurrent d i s t r i b u t i o n ................................ 43 VII VIII 34 IX S ta b il i ty of thermoviridin to various pH*s and temperatures...................................................................44 X The microbiological spectrum of thermoviridin * . . . . • 50 LIST OF FIGURES F ig u r e Page 1 Procedure for the recovery of thermoviridin 22 2 Thermoact inomy ce sv i r i d i s 26 ® • • • • • . • . . a f t e r 3 days incubation a t 50°C. 30 4 T. v i r i d i s a f t e r , 4 days incubation at 50°G. 30 5 .T• v irid is a f t e r 5 days incubation at 50°G. 31 6 T. v irid is a f t e r 6 days incubation at 50°G. 31 7 Thermoviridin production and pH changes a t 37° and 45°C.............................." .........................................37 8 U ltravio let absorption spectrum of thermo­ v ir id in ............................................................................................48 INTRODUCTION In the course of the continuing search for new and su­ perior a n t i b i o t i c s , the goal has been to iso la te cultures which produce a n tib io tic s d iffe r in g from those already known# For t h is reason some investig ato rs have attempted to obtain cultures from unusual sources, thereby increasing the chance of is o la tin g organisms which are unique. A second method of obtaining antibiotic-producing organisms which are unique is to in v estig ate some group of organisms which has not as yet been tapped as a source of a n tib io tic producers. The thermophilic actinomycetes comprise one such group. A search of the l i t e r a t u r e indicated th at these organisms have not been widely investigated as a source of a n tib io tic producers. Here then, is an area where any a n tib io tic - producing organisms isolated might well be d iffe re n t from those previously is o la te d . In addition to the apparent neglect of these organisms with respect to a n t i b i o ti c production, i t seemed advantageous to investigate them fo r two other reasons: 1. I t was con­ sidered possible th a t an a n tib io tic produced during a thermo­ p h ilic fermentation might be heat stab le . 2. Since the resp ira to ry rate of thermophilic organisms i s high, the time required fo r maximum a n tib io tic production should be shorter than In the case of mesophilic fermentations. 2 For these reasons a ^program fo r the screening of thermo­ p h ilic actinomycetes was established. After the iso la tio n and screening of 174 thermophilic cultures, chosen f o r fu rth e r in vestigation . one organism was This organism was found to be a new species in the genus Thermoactinomyces (Tsiklinsky)• The name Thermoactinomyces v i r i d i s was given to the organism, and the a n ti b i o ti c produced by i t was named thermoviridin. The work done on the selected organism and the a n tib io tic produced by i t is to be presented here. This includes the iso la tio n and c l a s s i f i c a ti o n of the organism, followed by studies on production media and conditions, recovery, p u r i f i ­ cation, chemical p ro p erties, of the antibio tico to x ic ity and microbial spectrum HISTORICAL The f i r s t reported investigation of the thermophilic actinomycetes was published in 1888 by Globig (15) . not the f i r s t He was in vestig ator to observe these organisms, however for he mentioned the previous observations of Geheimrath Koch, who influenced Globig to turn h is effo rts to the study of thes organisms• Globig found potato slices to be the most sa tisfac to ry medium for the is o la tio n of these organisms. After develop­ ing the technique for Iso latio n and cultiv atio n of the thermo­ p h ilic organisms, he began searching for them in a l l materials* types of In the course of his investigation he examined the feces of man, dog, guinea pig, rab b it, horse, pigeon and mouse He also examined the canal water and tap water, as well as bored samples obtained from the earth down to a depth of two meters. He found thermophilic organisms at a l l levels of depth te s te d . Soil cores from several foreign countries were also cultured. Globig1s curiosity concerning the possible h a b ita ts of these organisms also lead him to examine laboratory benches, flo o rs, dust and the h o t - a i r oven of the I n s t i t u t e of Hygeine In Berlin. The I n te r e s t aroused by Globig*s report concerning these novel, heat-loving organisms resu lte d in several subsequent publications dealing largely with the description of organisms 4 iso la te d and the sources from which they were obtained* ever, How­ there was no clear cut d is tin c tio n between the b ac teria and the actinomycetes* These early reports often refe r to the thermophilic actinomycetes as “b a c te r ia ” having thread-lik e forms • Rabinowitsch (27) examined the excrements of the horse, cow, dog, guinea pig, mouse and f i s h , actinomycetes in a l l cases. Manure suspended in water was incubated 18-24 hours at 62°C. to inoculate agar p la te s . and found thermophilic The suspension was then used Colonies appeared in 16-24 hours. Kedzior (18) iso lated a thermophilic actinomycete from sewage and described i t s growth on several media* The organ­ ism grew at temperatures from 35° to 65°C, with an optimum growth temperature of approximately 55°C* The spores pro­ duced by t h is organism were found to be r e s i s t a n t to heat, 5 % phenol, and to desiccation. Tsiklinsky various (34) described th ree organisms isolated from composts. The f i r s t organism, Thermoactinomyces v u lg a ris , was iso la te d from several sources including s o i l , hay, straw, manure and potato. The hyphae of th is organism were 0*5u in diameter, with round or oval spores borne singly on short conidia. for 20 minutes, The spores were found to survive at 100°C and to withstand 5% ' phenol for 24 hours. The optimal growth temperature for the organism was about 57°C. T. vulgaris grew well on a l l ordinary liqu id and solid media, and was found to be non-pathogenic to mice and guinea pigs. A second actinomycete culture possessed hyphae of 1.2 1.5u in diameter with spores borne in chains. The spores did 5 not survive for 5 minutes at 100°C. This organism resembled th at previously described by Kedzior in spore formation and sta in in g reactions* I t d iffe red , however, in that no growth occurred a t 35°C, and the culture had a marked odor. The th ird organism, Thermomyces lanuginosus, grew well on apple sprinkled with garden s o i l . Contaminating thermophilic b acteria were eliminated by tran sfe rrin g the organism to white bread, followed by a tr a n s f e r of spores to an agar p la te . procedure yielded a pure culture. This This organism grew well on the usual n u trie n t media, and had an optimum growth temperature of 5^»55°C. On on a dark t i n t . agar the mycelium was downy white, l a t e r taking The spores did not survive fo r one minute at 100°C. Sames (29) iso la te d a "thermotolerant” actinomycete from raw milk. This organism had an optimum growth temperature of approximately 55°C, and grew anaerobically to some extent as well as aerobically. The growth of the organism on various solid and liq u id media was described. Several thermophilic actinomycetes were isolated from various so ils by Gilbert (14). Actinomyces thermophilus grew well on potato medium at 55°C, possessed hyphae of 0.5 - 0.6u In diameter andspores of 0.8 - lu in diameter. of th is organism and several other iso la te s was The growth described* ft Schutze (33) described two thermophilic actinomycetes iso la te d from self-heated hay. Thermoactinomyces monospora. °ne of these organisms was This organism produced a gray- green a e r i a l mycelium, possessed hyphae of about lu in diameter, oval spores of 1.5 - 1.8u, and had an optimum temperature 6 range of 37° - 55°C. The organism being re-oorted in th is work resembled T. monospora in a l l but a few c h a rac te ristic s* Hoack (25) iso la te d a number of thermophilic actinomycetes and fungi from moist hay held at 45° - 46°C. Miehe (23) also iso lated many actinomycetes from hay but only a few of these were thermophilic* H© suggested th at thermophilic actin o­ mycetes probably a rise from self-h eatin g plant masses such as manure• Lieske (20) discussed the orig in of thermophilic actin o­ mycetes and th e i r a b i l i t y to survive -under conditions which are seldom optimum* He suggested th a t thermophiles might be mutants of mesophilic actinomycetes. Lieske thought th a t the origin of these thermophilic forms could be traced back to a sudden change in temperature requirement, ^e did not believe th a t thermophilic forms could be developed by gradual temperature r i s e . This sudden change or mutation involving temperature requirement was also be­ lieved to be re v e rsib le. Lieske concluded his discussion by s ta tin g th a t no conclusive evidence was available and th a t a re-examination of the theories expressed up to that time was in order* With two exceptions, p rior to 1910. the above reports were published Methods of is o la tio n , and descriptions of i s o la te s were in many cases incomplete, giving r is e to much confusion regarding the tru e id e n tity of the organisms is o ­ lated . The current publications dealing with the thermophilic actinomycetes are re la tiv e ly few in number. 7 A thermophilic actinomycete was iso la te d repeatedly from pasteurized cheese by Bernstein and korton (2). The organism had an optimum growth temperature of about 56°G. The name Actinomyces casei was suggested for th is organism* Katznelson (17) iso lated a thermophilic actinomycete from horse manure compost held a t 50°C* When the organism was grown on a starch-ammonium su lfa te agar medium, autolysis was noted a f t e r a certain period of incubation a t 5Q°C. After a study of th is a u to ly tic process, Katznelson concluded th at the autolytic mechanism was activated when the pH of the ■medium reached pH 6*0 - 6.5. No transm issible l y t i c agent could be demonstrated. Micromonospora vulgaris by Erikson (l2) (Tsiklinsky) has been isolated from composts made from lawn cuttings* The organism was found to have an optimum growth range of 45° 60°C. Heating a spore suspension of M * vulgaris for 5 minutes at 75° - 90°C induced growth in 18 hours, whereas unheated spores did not germinate during the same interval* Spores suspended in 1 % sucrose withstood 100°C for periods up to 45 minutes. Spores borne on the a e r ia l mycelium retained t h e i r v i a b i l it y and heat r e s i s t a n t properties a f t e r six months storage at 2°G. Erikson suggested th a t the organism survived periods of unfavorable growth conditions by means of i t s spores. Waksman, umb r e i t , and Cordon (42) studied the thermo­ p h ilic actinomycete and fungal population of so ils and com­ posts* They noted that thermophilic actinomycetes were pres­ ent in the s o i l in a l l seasons of the year* In winter 8 actinomycetes composed only a small percent of the thermophilic population, whereas in the spring they made up a large propor­ tio n of it# Thermophilic molds were fa r less abundant than the thermophilic actinomycetes at a l l seasons. The survival rate of thermophilic actinomycetes in the s o i l was determined by tr e a tin g portions of Sassafras sandy loam with the following: manure, s t e r i l i z e d and u n ste riliz e d fresh composted manure, and pure cultures of thermophilic actinomycetes. All samples were held at 28°C for 5 weeks# Plate counts at 3 and 5 weeks showed th a t the thermophilic population of the s o i l decreased rapidly at 28°C in a l l cases except where composts were added. Here the population i n ­ creased • In a second experiment the thermophilic population in horse manure was followed. Portions of fresh horse manure were incubated a t 50° and 65°C and sampled a t i n te r v a l s . At both temperatures the highest number of thermophilic actino­ mycetes reached nine b i l l i o n per gram of moist compost at 65°G, and three b i l l i o n per gram of moist compost a t 50°C. The thermophilic fungi numbered two hundred million per gram of moist compost a f t e r 10 days incubation at 50°C. The fungi found were fo r the most part members of the genus Thermomyces previously described by Tsiklinsky. In a third experiment s t e r i l e manure was inoculated with pure cultures of thermophilic b a c te ria , actinomycetes, and fungi in an attempt to determine the role of thermophiles In decomposition of manure. After 42 hours incubation at 50°C, r e s u lts indicated t h a t neither b acteria nor actinomycetes in 9 pure culture were as effective as the normal mixed population of manure in bringing about decomposition. The influence of temperature upon the microbial popula­ tio n and upon the r a te of decomposition of composting stable manure has been investigated by Waksxnsn, Cordon and Hulpoi (37). Portions of manure were held a t 28°, 50°, 65°, and 75°C. Chem­ i c a l analyses and counts on the number of organisms present were made periodically during 47 days of incubation. Results indicated th at the g re a te st amount of decomposi­ tion occurred in manure held a t 50°C. Microbiologically, at o 50 C the number of bac teria per gram of moist manure decreased quite rapidly early in the incubation period. This decrease was accompanied by an increase in the actinomycete and fungal population. At 65°C aerobic b acteria and fungi disappeared rapidly, whereas the actinomycete population increased. At 75°C only c e rta in spore-forming b ac teria survived. In 1953 Waksman and Corke (38) described a thermophilic actinomycete, Thermoactinomyces thalpophilus. The organism was iso lated from s o il and high temperature composts, and had an optimum temperature of approximately 50°G. c h a ra c te ristic s The growth of T. thalpophilus on various media were des cribed. The only previous report of an a n tib io tic produced by a It thermophilic actinomycete was published by Schone (32). The morphological and biochemical properties of the organism agreed well with Streptomyces therxnophilus, f i r s t reported by Gilbert (14). The organism had an optimum temperature of about 60°C. Schone called the a n tib io tic thermomycin. The b a c te ria l 10 spectrum of thermomycin was rath e r narrow* against 19 s tra in s I t was active of Corynebacterlum diphtheriae and in ­ h ib ite d L is te r ia monocytogenes only s l i g h t l y . All other organisms t e s te d were in se n sitiv e to thermomycin* The a n t i b i o t i c was produced in surface culture on a medium containing 10% blood serum and phosphate b u ffer. Maximum a n tib io tic a c t iv i t y was reached in 4 days a t 60°G. A s e r i a l d ilu tio n t i t e r the culture f i l t r a t e , of 256 units per ml was obtained in using C. diphtheriae PW8 as the assay organism* Extraction of the culture f i l t r a t e with ether followed by evaporation yielded a yellow powder, which In a concentra­ tion of 5 % inhibited _C. diphtheriae a t a d ilu tio n of 1:4096. Thermomycin could be completely salted out by one-half s a t ­ urated ammonium s u l f a t e . dialyzable. The a n tib io tic was found to be non- Regarding s t a b i l i t y , 15 minutes a t 75°G reduced the ^C. diphtheriae t i t e r 25%, whereas 15 minutes at 100°G destroyed a c t iv i t y completely. In jectio n of 2 ml of a n t i b i o ti c beer (256 units per ml) Into each of the 20 mice did not cause a toxic rea ctio n . There was also no i r r i t a t i o n when crude f i l t r a t e was placed on the cornea of a r a b b i t . Schone noted that S^. assmooth colonies, which thermophilus produced rough as well indicated d isso c ia tio n . Rough colon ies were characterized by an irre g u la rly folded surface and edge. Smooth colonies were c ir c u la r with an even edge, tiv e ly smooth surface, re la ­ and were smaller than the rough type. 11 Rough colonies produced an a n tib io tic t i t e r ml in the crude f i l t r a t e , tite r of 256 units per ml* of 4-8 units per whereas smooth colonies produced a MATERIALS AND METHODS Screening Methods Thermoactinomyces v i r i d i s was iso la te d from a composted manure p i l e on property belonging to the Michigan Department of Health. The material was sampled at a depth of 4 to 6 inches from the surface. One gram of sample was dilu ted 10”^ and 10”S, and d i r e c t platings of 0.5, were also made. 0.2 and 0.1 gram The d ilu tio n s and d ire c t samplings were plated out in duplicate using sodium caseinate agar and a medium which w ill be referred to as trypticase agar. This medium was very sim ilar to the n u trie n t agar for p e n i c il l in assay specified by the Pood and Drug Administration (5). Trypticase was used as the pancreatic dig est of casein, and the medium was modified by the addition of sodium chloride. This modified formulation was also used extensively in th is work as a liq u id medium. The formulae of these agar media are presented in Table I . I t has been found by Murray (24) and Erikson (12) that a water saturated atmosphere is necessary fo r the c u ltiv atio n of c e rtain thermophilic organisms on agar media. tio n plates were, therefore, The i s o l a ­ incubated at 50°C for 5-5 days in an atmosphere saturated with water. Colonies were picked and transferred to sodium caseinate agar s la n ts , which were then incubated in the same manner as the is o la tio n p la te s . The s la n t cultures were stored a t 25°C. T. v i r i d i s was 13 TABLE I AGAR MEDIA USED IN SCREENING Trypticase agar Component Sodium caseinate Agar Concentration per l i t e r Component Cone entration per l i t e r Bacto peptone 6*0 gm Sodium caseinate Trypticase 4.0 gm Glucose 1.0 gm 3.0 gm K2HP04 0.2 gm MgS04 .7Hg0 0.2 gm Bacto peptone 1.0 gm D ist. water to make 1000 ml Glycerine 5.0 gm pH 7.0 D ist. water to make 1000 ml Bacto beef extract Glucose Sodium chloride 1.0 gm 5.0 pH 7.3 2.0 gm 14 maintained on n u trie n t agar sla n ts since growth on sodium caseinate agar was poor. The n u tr ie n t agar contained Bacto peptone 1%, Bacto beef extract 0.5%, sodium chloride 0.5%, and had a pH of 6.8 - 7.0 . Antibiotic a c t i v i t y against 7 t e s t organisms was de­ termined by an agar cross-streak method sim ilar to t h a t of Waksmanfs (40). The actinomycete was streaked on n u trie n t agar and incubated f o r 3 days a t 50°C, at which time i t was cross-streaked with 7 t e s t organisms and re-incubated at 37°C. The t e s t organisms were Mycobacterium 607, Salmonella typhimurium, Micrococcus pyogenes var. aureus 209P, Escherichia c o li, E. coli 0111, Proteus v u lg a r is , and Bacillus s u b t i l i s 6633. Cross-streak re s u lts were read a f t e r 18-24 hours incuba­ tio n , except f o r Mycobacterium 607 r e s u lt s which were read a f t e r 36-48 hours incubation. U ltravio le t Ir ra d ia tio n Method A spore suspension of Thermoactinomyces v ir id is was sub­ jected to u l t r a v i o l e t i r r a d i a t i o n In an attempt to obtain a mutant which would give increased yields of thermoviridin. The growth from an agar sla n t culture of T. v i r i d i s was sus­ pended in 100 ml of s t e r i l e d i s t i l l e d water. The suspension was shaken vigorously for 5 minutes to d i s t r ib u t e the spores as evenly as possible. allowed t o s e t t l e , Large clumps and fragments were then and 21 ml of the suspension was carefully withdrawn and placed in a s t e r i l e P e tri dish. The spore suspension in the opened P e tri dish was exposed to the u l tr a v io l e t rays from a 15 watt General E le ctric 15 germicidal lamp placed 6 inch.es above the spore suspension* The suspension was s t i r r e d continuously throughout the ex­ posure period by means of* a magnetic s t i r r e r * Samples were withdrawn a fte r 0, 1, 2, 4, 8 and 16 minutes exposure. The samples were plated out in n u trie n t agar (Bacto peptone 1%, Bacto beef extract 0.5$, sodium chloride 0.5fo, pH 6.8 - 7 .0 ). After incubation at 45°C f o r 5 days, t o t a l counts were made. The exposure time of 2 minutes was chosen as the proper time for subsequent i r r a d i a t io n stu d ie s . After 2 minutes exposure using the above procedure, 4 ml of the ir ra d ia te d spore suspension was added to 100 ml of s t e r i l e d i s t i l l e d water. Forty ml of th is d ilu tio n was plated out in 1 ml amounts in n u trie n t agar. o The plates were incubated 4 days at 45 C and colonies were then picked and tra n sfe rre d to n u trie n t agar s l a n ts , afte r incubation these subcultures were tested for a n tib io tic a c tiv ity by the agar cross-streak method. Assay Methods In the course of th is work a n tib io tic a c tiv ity was de­ termined by three assay methods: 2) paper d is c , 1) s e r i a l d ilu tio n , and 3) agar d i lu tio n . Unfortunately, no one method was found to be s a tis fa c to ry for a l l cases in which an assay of a n tib io tic a c t iv i t y was desired. The thermoviridin unit was based cn the s e r i a l d ilu tio n te st. The unit was defined as the minimum amount of thermo­ v i r id i n in 1 ml which inhib ited the growth of a 1% inoculum of m. pyogenes v a r. aureus 209P in tryp ticase broth for 24 hours at 37° G• 16 The s e r i a l d i lu t i o n assay method was used for the d e te r­ mination of the b a c te r ia l spectrum of thermoviridin, and for following a n t i b i o ti c a c t i v i t y in studies on media development and p u r i f i c a t io n . The t e s t was performed following a proce­ dure sim ila r to th at of Schmidt and Moyer (51). Trypticase broth was inoculated with an 18-24 hour culture of the t e s t organism in the amount of 1% and then dispensed into tubes. In addition to the usual two-fold s e r ie s , ste rile a d ilu tio n se rie s which increased by increments of 10 units was frequently used. Controls for the t e s t included sample s t e r i l i t y , s te rility , and t e s t broth organism growth# Samples fo r assay were s t e r i l i z e d by f i l t r a t i o n (Seitz or f r i t t e d g l a s s ) , but in the case of solid preparations, samples were merely dissolved In methanol and assayed with­ out fu rth e r treatment. Methanol was found to in h ib it the growth of M. pyogenes var. aureus 209P, the only organism used under these conditions, up to a d ilu tio n of 1:8. Any in h ib itio n found in d ilu tio n s g reater than 1:8 was attrib u te d to thermoviridin. As the second method of choice, the paper disc assay was used as a means of following a n tib io tic a c t iv i t y during studies on media development, recovery and p u r ific a tio n . Assay plates contained 21 ml of trypticase agar as base coat and a 4 ml seed coat of the same medium. Seed coats were inoculated with an 18-24 hour culture of M. pyogenes var. aureus 209P in the amount of 1.4%, or with a B. s u b t i l i s 6633 spore suspension in the amount of 0.08%. The spore suspension contained 2 x 108 viable spores per ml. Seeded plates were stored a t 0°-5°C for no longer than 1 week, and 17 warmed before use. Paper discs 15 mm in diameter and capable o f absorbing up to 0.1 ml of sample were used. The discs were made from sheets of E and D f i l t e r paper $b23-.030. Assay plated seeded with B. s u b ti1is 6633 were incubated 18— 24 hours at room tem­ perature, and those seeded with M. pyogenes var. aureus 209P were incubated 18-24 hours at 37°C. a l l cases was The zone of in h ib itio n in considered to be the over-all diameter of the zone i n m illim eters. Thirdly, the agar d ilu tio n method of assay was selected as the means of te s tin g the s e n s i t iv i t y of several mycobacteria and pathogenic fungi to thermoviridin. The desired d ilu tions of the sample to be te s te d were made in the melted agar medium contained in s t e r i l e screw-capped tubes. had hardened, When the slanted tubes they were inoculated with the t e s t organism. Tubes inoculated with the mycobacteria were incubated at 37°G and read a f t e r 3 and 6 weeks incubation. Tests involving the fungi were incubated a t room temperature and read a f t e r 3 weeks incubation. Fermentation Methods I n i t i a l fermentation studies concerning thermoviridin production were carried out in stationary liq u id cu ltu res. Rectangular-shaped b o ttle s were placed in a horizontal posi­ tio n and p a r t i a l l y f i l l e d with 250 ml of culture medium. A layer of glass wool was added to insure adequate aeration and to support the mycelial growth. with r a th e r loosely f i t t i n g The bo ttles were plugged gauze-covered cotton plugs to allow free tr a n s f e r of gases. The b o ttle cultures s t e r i l i z e d by autoclaving one-half hour a t 121°C. were 18 Stationary liq u id cultures were inoculated by pooling the growth of several sla n t cultures of Thermoactinomyces v irid is in s t e r i l e d i s t i l l e d water* Five ml of th is suspension was d istrib u te d over the surface of the glass wool layer in each bottle* The cultures were then incubated 5 days a t 50°C. Most of the fermentation studies were carried out on a Gump rotary shaker in wide-mouth 500 ml Erlenmeyer f la s k s . The shaker operated a t about 250 rpm, each fla sk describing a two and one-quarter inch diameter c i r c l e . Each fla sk con­ tained 50 ml of medium and was plugged with gauze-covered cotton held in place over the l i p of the flask with a rubber band. The flasks were autoclaved 30 minutes at 121°C except when in solu ble p a r tic u la te matter, such as the various grain products, was present* -^n these cases the flasks were auto­ claved 45 minutes at 121°C* The inoculum for the fermentations was prepared by tra n s­ fe rrin g the growth from an agar slant culture of T. v irid is (not more than 10 days old) to a 1 l i t e r flask containing 100 ml of seed medium. at 45°C. The flask was shaken for about 48 hours A second fla sk of seed medium was inoculated with 2 ml of vegetative growth from the primary culture, for about 18 hours at 45°C. and shaken This secondary growth was used to inoculate the fermentation fla sk s , of inoculum per 50 ml of medium. each flask receiving 1 ml The procedure for inoculum preparation was also carried out a t 37°C, in which case the primary seed fla sk was shaken fo r 72 hours, and the secondary flask fo r 45 hours. The seed medium contained Bacto tryptone 2%, Bacto beef extract 0.5^, and had a pH of 6.S - 7.1. 19 Fermentations c a rrie d out a t 45°C were shaken 26-28 hours, whereas fermentations at 37°C were shaken 72 hours. Nutrients te ste d in the development of the fermentation medium were added to a basal medium a t levels of 0.5$, 1$, 2$, 4$ and sometimes 8%. The basal medium contained Bacto tryptone 4$, Bacto beef extra ct 0.5$, and had a pH of 6.9 - 7*1. Those materials which appeared t o enhance thermoviridin production were then added simultaneously to the basal medium. Each en­ hancing n u tr ie n t was added in i t s optimum concentration, and also a t higher and lower l e v e ls . By a lte r in g the concentra­ tion of a l l the supplementary constituents, many variations of the same medium were obtained. This a l te r a tio n of the balance of n u trie n ts was undertaken because the optimum con­ centration for a given supplement when present alone in the basal medium may not necessarily be i t s optimum when other supplementing n u trie n ts are present. Thermoviridin was also produced in a 30 l i t e r laboratory fermenter previously described by Olson, Jennings, Pisano and Junek (26). The fermenter contained 12 l i t e r s of fermentation medium and 150 ml (1.25$) of antifoam, which was mineral o i l containing 5$ octadecanol by weight. The fermenter containing a 12 l i t e r charge was autoclaved 3 hours at 126°C. The inoculum for the fermenter was 100 ml of a secondary culture obtained by the same procedure as for the preparation of inoculum for shake fla s k fermentations During the fermentation, (see page 18). the temperature was 45°C, the a i r flow 0.6 volume of a i r per volume of medium per minute, and the impeller speed 530 rpm. 20 Recovery Methods Recovery of thermoviridin. was f i r s t attempted by solvent extraction* Portions of a n ti b i o ti c beer were adjusted to pH 2, 4, 8 and 10. An equal amount of water immiscible solvent was added to each portio n. The so lv e n t-a n tib io tic beer mixtures were shaken f o r 1 minute and the layers allowed to separate. This shaking and separating process was repeated 3 times, and a f t e r the f i n a l separation both layers were assayed by the paper disc method. Solvents were evaporated from the discs at 37°C before being placed on the assay p l a t e s . tro ls Solvent con­ evaporated in lik e manner were also assayed. t h is procedure the following solvents were teste d : petroleum ether, aceta te , ethyl ether, Using benzene, chloroform, butanol, amyl and xylene. Solvent extractio n was found to be an unsuitable method of recovery. ta tio n method. The one selected may be called an acid p re c ip i­ After completion of the fermentation, the crude culture was adjusted to pH 5.5 - 6.5 and f i l t e r e d using c e l i t e 545 as a f i l t e r aid* An a lte rn a te means used in separating the mycelium from the a n ti b i o ti c beer was cen­ tr if u g a ti o n . The culture f i l t r a t e was adjusted to pH 3 with phosphoric acid and held at 0° - 5°C for at l e a s t 2 hours. Hydrochloric and s u lfu ric acids were also found to be equally e ffe c tiv e as p r e c ip ita tin g agents. The re s u ltin g p re c ip ita te was recovered and washed with water. Thermoviridin was extracted from the p r e c ip ita te with an amount of 80$ acetone equal to one-tenth the original 21 volume of the culture f i l t r a t e . The acetone e x tra ct was then concentrated by evaporation a t reduced pressure, and the water so lutio n remaining was dried from the frozen s t a t e . covery procedure i s i l l u s t r a t e d The r e ­ graphically in figure 1. I t was found th a t thermoviridin could be p re c ip ita te d by sa tu ra tin g the a n tib io tic beer with ammonium s u l f a t e . This method was not used, however, because of the convenience and sim plicity of the acid p re c ip ita tio n method. P u rifica tio n Methods Two methods were found to be of value in the p u rific a tio n of thermoviridin. The f i r s t method involved f ra c tio n a l p re c i­ p i t a t i o n of the active material from a methanol solution, ethyl ether as the p re c ip ita tin g agent. using Crude dry a n tib io tic m aterial was extracted with s u ff ic ie n t methanol to give an a n t i b i o ti c concentration of 300 units per ml. Extraction of the a n t i b i o ti c a c t i v i t y was effected during a 30 minute period of constant ag ita tio n with glass beads on a Gump ro tary shaker. A second portion of methanol equal in volume to the f i r s t , was added to the insoluble residue, and the extraction process repeated. The second methanol e x tra ct was separated from the in­ soluble residue and was combined with the f i r s t , giving a f i n a l a n tib io tic concentration of 150 units per ml of methanol. A quantity of ethyl ether equal to one-fourth the volume of the combined extracts was added to the methanol so lu tio n . The ethyl ether concentration was increased to one-half volume by the addition of a second one-fourth volume. In th is manner the ethyl ether content was increased to a t o t a l 22 Whole Crude Culture I Culture F i l t r a t e Mycelium (Discard) Adjust to pH 3 P re c ip ita te 1. HgO wash (Discard) 2* Residue (Discard) F iltra te (Discard) Extract with 80$ acetone 1 Acetone Extract Remove acetone and dry from frozen s t a te Dry Antibiotic Solid B 'igure ! • Procedure f o r th e reco v ery o f th e r m o v ir id in 23 concentration of 1, 2, 4, and 7 volumes. The p r e c ip ita te which formed with the addition of each increment of ethyl ether was recovered and dried under reduced pressure. The dried p re c ip ita te s were assayed by the s e r i a l d ilu tio n method. The counter current d i s tr ib u tio n method of Craig (6) was also of value in the p u r ific a tio n of thermoviridin. cedure was carrie d out in stoppered t e s t This pro­ tubes containing equal amounts of n-butanol and 0.04 M phosphate buffer at pH 6, each mutually saturated with the other beforehand. The pH a t which the material to be p u rifie d had a d i s ­ t r ib u tio n coe ffic ien t of approximately one was determined by assaying a butanol-water system containing the m aterial a f t e r i t s adjustment to several pH values. Craig (7) found that in a s e rie s of any given number of tubes, the purest f ra c tio n would appear in the middle of the serie s when the d i s t r ib u t i o n c o e ffic ie n t of the solute between the solvents approached one. The a n t i b i o t i c material to be purified was placed in tube number one containing equal volumes of butanol and b u ffer. The tube was shaken 25 times in a period of 30 seconds, and then centrifuged for 1 minute a t 1,500 rpm to e ff e c t complete separation of the two layers. The water layer of tube one was transferred to tube two, which con­ tained an equal volume of butanol. A fresh portion of buffer was then added to tube number one, and the shaking and cen­ tr if u g in g processes repeated. The water layers from tubes one and two were then tran sfe rred to the next consecutive tubes in the s e rie s (one to two, and two to th r e e ), and 24 buffer again added to tube number one. were continued u n t i l The above processes the material being p u rifie d was d i s t r i b uted in eight tubes. Dry weight determinations were made on both the butanol and bu ffer fra c tio n s co n tro ls. of a l l tubes, and on butanol and buffer All fra c tio n s were disc assayed and the unitage read from a standard curve* The degree of p u rific a tio n was then calculated from the dry weight d ata. Toxicity Study Method An acute to x ic ity study of thermoviridin was conducted using mice. Twenty gram White Swiss mice of the Webster s t r a i n from the Michigan Department of Health colony were injected in tra p e rito n e a lly with 800 units of thermoviridin. The so lid material used f o r th is study had an a n tib io tic a c t iv i t y of 25 units per mg. After in je c tio n the mice were weighed periodically and observed for 5 weeks. At th a t time the mice were sa crificed and an autopsy was performed. RESULTS Cultural Characteristics Thermoactinomyces v i r i d i s was found to have an optimum growth temperature of approximately 55°C. slowly a t 37gC and not at a l l a t 60°C. erature, The organism grew At the optimum temp­ growth on agar was f u lly developed in 3 days. At 37°C growth was not maximum even a f te r 14 days incubation. The hyphae were about 0.5u in diameter and bore oval conidia approximately l.Ou by 1.3u. on short sporophores. The conidia were borne singly The microscopic appearance of th is organism may be seen in figure 2. The preparation shown in the photomicrograph is a portion of a slide culture of Z* v i r i d i s prepared a f t e r the method of Riddell (28). Z* vJ-Pid-is is no^ acid f a s t and is gram negative. is This unusual since most actinomycetes are gram positive (36). However Lieske (20) found that certain thermophilic forms are gram negative. s t a in , Using Burkefs modification of the gram and with M. pyogenes var. aureus 209P mixed in the preparation as a control, T. v ir id is was gram negative, whereas the control organism gave the typical gram positive re a c tio n . The gram reaction did not depend on the age of the organism since a young vigorously growing shake flask culture and a fully grown shake flask culture both gave the gram negative reaction. Figure 2. ThermQactinomyces v ir id is 27 The growth c h a ra c te r is tic s T. v i r i d i s and biochemical reactions of on various organic media are presented in Table II* Results were read a f t e r 7 and 14 days incubation at 4^-50° C. Although growth on most media was fu lly developed a f t e r 7 days incubation, the cultures were incubated for an additional 7 days* A duplicate set of media were incubated at 37°C and the r e s u l t s read a f t e r 7, 14 and 21 days incuba­ tion* This was done in order to ascerta in whether or not the organism had been previously described by i t s c h a r a c te ris tic s at 37°C. I t was found, however, c u ltu ra l th a t re s u lts obtained at 37°and 50°C were identical* The glucose-peptone A and B media, glucose-asparagine agar, and starch agar A, were prepared according to formulae given by Waksxnan (36). The medium for the te s tin g of c e llu ­ lose digestion consisted of f i l t e r paper s t r i p s suspended in a solution of the s a l ts found in Czapek-Dox agar* The n u tr ie n t agar contained Bacto peptone 1%, Bacto beef extract 0*5$, sodium chloride 0*5$, and had a pH of 6.8 - 7*0. The c u ltu ra l c h a ra c te ristic s of T. v i r id i s on n u trie n t agar were taken as the standard description of the organism, host of these c h a ra c te ris tic s may be seen in figures 3-6. Figure 3 shows the degree of growth development a f t e r 3 days incubation a t 50°C. Figures 4-6 indicate the progressive development of the colonies on consecutive days t h e r e a f t e r . The color reproduction in these photographs is not e n tire ly accurate, but the series does serve to i l l u s t r a t e in general the various stages of growth on n u trie n t agar. 28 TABLE I I CULTURAL CHARACTERISTICS AND BIOCHEMICAL REACTIONS OP THERMOACTINOMYCES VIRIDIS ON VARIOUS ORGANIC MEDIA Medium Description of Growth or Biochemical Reaction a f t e r 14 days Incubation a t 45°C Nutrient agar Mycelial growth wrinkled and close to agar surface; blue-green a e r ia l mycelium; emerald-green water-soluble pigment present Nutrient broth Flocculent cream-colored submerged growth; Surface growth blue-green; green pigment present Glucose-peptone A agar Colorless colonies 1-2 mm in diameter; no a e r ia l mycelium Glucose-peptone A broth Flocculent cream-colored submerged growth Glucose-peptone B agar Wrinkled colorless growth; no a e r ia l mycelium Glucose-peptone B broth Flocculent cream-colored submerged growth Czapek-Dox agar No growth Sabouraud agar No growth Glucos e-asparagine agar No growth Corn meal agar Flat colorless colonies 1-2 mm in diameter Calcium malate agar Colorless colonies 1-2 mm in diameter; few colonies with blue-green a e ria l mycelium P o ta to p lu g No growth C arrot p lu g No growth B lo o d p l a t e Few large folded colonies; many small f l a t colonies; no a e ria l mycelium; hemolysis present 29 TABLE I I Medium C o n tin u e d Description of Growth or Biochemical Reaction a f t e r 14 days Incubation at 45°C Skim milk Coagulation followed by digestion N itra te reduction Not reduced to n i t r i t e s Cellulose dig estio n Negative Gelatin liq uefaction Positive Starch hydrolysis Positive Figure 3* T* v i r id i s a f t e r 3 days incubation at 50°C. Figure 4. T. v i r id i s a f t e r 4 days Incubation at 50°C, Figure 5# T* v i r i d i s a f t e r 5 days incubation a t 50°C* Figure 6. T. v i r i d i s a f t e r 6 days incubation a t 50°C* 52 The surface of the fu lly developed culture of T. v i r i d i s was bluish-green, whereas the reverse side of the culture was emerald-green# A water-soluble pigment was present which ’ appeared to be emerald-green in the agar, but more nearly fo rest-g re en when extracted into water# In naming th is considered# organism, s everal facto rs had to be The organism in many ways resembles Thermoactin- omyces monospora# described by Waksman (40) • The points of d iffe ren c e, however, are considered s i g n if ic a n t. T. monospcra possesses hyphae with a diameter of about l u , has an optimum growth range of 37° - 55°C, and does not coagulate milk# The hyphae of the organism reported here were approxi­ mately 0#5u in diameter, the optimum growth temperature was about 55°C, and milk was coagulated and digested. In addition a green water-soluble pigment was present# Bergeyfs Manual of Determinative Bacteriology (3) i n ­ cludes genus Thermoactinomyces in the genus Micromonospora# Waksman (37) has stated more recently th at thermophilic organisms which bear spores singly and produce an a e r i a l mycelium should be c la s s if ie d separately under the goius Thermoactinomyces• For the above reasons, the organism reported here is considered to be a new species for which the name Thermo­ actinomyces v i r i d i s is suggested# is The name thermoviridin suggested for the a n tib io tic produced by th is organism. U ltravio le t Ir ra d ia tio n The r e s u l t of exposing a spore suspension of T. v i r id i s to u l tr a v io l e t I r r a d ia tio n for 1-16 minutes i s shown in Table I I I . 35 TABLE I I I THE EFFECT OF ULTRAVIOLET IRRADIATION UPON THE VIABLE SPORE COUNT OP T. VIRIDIS Exposure period in minutes Viable spore count Percent k ill 0 150,000 1 15,000 90.00 2 6,700 95.50 4 700 99.53 8 170 99.89 16 40 99.97 Upon t e s t i n g the cultures iso la te d from a spore suspen­ sion which had "been irra d ia te d for 2 minutes, i t was found t h a t no culture produced s ig n ific a n tly more thermoviridin than did the parent s t r a i n . However, mutants which diffe red from the parent s t r a i n in pigment production, degree of sporulation, and colonial morphology were is o la te d . Fermentation Production of thermoviridin was f i r s t undertaken in sta tio n ary liquid c u ltu re s . A maximum a n tib io tic a c tiv ity of 4 units per ml was obtained a f t e r 5 days incubation a t 50°C with a medium containing Bacto peptone 2%, Bacto beef extract 0.5$, 7 .0 sodium chloride 0.5$, and having a pH of 6.8 - . F e r m e n ta tio n s t u d i e s u s in g sh ake f l a s k c u l t u r e s proved t o be m o st s u c c e s s f u l . B a c to t r y p t o n e was fo u n d t o be more 34 su ita b le than Bacto peptone f o r a n t i b i o ti c production, was therefore su b stitu ted for i t and in the fermentation medium. The medium then contained Bacto tryptone, Bacto beef e x tra c t, and sodium chloride. The balance of these constituents was a lte r e d to determine the optimum concentration of each n u trie n t. The formulation ■which gave maximum thermoviridin production is presented in Table IV. TABLE IV THYPTONE-BEEF EXTRACT FERMENTATION MEDIUM Constituent Concentration in grams per l i t e r Bacto tryptone 40 Bacto beef extract 5 pH 6.9 - 7.1 Using th is medium, an a n tib io tic level of 32 units per ml was obtained. Buffering the medium did not Increase thermo v i r id i n production. An attempt was made to increase the a n t i ­ b i o ti c potency by supplementing the tryptone-beef extract medium. A l i s t of the n u trie n ts added i s given in Table V. TABLE V NUTRIENTS TESTED IN THE TRYPTONE-BEEF EXTRACT MEDIUM FOR STIMULATION OF ANTIBIOTIC PRODUCTION Glycerol L-tryptophane Glucose DL-tyrosine Sucrose Casamino acids 35 TABLE V C o n tin u e d Maltose Bacto peptone"' Lactose Bacto tryptose’"' Com starch Soy peptone*' Com meal Soy bean meal Corn steep liquor Milk n u tr ie n t GG Ifriole wheat flour NZ amine Wheat germ BrewerTs yeast Bacto yeast extract w Nutrients showing some stimulation of a n t i b i o ti c production Several n u trie n ts caused a s l i g h t increase in a n tib io tic production, but the increase was not considered s ig n if ic a n t. The amino acids tryptophane and tyrosine were te s te d because these are the amino acids in Bacto tryptone which are present in su b stan tia l amounts, according to Difco la b o ra to rie s . Since in some cases in s u f fic ie n t trace elements may be the lim itin g factor in a n tib io tic production, several mineral s a l t s were added to the tryptone-beef extract medium. case of thermoviridin production, In the the addition of various mineral s a l t s had no enhancing e f f e c t . The ions, and the con centratlons in which they were added, are shown in Iable VI. Failure to improve upon the simple tryptone-beef extract medium to any marked extent indicates the rather sp e cific r e ­ quirements of T. v i r i d i s for a n tib io tic production. Growth and a n tib io tic a c t i v i t y have even been obtained using only a 4% tryptone solution as the fermentation medium. 36 TABLE V I METALLIC IONS ADDED TO THE TRYPTONE-BEEF EXTRACT MEDIUM WITHOUT EFFECT Ion Added Concentration of Ion in m g /lite r Ion Added Concentration of Ion in m g /lite r Ca (as CaClg) 72 and 144 Zn (as ZnS04 .7H20) 1 and 10 Mg (as MgS04 .7Hg0) 19.5 and 39 Fe (as FeS04 .7Hg0) 1 and 10 103 and 206 Cu (as CuS04) 0 .01 and 0.1 25.8 and 51*6 M n (as MnS04.H20) 1 and 10 K (as KC1) Na (as Na2HP04) Co (as CoCl2 .6H20) 0.1 and 1 Thermoviridin production at 37° and 45^0 have been compared using the tryptone-beef extract medium. A ntibiotic production was more rapid at 45°C, reaching a maximum in approximately 27 hours. At 37°C, about 48 hours were required fo r maximum production. The relationship of pH changes and thermoviridin production during fermentation a t 37° and 45°C may be seen in figure 7. Thermoviridin was also produced in a 30 l i t e r laboratory fermenter with the tryptone-beef extract medium. The growth obtained was good, although not so heavy as th at In shake f la s k s . A ntibiotic a c tiv ity did not exceed 4 units per ml. The low a n t i b i o ti c level obtained may have been caused by sub-optimal growth conditions. Because of the d i f f i c u l t i e s encountered and the expense of the medium, most of the a n t i ­ b io tic beer used for recovery and p u rific a tio n studies was produced by the shake f la s k method. 37 L9.0 pH CO < M CO o A - co o !» 0 p {> •P to > •H -P •P OJ o C 0) o to o n o3 to & O -P ol a O 45 o CO to CO 00 Xvi a©d O a) o >»o -P iQ c to *r—1 a £> £1 -P o •p 01 -P 01 O p. C Figure 7. Thermoviridin E-t production u d Q £ and p H changes at 37° and W -!> 38 Recovery I t was found th at the f i l t e r aid c e l i t e 545 adsorbed as much as 75fo of the a n ti b i o ti c a c tiv ity when the pH of the crude culture was pH 8,5 - S.7. By adjusting the pH to a range of pH 5.5 - 6.5 before f i l t r a t i o n , the amount of a c t iv i t y adsorbed was g rea tly reduced, but the problem was not e n tir e ly eliminated. Centrifugation was therefore the method of preference whenever the volume of crude culture to be tre a te d was not in excess of 3-4 l i t e r s . Although the solvent extraction method was found to be undesirable for the recovery of thermoviridin, obtained from the in vestigation of t h is given here. the r e s u lt s recovery method are Extraction of th em o v irid in from a water solu ­ tio n was accomplished with the use of n-butanol. The active m aterial was completely extracted with butanol only when the pH of the system was 5 or lower. Thermoviridin was not ex­ tra c te d from water by any other solvent teste d . Solid active material was also extracted with several so lv e n ts. Thermoviridin was found to be soluble in the following solvents, ethylene glycol, ethanol, in decreasing order of s o lu b ility : dihydroxyethyl ether, methanol, pyridine, 1,4-dioxane, and isoamyl alcohol. was insoluble in acetone, The a n t i b i o ti c ethyl acetate, diethylamine, 2- butanone, and cyclohexane. The selected recovery method has been outlined in figure 1 (see page 22). In order to effect complete precipi­ ta tio n of thermoviridin a t pH 3, i t was necessary to hold the a n tib io tic beer a t 0°-5°C for not less than 2 hours. Large 39 volumes of a n t i b i o ti c beer (8-10 l i t e r s ) were held overnight a t 0°-5°C. No p re c ip ita te was formed when a portion of s t e r i l e fermentation medium was adjusted to pH 3, indicating that the p r e c i p it a t e from the a n tib io tic beer was due to metabolic products of T. v i r i d i s . The washing of the acid p re c ip ita te with water did not cause a loss of a n tib io tic a c t i v i t y , as measured by a paper disc assay of the water wash. A ntibiotic a c tiv ity was not completely removed from the p r e c i p it a t e by extraction of the washed p re c ip ita te with a quantity of 80% acetone equal to one-tenth the orig inal volume of the a n tib io tic beer. A second extraction with 80% acetone fa ile d to increase the t o t a l recovery, th e re ­ fore the extraction was limited to one-tenth volume. Much of the p r e c ip ita te was insoluble in acetone and remained a f t e r ex tra ctio n . Approximately 80% of the a c tiv ity in the a n tib io tic beer was recovered in the acetone ex tra ct. After evaporation of the acetone, the remaining water fractio n was dried from the frozen s t a t e yielding a solid possessing up to 64 units per mg a c t i v i t y • Purification The solid material obtained from the recovery procedure was pu rified by fra c tio n a l p re c ip ita tio n or by countercur -rent d i s t r ib u t i o n . The degree of p u rific a tio n obtained by the f r a c tio n a l p re c ip ita tio n method may be seen in Table VII. 40 TABLE V I I PURIFICATION OF THERMOVIRIDIN BY FRACTIONAL PRECIPITATION % Activity Recovered i] Fraction Fraction mg in Fraction Units per mg S ta rtin g material** 192.0 20 3840 Residue 89.9 0 0 0 Units in Fraction P p t. from tg- vol • ether No ppt. P p t. from 1 v o l. ether 21.8 50 654 17.0 P pt• from 2 -vol • ether 16.4 80 1312 34.1 Ppt. from 4 vol. ether 18.6 30 558 14.5 P pt• from 7 v o l • ether 15.2 0 0 0 Total recovery 159.9 mg 2524 unitss 65 .6% * S u fficient methanol added to give an a n tib io tic con centration of 150 units per ml The presence.of a p re c ip ita te ^ i t h the addition of onefo urth and one-half volumes of ethyl ether depended upon the potency of the so lid m aterial being purified* When the start- ing material was crude, fo r example 5-10 units per mg, the one-fourth and one-half volume p re c ip ita te s were heavy. When the s t a r t i n g m aterial was of higher potency, the p r e c ip ita te in these fra c tio n s was l i g h t , or more often absent. In Table VII I t w ill be noted that the two volume f r a c ­ tio n showed a four-fold increase in purity over th a t of the 41 s t a r t i n g m a te ria l. I t was found th a t as the potency of the s t a r t i n g m aterial increased, the degree of p u rific a tio n effected decreased. The f a c t th at fewer eth e r-p re cip ita b le im purities were present in s ta rtin g material of higher potency is probably the reason for the above mentioned occurrence. In general, as the purity of the fractions increased, the color of the d ried p re c ip ita te s proceeded from tan to darkbrown, but i t was found by countercurrent d i s tr ib u tio n that the brown color was not associated with the a n tib io tic i t s e l f . F ractional p re c ip ita tio n from methaaol was also attempted using acetone, benzene, amyl acetate, te tra c h lo rid e and toluene, cyclohexane, carbon ^one of these solvents was found to be su ita b le for the p re c ip ita tio n of thermoviridin from me th an o l. Countercurrent d is tr ib u tio n was the second method used in the p u rific a tio n of thermoviridin. The number of tubes used in th is procedure was a r b i t r a r i l y established, but was based somewhat on the lim ita tio ns imposed by the time involved in making the tra n sfe rs from tube to tube with p ip e tte s . Sev­ e ra l pieces of apparatus for countercurrent d is tr ib u tio n have been described by Craig and Post (8), and with such equipment a large number of tran sfers may be made automatically, greatly simplifying the performance of the technique. In tra n s f e rr in g the water layers from tube to tube, p lete separation and t r a n s f e r were impossible. com­ Craig (7) has sta te d th a t th is does not markedly a ffe c t the f i n a l p u r i f i c a ­ tion when a large number of tra n sfe rs a r e used. Undoubtedly, with only eig ht tubes, p u rific a tio n was affected by the incorn- 42 p le te tr a n s f e r of the water layers. Visual observation of the se rie s of tubes a f t e r the completion of countercurrent d is tr ib u tio n showed the darkyellow color of the i n i t i a l water layer to have migrated to tube eight fo r the most p a rt, whereas the l ig h t e r yellow color in the butanol layer of the i n i t i a l d is tr ib u tio n r e ­ mained largely in tube one* The other tubes in the se rie s were progressively l ig h t e r in color proceding toward the middle tubes, which were colorless* The degree of p u rific a tio n obtained by the use of countercurrent d is tr ib u tio n i s presented in Table VIII* I t w ill be noted that in buffer fractio ns two and s ix a n t i b i o ti c a c t iv i t y i s present, but dry weight data Indicate t h a t no a c tiv e material is present in the fractions* These inconsistencies were probably caused by the use of 3 ml portions f o r dry weight determinations* The amount of solid m aterial in the portions was small, making errors in weighing sig n ific a n t• Aside from the above-mentioned discrepancies, these data demonstrate th a t much inactiv e material Is removed by countercurrent d i s t r ib u t i o n , and th a t considerable p u r i f i c a ­ tio n can be accomplished by th is method* Biological and Chemical Properties The s t a b i l i t y of thermoviridin in the a n t i b i o ti c beer has been te s te d up to 21 hours at several temperatures and pH values. The r e s u lts are presented in Table IX. Four portions of a n tib io tic beer were adjusted to pH values of 2, 4, 8 and 10* The four portions were then dispensed 43 TABLE VIII PURIFICATION OF THERMOVIRIDIN BY COUNTERCURRENT DISTRIBUTION Tube Number mg Active Material in Fraction Total Units Thermoviridin in Fraction Approximate Units per mg % Recovered in Fraction Butanol Fractions 1 3.80 0 0 0 2 1.68 84.0 50 5.4 3 1.31 171.0 130 11.0 4 0.57 153.9 270 9.9 5 1.74 0 0 0 6 2.32 0 0 0 7 6.55 0 0 0 8 1.14 0 0 0 1 3.50 0 0 0 2 0 99.7 3 7.31 121.8 17 7.9 4 7.41 171.0 23 11.0 5 2.32 133.4 58 8.6 6 0 92.8 7 5.83 0 0 0 8 1 1 .2 2 0 0 0 Buffer Fractions 6.4 6.0 Total recovery 66.2% Potency of s ta rtin g m aterial 40 units per mg 44 in t e s t tubes in 1 ml amounts and placed at the desired temperatures* At each time in te rv a l tubes were removed; those a t acid pH were neutralized with solid sodium bicarbonate. The amount of a n t i b i o t i c a c tiv ity remaining was then tested by the paper disc assay method* TABLE IX STABILITY OP THERMOVERIDIM TO VARIOUS pH VALUES MD TEMPERATURES Percent Antibiotic Activity Remaining Time 9C■c in Hrs • pH2 pH4 pH8 pHlO 24c*c 37° pH2 pH4 pH8 pHIO pH2 PH4 pH8 pHl< 1 100 100 100 100 100 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 100 100 100 100 4 100 100 100 100 100 100 100 100 100 100 100 100 8 100 100 100 100 100 100 100 75 100 100 100 50 21 100 100 100 100 100 100 100 50 100 100 100 25 The purpose of the s t a b i l i t y studies was to determine the length of time th at thermoviridin would withstand acid or alk alin e conditions a t several temperatures* This informa­ tio n was needed before the investigation of possible recov­ ery methods could be undertaken. was a r b i t r a r i l y A time lim it of 21 hours chosen because in most recovery processes there is no need to hold the a n tib io tic a t acid or alkaline pH values f o r longer periods. Thermoviridin has not been found to lose a c t iv i t y in the p r e s e n c e of sheep blood, nor does i t hemolyze sheep red blood 45 ce lls. This was determined by the paper disc assay method. The a n t i b i o ti c appears to be of the b a c t e r i o s t a t i c type* Subcultures were made from s e r i a l d ilu tio n assay tubes which showed in h ib itio n a f t e r 24 hours incubation, beginning with the highest d i lu t i o n showing in h ib itio n and proceeding toward the lowest. In a two-fold s e r ie s with in h ib itio n at 1:64, subcultures of the 1:64 and 1:32 d ilu tio n s grew out, but that of the 1:16 d ilu tio n did n ot. Failure to obtain growth in the subcultures from the 1:16 d ilu tio n and below, may have been due to an actual b a c te ric id a l effect at the lower d i l u ­ tio n s , or to a weakened physiological condition of the c e lls which might not allow the I n i t i a t i o n of growth in the s u b - * c u ltu r e . Several q u a lita tiv e biochemical t e s t s have been per­ formed using material having 30 units per mg a c tiv ity d i s ­ solved in s u ff ic ie n t water to give a concentration of 1 mg per ml. The biuret t e s t was negative, indicating the ab­ sence of peptide linkages in the preparation. The nin- hydrin t e s t was negative, also indicating the absence of proteins and related compounds. No yellow color was noted with the xanthoproteic reaction which would Indicate that no phenyl groups were present In the m aterial. The Molisch t e s t was also negative, suggesting the absence of carbo­ hydrates. Sulfur was not present in the active m aterial, as determined by the lead acetate t e s t . The above t e s t s were performed according to the methods described by Hawk, Oser and Summerson (16). 46 A water solu tio n containing 2 mg per ml of 30 unit per mg material contained 10*3$ nitrogen. The micro-Kjeldahl method of Ma and Zuazaga (21) was used for the determination. The above t e s t s are sig n ific a n t for nitrogen were negative. in th a t a l l but the t e s t In a preparation of only par­ t i a l p u rity , p o sitiv e t e s t s do not n ecessarily give any in ­ dica tio n of the chemical nature of the a n tib io tic i t s e l f , since impurities may well be responsible fo r the p o sitiv e reaction* Thermo v ir id in could be characterized as a chemical compound only i f a c r y s ta l li n e product were available, but in the l i g h t of the above data and also s o l u b i li t y data, several generalizations concerning i t s chemical nature can be made. According to Florey et_. a l . (13), a compound which has p r e f e r e n t ia l s o l u b i li t y in an organic solvent under acid conditions, is i t s e l f acid in nature. This Is true of thermo v i r id i n , which in the presence of both water and butanol favors th e butanol phase a t acid pH and the water phase at alk aline pH. Thus i t appears t h a t thermo v irid in is an organic acid which contains no su lfu r or phenyl groups, but which may contain nitrogen. The free acid was found to have only lim ited s o lu b ility in water, whereas the sodium s a l t was soluble to the extent of a t l e a s t 300 units per' ml. beyond th is le v e l. Its s o lu b ility was not teste d 47 Thermoviridin was also found to be of such molecular size that i t was d ialy za b le . system. This was demonstrated i n a water Dialysis was carried out a t room temperature for a 24 hour period. Toluene was added as a p reservativ e. The u l t r a v i o l e t absorption spectrum of thermoviridin was determined in a Beckman quartz spectrophotometer. Two prepara tions from the same experiment but having 30 units per mg and 80 u n its per mg a c tiv ity respectively were t e s t e d . A water solutio n of each preparation containing 0.04 mg per ml was used. Therefore, the f i r s t solution contained 1.2 units of thermoviridin per ml, and the second, 3.2 units per ml. It was necessary to use a solution of low concentration so th a t there would be s u f fic ie n t l ig h t transmission for a reading in the lower portion of the u l tr a v io l e t spectrum. The u l tr a v io l e t absorption spectrum of a th ir d prepara­ tion having 30 units per mg a c t iv i t y was also determined. The water solution tested contained 0.25 mg of material per ml, or 7.5 units per ml. The re s u lts of the three determina­ tions are found in figure 8. The r e s u lt s show t h a t absorption increases with an i n ­ crease in thermoviridin concentration. had 1.2 and 3.2 units The preparations which a c tiv ity per ml showed a maximum ab­ sorption peak at a wave length of 268mu,, whereas the solu­ tio n contained 7.5 units per ml showed maximum absorption at 272mu. This difference is not considered s ig n if ic a n t, and may have been due in part to the degree of s e n s i t i v i t y of the spectrophotometer. The absorption shown is a t t r i b ­ uted to thermoviridin and Is considered to be c h a ra c te r is tic of i t . 48 0.70 Thermoviridin concentration A 1.2 units per ml C 7.5 units per ml 280 300 0.60 Optical density 0.40 0.50 _ 0.20 _ 0.10 0.08 220 240 260 320 Wave length in millimicrons Figure 8. U ltraviolet absorption spectrum of thermoviridin 340 49 The microbiological spectrum of thermoviridin is pre­ sented in Table X. The s e n s i t iv i t y of M. pyogenes var. aureus 2Q9P was taken as the standard to which the s e n s i t iv ­ i ty of a l l other organisms was compared* I t w ill be noted th a t two of the micrococcus s tr a in s are p e n i c il l in re s is ta n t* S train 193 was found to require 200 times as much p e n i c il l in fo r in h ib itio n as the 209P s tr a i n , whereas the 218 s t r a i n r e ­ quired 1600 times as much as the 209P strain* Table X shows th a t a four-fold Increase in the concentration of thermo­ v i r id in was s u f f i c i e n t for the in h ib itio n of both r e s is t a n t stra in s• The s e n s i t i v i t y of a l l b ac terial cultures except the mycobacteria was tested using the two-fold s e r i a l d ilu tio n method. The agar d ilu tio n method was used in testin g the s e n s i t i v i t y of the mycobacteria and fungi. The streptococci and D. pneumoniae type I I I were grown in Felton* s broth. infusion broth, e x tra c t agar. _C. diphtheriae PW B was grown in veal and the mycobacteria, on glycerol-beef The remaining bacteria and fungi were grown on try p tle a se broth and agar respectively. Anaerobic conditions were obtained for the growth of Clostridium t e t a n i (Pease) and Vibrion seoticum by the use of germinating oats in a desiccator (22)• The pressure within the desiccator was reduced by means of a vacuum pump, thereby hastening the attainment of anaerobic conditions. The values given fo r a l l bacteria in Table X except those fo r the mycobacteria are based on the degree of in h i­ b itio n a f t e r 18-24 hours incubation at 37°C. The values 50 TABLE X THE MICROBIOLOGICAL SPECTRUM OP THERMOVIRIDIN u/ml required for in h ib itio n Organism Micrococcus pyogenes v ar. aureus 209P 1 218* 4 it 1 9 3 '* 4 tt B314 4 tt SM 4 tt » n 16 Streptococcus viridans 31 tt n 143 16 4 Streptococcus hemolyticus SPIV " M K64C 4 » » 316A 16 ** » 314B 16 4 Diplococcus pneumoniae type I I I 16 Corynebacterium diphtheriae PW 8 Sarcina lu te a PCI-1001W ♦25 Bacillus s u b t i l i s .5 231 Clostridium t e t a n i Pease s t r • .03 Vibrion septicum Proteus vulgaris 1414 64 E sc h e r ic h ia c o l i PDA 64 64 Shigella paradysenteriae 15001 S a l m o n e l l a typhimurium Edward s t r . M y c o b a c t e r i u m tuberculosis var. h o m i n i s H37 9 64 > 26 51 TABLE X Continued Organism Mycobacterium phlei 1201 u/ml required for in h ib itio n > 26 Trichophyton mentagrophytes >26 Histoplasma capsulatum >26 Blastomyces derm atiditis >26 Nocardia asteroides >26 Aspergillus >26 fumigatus Candida albicans p en ic illin r e s is ta n t >26 52 given for the mycobacteria are based on the degree of com­ plete in h ib itio n a f te r 6 weeks incubation at 37°C, and those f o r th e fungi, on the degree of complete in h ib itio n a f t e r 3 weeks incubation at room temperature. Toxici ty In jectio n of 32 mg of material containing 25 units per mg a c t iv i t y into each of 3 mice by the in tra p e rito n e a l route showed no l a s ti n g toxic e f f e c t . During the f i r s t 24 hours following in je c tio n the mice were sluggish and generally i n ­ ac tiv e , but l a t e r appeared healthy and a c tiv e . The mice were observed for a three-week period following in je ctio n and during that time increased t h e i r weight from approx­ imately 20 grams to 27 grams. Following the period of observation, the mice were sacrificed and an autopsy was performed. was normal. The gross appearance of the in te rn a l organs DISCUSSION In considering the production of thermoviridin, i t is unusual th a t the lev e l of a n tib io tic a c tiv ity obtained with the tryptone-beef extract medium could not be improved s ig ­ n ific a n tly . Throughout the media stu d ies, media which con­ tained various peptone-like products in combination with beef ex tra ct were consistently most successful. In the case of thermoviridin, the factor which limited a n t i b i o ti c production was not found. The deficiency, was the case, may have been in the carbon, nitrogen, or mineral source. i f such vitamin, With the correction of such a deficiency, i t would seem fe a s ib le that thermoviridin production could be g reatly increased. Waksman (36) has stated th a t, as a r u le , actlnomycetes p refe r proteins to carbohydrates as a carbon source. Tryp- tone serves as a' source of both carbon and nitrogen for Thermoactinomyces v i r i d i s . This is supported by the f a c t th a t growth has been obtained using only a 4% tryptone solu­ tio n as the fermentation medium. The beef extract is be­ lieved to serve as a source of supplementary growth factors and minerals. Of the n u trie n ts added to the tryptone-beef extract fermentation medium, whole wheat and glycerol stimulated the growth of T. v i r i d i s to some extent, although there was no s ig n if ic a n t enhancement of thermoviridin production. 54 Neither of these m aterials was therefore included as a permanent constituent of the fermentation medium. Most a n t i b i o ti c fermentations require approximately 72 hours to reach maximum production, whereas only 27 hours were required fo r maximum thermoviridin production at 45° C. P u rific a tio n of the dry a n tib io tic m aterial obtained from th e acetone e x tra c t was only p a r t i a l l y achieved. Countercurrent d is trib u tio n was found to be the most effec­ tiv e of the two methods used. Had commercial equipment been available so that many more tra n s fe rs could have been made, a much purer product could lik e ly have been obtained. Battersby and Craig (1) have reported the is o la tio n of c r y s t a l l i n e tyrocidine by th is method, so th a t even a c r y s ta l li n e product is possible. *Countercurrent d is tr ib u tio n as described by Craig, and f r a c tio n a l p re c ip ita tio n are primarily laboratory to o ls, are not p ra c tic a l fo r large scale p u rific a tio n . and Such methods are of great value in purifying an a n tib io tic and are a great aid in reaching the ultimate goal of p u rific a tio n , a c r y s ta l ­ l in e product. The s t a b i l i t y of thermoviridin at temperatures above 37°C was not te s te d system atically. However, autoclaving 10 ml of a n t i b i o ti c beer for 5 minutes at 121°C caused a reduction in a c t iv i t y of approximately 50$. A more thorough investigation of s t a b i l i t y a t temperatures above 57°C was considered important only i f a c ry s ta llin e product were a v a ila b le , which was not the case. Vfith the exception of the u l t r a v i o l e t absorption spec­ trum, the chemical c h a ra c te ristic s of thermoviridin have 55 been determined f o r the most part by in d ir e c t evidence* The negative q u a l i t a t iv e color t e s ts indicate the absence of proteins and carbohydrates in the preparation tested# The s o l u b i li t y c h a ra c te ris tic s of thermoviridin under acid and alk alin e conditions indicate th a t the a n t i b i o ti c is an acid# More d e f i n i te conclusions concerning the chemical nature of th is a n t i b i o ti c cannot be drawn from the data presented* The microbiological spectrum of thermoviridin is not a t a l l unusual or unique. I t is primarily the gram positive b a c te ria which are inhibited by the antibio tic* The fungi were found to be in se n sitiv e to thermoviridin at the levels tested# However, Histoplasma capsulatum and Blastomyces derm atiditis showed some s e n s i t iv i t y to 26 units per ml of thermoviridin when assayed i n i t i a l l y , but did not appear s e n s itiv e when the assays were repeated. levels Thus, a t higher of concentration thermoviridin might be more effec­ tiv e against these organisms# The limited supply of the a n t i b i o t i c prevented such testing# Speculation as to the possible chemotheraputic value of thermoviridin i s perhaps in order. Since i t does I n h ib it p e n i c i l l i n r e s i s t a n t micrococci in rather low concentrations, the possible value of thermoviridin would be i t s use as an adjunct to p e n i c i l l i n . I f the two fungi mentioned above were found to be se n sitiv e to thermoviridin a t higher concentra­ tio n s , the future of the a n tib io tic would then be more promising• SUM M ARY A thermophilic actinomycete was iso la te d from a composted manure p i l e . Since no previous descriptio n of th is organism was found, i t was considered a new species of the genus Thermoa ct inomy ce s (Tsiklinsky)* v irid is The name Thermoactinomyces is suggested fo r the organism, and the name thermo­ v i r id i n fo r the a n t i b i o ti c produced by i t . Thermoviridin was produced i n i t i a l l y b o t tl e cu ltu res. in stationary Subsequent production was carried out on a Gump ro tary shaker for the most p a rt, and also in a 30 l i t e r laboratory fermenter. The fermentation medium consisted of Bacto tryptone 4$, Bacto beef extract 0*5$, and had a pH of 6.9 - 7.1* The addition of a number of n u trie n ts to this medium did not increase a n t i b i o ti c potency above the 32 units per ml ob­ tained in the basal medium. Maximum a n tib io tic production was reached In 27 hours a t 45°G. Recovery of thermoviridin was accomplished by acid p r e c ip ita tio n a t pH 3. The p re c ip ita te was washed with water and then extracted with a quantity of 80$ acetone equal to one-tenth the o rigin al volume of the a n tib io tic beer. The acetone e x tra ct was evaporated, and the remain­ ing water residue was dried from the frozen s t a t e . The solid material obtained by t h is procedure had 64 units per mg a c t iv i t y against M. pyogenes var. aureus 209P. 57 Some p u rific a tio n of the active solid m aterial was accomplished by fr a c tio n a l p re c ip ita tio n from methanol using ethyl ether as the p re c ip ita tin g agent. The countercurrent d i s t r ib u t i o n method of Craig was also an e ffe ctiv e method of p u rific a tio n . A preparation having 270 units per mg a c tiv ity was obtained by t h i s method, using a butanol-water system buffered at pH 6. Thermoviridin was found to be stable at pH 2 for 21 hours a t 57°C, but at pH 10 for the same time period approx­ imately 7bfo of the a n t i b i o t i c a c tiv ity was l o s t . The s t a ­ b i l i t y of thermoviridin to temperatures higher than 37°C was not in v estig ate d . Chemical t e s t s and s o lu b ility data indicate th at thermo­ v ir id in i s groups. an organic acid which contains no su lfu r or phenyl The active material appeared to be non-protein and non-carbohydrate in nature. Thermoviridin was dialyzable and could be prec ip itate d with saturated ammonium s u lf a te . The a n t i b i o ti c preparations tested showed maximum ab­ sorption in the range of 268-272mu. Thermoviridin was primarily active against the gram positiv e b acteria te s te d . mice, consisting of a single in tra p erito n e al in je ctio n of 52 mg of material case. Preliminary to x ic i ty te s ts in (800 u n its ) , did not cause death in any An autopsy of the mice showed no abnormalities in the gross appearance of the organs. REFERENCES 1* 2. Battersby, A . R . , and I»* C. Craig ( 1 9 5 2 ) The c h e m i s t r y oftyrocidine* I. Iso latio n and characterizatio n of a single peptide. Jour. Amer. Chem. Soc. 74: 4019 Bernstein, A., and H* E. Morton (1934) A new thermo­ p h ilic actinomyces. Jour. Bact. 2^7: 625 3* Breed, R. S ., E. G. D. Murray, and A. P. Hitchens (1948) BergeyTs manual of determinative bacteriology, ed. 6, Williams and Wilkins Co., Baltimore 4. Burke, V. (1922) Notes on the gram s ta in with descrip­ tio n of a new method. Jour. Bact. 7: 159 5. Compilation of regulations for t e s t s and methods of assay and c e r t i f i c a t i o n of a n tib io tic and a n t i b i o ti c containing drugs, vol. I , U. S. Dept, of Health, Education, and Welfare; Pood and Drug Administration 6 . Craig, L. C. (1944) Id e n tific a tio n of small amounts of organic compounds by d is trib u tio n stu dies, I I . Separa­ tio n by counter-current d i s t r ib u t i o n . Jour. Biol. Chem. 155: 519 7. _____________ (1950) P a rtitio n chromatography and countercurrent d i s t r ib u t i o n . Anal. Chem. 22: 1346 8. ______________, and 0. Post (1949) Apparatus for countercurrent d i s t r i b u t i o n . Anal. Chem. 21: 500 9. 10 . 11. ____________, W. Hausmann, E. H. Ahrens J r . , and E. J . Harfenist (1951) Automatic countercurrent d i s t r i b u ­ tion equipment. Anal. Chem. 23: 1236 de Beer, E. J . , and M. B. Sherwood (1945) The paperdisc agar-plate method f o r the assay of a n tib io tic substances. Jour. Bact. J50: 4-59 Erickson, D. (1941) Micromonospora. Studies on some lake-mud s tra in s of Jour. Bact. 41: 277 ________, (1952) Temperature/growth relationsh ip s of a thermophilic actinomycete, Micromonospora v u lg a ris. Jour. Gen. Microbiol. 6: 286 59 13. Florey, H. W., E. Chain, N. G. Heatley, M. A. Jennings, A. 0* Sanders, E. P. Abrahan. , and M. B. Florey (1949) A n tibio tics; a survey of p e n i c i l l i n , streptomycin, and other antimicrobial substances from fungi, actinomycetes, b a c te ria , and p la n ts, vol. I , Oxford Univer­ s i t y Press, London 14 * G ilb ert, A. (1904) Uber Actinomyces thermophilus und andere Actinomyceten. Z ietschr. Hyg. 47: 383 15. Globig, L. (1888) Bakterien-Wachs turn bei 50 bis 70°. Z eitsch r. Hyg. 3: 294 16. Hawk, P. B., B. L. Oser, and W. H* Surmnerson (1947) P ra c tic a l physiological chemistry, ed. 12, Blakiston Co. , Philadelphia 17. Katznelson, L. (1940) Autolysis of a thermophilic aetinomyces. Soil Sci. 49: 83 18. Kedzior, D. £1896) Uber eine thermophile Cladothrix. Archiv fur Hyg* 27: 328 19. Kelner, A. (1948) Mutation in Streptomyces flaveolus induced by X-rays and u l tr a v io l e t l i g h t . Jour. Bact. 56: 457 20. Lieske, R. (1921) Morphologie und Biologie der S trahle np ilze. Leipzig Borntraeger 21. Ma, T. S ., and G. Zuazaga (1942) Micro-Kjeldahl determina­ tion of nitrogen, a new in dicato r and an improved rapid method. Xnd# Eng. Chem., Anal. M . 14 280 22. Manual of methods for pure culture study of b a c te ria . Biotech Publications, Geneva, U. Y. 25. Miehe, H. 24. Murray, H. C. (1944) Aerobic decomposition of cellulose by thermophilic b a c te ria . Jour. Bact. 47: 117 25. Noack, K. (1912) Beitrage zur Biologie der thermophilen Organismen . Jahrb. Wiss. Bot. 51: 593 26. Olson, B. H., J. C. Jennings, M. Pisano, and A. J. Junek (1954) Production, recovery, and p u rific a tio n of synnematin A. and B. A ntibio t. and Chemother. 4: 1 27. Rabinowitsch, L. (1895) Ueber die thermophilen Bakterien. Z eitsch r. Hyg. 20: 154 (1907) Die Selbsterhitzung des Heues, Jena 60 28* Riddell, R. W. (1950) Permanent stained mycological preparations obtained by slid e culture* Mycologia 42: 265 29* Sames, T. (1900) Zur Kenntnis der bei hoher Temperatur wachsenden Bakterien und S tre p to th rix arten . Z eitschr. Hyg. 33: 313 30. Savage, G. M. (1949) Improvement in streptomycin-produc­ ing s tra in s of Streptomyces griseus by u l tr a v io l e t and X-ray energy. Jour. Bact. 57: 429 31. Schmidt, W * H., and A. J. Moyer (1944) P e n ic illin ; Methods of assay. Jour. Bact. 47: 199 32* Schone, R. (1951) An a n tib io tic which In h ib its Corynebacterium diphtherias produced by the S form of Streptomyces thermophilus. A ntibiot. and Chemother. 1: 176 33. Schutze, H. (1908) Beitrage zur Kenntnis der thermophilen Aktinomyzeten und iher Sporenbildung. Archiv fur Hyg. 67: 35 34. Tsiklinsky, P. Ann. I n s t . 35. Waksman, S. A. (1940) On the c la s s if ic a tio n cetes. Jour. Bact. 39: 549 36. ___________ (1950) The actlnomycetes, t h e i r nature, occurence, a c t i v i t i e s , and importance. Chronica Botanica Co., Waltham, Mas s • 37. ___________, f . C* Cordon, and N. Hulpoi (1939) Influence of temperature upon the microbiological population and decomposition in composts of stab le manure. Soil Sci. 47: 83 38. , and C. T. Corke (1953) Ther moa ct i n omyce s Tsiklinsky, a genus of thermophilic actinomycetes. Jour. Bact. 66p 377 39* , and A. T. Henrici (1943) The nomenclature and c l a s s i f i c a ti o n of the actinomycetes. Jour. Bact. 46: 337 40. , E. S. Horning, M. Welsch, and H. B. Woodruff (1942) D istribution of antagonistic actinomycetes in nature. Soil Sci. 54: 282 41. I* IT IT ' tt (1899) Sur les mucedinesthermophiles. Pasteur 13: 500 of actinomy- , and II. A. Lechevalier (1953) Guide to the c l a s s i f ie a tio n and id e n tif ic a tio n of the actinomycetes and t h e i r a n t i b i o ti c s . Williams and Wilkins Co., Baltimore 61 42. __________, W. W. Umbreit, and T. C. Cordon (1959) Thermo­ p h ilic actinomycetes and fungi in s o ils and in com­ p o sts. Soil Sci. 47: 57 45. ■iifest, E. S ., and V tf. R. Todd (1952) Textbook of bio­ chemistry. Macmillan Co., N. Y.