Antibiotic substances from yeasts

Citation preview

ANTIBIOTIC SUBSTANCES PROM YEASTS

A T h e s is P re s e n te d to t h e F a c u lty o f t h e D e p artm en t o f B a c t e r i o l o g y U n iv e r s ity o f S o u th e rn C a li f o r n ia

In P a r t i a l F u lfillm e n t o f t h e R e q u ire m e n t f o r t h e D egree o f M a ste r o f S c ie n c e

by M i t s u r u N a k a m u ra Ju n e 1950

UMI Number: EP55015

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.

Dissertation Publishing

UMI EP55015 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code

ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 40106 *1346

This thesis, written by

MITSURU ..NAKAMURA under the guidance of h%3.... Faculty Committee, and ap pro ved by all its members, has been presented to and accepted by the Council on Graduate S tu dy and Research in partial fulfill­ ment of the requirements for the degree of

MASTER OF SCIENCE

Date

1 M M ..1 9 5 0

Faculty Committee

TABLE OF CONTENTS

CHAPTER I.

II. III.

THE

PAGE PROBLEM

.........................................

1

S t a t e m e n t o f t h e p r o b l e m ...............................

1

I m p o r t a n c e o f t h e s t u d y ....................................

1

REVIEW OF THE L I T E R A T U R E ..........................

3

METHODS AND R E S U L T S ...............................

15

E X P E R IM E N T S ................................................ S c re e n in g o f y e a s ts f o r a n t i b i o t i c

15 a c ti­

v i t y b y a d j u s t i n g t h e p H .............................

16

B est carb o n so u rce f o r a n t i b i o t i c p ro ­ d u c tio n by T o ru la u t i l i s

. . . . . .

19

B est n itro g e n so u rce f o r a n ti b i o ti c p r o d u c t i o n b y T . u t i l i s ..............................

20

E f f e c t o f s h a k in g t h e c u l t u r e on a n t i b i ­ o tic p ro d u c tio n

....................................

E x tra c tio n o f th e a n t i b i o t i c

22

su b s ta n c e

f r o m t h e m e d i u m ........................................ U se o f s y n t h e t i c m e d i a . . . . . . . .

23 26

O b ta in in g a b a c t e r i a l sp e ctru m f o r th e a n tib io tic

. . . . .

T e s tin g f o r h e a t s t a b i l i t y a n tib io tic T o x ic ity t e s t s

31

o f th e . . . . .

o n r a t s ....................................

31 34

Hi CHAPTER XV.

PAGE

SUMMARY AND CONCLUSIONS'

BIBLIOGRAPHY

. . . . . . . . . . . .

.............................................................................................

35 38

L IST OP TABLES

TABLE I. II. III.

PAGE The C o m p o s i t i o n o f U n i v e r s a l y e a s t M e d ium *. .

15

Y e a sts S c re e n e d f o r

15

A n tib io tic A c tiv ity

. . .

A n t i b i o t i c A c t iv i t y o f F i l t r a t e s o f Y e a s ts Grown a t V a r y i n g PH L e v e l s A g a i n s t S . a u r e u s

IV .

A n tib io tic A c tiv ity of F i l t r a t e s

o f Y e a sts

Grown a t V a r y i n g PH L e v e l s A g a i n s t E . o o l i V.

X II.

20

.

.....................

22

m edium

. . . . . . .

24

• • • • • • • • • •

27

E n d -P ro d u c ts o f Y east F e rm e n ta tio n o f S ugar in R e la tiv e P ro p o rtio n s

X I.

. . . . . . .

C o m p o s i t i o n s o f S y n t h e t i c M e d ia U sed f o r A n tib io tic P ro d u c tio n

X.

18

M easu rem en t o f I n h i b i t i o n P ro d u c e d by E t h e r E x t r a c t o f T. u t i l i s

IX .

.

E f f e c t o f N itro g e n S ource on A n t i b i o t i c P ro d u c tio n by T o ru la u t i l i s

V III.

.

E f f e c t o f C arbon S o u rc e on A n t i b i o t i c P r o d u c t i o n by T o r u l a u t i l i s

V II.

17

F u r t h e r S t u d i e s on t h e E f f e c t o f PH o n t h e P ro d u c tio n o f A n t i b a c t e r i a l S u b sta n c e s

V I.

17

U su a lly P r e s e n t

. . .

P r o p e r t i e s o f E n d - P r o d u c t s ................................... ...

50 .

50

B a c t e r i a l S p e c tru m O b ta in e d from a n A n t i b i o t i c P ro d u ced by T. u t i l i s

on S y n t h e t i c Medium

52

CHAPTER I

THE PROBLEM A n tib io tic s a re p la y in g an im p o rta n t r o le o f m e d ic a l s c ie n c e s to d a y .

and stre p to m y c e s, of

s u b s ta n c e from y e a s t s .

S ta te m e n t o f th e p ro b le m ,

The p u r p o s e o f t h i s

stu d y

surv ey te n y e a s ts f o r th e p ro d u c tio n o f an a n t i b i o t i c

p rin c ip le . a g e n t,

m o ld s,

w o rk h a s b e e n r e p o r t e d o n t h e p o s s i b i l i t y

p ro d u c in g an a n t i b i o t i c

w as t o

fie ld

Many a n t i b i o t i c s h a v e b e e n i s o ­

l a t e d a n d d e s c r i b e d from b a c t e r i a , b u t v ery l i t t l e

in th e

I f a y e a s t w as f o u n d t h a t f o r m e d a n a n t i b a c t e r i a l

t h e a im w as t o d e t e r m i n e t h e o p tim u m c o n d i t i o n s

maximum a n t i b i o t i c p r o d u c t i o n , th a t d e ta ile d stu d ie s o f i t s

and to i s o l a t e

th e a g e n t so

sp e c tru m c o u ld be o b t a i n e d .

Im p o rta n c e o f th e s t u d y . a n t i b i o ti c s have been d isc o v e re d , m e d ic in e .

D e s p i t e t h e f a c t t h a t m any re la tiv e ly

few a r e u s e d i n

U n le s s t h e n e w ly d i s c o v e r e d a g e n t d e m o n s tr a te s a

m a rk e d ly d i f f e r e n t sp e c tru m a g a i n s t p a th o g e n s i t fe a sib le

fo r

to d e v e lo p i t

is not

c o m m e rc ia lly i n c o m p e titio n w ith

com pounds f o r w h ic h t h e

f a c t o r s o f p r o d u c t i o n a r e known.

The f a c t o r s c o n t r o l l i n g t h e p r o d u c t i o n o f y e a s t s a r e m o re f u l l y u n d e r s t o o d t h a n f o r a n y o t h e r g r o u p o f o r g a n i s m s . A good a n t i b i o t i c

fro m t h i s

and i n la r g e q u a n t i t i e s .

so u rc e c o u ld be p ro d u ce d c h e a p ly

I t was h o p e d t h a t i n

se a rc h in g f o r

2 a new a n t i b i o t i c be e f f e c tiv e

fro n r y e a s t ,

o n e m i g h t be* f o u n d t h a t w o u l d

a g a i n s t a w id e v a r i e t y

o f o rg a n ism s.

CHAPTER I I

REVIEW OP THE LITERATURE F o r m any y e a r s t h e r e h a s h e e n a p o p u l a r t h e o r y t h a t a b s c e s s e s a n d p i m p l e s w i l l d i s a p p e a r w hen y e a s t i s a n d t h a t i t w as o f i m p o r t a n c e a l s o sk in d is e a s e s .

in g e s te d

i n th e tre a tm e n t o f o th e r

L ong b e f o r e s c i e n t i s t s

re a liz e d

c o n t a i n e d v i t a m i n s , p h y s i c i a n s w ere u s i n g i t

th a t y east

f o r th e ra p y

a g a in s t sk in d is o r d e r s . The a n t i b a c t e r i a l a c t i v i t y

o f t h e y e a s t w as f i r s t

r e p o r t e d b y S e r g e n t ( 1 9 0 3 ) , who r e p o r t e d a s u b s t a n c e p r o ­ d u c e d b y y e a s t w h i c h w as t o x i c t o B u rn e tt

(1905) r e p o r t e d

S ta p h y lo c o c c u s. a u re u s .

th e use o f y e a s t as a d i s i n f e c t a n t .

H ayduck (1 9 0 9 ) m e n tio n s i n h i s p a p e r t h a t L ange, H e n n eb e rg , an d H ayduck h a v e d e t e c t e d

th e p re s e n c e in

c e rta in c e re a ls

o f s u b s t a n c e s t h a t a r e p o is o n o u s t o y e a s t w hen d i s s o l v e d to g e th e r w ith su g a r in d i s t i l l e d w a te r.

He w as a b l e t o

o b t a i n a s i m i l a r s u b s t a n c e f ro m d i s t i l l e r ’ s y e a s t w hen i t w as d r i e d r a p i d l y a t h i g h t e m p e r a t u r e s , a n d e x t r a c t e d w i t h w a te r c o n ta in in g 0 .1 p e r c e n t h y d ro c h lo ric a c id . t h a t no s u c h p o is o n o u s e x t r a c t i s t h a t h as been d r ie d slo w ly , try p tic

o b ta in a b le

o w in g p r o b a b l y t o

enzym es upon th e p o is o n o u s p r o t e i n .

He f o u n d

from y e a s t th e a c tio n of He e x t r a c t e d a t

b o t h 2 5 ° C. a n d a t 5 0 ° G ., b u t f o u n d t h a t a n e x t r a c t i o n a t 5 0 ° C. g a v e b e t t e r r e s u l t s .

A t 50° C .,

th e e x tr a c tio n

is

4 c o m p l e t e d i n tw o h o u r s .

H is e x p e r im e n ts i n d i c a t e d

su lfu ric

a c id can re p la c e h y d ro c h lo ric

a c id n o r a c e tic

in th is e x tra c tio n p ro cess. from b re w e r* s y e a s t , some o f i t s p ro p er.

th a t n e ith e r a c id

To p r e p a r e a p o i s o n o u s e x t r a c t

h e f o u n d t h a t i t w as n e c e s s a r y t o re m o v e

lim e by d i l u t e

a c id p re v io u s to

th e e x tr a c tio n

C a lc iu m s a l t s p r e v e n t th e p o i s o n o u s e f f e c t .

y e a s t w as w e a k e n e d b y s t o r a g e b e f o r e b e i n g d r i e d , p o iso n o u s e x t r a c t r e s u l t e d .

The a c t i o n o f t h i s

I f th e

a le ss

s o l u t i o n w as

i n t e n s i f i e d by d i a l y s i s . L a te r in

t h e sam e y e a r ,

F ernb aeh

(1 909),

m a t e l y t h e sam e p r o c e d u r e a s H a y d u c k ( 1 9 0 9 ) ,

u sin g a p p ro x i­

fo u n d an a c t i v e

su b s ta n c e t h a t c o u ld k i l l b a c t e r i a .

He e x t r a c t e d a m a c e r a ­

tio n o f d e s s ic a te d y e a s t w ith d ilu te

(1 :1 0 0 0 ) h y d r o c h lo r ic

a c i d f o r 19 h o u r s a t 3 7 ° C . , a n d f i l t e r e d n e u tra liz a tio n ,

th e f i l t r a t e

y e a s t i t s e l f as w e ll as to b a c te ria .

th e

s o lu tio n .

e x h i b i t e d m arked t o x i c i t y

A fte r to th e

v a r io u s o t h e r s t r a i n s and to

In h i s e x p e rim e n ts he u se d one B a c te riu m c o l l and

one S ta p h y lo c o c c u s a u re u s pyogenes s t r a i n .

The s o l u t i o n

p rov ed to be b a c t e r i c i d a l to b o th o f th e s e ,

t h o u g h so m e w h a t

m o re i n t e n s e a g a i n s t t h e l a t t e r . v o la tile .

Upon d i s t i l l i n g

He f o u n d t h e t o x i n t o b e

th e f i l t r a t e

under d im in ish e d

p r e s s u r e such t h a t th e te m p e ra tu re d id n o t r i s e t h e t o x i c i t y w as t r a n s f e r r e d r e s i d u a l l i q u i d w as i n a c t i v e .

to th e d i s t i l l a t e

a b o v e 4 0 ° C. , w h ile th e

No r e a c t i o n s w e r e o b t a i n e d f o r

a l d e h y d e s —n o t a b l y f o r m a l d e h y d e - - to w h ic h b a c t e r i c i d a l a c t i o n

5 m ig h t h e a t t r i b u t e d *

H a y d u c k ( 1 9 0 9 ) r e p e a t e d som e o f F e r n -

b a c h 1s w o r k b u t ' c o u l d n o t o b t a i n t h e F e m b a c h and V u lq u in a c t i o n ' i s .d u e t o a v o l a t i l e th e a m in e s. c ry sta ls

to x ic e x tr a c t.

(1909) s a i d t h a t th e a n t i b a c t e r i a l s u b s t a n c e I n s v in g t h e r e a c t i o n

T hey s a i d t h a t t h e t o x i c s u b s t a n c e i s o l a t e d

from h y d r o c h l o r i c

a c id re se m b le s th e

of as

c o m p le x a m i n e s

b e c a u s e t h e y h a v e o b s e rv e d no a n t i b a c t e r i a l pow er i n t h e s im p le a m in e s , n e i t h e r t h o s e am in es c o n t a i n i n g a s im p le a lc o h o l r a d i c a l , tim e s.

n o r th o se

s u b s titu te d one,

tw o , o r t h r e e

They r e p o r t i n a n o t h e r p a p e r ( 1 9 1 0 ) t h a t t h e t o x i c

su b s ta n c e s e x tr a c te d

from m a sh es o f y e a s t a r e d i f f e r e n t fro m

th o s e e x t r a c t e d from c e r e a l s . fro m w h e a t m ash w as a c t i v e

T hey s a i d t h a t t h e d i s t i l l a t e

i n s u p p r e s s i n g y e a s t g ro w th o n ly

i n th e p re s e n c e o f su g a r w h ile th e d i s t i l l a t e

f r o m y e a s t m ash

w as i n d e p e n d e n t o f a s o u r c e o f s u g a r . F ern b ach

(19 1 0 ) fo u n d a t o x i c

su b s ta n c e u sin g a

s l i g h t l y d i f f e r e n t t e c h n i q u e fro m t h e one h e u s e d e a r l i e r . He d r i e d

t h e y e a s t r a p i d l y a t 7 0 ° C. t r e a t e d w i t h v e r y d i l u t e

h y d ro c h lo ric a c id .

He a l s o o b t a i n e d a t o x i c

su b s ta n c e

from

,4

y e a s t t h a t h a d b e e n d r i e d a t o n l y 3 7 ° C. b y h e a t i n g 2 g ra m o f y e a s t w i t h 2 0 0 m l . o f 0 . 1 p e r c e n t h y d r o c h l o r i c a c i d f o r 19 h o u r s a t 3 7 ° C.

The t o x i c

s u b s t a n c e was d e s t r o y e d a t 1 0 0 ° C . ,

b u t w as v o l a t i l e w i t h s t e a m u n d e r r e d u c e d p r e s s u r e a t a t e m p e r a t u r e n o t e x c e e d i n g 4 0 ° C.

6 K ra e m e r ( 1 9 1 6 ) m e n t i o n e d a c o m m e r c i a l y e a s t p r o d u c t f o r t h e t r e a t m e n t o f p u t r i d w ounds, u n d e r t h e name X e r a s e ♦

u lc e rs,

e tc .,

m a rk e te d

T h e. fo rm u la c o n s i s t e d o f d r i e d y e a s t ,

100 p a r t s ;

d e x tro s e ,

2 0 p a r t s * w h i t e c l a y o r a lu m in u m s i l i c a t e ,

125 p a r t s ;

n u trie n t s a lts ,

Hawk, K n o w le s ,

3 p a rts*

R eh fuse,

and C la r k e

(1917) fo u n d t h a t

P le is c h m a n n * s C o m p resse d Y e a s t o r b a k e r ’ s y e a s t was a u s e f u l re m e d y i n t h e t r e a t m e n t o f f u r u n c u l o s i s , a c n e v u l g a r ! e s , acne ro s a c e a ,

c o n stip a tio n ,

and c e r t a i n o t h e r c u ta n e o u s and

g a s tr o - in te s tin a l c o n d itio n s .

T hey w e r e n o t s u r e w h e t h e r t h e

■ b e n e f i c i a l e f f e c t o f y e a s t w as d u e a l o n e t o i t s a c t i o n o r t o some f i x e d e f f e c t o n t h e

la x a tiv e

in te s tin a l tra c t.

B o th

t h e f r e s h y e a s t a n d t h a t k i l l e d b y b o i l i n g w a t e r sh o w e d l a x a ­ t i v e p o w ers.

M u rlin e and M a t t i l l

(1925) u n d e rto o k th e i n v e s ­

t i g a t i o n t o s e e w h e t h e r y e a s t w h en t a k e n u n d e r c o n t r o l l e d c o n d itio n s has a tr u e in d ic a te d th a t i t

la x a tiv e a c tio n .

d id have a la x a tiv e a c tio n .

w as n o i n c r e a s e d m o i s t u r e y e a st,

T h e i r e x t e n s i v e w o rk A lth o u g h t h e r e

c o n te n t as th e r e s u l t o f ta k in g

t h e r e w as a m e a s u r a b l e i n c r e a s e i n t h e b u l k o f s t o o l . S c h ille r

(1 9 2 4 ) d i s t i n g u i s h e d b e tw e e n n a t u r a l a n t a ­

g o n i s m a n d t h e a n t a g o n i s m i n d u c e d w hen tw o o r g a n i s m s a r e c u ltu re d to g e th e r.

When w i n e o r b e e r y e a s t a r e g ro w n w i t h

b a c t e r i a i n th e p r e s e n c e o f s u g a r and no p r o t e i n ,

th e y e a s ts

f u n c t i o n a s a n ta g o n is ts and th e b a c t e r i a a r e d ig e s te d by a p ro te o ly tic

su b s ta n c e .

The a c t i o n d i s a p p e a r s i f

th e te m p e ra tu re

7 e x c e e d s 6 0 ° C. s o t h a t o n e may c o n c l u d e t h a t t h e s u b s t a n c e s a re th e rm o la b ile .

S c h ille r

(1 9 2 7 ) s a y s t h a t t h e l y t i c

sub­

s ta n c e i n in d u c e d a n ta g o n ism i s n e v e r p ro d u c e d by an o rg a n ism i n p u re c u l t u r e w h e rea s i n n a t u r a l a n ta g o n is m i t

a lw a y s i s .

T hree e x p e r im e n ts a r e m e n tio n e d i n w h ic h t h e l y t i c p ro d u c e d by y e a s t , b a c illi, p ig s.

s u b s ta n c e

g ro w in g i n a s s o c i a t i o n w i t h t u b e r c l e

w as i n j e c t e d t o g e t h e r w i t h t h e p a t h o g e n i n t o

The e x p e r i m e n t a l r e s u l t s

i n d i c a t e d t h a t t h e r e was a

s lo w e r p r o g r e s s o f th e in d u c e d d i s e a s e th e u s u a l s e q u e la .

g u in e a

i n c o m p a ris o n w ith

In 1925, he m e n tio n s th e f a c t t h a t

b a c t e r i a may p r o d u c e a l y t i c

s u b s ta n c e f o r th e y e a s ts o f

b e e r and w in e . S c h ille r

(1 9 3 0 ) e l a b o r a t e d - on h i s

d e s c r i b e d some o f t h e

e a r l i e r w o rk a n d

" f o r c e d a n ta g o n is m " b e tw e e n y e a s t s and

H e m o p h il u s i n f l u e n z a e .

He f o u n d t h a t y e a s t s d i g e s t e d t h e

l i v i n g K o c h 's b a c i l l u s

(H. I n f l u e n z a e ) w hen t h e o r g a n i s m s

a r e g ro w n t o g e t h e r i n a m edium d e p r i v e d o f o t h e r n i t r o g e n o u s su b sta n c e s,

and c o n ta in in g o n ly g lu c o s e o r s a c c h a ro s e i n

s o l u t io n in th e d i s t i l l e d th e b a c t e r i o l y t i c

w a te r.

The y e a s t c e l l s

se c re te d

s u b s t a n c e s , w h ic h d i s s o l v e d t h e b a c i l l i .

He s t u d i e d t h e a n t a g o n i s m i n d u c e d b e t w e e n v a r i o u s p r o t e o l y t i c b a c t e r i a a n d H. I n f l u e n z a e i n

th e l i v i n g

c o n d itio n w ith th e

i n t e n t i o n o f o b t a i n i n g t h e b a c t e r i o l y s l n o n H. I n f l u e n z a e a p a r t from th e c e l l .

He f o u n d t h a t H. i n f l u e n z a e i s

e x a c tin g w ith r e g a r d to i t s

n o u ris h m e n t, how ever,

if

v ery it

is

p l a c e d i n a p o o r n u t r i t i o n a l m edium , w ill d ig e st i t .

th e p r o t e o l y t i c 'b a c te r ia

T h i s d i g e s t i o n w as o b s e r v e d b o t h i n d i s t i l l e d

w a te r and on a g a r .

The H. i n f l u e n z a e s e r v e s a s t h e n i t r o g e n

s o u r c e f o r t h e p r o t e o l y t i c b a c t e r i a w h ich a t t a c k i t . D e s p i t e t h e s e many r e p o r t s o f a n a n t i b i o t i c fro m y e a s t ,

H e n ric i

(1930, p . 231) s t a t e s

d e m o n s tra b le a n t i b a c t e r i a l . a c t i o n .

su b s ta n c e ;

t h a t y e a s t s h a v e no

He d o e s n o t . m e n t i o n t h e

i n v e s t i g a t i o n s o f F e m b a c h and H ayduck o r o t h e r i n v e s t i g a t o r s , n o r d o es he o f f e r any e x p e rim e n ta l e v id e n c e i n e x p la n a tio n o f th is

sta te m e n t. B achm ann a n d O g a i t ( 1 9 3 5 ) r e p o r t e d t h a t t h e a n t i b a c t e r i a l

a c t i o n o f y e a s t s w as d u e t o produced.

t h e l a r g e a m o u n ts o f a e e t a l d e h y d e

T ik k a an d I t k o n e n

( 1 9 4 1 ) f o u n d t h a t b a k e r 1s y e a s t

f o r m e d t h e r m o l a b i l e f e r m e n t s w hen g ro w n i n r y e d e c o c t i o n . T hese f e r m e n ts h a d a s t r o n g l y b a c t e r i c i d a l a c t i o n on A e r o b a c te r a e ro g e n e s and e x e r te d a c o n s id e r a b le i n h i b i t o r y t h e i r g ro w th .

A y e a s t s o l u t i o n w as m ade b y s u s p e n d i n g r y e

f l o u r i n 0 .2 5 M p h o sp h a te b u f f e r c e n t g lu c o se ,

ste riliz in g ,

y e a st c u ltu re ,

(pH 6 . 0 )

c o n ta in in g 2 p e r

and i n o c u l a t i n g w i t h a p r e s s e d

a n d t h e n i n c u b a t i n g a t 3 7 ° G♦ f o r 48 h o u r s .

T h i s g r o w t h was c e n t r i f u g e d ste rile

a c t io n on

and th e s u p e r n a t a n t f i l t e r e d

under

c o n d itio n s a f t e r th e a d d itio n o f 0 .5 p e r c e n t g lu c o s e .

The f i l t r a t e

w as d i v i d e d i n t o tw o p a r t s

a n d o n e p a r t w as

h e a t e d u n d e r r e f l u x on a b o i l i n g w a t e r b a t h f o r one h o u r and c o o le d ,

a f t e r w h ic h b o th s o l u t i o n s w ere t r e a t e d w ith f i v e m l.

9 o f a f r e s h c u l t u r e o f A. a e r o g e n e s # w e ll in th e h e a te d s o l u t io n ,

The t e s t o r g a n i s m g r e w

and f a i l e d

t o g ro w a t a l l . i n t h e

u n h e a te d # M a is in and P o u r b a ix

(1941) r e p o r t e d a b e n e f i c i a l

e f f e c t o f f r e s h b ak er* s y e a s t 'a g a i n s t b e n z o p y re n e c a n c e r i n m ic e .

S u g iu r a and Rhoads

liv e r cancer in ra ts y e llo w )

is

(1 9 4 1 ) s a i d

t h a t th e d e v elo p m e n t o f

fe d b e ta -d im e th y la m in o -a z o b e n z e n e

(b u tte r

i n h i b i t e d by th e d a i l y i n g e s t i o n o f a s m a ll am ount

o f e th y l a lc o h o l e x tr a c tio n o f y e a s t.

T hese r a t s d i d n o t

d e v e l o p l i v e r c a n c e r w hen f e d a d i e t o f u n p o l i s h e d r i c e

con­

t a i n i n g 15 p e r c e n t b r e w e r *s y e a s t . T akahashi

(1 9 4 2 ) d i s c o v e r e d a v i r u s i n a c t i v a t o r w h ic h

was e x t r a c t e d f r o m y e a s t b y a u t o l y s i s , a n d b y a u t o c l a v i n g . H is m e t h o d c o n s i s t e d o f m i x i n g o n e k i l o o f f r o z e n b a k e r ’ s o r b re w e r’s y e a s t cake w ith f o u r l i t e r s

of d is tille d

a u t o c l a v i n g f o r 3 0 m i n u t e s a t 15 p o u n d s p r e s s u r e . c l a v e d m a t e r i a l w as f i l t e r e d The f i l t r a t e

The a u t o -

th ro u g h a pad o f c e l i t e

N o. 505#

was t r e a t e d w i t h a c e t o n e a n d t h e p r e c i p i t a t e

f o r m e d w as s e p a r a t e d b y c e n t r i f u g a t i o n . d isso lv e d

w a te r and

i n a v o lu m e o f d i s t i l l e d

o rig in a l f i l t r a t e .

The p r e c i p i t a t e w as

w a te r e q u a l to

In a l a t e r p ap er

t h a t o f th e

(1 9 4 6 ), he s a id t h a t t h i s

i n a c t i v a t i o n was b r o u g h t a b o u t b y a r e a c t i o n i n v o l v i n g t h e i n a c t i v a t o r a n d some g r o u p i n t h e v i r u s p a r t i c l e n ecessary fo r i t s B ari

w h i c h w as

in fe c tiv ity .

(1 9 4 0 ) s a i d t h a t a m e d i c i n a l y e a s t c a n be p r e p a r e d

10 f r o m bo t t o m - f e m e n t a t i o n w a s t e y e a s t b y m a k i n g a p u l p o f i t and s ie v in g i t

tw ic e w ith f r e s h c o ld w a te r .

To r e m o v e t h e

b i t t e r s u b s t a n c e s t h e p u r i f i e d y e a s t was t r e a t e d w i t h f o u r v o l u m e s o f c o l d 2 p e r c e n t s o d iu m c a r b o n a t e s o l u t i o n .

He

s a i d t h a t t h e p r o d u c t m u s t b e d r i e d b e l o w 3 5 ° C. t o 5 0 ° C. Cook a n d K r e k e ( 1 9 4 0 ) d e s c r i b e d a c r u d e y e a s t t h a t c o n ta in e d .fa c to rs and h i s

in h ib ito ry

co -w o rk ers

t o m o ld g r o w t h .

L a te r,

Cook

(1 9 4 1 ) w ere a b le t o d e t e c t a g r o w th -

d e p r e s s a n t s u b s t a n c e f r o m y e a s t . * T hey f o u n d t h e i r s u b s t a n c e to be in h ib ito r y P e n ic iIliu m

to th e g ro w th o f A s p e r g i l lu s n i g e r and

g lab ru m .

The p r e p a r a t i o n o f t h e y e a s t e x t r a c t

t h a t t h e y u s e d i n t h e i r e x p e r i m e n t s w as d e s c r i b e d i n 1 9 3 8 . One l i t e r o f a c o n c e n t r a t e d a l c o h o l i c e x t r a c t o f 2 0 p o u n d s o f b a k e r * s y e a s t w as f r a c t i o n a l l y p r e c i p i t a t e d w i t h t h r e e p o r tio n s o f a c e to n e , a c e to n e a d d itio n .

c o n c e n t r a t i n g th e s o l u t i o n b e tw e e n e a c h

The f i n a l a c e t o n e - s o l u b l e p o r t i o n w as

a b o u t 50 tim e s a s a c t i v e

a s th e o r i g i n a l e x t r a c t in d e p re s s in g

t h e g r o w t h o f b o t h A. n l g e r a n d P . g l a b r u m w h en a d d e d t o t h e c u l t u r e m edium i n c o n c e n t r a t i o n s o f f r o m 0 . 0 5 p e r c e n t t o 1 .0 p e r c e n t.

T h i s f r a c t i o n w as a l s o t e s t e d

on th e g ro w th o f E. c o l l and S. a u r e u s . n o t o b ta in e d in e i t h e r c a s e , b u t th e r a t e

fo r its

effect

C o m p le t e k i l l i n g w as o f g ro w th was

g r e a t l y d e p re s s e d by a d d i t i o n o f a b o u t 2 p e r c e n t o r g r e a t e r o f th e f r a c t i o n s . in

T h is d e p r e s s o r seem ed t o a f f e c t t h e o rg a n is m

t h a t t h e form o f c o lo n y o f E. c o l l on n u t r i e n t a g a r

11

c o n t a i n i n g t h e d e p r e s s a n t was c h a n g e d f r o m s m o o t h t o r o u g h t o g ra in y w ith in c r e a s in g c o n c e n tr a tio n s .

M ic ro sc o p ic a lly t h i s

c h a n g e was a s s o c i a t e d w i t h a g r e a t i n c r e a s e t h e E* c o l l stru c tu re s.

in th e le n g th o f

c e l l and;, t h e d e v e l o p m e n t o f t a n g l e d f i l a m e n t o u s T h i s d e p r e s s a n t was n o n - v o l a t i l e ,

re a d ily

so lu b le

i n w a t e r , 95 p e r c e n t a lc o h o l and a c e to n e c o n t a i n i n g a t r a c e o f w a te r,

and i s

s t a b l e a t 1 0 0 ° C. a t pH 7*3*

C a rp e n te r

(1945) fo u n d t h a t T o r u lo s p o r a u t i l i s

ma.1 o r h a d som e a n t i b a c t e r i a l a c t i v i t y . g ro w n o n a l i q u i d s y n t h e t i c m e d iu m .

T h i s o r g a n i s m was

T he c e l l s w e r e p r e c i p i ­

t a t e d by c e n t r i f u g a t i o n and th e c e l l s

th e m s e lv e s and th e

s u p e r n a t a n t l i q u i d was t e s t e d f o r a n t i b i o t i c

a c tiv ity

b o t h w ere fo u n d t o be a c t i v e a g a i n s t S . a u r e u s . was d e s t r o y e d b y h e a t i n g t h e

v ar.

and

T h is a c t i v i t y

c e l l s a t 7 5 ° C. f o r 5 m i n u t e s .

C a r p e n t e r ' t h o u g h t t h a t t h i s w as d u e t o t h e f a c t t h a t e i t h e r t h e a n t i b a c t e r i a l s u b s t a n c e o f t h e y e a s t w as v o l a t i l e , h e a t la b ile ,

o r th a t liv in g

tio n of in h ib itio n .

c e l l s w ere e s s e n t i a l to th e m a n if e s ta ­

He m e n t i o n e d t h a t t h e c l i n i c a l u s e o f

d y n am ic y e a s t c u l t u r e s

in fiv e

cases of p u ru le n t in fe c tio n

had i n d ic a t e d t h a t th e m a te r ia l.w a s n o t o n ly n o n -to x ic b u t i s rem a rk ab ly e f f i c i e n t i n t o p i c a l a p p l i c a t i o n s . S e ig n e u rin , a b a c te rio sta tic

C arraz,

R oux, a n d S y n d i c o

a g e n t from b e e r . y e a s t .

t i o n in c lu d e d d ry in g o f th e y e a s t,

(1947) d e s c r i b e d

The m e th o d o f e x t r a c ­

th e n d e h y d r a tin g by a lc o h o l

a n d e x t r a c t i n g b y o r g a n i c s o l v e n t s i n a n a c i d pH .

T h i s was

12 e v a p o ra te d u n d er red u c ed p re s s u re a t a te m p e ra tu re n o t o v e r 4 5 ° C.

The d i s t i l l a t i o n

c e n ta g e o f th e re s id u e

w as e x t e n d e d u n t i l a c e r t a i n p e r ­

l i q u i d w as o b t a i n e d .

r e s i d u e was p l a c e d i n - t h e in to

tw o l a y e r s ,

so lid .

ic e b o x .

th e one i n a c t i v e

T h is l i q u i d

The r e s i d u e s o o n s e p a r a t e d liq u id ,

th e o th e r a c tiv e

The p r e c i p i t a t e w as r e c o v e r e d b y a n o r g a n i c s o l v e n t

i n a n a c i d pH, a n d b y t h e m e th o d o f c h r o m a t o g r a p h y o n m ag n esiu m . When t h i s

The p r e c i p i t a t e

s u b s t a n c e w as e x t r a c t e d

w as no a c t i v i t y it

was t h e n t a k e n " u p a g a i n b y w a t e r . i n a n a l k a l i n e pH t h e r e

o b s e r v e d , b u t when e x t r a c t e d i n a n a c i d pH,

was f o u n d t o b e a c t i v e a g a i n s t S .

p n e u m o n iae , S tr e p to c o c c u s s p . , I t w as c o m p l e t e l y i n a c t i v e

a u r e u s . D ip lo c o c c u s

and G o ry n e b a c te riu m d i p h t h e r i a e .

a g a in s t S a lm o n e lla , S h ig e lla

p a r a d y s e n t e r l a e A, S h . p a r a d y s e n t e r ! a e By P r o t e u s v u l g a r i s , and E. c o l i . S a r t o r y and M eyer (1 9 4 6 ) fo u n d t h a t f i l t r a t e s

of fresh

b a k e r ’ s y e a s t o b ta in e d by p la s m o ly s is c o n ta in e d a f a c t o r t h a t in h ib its tio n s .

th e g ro w th o f P . v u l g a r i s and E. c o l i a t h ig h d i l u ­ D rie d baker* s y e a s t" had no e f f e c t on th e b a c t e r i a .

Uroma a n d V i r t a n e n

( 1 9 4 ? ) r e p e a t e d H a y d u c k * s w o rk a n d

f o u n d 't h e a c t i v e s u b s ta n c e s t h a t i n h i b i t e d S. a u re u s .

t h e g ro w th o f

T hey l e a r n e d * t h a t b y e x t r a c t i n g w i t h a l c o h o l ,

e th e r,

a c e to n e ,

am yl a c e t a t e o r o t h e r l i p o i d

a c tiv e

s u b s ta n c e s c o u ld be i s o l a t e d

s o lv e n ts ,

from y e a s t c e l l s

g r e a t e r am ount th a n by H ayduck*s m eth o d .

th e

in a

The b e s t m e t h o d ,

13 h o w e v e r , w as b y t h e u s e o f a c e t o n e a c i d i f i e d w i t h h y d r o ­ c h lo ric

a cid #

One k i l o g r a m

w i t h tw o l i t e r s

o f a c e to n e

h y d ro c h lo ric a c id .

o f f r e s h b a k e r * s y e a s t w as m ix e d c o n t a i n i n g 2 m l. o f c o n c e n t r a t e d

The y e a s t w as c r u s h e d a n d t h e s o l u t i o n

shaken f o r s ix h o u rs.

A fte r f i l t r a t i o n ,

th e c le a r r e d d is h -

b ro w n a c e t o n e a q u e o u s s o l u t i o n p o s s e s s e d t h e b a c t e r i o s t a t i c a c tiv ity .

T h i s . a c e t o n e a q u e o u s s o l u t i o n w as e v a p o r a t e d i n a

v acu um t o g e t r i d o f t h e a c e t o n e a n d t o c o n c e n t r a t e t h e a n t i ­ b io tic

a g e n t.

The r e m a i n i n g l i q u i d w a s r e e x t r a c t e d w i t h

e th e r and th e e th e r th e n e v a p o ra te d . o il.

T h e r e r e m a i n e d a b ro w n

T h is o i l h a d v e ry s t r o n g b a c t e r i o s t a t i c

a c tiv ity .

T h i s s u b s t a n c e was q u i t e a c t i v e a g a i n s t S t r e p t o c o c c u s p y o g e n e s , C o ry n e b a c te rlu m p s e u d o d l p h th e r la e , B a c i l l u s m o n i a e . a n d C. d i p h t h e r i a e .

T h ey c o n c l u d e t h a t t h e a n t i b i o t i c

a c t i v i t y w as d u e t o t h e u n s a t u r a t e d p ro d u c t and c la im

c e r e u s . D. p n e u ­

fa tty

a c id s in t h e i r f in a l

t h a t m o s t o f t h e a n t i b i o t i c s d e s c r i b e d fr o m

y e a s t s w ere u n s a t u r a t e d f a t t y a c i d s . As i t

is

c le a rly

s e e n in th e f o r e g o in g re v ie w ,

th e re

a r e o b s e r v a t i o n s and e x p e r i m e n t a l e v id e n c e t h a t i n d i c a t e y e a sts

are p ro d u cers of a n tib io tic

a g e n ts.

T h is seem ed t o

w a r r a n t a m o re e x h a u s t i v e s t u d y o f t h e y e a s t s d e t e r m i n e w h ich y e a s t s e l a b o r a t e d

th a t

in o rd e r to

a n ti b a c t e r i a l su b s ta n c e s.

T h i s w o rk was n o t ’ u n d e r t a k e n w i t h t h e p u r p o s e o f r e e x a m i n i n g e a r l y w ork o r r e p e a t i n g w ork d o n e b y o t h e r m en.

The w o rk

w as s t a r t e d w i t h t h e i n t e n t i o n o f m a k i n g a g e n e r a l s u r v e y o f

a v a r i e t y o f y e a s t s p e c i e s to d e te r m in e w h ic h o n es p ro d u c e a n tib io tic

a c tiv ity

and u n d e r w hat e n v iro n m e n ta l c o n d itio n s

CHAPTER I I I

METHODS AND RESULTS S i n c e t h e e a r l y p o r t i o n o f t h i s w o rk w a s t o g e n e r a l s u r v e y o f som e o f t h e y e a s t s ,

in c lu d e a

a u n i v e r s a l y e a s t m edium

o f W ic k e rh a m was u s e d i n o r d e r t o g ro w t h e y e a s t s *

(T a b le I )

T ab le I . THE COMPOSITION OF UNIVERSAL YEAST MEDIUM P e p to n e (D ifc o ) Y east E x tra c t M a lt E x t r a c t D e x tro se A gar D i s t i l l e d W ater

5*0 3*0 3*0 1 0 .0 15*0 1 0 0 0 .0

gnu gnu gnu gm. gm. m l.

A v a r i e t y o f y e a s t s w e re s c r e e n e d t o d e t e r m i n e r o u g h l y w h ic h m ig h t be p ro m is in g a s p r o d u c e r s o f a n t i b i o t i c

a g e n ts.

TABLE I I YEASTS SCREENED FOR ANTIBIOTIC ACTIVITY C a n d id a l l p o l i t i c u s D ebarom yces g lo b o s u s H a n s e n u la a n o m a la M ycoderm a c e r e v l s i a e P ic h ia k lu y v e rl H h o d o to ru la f l a v a S acch aro m y ces c a r l s b e r g e n s i s Sac ch aro m y ce s c e r e v l s i a e T o r u l a u t i l i s (u . S . C . ) Z y g o sacch aro m y ces p r lo r la n u m #53 S to c k c u l t u r e s o f t h e s e y e a s t s p e c i e s w ere k e p t i n r e f r i g e r a t o r o n u n i v e r s a l y e a s t m edium s l a n t s .

th e

(T a b le

II)

16 EXPERIMENT I .

S c re e n in g o f y e a s t f o r a n t i b i o t i c

a c tiv ity

b y a d j u s t i n g t h e pH. E l e v e n h u n d r e d m l . o f l i q u i d u n i v e r s a l y e a s t b r o t h w as p rep ared ,

u s in g th e fo rm u la g iv e n i n

o m itte d .

T h i s v o lu m e w as d i v i d e d i n t o

110 m l. a p i e c e •

T ab le I , w ith th e a g a r 10 e q u a l p o r t i o n s o f

E ach o f t h e s e p o r t i o n s w ere a d j u s t e d t o

v a r i o u s pH v a l u e s b y t i t r a t i n g

w i t h 0 . 1 N NaOH a n d 0 . 1 N HC1.

The pH w as d e t e r m i n e d e l e c t r o m e t r i c a l l y b y u s i n g t h e B e ck m an pH m e t e r . 6 .5 >

7*0,

The pH v a l u e s u s e d w e r e : 7*5* 8 . 0 ,

and 8 .5 *

a t e a c h o f t h e s e pH v a l u e s . in to

4 .0 ,

4 .5 *

T h e r e w as 1 1 0 m l . o f y e a s t b r o t h The 110 m l . w as t h e n d i s p e n s e d

11 t e s t t u b e s so t h a t e a c h t e s t

t u b e c o n t a i n e d 10 m l .

T h e r e w e r e 11 t e s t t u b e s o f e a c h pH a v a i l a b l e sc re e n in g t e s t .

5*0, 5*5, 6 .0 ,

fo r th is

E ach o f t h e t e n y e a s t s w ere i n o c u l a t e d o n to

e a c h o f t h e v a r i o u s pH v a l u e s .

One t u b e f r o m e a c h pH v a l u e

was k e p t a s c o n t r o l .

The i n o c u l a t e d

3 0 ° C. f o r 72 h o u r s .

At th e end o f th e in c u b a tio n p e rio d ,

t u b e s w ere a g i t a t e d ,

tu b e s w ere i n c u b a te d a t

th e c o n te n ts f i l t e r e d

th e

t h r o u g h a N o. 1

f i l t e r p a p e r and a l i q u o t s p la c e d i n O x fo rd cu p s on a g a r p o u r p l a t e s o f t h e tw o t e s t o r g a n i s m s ,

S. a u re u s and E. c o l i *

The p o u r p l a t e s w e r e p r e p a r e d b y s u s p e n d i n g 3 m l . o f a 2 4 - h o u r n u t r i e n t b r o t h c u l t u r e o f t h e t e s t o r g a n i s m s i n 15 m l . o f m e l t e d a n d c o o l e d n u t r i e n t a g a r w h i c h w as p o u r e d i n t o s te rile

p e tri d ish .

a t 3 7 ° G. a n d r e a d .

a

T hese p l a t e s w e re i n c u b a t e d f o r 24 h o u r s R e la tiv e

z o n e s o f i n h i b i t i o n w ere r e c o r d e d

17 as /

to / / / / .

No i n h i b i t i o n

o b s e r v e d was r e c o r d e d a s . - .

The

r e s u l t s may b e s e e n i n T a b l e s I I I a n d I V . TABLE I I I ANTIBIOTIC ACTIVITY OF FILTRATES OF YEASTS GROWN AT VARYING PH LEVELS AGAINST S . AUREUS pH 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 * 0 7 * 5 8 . 0 8 . 5 G. l i p o l i t i c u s B. g l o b o s u s H. a n o m a l a M. c e r e v l s i a e P. k lu y v e rl R. f l a v a S. e a rlb e rg e n s is S. c e re v ls ia e T. u t l X l s ( u T s . c . ) Z. p r i o r i anum C o n tro l

*

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

/ / / / / - / / / / / / - - -

-

-

-

-

-

-

-

-

-

- = 0 mm. i n h i b i t i o n ; / * 0 - 15 mm. i n h i b i t i o n ; / / 2 0 mm. i n h i b i t i o n ; / / / * 2 0 - 30 mm. i n h i b i t i o n .

* 15 -

TABLE IV ANTIBIOTIC ACTIVITY OF:FILTRATES OF YEASTS GROWN AT VARYING PH LEVELS AGAINST E . COLI _______________________pH 4 . 0 4 , 5 5 * 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0 8 . 5 C. l i p o l i t i c u s D. g l o b o s u s H. a n o m a l a M. c e r e v l s i a e P. k lu y v e rl — R. f l a v a S. c a rlb e rg e n s is S. c e re v ls ia e T . u t l l l s ( U T S . C .) Z. p r l o r i a n u m C o n tro l *

-

-

-

-

-

-

-

-

-

-

.......................................................... / / / / / / / / - / / / /// // -

- * O mm. I n h i b i t i o n ; / = 0 - 1 5 mm. i n h i b i t i o n ; mm. i n h i b i t i o n ; / / / z 2 0 - 3 0 mm. i n h i b i t i o n .

//

* 1 5 -20

18

Prom T a b l e s I I I

a n d IV ,

i t was c l e a r t h a t t h r e e

a p p ea re d to be p o t e n t i a l a n t i b i o t i c M. c e r e v l s i a e , T . u t i l i s . y e a sts a t th is

tim e ,

p r o d u c e r s ; H. a n o m a l a ,

B a th e r th a n s c r e e n in g a d d i t i o n a l

t h e d e c i s i o n w a s m ade t o

t i o n s h i p b e t w e e n pH a n d a n t i b i o t i c d e ta il.

y e a sts

stu d y th e r e l a ­

e la b o ra tio n in g re a te r

T h e r e f o r e i n t h e new e x p e r i m e n t s t h e m edium w a s

a d ju s te d so t h a t s m a lle r d if f e r e n c e s w ith in th e e f f e c t i v e ra n g e c o u ld be s t u d i e d .

R e su lts o f t h i s

m e d i a w i t h a pH r a n g e v a l u e s o f 7 * 0 , 8 . 25, 8 .5 ,

a n d 8 . 7 5 may b e s e e n i n

te c h n iq u e u s in g

7*25,

7 .5 ,

7*75, 8 .0 ,

T a b l e V.

TABLE V FURTHER STUDIES ON THE EFFECT OF PH ON THE PRODUCTION OF ANTIBACTERIAL SUBSTANCES Y east used to p rod uce f i l t r a t e .

A c tiv ity o f f i l t r a t e . T e s te d a g a in s t S. au reu s ’ pH

H. a n o m a l a M. c e r e v l s i a e T. u t i l i s (U T S .C .)

7 .0 7 .2 5 7 .5 7 .7 5 8 .0 8 .2 5 8 .5 8 .7 5 -*

/

-

//

/

/

//

/

//

T e s te d a g a i n s t E. pH H. a n o m a la M. c e r e v l s i a e t . u t i l i s (uTs. C•') *

/

-

/ / /

/

c o li

7 .0 7 .2 5 7 .5 7 .7 5 8 .0 8 .2 5 8 . 5 8 .7 5 —

—

-

//

/ / . //

//

/.

/ .// . / / //// /

—

- r 0 mm. i n h i b i t i o n ; / = 0 - 1 5 mm. i n h i b i t i o n ; / / a 1 5 - 2 0 mm. I n h i b i t i o n ; / / / « 2 0 - 3 0 mm. i n h i b i t i o n ; / / / / « 3 0 - 4 0 mm. I n h i b i t i o n .

19 From t h e d a t a i n T a b l e V, T. u t i l i s

w as s e l e c t e d a s t h e

m o st p r o m is in g y e a s t w ith w h ic h to c o n tin u e t h i s

research .

A

pH o f 8 . 2 5 w as c o n s i d e r e d t h e o p tim u m f o r a n t i b i o t i c p r o d u c t i o n b y T. u t i l i s

a n d w as u s e d t h r o u g h o u t t h e e x p e r i m e n t s .

EXPERIMENT I I .

B est carb o n so u re e f o r a n t i b i o t i c p ro d u c tio n

b y T. u t i l i s . The n e x t f a c t o r t o b e c o n s i d e r e d w as t h e s u g a r o r t h e c a r b o n s o u r c e t h a t w o u l d g i v e maximum p r o d u c t i o n o f t h i s b io tic

s u b s ta n c e as m easu red by th e d ia m e te r o f th e zo n es o f

in h ib itio n . T ab le I ,

U s i n g t h e u n i v e r s a l y e a s t m edium d e s c r i b e d i n

v a r i o u s m ed ia w ere p r e p a r e d ,

p re se n t.

D e x tro se ,

m edium w a s a d j u s t e d

filtra tio n

fru c to se ,

sugar so u rce.

tw o p o r t i o n s ;

f o r 15 m in u te s

a n d t h e o t h e r w h i c h w as s t e r i l i z e d

some s u g a r s w i l l f o r m t o x i c

and

The pH o f e a c h

a t 15 p o u n d s p r e s s u r e

th ro u g h th e S e itz f i l t e r .

e x c e ssiv e h e a t.

g a la c to se ,

to 8 .2 5 an d e a c h d i v i d e d i n t o

o n e , w h i c h w as s t e r i l i z e d th e a u to c la v e ,

a l t e r i n g o n ly th e su g a r

la c to s e , m a lto se ,

s u c r o s e w ere s u b s t i t u t e d a s t h e

in

a n ti­

by

T h i s w as d o n e b e c a u s e

s u b s t a n c e s w hen s t e r i l i z e d

by

T h e r e w as no d i f f e r e n c e o b s e r v e d i n t h e

p r o d u c t i o n o f a n t i b i o t i c s b y T. u t i l i s

as th e r e s u l t o f

m eth o d o f s t e r i l i z a t i o n . 2 * u t i l i s . w as i n o c u l a t e d i n t o

th e

12 m e d i a w h i c h c o n ­

t a i n e d d i f f e r e n t s u g a r s as c a rb o n s o u rc e s and in c u b a te d f o r 72 h o u r s a t 3 0 ° G*

G r o w th o c c u r r e d i n a l l o f t h e m e d i a ,

g lu c o se g iv in g th e m ost r a p id and a b u n d a n t.

A fte r th e

20 in c u b a tio n p e rio d ,

t h e b r o t h w as f i l t e r e d

p a p e r and th e f i l t r a t e

t h r o u g h No. 1 f i l t e r

t e s t e d a g a i n s t t h e tw o t e s t o r g a n i s m s

t o m easure d e g re e s o f i n h i b i t i o n .

T a b l e VI sh o w s t h e r e s u l t s

o b ta in e d . T h is s e r i e s o f e x p e r im e n ts i n d i c a t e d

t h a t d e x t r o s e was

th e b e s t su g a r used f o r th e p ro d u c tio n o f th e a n t i b a c t e r i a l a g e n t.

I t may b e t h a t t h e o t h e r s u g a r m e d i a w o u l d h a v e

y i e l d e d some ( o r m o r e ) a n t i b i o t i c

if

s u f f i c i e n t tim e h ad b e e n

a l l o w e d f o r maximum g r o w t h t o o c c u r .

Two f a c t o r s t h a t r e s u l t

i n maximum z o n e s o f i n h i b i t i o n a r e t h e r e f o r e and d e x tr o s e as th e

a pH o f 8 . 2 5

carb o n s o u rc e . TABLE VI

EFFECT OF CARBON SOURCE ON ANTIBIOTIC PRODUCTION- BY TORULA U T IL IS S u g a r u s e d i n m edium

A n tib io tic a c tiv ity of f i l t r a t e v ersu s S. au reu s E. c o l i

D e x tro se F ru c to se O a la c to se L a c to se M a lto se S ucrose *

//// /

*

//// /

- = 0 mm. i n h i b i t i o n ; / * 0 - 1 5 mm. i n h i b i t i o n ; / / / / « 3 0 - 4 0 mm. i n h i b i t i o n .

EXPERIMENT I I I .

B est n itro g e n so u rce f o r a n ti b i o ti c

p ro d u c­

t i o n b y T. u t l l i s . In a n t i b i o t i c p ro d u c tio n th e n itr o g e n so u rce i s to be an im p o rta n t v a r i a b l e .

known

The n i t r o g e n s o u r c e o f t h e

21 u n i v e r s a l y e a s t b r o t h m edium w a s r e p l a c e d b y p e p t o n e , o x a la te ,

ammonium p h o s p h a t e , amm onium n i t r a t e ,

d e ad c e l l s o f S . a u r e u s , d e a d E. c e lls ,

and v ia b le E. o o li c e lls #

c o ll c e lls . The v i a b l e

E . c o l l c e l l s w ere u s e d to o b s e r v e t h e phenom enon d e s c r i b e d by S c h i l l e r

ammonium

p o ta ssiu m n i t r a t e ,

v ia b le 8 . au reu s S. a u re u s and

" f o r c e d a n t a g o n i s m 11

(1 9 2 4 ).

The d e a d c e l l s w e r e

p r e p a r e d by g ro w in g th e o rg a n ism s i n an a g a r s l a n t . i n a b o t t l e and h a r v e s t i n g th e so lu tio n . s a lin e ,

c e l l s by w a sh in g th e

c e l l s o f f w ith s a l in e

T h i s s u s p e n s i o n was c e n t r i f u g e d ,

and rew ash ed f o u r tim e s .

b y h e a t i n g 3 0 m i n u t e s a t 1 0 0 ° C. w as a d j u s t e d t o a pH o f 8 . 2 5 . th e v a r io u s m ed ia,

resu sp en d ed in

The c e l l s w e r e t h e n k i l l e d A l l o f th e m ed ia p r e p a r e d

T. u t i l l s

was I n o c u l a t e d i n t o

i n c u b a t e d f o r 72 h o u r s a t 3 0 ° C.

G ro w th

o c c u r r e d i n m ed ia c o n t a i n i n g p e p to n e ,

ammonium o x a l a t e ,

ammonium p h o s p h a t e ,

and p o ta s s iu m n i t r a t e

as n itro g e n so u rc e s.

ammonium n i t r a t e ,

T h i s w as f i l t e r e d

t h e two t e s t o r g a n i s m s .

The c o n t r o l s

and t e s t e d a g a i n s t

t h a t w ere u s e d i n t h i s

s e r i e s o f e x p e r i m e n t s w ere a l i q u o t s o f t h e u n i n o c u l a t e d m e d ia . The d i a m e t e r o f t h e z o n e s o f i n h i b i t i o n w as m e a s u r e d i n mm. H o w e v e r,

i t m u st b e rem em b ered t h a t t h e d i a m e t e r o f t h e

O x f o r d c u p i t s e l f i s 8 mm.,

so a n i n h i b i t i o n

i n d i c a t e s a z o n e o f 1 mm. s u r r o u n d i n g t h e

z o n e o f 10 mm.

cup.

The r e s u l t s

o f v a r y i n g t h e n i t r o g e n s o u r c e a r e sh o w n i n T a b l e V I I . P e p to n e a p p e a re d to be th e b e s t n i t r o g e n s o u rc e f o r 1*

u tills

f o r th e p r o d u c tio n o f t h i s

a n ta g o n is tic

su b s ta n c e .

22 TABLE V II

EFFECT OF NITROGEN SOURCE ON ANTIBIOTIC PRODUCTION BY TORULA U T ILIS N itro g e n so u rc e

A n tib io tic a c tiv ity o f f i l t r a t e Z o n e s i n mm. S. au reu s

P e p to n e Ammonium o x a l a t e Ammonium p h o s p h a t e P o ta ssiu m n i t r a t e Ammonium n i t r a t e D ead S* a u r e u s c e l l s D ead E . c o l i c e l l s L iv e S . a u re u s c e l l s L iv e E . c o l l c e l l s

E.

18 13 11 0 0 0 0 0 0

c o li 16 15 11 0 0 0 0 0 0

The 11f o r c e d a n t a g o n i s m ” p h e n o m e n o n w as n o t o b s e r v e d *

N e ith e r

t h e y e a s t n o r t h e t e s t o r g a n i s m g re w w hen p l a c e d t o g e t h e r i n a m edium c o n t a i n i n g c o m p o n e n t s o f t h e b a c t e r i a l c e l l a s n i t r o gen so u rces*

T h erefo re,

t h e y e a s t was u n a b l e t o u s e t h e

S. a u re u s o r E . c o l i c e l l s

fo r n itro g e n so u rce.

Ammonium

o x a la te used as th e n itr o g e n

so u rce a ls o

se e m e d t o p e r m i t t h e

d e v elo p m e n t o f a n . a n t i b i o t i c

a s d i d ammonium p h o s p h a t e .

It

s e e m s t h a t ammonium i o n s w o u l d p e r m i t some a n t i b i o t i c p r o d u c ­ t i o n b u t to a l e s s e r d e g r e e a s co m p ared w ith p e p to n e .

The

e x p e r im e n ts w ere c o n ti n u e d u s i n g p e p to n e a s t h e n i t r o g e n so u rce i n t h is

u n i v e r s a l y e a s t m e d iu m .

EXPERIMENT I V .

E f f e c t o f sh a k in g th e

c u l t u r e on a n t i b i o t i c

p ro d u c tio n * In o r d e r to d e te rm in e w h e th e r sh a k in g o r d i s r u p t i n g

23 t h e p e l l i c l e h a d a n y e f f e c t on t h e q u a n t i t y o f t h e a n t i b i o t i c p r i n c i p l e p r o d u c e d , tw o f l a s k s o f t h e m edium w i t h a l l c o n d i ­ tio n s

a d j u s t e d f o r o p tim u m p r o d u c t i o n was i n o c u l a t e d .

One

f l a s k w as u s e d a s t h e c o n t r o l w i t h n o s h a k i n g o r a g i t a t i o n , t h e o t h e r f l a s k w as c o n s t a n t l y

a g i t a t e d by a m e c h a n ic a l

sh a k in g d e v ic e f o r th r e e d a y s . f l a s k d i d n o t form a p e l l i c l e

The g r o w t h i n t h e a g i t a t e d and th e b r o th a p p e a re d t u r b i d .

A fte r th e in c u b a tio n p e rio d , b ro th f i l t r a t e s

fro m b o th o f

t h e f l a s k s w ere t e s t e d a g a i n s t S . a u r e u s and E.

c o li.

It

w as e v i d e n t t h a t a g i t a t i o n o r s h a k i n g h a d a n u n f a v o r a b l e e f f e c t on th e p r o d u c tio n o f t h i s

a n tib io tic

s u b s ta n c e b ecau se

t h e r e w as o n l y a s m a l l z o n e o f i n h i b i t i o n p r o d u c e d b y t h e bro th f i I t r a t e tra te

f rom t h e a g i t a t e d c u l t u r e , whe r e a s , t h e

fro m t h e s t i l l

c u ltu re

sh o w e d l a r g e z o n e s o f i n h i b i t i o n .

A ll o f th e fe rm e n ta tio n beyond t h i s w ith o u t th e a g i t a t i o n .

It

f i 1-

p o i n t was c a r r i e d on

ap p eared t h a t th e p e l l i c l e

p ro duced

by t h e u n a g i t a t e d g ro w th h a d a d e c i d e d e f f e c t on th e q u a n t i t y o f th e a n tib io tic EXPERIMENT V.

a g e n t e la b o r a te d by th e y e a s t c e l l s .

E x tr a c tio n o f th e a n t i b i o t i c

su b s ta n c e

from

t h e m e d iu m . The n e x t p r o c e d u r e i n v o l v e d t h e e x t r a c t i o n o f t h i s a n tib a c te ria l p rin c ip le

from t h e b r o t h a f t e r g ro w in g t h e y e a s t

u n d e r c o n d i t i o n s w h i c h w e r e f o u n d t o y i e l d maximum i n h i b i t i o n zones.

The e x t r a c t i o n w as c a r r i e d o u t b y m i x i n g tw o p a r t s o f

th e f e r m e n ta tio n b r o t h w ith one p a r t o f s o l v e n t . shaken and s e p a ra te d in a s e p a ra to ry fu n n e l. p o r t i o n w as re m o v e d a n d t h e

T h i s w as

The a q u e o u s

s o l v e n t p o r t i o n was t h e n d i s t i l l e d

i n v a c u o t o rem o v e t h e s o l v e n t .

S im u lta n e o u sly w ith th e

e x tr a c tio n o f th e fe rm e n ta tio n b r o th ,

th e u n in o c u la te d y e a s t

b r o t h w as e x t r a c t e d a s a c o n t r o l . S o lv e n ts su ch as c h lo ro fo rm , e th y l a lc o h o l,

carbo n t e t r a c h l o r i d e ,

and a c e to n e w ere u s e l e s s

in th e e x t r a c t i o n and

no a c t i v e s u b s t a n c e c o u ld b e e x t r a c t e d . e x t r a c t i o n was s u c c e s s f u l . d riv e n o ff , w as l e f t .

H o w e v e r,

e th e r

When a l l o f t h e e t h e r h a d b e e n

a s m a l l v o lu m e o f a n o i l y ,

v isc o u s,

c le a r liq u id

The c o n t r o l b r o t h e x t r a c t i o n a l s o y i e l d e d a l i q u i d ,

b u t t h i s w as d a r k e r and gummy. te s te d fo r a n tib io tic

B o th o f t h e s e p o r t i o n s w ere

a c tiv ity .

The d i a m e t e r s o f i n h i b i t i o n

can be s e e n i n T ab le V I I I . TABLE V I I I MEASUREMENT OF INHIBITION PRODUCED BY ETHER EXTRACT OF T . U T IL IS MEDIUM Zone o f i n h i b i t i o n i n mm. F ra c tio n S* a u r e u s

E. c o ll

E th e r e x tr a c te d f r a c t i o n from f e r m e n t a t i o n b r o t h

43

36

E th e r e x tra c te d f r a c tio n from u n i n o c u l a t e d c o n t r o l b ro th

35

29

25 The r e s u l t s

show t h a t t h e a n t i b i o t i c

s o lu b le in e th e r and can be e x tr a c te d

i n . e t h e r i n a way t h a t

a c o n c e n tra te d su b s ta n c e c a n be o b ta in e d . b i t i o n w ere q u i t e

im p re ss iv e .

zones o f i n h i b i t i o n .

fra c tio n is

H ow ever,

The z o n e s . o f i n h i ­

th e c o n tr o l a ls o

gave

T h i s w as a v e r y d i s t u r b i n g f a c t o r .

H ow ever, t h e z o n e s o f i n h i b i t i o n p r o d u c e d by t h e e t h e r e x t r a c t o f t h e f e r m e n t a t i o n b r o t h w as g r e a t e r t h a n t h e z o n e s o f i n h i b i t i o n p ro d u c e d by t h e e t h e r e x t r a c t o f t h e u n i n o c u l a t e d c o n tro l b ro th . a n tib io tic

T h i s w o u l d seem t o i n d i c a t e

t h a t t h e r e w as a n

f r a c t i o n p r e s e n t in t h e . e t h e r e x t r a c t o f th e ferm en ­

ta tio n b ro th . The n e x t s t u d i e s w e r e m ade t o d e t e r m i n e ,

th e re fo re ,

th e

c au se o f th e zones o f i n h i b i t i o n p ro d u ce d by e t h e r e x t r a c t e d m a t e r i a l fro m t h e u n i n o c u l a t e d some f r a c t i o n i n e th e r.

c o n tro l.

I t w as t h o u g h t t h a t

t h e m edium w as b a c t e r i c i d a l w hen t r e a t e d w i t h

Chapman ( 1 9 5 0 ) s a y s t h a t w h e n c o m m e r c i a l p e p t o n e s a r e

d ia ly se d ,

th e d ia ly s e e

b a c te rio sta tic ,

(n o n -d ia ly z a b le f r a c tio n ) i s d e c id e d ly

w h ile th e d i a l y s a te

su p p o rts b a c te r ia l.g r o w th .

In o r d e r to d e te rm in e w hat f r a c t i o n i n

t h e m edium u s e d f o r

y e a s t g ro w th p o s s e s s e d t h i s b a c t e r i o s t a t i c o f v a r i o u s c o m p o s i t i o n s w e r e made u p . c o m p l e t e m e d iu m . e n t w as e l i m i n a t e d (3 ) m a lt e x t r a c t ,

su b s ta n c e ,

m ed ia

The c o n t r o l w as t h e

O t h e r m e d i a w e r e m ade i n w h i c h o n e c o n s t i t u ­ from e a c h :

( 1 ) p e p to n e ,

and ( 4 ) . d e x tr o s e .

(2 ) y e a st e x tr a c t,

E a c h m edium w as e x t r a c t e d

w ith e th e r under i d e n t i c a l c o n d itio n s .

The e t h e r - e x t r a c t e d

26 f r a c t i o n s w ere t h e n t e s t e d

fo r th e ir a c tiv ity

a g a in s t S. au reu s

a n d E . c o l i a n d i t w as f o u n d t h a t t h e a n t a g o n i s t i c p h e n o m e n o n was d u e t o t h e p e p t o n e i n t h e m ediu m b e c a u s e t h e m edium w i t h ­ o u t t h e p e p t o n e w as t h e o n l y m edium t h a t d i d n o t show i n h i b i ­ tio n

zones♦

A l l o f t h e o t h e r s c o n t a i n i n g p e p t o n e sh o w e d

i n h i b i t i o n zones o f s im ila r d ia m e te rs . t h a t when p e p to n e i s to x ic

su b sta n c e i s

EXPERIMENT V I .

I t ap p eared ,

th e n ,

t r e a t e d w ith e t h e r a s m a ll am ount o f

e x tra c te d .

U se o f s y n t h e t i c m e d i a .

I n o r d e r t o o b t a i n t h e a n t i b i o t i c p r o d u c e d by T . u t i l l s w ith o u t th e i n t e r f e r e n c e from th e e t h e r e x t r a c t o f p e p to n e , th re e

s y n t h e t i c m ed ia s u i t a b l e

f o r y e a s t g r o w th w ere u s e d .

The c o m p o s i t i o n s a p p e a r i n T a b l e IX . a c ts as a b u ffe r, d e sire d

le v e l•

was r e m o v e d i n o r d e r t o a d j u s t t h e pH t o t h e

S u ero se and d e x tro s e

s y n t h e t i c m e d ia w ere s t e r i l i z e d a s e p tic a lly

C a lc iu m c a r b o n a t e , w h ic h

s o lu tio n s used i n th e

by S e i t z f i l t r a t i o n

and added

t o t h e r e m a i n d e r o f t h e m edium t h a t h a d b e e n

ste riliz e d

i n t h e a u t o c l a v e f o r 3 0 m i n u t e s a t 15 p o u n d s

p ressu re.

The m e d i a w e r e t h e n a d j u s t e d t o a pH o f 8 . 0

and i n o c u l a t e d . w i t h T. u t l l i s .

to 8 .2 5

The f e r m e n t a t i o n f l a s k s w e r e

i n c u b a t e d a t 3 0 ° G. a n d a t t h e e n d o f 3 d a y s ,

2 5 .0 m l.

of

t h e f e r m e n t a t i o n l i q u o r w a s a s e p t i c a l l y w i t h d r a w n fr o m e a c h o f t h e m ed ia and e x t r a c t e d w i t h e t h e r . te s te d

a g a in s t th e t e s t o rg a n ism s.

w as o b s e r v e d .

The e x t r a c t s w e r e

No a n t i b i o t i c

A s i m i l a r p r o c e d u r e w as c a r r i e d

a c tiv ity

on a f t e r 5 d a y s ,

27 TABLE IX

COMPOSITIONS OF SYNTHETIC MEDIA USED FOR ANTIBIOTIC PRODUCTION Wi 1 6 1 © rs M edi um (1 9 0 1 ) S u cro se MgS04 KC1 NH4 C I N&2HP0 4 CaC03 W ater

1 0 .0 0 .2 5 O .2 5 0 .2 5 0 .2 5 0 .2 5 1 0 0 .0

gm. gm. gm. gm. gm. gm. m l.

P r i n g s & e l m Medium (1906) D e x tro se (NH4 ) 2 S0 4 MgS04 K2HPO4 W ater

5 .0 5 .0 0 .1 8 0 .7 5 1 0 0 .0

gm. gm. gm. gm. m l.

F u l m e r a n d N e l s o n Medium (1 9 2 3 ) D e x tro se K2 HPO4 NH4 C I C a C ls CaCOj W ater

1 0 . 0 gm 0 . 1 gm 0 .1 8 8 g m 0 . 1 0 gm 0 . 0 4 gm 1 0 0 .0 ml

28

and a g a i n no a n t i b i o t i c th e 7 th d ay,

a c tiv ity

w as o b s e r v e d .

H ow ever, on

th e y e a s t- t h a t h ad b e e n g ro w in g on th e F u lm e r-

N e l s o n m edium h a d p r o d u c e d e n o u g h o f t h e a n t i b i o t i c p r i n c i p l e to

g i v e z o n e s o f i n h i b i t i o n w hen t e s t e d a g a i n s t t h e t e s t

o rg a n ism s .

The P r i n g s h e i m m edium , a n d t h e W i l d i e r s m edium

show ed no s u c h a c t i v i t y . A new b a t c h o f t h e F u l m e r - N e l s o n m edium was m ade a n d i n o c u l a t e d i n o r d e r t o r e p e a t t h e w ork and t o o b t a i n a l a r g e r am ount o f th e a n t i b i o t i c a g e n t .

The a n t i b i o t i c

f r a c t i o n w as

s e p a r a t e d from t h e f e r m e n t a t i o n l i q u o r u s i n g f i v e 20 m l. p o rtio n s o f e th y l e th e r. 200 m l.

se p a ra to ry fu n n e l.

The s e p a r a t i o n w a s p e r f o r m e d i n a The e t h e r p o r t i o n w as t h e n p l a c e d

in a f l a s k and s e t i n a c o n s ta n t w a te r b a th w ith th e te m p e ra ­ tu re

k e p t a t 4 0 ° G.

The e t h e r w as d r i v e n o f f i n o n e h o u r ,

b u t t h e f l a s k was k e p t i n t h i s w a t e r b a t h f o r t h r e e h o u r s t o in s u r e th e e lim in a tio n o f a l l o f th e e th e r . th e e x tra c tio n ,

a s m a l l v o lu m e o f a n o i l y ,

l i q u i d w as f o u n d i n t h e f l a s k . e x tr a c te d sim u lta n e o u s ly , liq u id o r c ry s ta ls th e a c t i v i t y d is tille d

At th e end o f v isc o u s,

The c o n t r o l ,

w h ic h was

w as e v a p o r a t e d t o d r y n e s s .

re m a in e d i n th e f l a s k *

o f th e a n t i b i o t i c

fra c tio n ,

w a t e r was a d d e d t o t h e o i l y

so lu b le in w a te r.

No

In o r d e r to

te st

5 ,0 m l. o f s t e r i l e ,

liq u id

and on s h a k in g

a t u r b i d s u s p e n s i o n w as f o r m e d i n d i c a t i n g t h a t t h i s w as o n l y s l i g h t l y

c le a r

s u b s ta n c e

A s i m i l a r v o lu m e o f w a t e r

was a l s o a d d ed t o t h e c o n t r o l f l a s k an d s h a k e n t o o b t a i n a

29 c o n t r o l s o l u t i o n f r o m t h e u n i n o c u l a t e d m edium '. Each o f t h e s e

s o l u t i o n s w ere t e s t e d

a g a in s t a po u r-

p l a t e m ade f r o m 2 4 - h o u r b r o t h c u l t u r e s o f S . a u r e u s a n d E . c o li.

The f r a c t i o n e x t r a c t e d f r o m t h e i n o c u l a t e d m edium

sh o w e d z o n e s o f i n h i b i t i o n

t h a t a m o u n te d t o 2 3 mm. f o r S .

a u r e u s a n d 4 0 mm. f o r E . c o l l . w ith o u t i n h ib itio n z o n es.

T hus,

The c o n t r o l f r a c t i o n w as th e a n t i b i o t i c

s u b s ta n c e

w as p r o d u c e d b y t h e g r o w t h o f t h e y e a s t o n a s y n t h e t i c m ediu m a n d i s o l a t e d b y m e a n s o f e t h e r e x t r a c t i o n . I t is

known t h a t S . c e r e v i s l a e i n t h e a n a e r o b i c

fe rm e n ta tio n o f su g a r y ie ld s p ro d u c ts som e o f w h i c h may b e i n h i b i t o r y in

t h i s w ork.

(lis te d

i n T a b l e X)

to th e t e s t o rg a n ism s u se d

T h e r e i s n o t m uch know n a b o u t t h e f e r m e n t a t i o n

o f s u g a rs by T. u t i l i s

and th e b y - p r o d u c ts p ro d u c e d .

i t w as t h o u g h t d e s i r a b l e

to e lim in a te

th e p o s s i b i l i t y

e n d - p r o d u c t s o f th e n a t u r e p ro d u c e d by S . c e r e v i s i a e r e s p o n s ib le f o r th e o b se rv e d e f f e c t . co u rse,

I t is

how ever,

th a t are

re a liz e d ,

t h a t c o n d i t i o n s o f g ro w th a r e e s s e n t i a l l y

th e f l a s k s ,

H o w e v e r,

of

a e ro b ic in

som e a n a e r o b i c d i s s i m i l a t i o n may b e

ta k in g p la c e . The p r o p e r t i e s o f t h e p o t e n t i a l e n d - p r o d u c t s l i s t e d in

T a b le X w ere s t u d i e d .

T hey a r e l i s t e d

in

T ab le X I.

T here

a re s e v e r a l c o m p o u n d s .th a t a p p e a r i n th e e t h e r e x t r a c t e d p o r t i o n by v i r t u e o f t h e i r s o l u b i l i t y e th y l a lc o h o l,

a c e ta ld e h y d e ,

in e th e r .

a c e tic a c id ,

T hey a r e

and 2 ,3 - b u ty le n e

30 TABLE X

END-PRODUCTS OF YEAST FERMENTATION OF SUGAR IN RELATIVE PROPORTIONS USUALLY PRESENT 4 8 .0 0 $ 4 7 .6 $ 0.2 $ 0 .6 5 $ 0 10 2 .7 5 $ 0 .4 0 $ 0 .0 3 $

A lc o h o l, e t h y l C arbon d i o x i d e A c e ta ld e h y d e A c e tic a c id 2 ,3 -B u ty le n e g ly c o l G ly c e ro l L a c tic a c id S u c c in ic a c id

. $

TABLE XI PROPERTIES OF END-PRODUCTS E n d-P rod uct

S o lu b ility in e th e r

S o lu b ility in w a te r

E th y l a lc o h o l

S o l. In a l l p ro p o rtio n s S o l. in a l l p ro p o rtio n s S o l. in a l l p ro p o rtio n s S o l. in a l l p ro p o rtio n s In so lu b le

S o l. in a l l p ro p o rtio n s 7 8 .3 7 S o l• in a l l 2 1 .0 p ro p o rtio n s S o l• in a ll p ro p o rtio n s 1 1 8 .1 S o l. in a ll p ro p o rtio n s 1 9 2 .0 S o l. In a l l p ro p o rtio n s 2 9 0 .0 S o l. in a l l p ro p o rtio n s D ecom poses 6 . 8 g m ./lO O m l.D e c o m p o s e s

A c e ta ld e h y d e A c e tic a c id 2 , 3 -B u ty le n e g ly c o l G ly c e ro l L a c tic a c id S u c c in ic a c id

g ly c o l.

S o l. in a l l p ro p o rtio n s 0 . 3 g m ./lO O m l .

B o ilin g ^ p o in t, C.

When t h e e t h e r e x t r a c t e d f r a c t i o n w as h e a t e d

m i n u t e s a t 1 0 0 ° C. t h e

a n tib io tic

a c tiv ity

t h a t t h e s u b s t a n c e w as h e a t s t a b l e . e th y l a lc o h o l,

and a c e ta ld e h y d e ,

When s m a l l c o n c e n t r a t i o n s

re m a in e d ,

f o r 10 in d ic a tin g

A lso , by t h i s h e a t in g ,

i f p re se n t,

(0 .5 $ i n w a te r)

w e re , d r i v e n o f f .

o f a c e tic

a c id and

2 , 3 - b u t y l e n e g l y c o l w ere p la c e d I n O xford cups to t e s t

th e

31 to x ic ity

o f t h e s e tw o s u b s t a n c e s a g a i n s t S . a u r e u s a n d E . c o l i ,

n o i n h i b i t i o n w as o b s e r v e d , b i t i o n w ere n o t due t o b u t to

in d ic a tin g t h a t th e zones o f i n h i ­

th e e n d -p ro d u c ts o f th e fe rm e n ta tio n

a sp e c ific a n tib io tic

a g e n t produced i n th e fe rm e n ta ­

tio n ,

EXPERIMENT VII,

O b ta in in g a b a c t e r i a l sp e c tru m f o r th e

a n tib io tic , The a n t i b i o t i c

s o l u t i o n s m ade up a s d e s c r i b e d i n

E x p e r i m e n t VI w as t e s t e d b y p l a c i n g s t a n d a r d a m o u n ts

(0 .1 m l.)

i n O xford cups on p o u r - p l a t e s o f a v a r i e t y o f o rg a n is m s . The a c t i v i t y

o r la c k o f a c t i v i t y ,

w h ic h c o m p ris e th e

b a c t e r i a l sp e ctru m f o r th e a n t i b i o t i c ,

XII.

may b e s e e n i n

Two t y p e s o f i n h i b i t i o n w e r e n o t i c e d .

c o m p le te c l e a r i n g i l l u s t r a t e d

in F ig u re l a ,

o f t h e e x t r a c t a g a i n s t S a r c i n a l u t e a .. c le a r i n g as can be se e n i n F ig u re l b , o r g a n i s m w as A e r o b a c t e r a e r o g e n e s .

T ab le

One was a sh o w in g t h e e f f e c t

The o t h e r w as a p a r t i a l i n w h ich th e t e s t

A 2 4 - h o u r g r o w t h Is

re p re se n te d in each. EXPERIMENT V I I I .

T e s tin g f o r h e a t s t a b i l i t y

o f th e a n t i b i o t i c .

I n o r d e r t o s tu d y th e e f f e c t o f h e a t on t h i s o tic ,

o n e - h a l f m l. o f t h e s u s p e n s io n ,

p r e p a r e d by m ix in g 1 m l.

o f t h e e t h e r e x t r a c t e d o i l w i t h 5 ml. w a t e r , each of fiv e

sm a ll t e s t

6 0 ° C. f o r f i v e m i n u t e s ,

tu b e s.

a n tib i­

The f i r s t

w as p l a c e d i n t o

t u b e w as h e a t e d a t

th e se co n d tu b e .w a s h e a te d f o r f iv e

32 TABLE X I I

BACTERIAL SPECTRUM OBTAINED FROM AN ANTIBIOTIC PRO DUCED BY TORULA U T IL IS ON SYNTHETIC MEDIUM T e s t o rg an ism A e ro b a c te r a ero g e n e s A e ro b a c te r c lo a c a e A lc a lig e n e s f e c a l i s A lte rn a rla sp. A s p e r g illu s fu m ig a tu s A z o to b a c te r v i n e la n d il B a c illu s .b re v is B a c illu s m e tie n s B a c i l l u s m y c o ld e s B a c i l l u s p o ly m y x a B a c illu s s u b t l ll s B a c illu s th e rm o a c id u ra n s E sc h e ric h ia c o li K u rth ia z o p f li L a c to b a c illu s a ra b ln o sa M u c o r m u ce d o M y c o b ac teriu m l a c t i c o l a N e isse ria c a ta r r h a l! s O ospora o d i u r a . P ro te u s v u l g a r i s Pseudom onas a e r u g i n o s a S a lm o n e lla n e w p o rtil S a rc ln a l u te a S e r r a t l a m arcescen a S h ig e lla p a ra d y se n te rla e S h ig e lla sonnei S ta p h y lo c o c c u s a u re u s S tre p to c o c c u s f e c a l i s S tre p to m y c e s la v e n d u la e T h a m n ld iu m e l e g a n s *

p s p a r tia l c le a rin g ;

D ia m e te r o f i n h i b i t i o n z o n e i n mm« 25 21 0 0 0 0 u 12 24 21 16 0 40 13 0 0 0 23 0 0 0 12 27 0 10 10 23 0 0 0

P P

c c c c c c

c

p c p p c

c s c o m p lete c l e a r i n g .

33

ACTION OF AN ANTIBIOTIC ISOLATED FROM TORULA LTTILIS ON ( a ) SARCINA LUTEA AND ( b ) AEROBACTER AEROGENES

34 m i n u t e s a t .70° C . , th e

fo u rth tu b e ,

th e th ir d tu b e ,

f i v e m in u te s a t 8 0 ° C .,

f i v e m in u te s a t 90° C .,

f i v e m i n u t e s a t 1 0 0 ° C.

A fte r th e h e a tin g tre a tm e n t,

p o r t i o n w as t e s t e d f o r t h e i r a n t i b i o t i c t e s t o rg an ism s*

s u b s ta n c e i s h e a t s t a b le

p re v io u s e x p e rim e n ts EXPERIMENT I X .

S ix w h ite r a t s ,

sio n ,

each

o r decrease

in th e

T h is p ro v e s t h a t th e

a s w as i n d i c a t e d

in

(p ag e 3 0 ) .

T o x ic ity t e s t s

in tra p e rito n e a lly

tu b e ,

p o te n c y a g a in s t th e

T h e r e w as n o i n a c t i v a t i o n

zo nes o f i n h i b i t i o n due to th e h e a t in g . a n tib io tic

and th e f i f t h

on r a t s .

w e ig h in g l e s s

t h a n 4 0 g m ., w e r e i n j e c t e d

w ith o n e - h a l f m l. o f th e a n t i b i o t i c

suspen­

p r e p a r e d by s u s p e n d in g 1 m l. o f th e e t h e r e x t r a c t e d o i l

i n 5 m l. o f d i s t i l l e d th e a n tib io tic

w a te r,

in a n im a ls.

fo r d e ta ile d

of

The r a t s w e r e o b s e r v e d c o n s t a n t l y ,

a n d a t t h e e n d o f 48 h o u r s , d e a th o c cu rred .

in o rd e r to t e s t f o r t o x ic i ty

one o f th e r a t s d i e d .

No f u r t h e r

A l t h o u g h t h i s e x p e r i m e n t w as n o t b r o a d e n o u g h

c o n c lu sio n s,

th e r e s u l t s

b i o t i c w as n o t e x t r e m e l y t o x i c .

in d ic a te

th a t th is a n ti­

CHAPTER IV

SUMMARY AND CONCLUSIONS T h i s p r o j e c t w as u n d e r t a k e n t o d e t e r m i n e t h e p o s s i b i l i ­ tie s

o f p ro d u c in g a n t i b i o t i c s

v o lu m e o f l i t e r a t u r e m o ld s ,

from y e a s t s *

o n new a n t i b i o t i c

and s tr e p to m y e e s ,

D e sp ite th e g r e a t

a g e n t s from b a c t e r i a ,

th e re has been r e l a ti v e ly

little

w ork d one on t h e y e a s t s a s a p o t e n t i a l s o u r c e o f a n t i b i o t i c s * A su rv ey o f th e l i t e r a t u r e

in d ic a te d th a t th e re had been

sp o ra d ic r e p o r ts o f a n ta g o n is tic

s u b s ta n c e s p rod uced by

y e a sts. T h is w ork s u r v e y e d t e n y e a s t s , som e s o r t o f a n t i b i o t i e

su b sta n c e s*

M. c e r e v i s i a e , a n d T . u t i l l s *

It

o f w h ic h t h r e e p ro d u c e d T hey w e r e H. a n o m a l a »

a p p e a r e d t h a t pH w as a n

im p o rta n t f a c t o r in th e a n t i b i o t i c s

pro du ced by y e a s t s •

When g ro w n i n a s u i t a b l e m edium w i t h t h e pH a d j u s t e d b e t w e e n 7*5 and 8 * 5 ,

th e th r e e y e a s t w i l l produce a n t i b i o t i c s ,

w h en g ro w n a t a pH b e l o w 7 * 0 , n o a n t i b i o t i c Of t h e

t h r e e y e a s t s m e n tio n e d a b o v e,

to be th e m ost p ro m is in g ,

is

w h ile

e la b o ra te d *

T. u t i l i s

ap p eared

a n d a l l o f t h e r e m a i n i n g w o r k w as

c a r r i e d o u t w ith t h i s o rg a n ism and th e a n t i b i o t i c p ro d u c e d by t h i s y e a s t . The pH f o r o p tim u m a n t i b i o t i c

p r o d u c t i o n w as 8 . 2 5 *

When t h e f e r m e n t a t i o n m edium w as t h e u n i v e r s a l y e a s t m e d iu m , d e x t r o s e w as t h e b e s t s u g a r o r c a r b o n s o u r c e ,

o f th o se

te s te d ,

36 fo r a n tib io tic

p ro d u c tio n .

F o r o p tim u m p r o d u c t i o n ,

p e p to n e

i n t h e m edium w as b e t t e r t h a n a n y o t h e r n i t r o g e n s o u r c e su b s titu te d .

I t a ls o

a p p e a r e d t h a t a s h a k e c u l t u r e w as u n ­

fa v o ra b le f o r th e e la b o r a tio n o f th e t h a t w hen t h e f e r m e n t a t i o n f l a s k

is

a n tib io tic

s u b s ta n c e and

a llo w e d to

s i t q u ie tly ,

maximum z o n e s o f i n h i b i t i o n w e r e o b t a i n e d • When e x t r a c t i o n o f t h e a n t i b i o t i c f r o m t h e f e r m e n t a ­ t i o n l i q u o r w as a t t e m p t e d , e th y l a lc o h o l,

c h lo ro fo rm ,

carb o n t e t r a c h l o r i d e ,

and a c e to n e p ro v ed u n s u c c e s s f u l.

e x t r a c t i o n s w ere p o s s i b l e w ith e t h y l e t h e r .

S u ccessfu l

An o i l y

liq u id

w as o b t a i n e d b y t h i s e x t r a c t i o n t h a t p o s s e s s e d a n t i b a c t e r i a l p ro p e rtie s.

But th e u n in o c u la te d ,

an a n ta g o n is tic

c o n tro l b ro th a ls o y ie ld e d

s u b s t a n c e w hen e x t r a c t e d w i t h e t h e r .

The

e x p e r i m e n t s p e r f o r m e d i n d i c a t e d t h a t t h i s was d u e t o som e c o m p o n e n t i n t h e p e p t o n e p o r t i o n o f t h e m e d iu m . When T . u t i l l s pH a d j u s t e d t o 8 . 0 , o b ta in e d .

w a s g ro w n o n s y n t h e t i c m e d i a ,

a sim ila r o ily ,

a n tib io tic

w ith th e

s u b s t a n c e w as

The c o n t r o l s y n t h e t i c m e d iu m , u n i n o c u l a t e d ,

fa ile d

t o g i v e t h e c o m p l i c a t i o n t h a t t h e u n i v e r s a l y e a s t m edium gave. The o i l y

liq u id ,

o b t a i n e d fro m a n e t h e r e x t r a c t o f th e

c u l t u r e m e d iu m , w as m ix e d w i t h 5 m l . o f s t e r i l e , w a te r.

T h is s u s p e n s i o n was t e s t e d

o rg a n ism s.

a g a in s t a v a r i e ty o f m ic ro ­

I t p ro v ed c o m p le te ly i n e f f e c t i v e

m o ld s t e s t e d .

H o w e v e r,

it

d is tille d

a g a in st th e fiv e

d i d show p r o n o u n c e d a n t i b i o t i c

37 e f f e c t a g a i n s t m any Gram p o s i t i v e a n d Gram n e g a t i v e o r g a n i s m s * The o r g a n i s m s i n h i b i t e d a n d t h e d e g r e e o f i n h i b i t i o n

can be

s e e n i n T a b le X II* T h is a n t i b i o t i c

su b s ta n c e p ro v e d to be h e a t s t a b l e .

H e a t i n g t o 1 0 0 ° C. d i d n o t i n a c t i v a t e T o x ic ity t e s t s to x ic ity is

a re n o t c o n c lu siv e ,

b u t a m eager

in d ic a te d .

I t i s hoped t h a t t h i s a d e fin ite

th e a g e n t.

w o rk w i l l h e l p i n g i v i n g y e a s t s

p la c e i n th e a n t i b i o t i c

fie ld .

h a s b e e n sh o w n t o p r o d u c e a n a n t i b i o t i c

T o ru la u t i l i s

su b s ta n c e .

BIBLIOGRAPHY

BIBLIOGRAPHY

B a c h m a n n , W. a n d O g a i t . 1 93 5 * " E x p e r i m e n t e l l e r B e i t r a g z u r a n aero b en O b e r f la c h e n k u ltu r . 2 . U n te rsu c h u n g g a s f o r m ig e r S to ffw e c h s e lp ro d u k te von S a u r e s t o f f z e h r e r n (B a c k e rh e fe , M ilc h h e fe , S ta p h y lo c o c c u s a u r e u s , B a c te riu m c o l l , B a c te riu m p ro d ig io s u m ) a u f G e b h a lt an A z e ta ld e h y d and d a m it v e r b u n d e n e * b a k t e r i z l d e F e r n w ir k u n g ." Z e n t r a l b l . B a k t. 1 A b t. O r i g . , 1 3 4 :2 8 1 -2 8 8 . (A s r e v i e w e d i n B i o l . A b s ., 1 1 :9 0 8 4 , 1937*) B a ri,

Z. 1 9 4 0 . B e r . U n g a r. P h arm . G e s . , 1 6 :9 5 -1 1 3 * ("T he p r o d u c tio n o f m e d ic in a l and p h a rm a c e u tic a l a p p l i c a t i o n o f b e e r y e a s t , d r i e d y e a s t , a n d y e a s t e x t r a c t . ” R e v ie w e d i n Chem. A b s t r . , 3 4 : 4 2 2 3 , 1 9 4 0 . )

B u rn e tt,

E . A. 1905* A n n . R e p t . N e b r . A g r . E x p . S t a . , p . 1 0 .

C a r p e n t e r , C. W. 194 5* 11A n t i b a c t e r i a l p r o p e r t i e s o f y e a s t s , F u s a r i u m S p . , O n io n a n d g a r l i c . ” H a w a i i a n P l a n t e r s ' R ecord, 4 9 :4 1 - 6 7 . C h apm an, G. H. 1 9 5 0 .

( P e r s o n a l C o m m u n ic a tio n s.)

C ook, E . S . a n d C. W. K r e k e . 1 9 4 0 . " A b i l i t y o f r e s p i r a t o r y s t i m u l a t i n g f a c t o r s t o o v erco m e t o x i c a c t i o n o f g e r m i­ c id e s and m o ld s .” N a tu re , 1 4 6 :6 8 8 . C ook , E . S . , C. W. K r e k e , a n d L . G. N u t i n l . 1938* " F r a c t i o n s from y e a s t w h ic h s t i m u l a t e t h e r e s p i r a t i o n o f y e a s t and a n i m a l t i s s u e s . " S t u d i e s o f t h e I n s t i t u t u r n D i v i T h om ae, 2 :2 3 -3 7 * C ook,

E . S . , C. ¥ . K r e k e , S r . M. C. G i e r s e h , a n d S r . M. P . S e h r o e d e r . 1 9 4 1 . ”A g r o w t h - d e p r e s s a n t s u b s t a n c e f r o m y e a s t . ” S c ie n c e , 9 3 :6 1 6 -6 1 7 *

F e r n b a c h , A. 1909* tfS u r u n p o i s o n e l a b o r e p a r l a l e v u r e . ” C o m p t. R e n d , d e s S e a n c e s d e 1 * A c a d , d e s S c i e n c e s , 1 4 9 :4 3 7 -4 3 9 * F e r n b a c h , A. 1 9 1 0 . A n n . B r a s s * D i s t . , 1 2 : 3 6 1 . ( " T o x i c s u b s t a n c e s e l a b o r a t e d b y y e a s t . ” R e v ie w e d I n Chem. A b s tr ., 4 :9 4 8 , 1 9 1 0 .) F e r n b a c h , A. a n d E . V u l q u i n . 1 9 0 9 . C o m p t. R e n d . S o c . d e B io l* , 6 7 :6 9 8 -7 0 1 . F e r n b a c h , A. a n d £♦ V u l q u i n . 1 9 1 0 . " S u r l e p o u v o i r m i c r o b i e id e d e s m a c e r a tio n s de l e v u r e e t d e s m a c e r a tio n s de c e r e a l e s . “ C o m p t. R e n d , d e s S e a n c e s d e l * A c a d . d e s S c ie n c e s . 1 5 1 :6 5 6 -6 5 8 .

40 F u l m e r , E . I . a n d V. N. N e l s o n . 1 9 2 3 . " S t u d i e s o n y e a s t . V I . On t h e c o n t i n u o u s g r o w t h o f S a c c h a r o m y e e s c e r e v i s i a e i n s y n t h e t i c m e d iu m .” J . I n f e c t . D i s . , 3 3 ; 1 3 0 -1 3 3 • H ayduck, F . 1 9 0 9 . A llg em . Z. B i e r b r . M a l z f a b r ., 3 6 :5 0 5 -5 0 7 , 5 1 3 -5 1 6 . W ochschr. B r a u ., 2 6 :1 7 7 -1 7 9 , 1 8 9 -1 9 0 . ( ”Y e a st, p o i s o n s i n g r a i n s , w o r t s , a n d y e a s t s a n d t h e d ep en d en ce o f t h e i r a c t i o n i n m in e r a l s a l t s , and y e a s t p o i s o n s i n y e a s t . ” R e v ie w e d i n Chem. A b s t r . , 3 : 9 3 9 , 1437, 1 9 1 0 .} H e n r i c i , A r t h u r T . 1 9 3 0 . M o ld s , y e a s t s , J o h n W i l e y & S o n s , I n c . , New Y o r k ,

and a c t ln o m y c e te s . 296 p p .

H odgm an, C h a r l e s D. a n d H a r r y N. H o l m e s . 1 9 4 1 . H a n d b o o k o f C h e m ls try and P h y s i c s . C h e m ica l R ubber P u b l i s h i n g C o ., C le v e la n d , 2503 p p . K r a e m e r , H e n r y . 1 9 1 6 . A p p l i e d a n d e c o n o m ic b o t a n y . S e c o n d E d . , J o h n W il e y & S o n s , I n c . , New Y o r k , 6 2 2 p p . M a i s i n , J . a n d Y. P o u r b a i x . 1 9 4 0 . B u l l . A s s o c . F r a n c . E t u d e . C an cer, 2 9 :2 2 3 -5 1 . ( " A n t i b l a s t i e f a c t o r s o f a lim e n ta r y o r i g i n . ” R e v ie w e d i n Chem. A b s t r . 3 7 : 4 4 4 3 , 1 9 4 3 . ) P r i n g s h e i m , H. 1 9 0 6 . " U e b e r d i e S o g e n n a n t e B i o s F r a g e u n d d i e gew ohnung d e r H efe a n g e z u c k e r t e m i n e r a l s a l z N a h ru n g s lo s o n g e n .” C e n tr . B a k t . , 1 6 :1 1 1 -1 1 9 . P r a t t , R o b e rtso n and J e a n D u fre n o y . 1949. A n t i b i o t i c s . J . B . L i p p e n c o t t C o . , P h i l a d e l p h i a , 2 5 5 PP* S a r t o r y , A. a n d J . M e y e r . 1 9 4 6 . " E s s a i s d e m i s e e n e v i d e n c e e t de d o sag e de f a c t e u r s p h y to - e t b a c t e r i o s t a t i q u e s e l a b o r e s p a r d e s l e v u r e s . ” B u l l . A c a d . M e d ., 1 3 0 : 1 1 9 - 1 2 2 . S c h i l l e r , I . 1 9 2 4 . C e n tr . B a k t. P a r a s i t e n k . I . A b t. O r i g . , 9 2 : 1 2 4 - 1 2 8 . ( “ A c q u i r e d a n t a g o n i s m . ” R e v ie w e d i n Chem. A b s t r .

1 8 : 3 6 2 0 .)

S c h i l l e r , I . 1 9 2 5 . C e n tr . B a k t. P a r a s i t e n k . I . A b t. O r i g . , 9 4 : 6 4 - 6 6 . ( " B a c t e r i a l a n d y e a s t a n t a g o n i s m . ” R e v ie w e d i n Chem. A b s t r . , 1 9 : 2 6 8 5 , 1 9 2 5 . ) S c h i l l e r , I . 1927. C e n tr . B a k t. P a r a s i t e n k . I . A b t. O r i g ., 1 0 3 : 3 0 4 - 3 1 4 . ( “ i n d u c e d a n t a g o n i s m . ” R e v ie w e d i n B i o l * A b s tr., 2 :11 , 6 0 3 .)

41 S c h i l l e r , I . 1930* " S u r 1 *a n t a g o n i s m e p r o v o q u e b a c t e r l e s p r o t e o l y t i q u e s . ” C o m p t. R e n d . H e b d o m a d a l r e s d e l a S o c i e t e de B io lo g ie , 1 0 5 :4 2 3 -4 2 5 • S e i g n e u r i n , R . , G. C a r r a z , A. R o u x , a n d S . S y n d i c © ♦ 1 9 4 7 . "De l * a c t i o n b a c t e r i o s t a t i q u e . i n v i t r o d e s e x t r a i t s d e l e v u r e de b i e r e e t de b a e t e r i e s - d u v i n a l g r e . ” A n n a les de l * I n s t i t u t P a s te u r , 73581- 8 2 . S e rg e n t,

E . 1 9 0 3 . Ann. I n s t .

P a ste u r,

17.

S u g i u r a , K. a n d C. P . R h o a d s . 1 9 4 1 . ” E x p e r i m e n t a l l i v e r c a n c e r i n r a t s and I t s i n h i b i t i o n by r i c e - b r a n e x t r a c t , y e a s t , a n d y e a s t e x t r a c t . 11 C a n c e r R e s e a r c h , 1 : 3 - 1 6 . T a k a h a s h i , W. W. 1 9 4 2 . "A v i r u s i n a c t i v a t o r f r o m y e a s t . " S c ie n c e , 95 5 586-587. T a k a h a s h i , W. N. 1 9 4 6 . " P r o p e r t i e s o f a v i r u s fro m y e a s t . ” S c i e n c e , 1 0 4 :3 7 7 *

in a c tiv a to r

T i k k a , J . a n d J . I t k o n e n . 1 9 4 1 . Suom en K e m i s t i l e h t i 1 4 b . ( " B i o l o g i c a l p r o c e s s e s i n d o u g h . I . The a n t a g o n i s m s b e t w e e n m i c r o o r g a n i s m s . ” R e v ie w e d i n Chem. A b s t r . , 35 5 4 8 6 0 . ) U rom a, E . a n d 0 . E . V i r t a n e n . 1 9 4 7 . " A n t i b i o t i c s o f y e a s t s . ” A n n a les M e d ic in a e E x p e r im e n ta l! s e t B i o lo g i a e F e n n ia e , 2 5 53 6 - 4 7 . W aksman, S e lm a n A . 1 947* M i c r o b i a l a n t a g o n i s m s a n d a n t i b i o t i c s u b s t a n c e s . T he C om m onw ealth F u n d ; Hew Y o r k , 4 1 5 p p . W i l d i e r s , E . 1 9 0 1 . "Une n o u v e l l e s u b s t a n c e i n d i s p e n s i b l e a u d e v e l o p m e n t d e l a l e v u r e . " L a C o 'l u l e , 1 8 : 3 1 3 .