Antibiotics from Bacillus Polymyxa and Bacillus Subtilis

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P U R D U E U N IV E R SIT Y

THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION

Ralph Franklin Uachter

BY

e n title d

Antibiotics from Bacillus Folymyxa

and

Bacillus

Subtilis

COMPETES WITH THE UNIVERSITY REGULATIONS ON GRADUATION THESES

AND IS APPROVED BY ME AS FULFTLUING THIS PART OF THE REQUIREMENTS

FOR THE DEGREE OF

Doctor of Philosophy

P r o f e s s o r in C h a r g e o f T h e s is

H ear of S chool or D epartm ent

TO THE LIBRARIAN:----FS— THIS THESIS IS NOT TO BE REGARDED AS CONFIDENTIAL.

PR O FESSO B D î OHABGE

GKAD.

SCHOOL FORM

9 —3 - 4 9 —1M

ANTIBIOTICS FROM BACILLUS POLÏMYXA AND BACILLUS S U B T IL IS A T h esis S ubm itted

to

the F a c u lty

of Purdue U n i v e r s ity

by R alph F r a n k lin 7 /ac h te r In P a r t i a l F u lfillm e n t

of th e

R eq u irem en ts f o r th e D egree of D octor of P h ilo so p h y A ugust,

1950

ProQuest Number: 27714189

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 27714189 Published by ProQuest LLC (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346

ACKNOWLEDGMENT The w r i t e r w i s h e s t o e x p r e s s h i s a p p r e c i a t i o n

for

a d v i c e a n d d i r e c t i o n g i v e n b y D r . N. B o h o n o s t h r o u g h first

part

of th is

th ro u g h th e

latter

r e s e a r c h and by D r. F . p a rt.

v e ry kind a s s i s t a n c e

He a l s o w i s h e s

rendered

lan d ,

in c o n firm in g the

(M arburg) Company,

id en tity

i n com paring th e B a c i l l u s

k nown p o l y m y x i n s ,

to

acknow ledge

su b tilis

polym yxa a n t i b i o t i c w i t h

a nd b y M i s s D o r i s E .

stra in s

M ary­

o f th e Pitm an-M oore

B u n n ell,

Purdue V e t e r i n a r y D e p a rtm e n t, in a s s a y in g th e ag ain st v iru le n t

the

of the

B e ltsv ille ,

of th e B a c illu s

c u l t u r e , by D r. H a ro ld N ash,

the

W. Q u a c k e n b u s h

b y D r . N. G-. S m i t h ,

U n ited S t a te s D epartm ent o f A g r i c u l t u r e ,

the

of B ru cella

su is.

of th e

a n tib io tic s

table

of

contents

Page ABSTRACT...............................................................................................................................

i

INTRODUCTION.....................................................................................................................

1

EXPERIMENTA L .....................................................................................................................

7

I.

II.

E stab lish m en t of a C o lle c tio n of A n ta g o n istic M i c r o o r g a n i s m s ...............................................................

7

A.

C r o w d e d P l a t e M e t h o d ....................................................................

7

B.

B a c t e r i a l A g a r P l a t e M e t h o d .................................................

7

C.

B a c t e r i a l S p r a y M e t h o d ...............................................................

8

D.

A n t i b a c t e r i a l S p e c t r a .................................................................

8

E.

B l o o d L y s i s ...........................................................................................

9

The S t u d y o f C u l t u r e

77

( B a c i l l u s p o l y m y x a ) ...................

9

A.

S i n g l e C o l o n y I s o l a t e s ..............................................................

9

B.

S electio n

9

o f a T e s t O r g a n i s m ......................................

C. D e t e r m i n a t i o n o f B e s t M e d i 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 . . . . , .........................................................

10

D. R e l a t i o n o f N i t r o g e n S o u r c e t o A n t i b i o t i c P r o ­ d u c t i o n ....................................................................................................... 16 E.

T h e O b t a i n i n g o f a n A n t i b i o t i c .........................................

17

1 . C a r b o n A d s o r p t i o n ..................................................................... 1 7 2. D e term in atio n

17

3. A p p l i c a t i o n o f A d s o r p tio n and E l u t i o n T e c h n i q u e s .......................................................................................

18

4.

20

S ep aratio n

a s t h e H y d r o c h l o r i d e ................................

5. P u r i f i c a t i o n F.

of B est E lu tio n P r o c e d u r e . . . . .

P ro p e rtie s

t h r o u g h P i c r a t e F o r m a t i o n .............. 22

o f t h e A n t i b i o t i c ............................................... 2 2

1 . S t a b i l i t y ..........................................................................................

22

2 . S p e c ific ity

23

o f A c t i o n ..........................................................

TABLE OE CONTENTS ( C o n t i n u e d ) Page 3.

T o x i c i t y ..........................................................................................

4• S o l u b i l i t y

R e a c tio n s....

25

C o m p a r i s o n w i t h Known P o l y m y x i n s .................................

25

T h e S t u d y o f C u l t u r e 37 ( B a c i l l u s s u b t i l i s M a r b u r g ) ..........................................................................................................

26

A.

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

26

B. S e l e c t i o n o f a M e d i 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 .............................................................................................................

27

1 . C o m p a r i s o n o f M e d i a .............................................................

27

2 . E ffect

of M e tallic

I o n s ..................................................

29

3. E f f e c t

o f Y e a s t E x t r a c t ...................................................

29

to D eterm ine a S e p a r a t io n P r o c e d u r e . . .

29

G-. III.

C.

and C l a s s i f i c a t i o n

24

Id en tific atio n

S tu d ies 1.

S ta b ility

o f F e r m e n t a t i o n L i q u o r s ........................

29

2.

C a r b o n A d s o r p t i o n a n d E l u t i o n ...................................

30

3.

P r e c i p i t a t i o n E x p e r i m e n t s .............................................

30

4. E x tra ctio n s

o f t h e A c i d P r e c i p i t a t e ...................

31

D. The O b t a i n i n g o f a n A n t i b i o t i c o f C u l t u r e 37 i n a S t a b l e F o r m .............................................................................

32

E.

S o l u b i l i t y a n d S t a b i l i t y ......................................................

35

F.

D eterm in atio n

36

G-.

R elatio n of In h ib itio n

H.

E ffect

o f S e r u m on A n t i b i o t i c A c t i v i t y .................

37

I.

T o x i c i t y o f X a n t h e l l i n ............................................................

38

J*

I n v e s t i g a t i o n s t o D e te rm in e an A p p l i c a b le C h r o m a t o g r a p h i c M e t h o d .............................................................

38

1C. P a p e r C h r o m a t o g r a p h y a n d P a r t i t i o n on C e l l u ­ l o s e ............................................................................................................

39

of th e A n t i b a c t e r i a l S pectrum . . . t o I n c u b a t i o n T im e

37

TABLE OF CONTENTS ( C o n t i n u e d ) Page L . L i g h t A b s o r p t i o n C h a r a c t e r i s t i c s ........................................

43

M. P r o p e r t i e s

o f t h e A n t i b i o t i c ................................................

44

DISCUSSION.......................................................................................................................

47

SUMMARY...............................................................................................................................

50

BIBLIOGRAPHY..................................................................................................................

52

LIST OF TABLES AND FIGURES L ist

of T ables

T able 1.

Page The a n t i b a c t e r i a l s p e c t r a a n d t e n d e n c y t o b l o o d l y s i s o f th e c o l l e c t i o n of a n ta g o n is ti c m i c r o o r g a n i s m s ........................................................................................

2.

S p e c if ic ity of a c tio n

77.

60

3.

E f f e c t o f g l u c o s e c o n c e n t r a t i o n i n a n NZ a m i n e * m e d i u m on a n t i b i o t i c p r o d u c t i o n b y c u l t u r e 7 7 . .

61

4. 5. 6• 7* 8* 9. 10. 11. 12. 13. 14*

15.

of the liq u o r of c u ltu re

54

V a r i a t i o n o f a n t i b i o t i c p r o d u c t i o n b y c u l t u r e 77 w i t h d i f f e r e n t m e d i a ( G r o u p 1 ) ..............................................

62

V a r i a t i o n o f a n t i b i o t i c p r o d u c t i o n b y c u l t u r e 77 w i t h d i f f e r e n t m e d i a ( G r o u p 2 ) ..............................................

63

V a r i a t i o n o f a n t i b i o t i c p r o d u c t i o n b y c u l t u r e 77 w i t h d i f f e r e n t m e d i a ( G r o u p 3 ) ..............................................

64

E f f e c t o f n i t r o g e n s o u r c e on a n t i b i o t i c p r o d u c t i o n b y c u l t u r e 7 7 ..........................................................................................

65

E f f e c t o f y e a s t c o n c e n t r a t i o n on a n t i b i o t i c p r o ­ d u c t i o n b y c u l t u r e 7 7 .....................................................................

66

E f f e c t o f v a r i o u s c o m m e r c i a l p e p t o n e s on t h e a n t i ­ b i o t i c p r o d u c t i o n o f c u l t u r e 7 7 ...........................................

67

E f f e c t o f a m ino a c i d m i x t u r e s on t h e a n t i b i o t i c p r o d u c t i o n o f c u l t u r e 7 7 .............................................................

68

E f f e c t o f c a r b o n c o n c e n t r a t i o n a n d pH o n t h e a d ­ s o r p t i o n o f t h e a n t i b i o t i c o f c u l t u r e 7 7 ...................

69

C om parison of e l u a n t s f o r rem o val o f th e a n t i ­ b i o t i c o f c u l t u r e 77 f r o m c a r b o n ........................................

70

E f f e c t o f v a r i a t i o n o f c o n c e n t r a t i o n a nd a c i d i t y o n e l u t i o n ................................................................................................... Z ones o f i n h i b i t i o n used f o r th e c o n s t r u c t i o n o f a b a t c h c u r v e and f o r t h e d e t e r m i n a t i o n o f t h e a c t i v i t y o f c a r b o n e l u a t e s ...................................................... E f f e c t o f t e m p e r a t u r e a n d pH on t h e a c t i v i t y o f t h e a n t i b i o t i c o f c u l t u r e 7 7 ...................................................

71

72 73

L ist

o f T ab les

(C on tin u ed )

T able 16. 17. 18.

Page E f f e c t o f m e t a l l i c i o n s on t h e a n t i b i o t i c p r o d u c ­ t i o n o f c u l t u r e 37 i n a t r y p t i c a s e s o y m e d i u m . . .

74

The s t a b i l i t y o f t h e a n t i b i o t i c ( s) i n t h e f e r m e n ­ t a t i o n l i q u o r s o f c u l t u r e 3 7 ...................................................

75

The s t a b i l i t y o f a q u e o u s s o l u t i o n s o f t h e so d iu m s a l t o f x a n t h e l l i n .............................................................................

76

19.

T he a n t i b a c t e r i a l

s p e c t r u m o f x a n t h e l l i n ..........................

77

20.

V a r ia tio n of i n h i b i t i n g c o n c e n tr â tion of x a n th e l­ l i n w ith i n c u b a t i o n tim e i n b r o th d i l u t i o n a s s a y ...............................................................................................................

78

L ist

of F ig u res

F ig u re 1.

2.

3.

Page

The u l t r a v i o l e t a b s o r p t i o n s p e c t r a o f f r a c t i o n s o b t a i n e d from a x a n t h e l l i n sam ple f o l l o w i n g p a r t i t i o n o n c e l l u l o s e ...............................................................

79

The l i g h t a b s o r p t i o n s p e c t r u m o f x a n t h e l l i n ( a ) a b s o r p t i o n i n t h e v i s i b l e r e g i o n ........................... ( b ) a b s o r p t i o n i n t h e u l t r a v i o l e t r e g i o n ................

80 81

Changes i n th e u l t r a v i o l e t a b s o r p t i o n s p e c tru m of x a n t h e l l i n w i t h o x i d a t i o n .......................................................

82

The t i t r a t i o n

curve of x a n t h e l l i n

83

ABSTRACT In a program d e sig n e d b acterial

source,

of In d ian a and f o r

so ils,

to

fin d

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

a number o f c u l t u r e s , w ere sc re e n e d

tendency to

cause

ly sis

r e s e a r c h was b eg u n b a c t e r i a

iso lated

from a

from sam p les

for a n tib ac terial

sp ecificity

of b lo od .

tim e t h i s

At th e

had n o t b e e n i n v e s t i g a t e d

as

e x t e n s i v e l y a s had m o ld s and a c t i n o m y c e t e s a s s o u r c e s

of

a n tib io tic s. From t h e chosen f o r the

c u ltu re

filtra te s

stu d y a w hite

bacteriu m

am orphous p r o d u c t was o b t a i n e d

form o f a h y d r o c h l o r i d e .

a h ig h degree

o f the f i r s t

of a c t i v i t y

in

T h is p r o d u c t , w hich e x h ib ite d

ag ainst

g r a m - n e g a tiv e b a c t e r i a , was

found t o be a m ix tu r e o f p o ly m y x in s , w ith th e m a jo r com ponent corresp o n d in g

t o p o ly m y x in D when com pared by p a p e r c h r o m a t o ­

graphy. From t h e selected in

the

th is

for

cu ltu re

sodium s a l t .

a n t i b i o t i c was i d e n t i f i e d (M arburg)

and

an a u t h o r i t y on s o i l p a rtitio n

the

in

b a c illi.

the p ro d u c t.

two y e l l o w f r a c t i o n s

caused by a d if f e r e n c e form o f th e

free

The b a c t e r i u m p r o d u c i n g as

a stra in

P a p e r c h ro m a to g ra p h y and showed t h e p r e s e n c e S ep aratio n

in p a r titio n

in the m o b ility

acid

of B a cillu s

i d e n t i f i c a t i o n was c o n firm e d by

on p o w d e r e d c e l l u l o s e

one a n t i b i o t i c

the

of th e second b a c te riu m

s t u d y a y e llo w am orphous p r o d u c t was o b t a i n e d

form o f t h e

su b tilis

in to

filtra te s

and i n t h e

of the

of only

su b stan ce

on c e l l u l o s e was of th e

su b stan ce

form o f th e

sa lt.

in

ii C h e m ic a l and p h y s i c a l p r o p e r t i e s was d i f f e r e n t

in d icated

from o t h e r r e p o r t e d

B.

su b tilis

a n d t h e na me " x a n t h e l l i n " w a s a s s i g n e d X a n t h e l l i n was fo u n d w eight

of about 1000, to

u n satu rated

lin k ag es,

tio n

u ltra v io le t

in

the

a n tib io tic

c h aracteristics

c o n ta in

lu te a

a sharp decrease a g a in s t B r.

su is.

in

to

stro n g lig h t

co n tain absorp­

v isib le .

o r a i r had a m arked e f f e c t

a n d on t h e

stra in s

to m ic e . the

an e q u i v a l e n t

lig h t

a b so rp tio n

su b stan ce.

and v i r u l e n t

r e l a t i v e l y n o n -to x ic

a n tib io tic s,

reg io n as w ell as in th e

a c tiv ity

su b stan ce

it.

X a n t h e l l i n e x h i b i t e d maximum a c t i v i t y , S arcina

th e

7 «8% n i t r o g e n ,

and t o e x h i b i t

of the

to

t o be an a c i d w i t h

O x id a tio n by h y d ro g e n p e r o x id e on t h e

th at

in v i t r o , ag ain st

of B ru cella

How ever,

blood

in h ib ito ry e ffe c t

s u i s , and was serum cau sed

of th e

a n tib io tic

1 ANTIBIOTICS FROM BACILLUS POLYMYXA AND BACILLUS S U B T I L I S

INTRODUCTION An a n t i b i o t i c deriv ed

has been d e fin e d

from or p ro d u ced

cap ab le, in

by l i v i n g

sm all c o n c e n tr a tio n ,

processes

of m icro o rg an ism s"

b e n efite d

from a n t i b i o t i c s

an tib io tic

a s a " c h e m i c a l compound o rg a n is m s , w hich i s

of in h ib itin g

(l).

A lthough th e C hinese

was i s o l a t e d

not u n til about 1942, w ith th e r e a liz a tio n

a field

of p e n ic illin

in v o lv in g

in 1896, i t o f the

th at a n tib io tic

l a r g e num bers o f w o r k e r s .

a p p r o x i m a t e l y 1 5 0 now kn o w n o n l y s e v e n a r e

m ycin,

p e n ic illin ,

C hlorom ycetin,

stre p to m y cin ,

terram y cin ,

i n te r m s o f volume t h e

p ro d u ctio n

a ll

It

sale

o th er m ed icin als. of p e n i c i l l i n

o ne h a l f

of so u rce, i . e . , by b a c t e r i a , p e n ic illin ,

co m m ercially

of a n tib io tic s th at

t h o s e prod u ced by m o lds,

surpasses

f o r more t h a n

(aureom ycin,

and te r r a m y c in )

are a l l

the

on t h e b a s i s

Except fo r

co m m ercially

strep to m y cin ,

ob tain ed

(2 ).

by a c tin o m y c e te s ,

by o rg a n is m s o t h e r t h a n m i c r o b i a l . a m old m e t a b o l i t e ,

H ow ever,

i n 1948 th e

c a n c o n v e n i e n t l y be c l a s s i f i e d

w hich i s

aureo-

and t y r o t h r i c i n .

and s tr e p to m y c in a c c o u n te d

im portant a n t i b i o t i c s m y cetin ,

of the

o f m a n u f a c t u r e r s 1 incom e fro m m e d i c i n a l s

A n tib io tics

became

B etw een 1942

b a citra cin ,

has been s ta te d

was

c lin ica l

research

and 1949 o v e r 120 a n t i b i o t i c s w e r e r e p o r t e d , b u t

av ailab le:

life

2500 y e a r s a g o , and a c r y s t a l l i n e

(m ycophenolic a c id )

p o ssib ilitie s

the

C hloro­

from a c t i n o m y c e t e s .

2 Many e x t r e m e l y a c t i v e

a n tib io tic s

have been

iso lated

from

a c t i n o m y c e t e s b a t m o st o f them h a v e h i g h t o x i c i t y . The r e s e a r c h b e i n g r e p o r t e d cerned w ith because Today,

in

s e e k in g an a n t i b i o t i c

th is

source appeared

about s ix ty

of b a c te r ia l

of the

(4 ),

from b a c t e r i a , m a in ly

t o be th e

a re produced o th er

T y ro th ricin ,

lim ited

to x ic,

lea st

Of t h e s e ,

much s m a l l e r v o l u m e t h a n t h e b ein g

t h e s i s was con­

known a n t i b i o t i c s

ferm e n ta tio n s.

( 3 ) and t y r o t h r i c i n

th is

is

in v estig ate d . are p ro d u cts

o n ly tw o,

b a citra cin

com m ercial a n t i b i o t i c s . to

to p ic al

usage,

b a c i t r a c i n h a s n o t become c o m p l e t e l y e s t a b l i s h e d The p o l y m y x i n s had n o t

(5,

the v e ry

m y cetin ,

6 , 7,

fine

and

c lin ic a lly .

8 ) p r o b a b l y w o u ld be i n u s e t o d a y

a n tib io tic s,

a u r e o m y c i n and

C hloro­

been d isc o v e re d .

B acitracin ,

ty ro th ricin ,

of the b a c t e r i a l a n t i b i o t i c s

th e are

polym yxins, p ro d u cts

f o r m i n g b a c i l l i w h o s e common h a b i t a t b io tic s

in

c o m m e r c ia lly

is

from s p o re b e a r i n g b a c i l l i a r e ,

com plex c h e m ic a l

(l).

only fo r g ram icid in S (9 ),

th ric in

( g r a m i c i d i n and t y r o c i d i n ) ,

S o i l sa m p le s from d i f f e r e n t

lected

for

g en eral,

th e

of

com ponents of t y r o ­

and t h e p o l y m y x i n s .

c o u n ties

of In d ian a

of th e a n tib io tic -p ro d u c in g b a c te ria

th is p ro je c t.

The f i r s t

was p ro d u c e d by a g r a m - n e g a t i v e be a s t r a i n

A n ti­

C hem ical fo rm u la e have so f a r been

offered

source

so il.

spore-

s t r u c t u r e w h i c h t e n d s t o make t h e i r p u r i ­

fic a tio n d if f ic u lt

as the

of a e r o b i c ,

th e in

and many m ore

of B a c illu s

a n tib io tic

c o l­

obtained

b a c i l l u s w hich proved

p o ly m y x a . and t h e

served

to

second a n t i b i o t i c

3 is

a m etab o lite

of a

g r a m —p

id e n tifie d , as B a c illu s W hile r e s e a r c h p ro g ress, th ree

o sitiv e

su b tilis

on t h e

B.

lab o ra to ries

b a c i l l u s "which h a s b e e n

(M arburg) .

polym yxa a n t i b i o t i c w as i n in

th e U nited

E n g l a n d w e r e a l s o w o r k i n g on a n t i b i o t i c s B.

a nd

from s t r a i n s

of

polym yxa, th e N o rth e rn R e g io n a l L a b o r a to r y o f th e

D epartm ent

of A g ric u ltu re

Co m p any ( 5 ) ,

two g r o u p s

carried

at

of th e

e ig h te e n p a p e r s by f o r ty - o n e e v e r y t h i n g k nown a b o u t t h e

fo llo w ed , th e

called

jo in t

tim e.

a n tib io tics

O rig in ally ,

"aero sp o rin "

fin d in g s

au th o rs

iso lated

of

"p o ly m y x in .”

of B.

of the

polym yxins a re l i s t e d , a l l

sim ila r s t r u c t u r e , desig nated

polym yxins

" a e r o s p o r i n ” became p o ly m y x in A and

" p o l y m y x i n ” b e c a m e p o l y m y x i n D.

A ll o f th e polym yxins con­

t a i n L - th r e o n in e , L -o f,/-d iam in o b u ty ric acid ; a l l

of

i n E n g la n d was

polym yxa w e r e shown t o b e s t r a i n s fiv e

(7)

set fo rth .

( from B a c i l l u s a e r o s p o r u s ) and t h a t

cy clic

o c ta n o ic

the

k n ow n a n d c a n be d e s c r i b e d

the a n t i b i o t i c

At p r e s e n t

G, D, a n d E ;

concerned,

a n d f r o m some w o r k w h i c h

fa c ts are

same o r g a n i s m .

B,

a co m p letio n of

in w hich n e a r l y

t h e A m e r i c a n C y a n a m i d Com pany w a s c a l l e d

p ep tid es

The l a t t e r

in a c o lle c tio n

polym yxins i s

researches

e sse n tia l

a e r o s p o r u s a n d B.

(6 ) a l l m aking

o f t h e New Y o r k A c a d e m y o f S c i e n c e s

c o n ta in in g an account of t h e i r

From t h e s e

same

th e re s e a rc h through to

1949, A nnals

b riefly .

t h e A m e r ic a n C yanam id

about the

the ch em ical s t r u c t u r e s lune,

(10),

and t h e W ellcom e L a b o r a t o r i e s

p relim in ary re p o rts

A,

S ta te s

acid ,

and 6 -m ethy 1 -

except C c o n tain D -le u c in e ;

k

L -p h en y lalanin e D—s e r i n e

is

c irc u lin

(11),

is

present

present

i n p o l y m y x i n s B a n d C,

i n polym yxin D o n ly .

from B aci l l n s

guished

from i t

by v i r t u e

Tlie a n t i b i o t i c ,

c i r c u l a n s t h as the

c h e m ic a l c o m p o s itio n a s polym yxin A b ut of its

and

same

can be d i s t i n ­

in a c ti v a ti o n by a l i ­

pase • M ore a n t i b i o t i c s B.

su b tilis

have been re p o rte d

th a n from any o th e r

from s t r a i n s

organism

th e se has been co m p letely d efin ed

(1).

None o f

c h e m i c a l l y and f o r

o f them e s s e n t i a l l y no c h e m i s t r y h a s b e e n r e p o r t e d . b e t t e r know n o n e s a r e c illin

(1 3 ),

b a cillo m y cin and S n e l l

eum ycin (17),

(IB ).

been s tu d ie d b a citra cin

su b tilin (1 4 ),

and t h e

Of t h e s e ,

su b tily sin

gested

for

su b te n o lin

a n tifu n g al fa c to rs su b tilin

and s u b t i l i n

use i n canned

foods.

the M ic h e n e r-S n e ll f a c t o r s a re ag ain st

(1 6 ),

o f M iche n e r

and b a c i t r a c i n

have

research

some

h av in g r e c e n t l y been su g ­ E um ycin, rep o rted

The B . s u b t i l i s

b acillo m y cin ,

and

t o be m o st e f f e c ­

f u n g i and to have r e l a t i v e l y

b acterial a c tiv ity .

resp ects

(1 5),

The

{3 )> b a -

b e in g p ro d u ce d c o m m e rc ia lly and used t o

c lin ic a lly ,

in t h is

b a c itra c in

some

t o a m uch g r e a t e r d e g r e e t h a n t h e r e s t ,

ex ten t

tiv e

(1 2 ),

of

little

a n tib io tic

a n ti­ o b tain ed

r e s e m b l e s eum ycin and s u b t e n o l i n

in

some

b u t a l s o e x h i b i t s m arked d i f f e r e n c e s .

The e x p e r i m e n t a l p a r t t h r e e m ain d i v i s i o n s , a n ta g o n istic c u l t u r e s , th e

of t h i s

th e f i r s t

c u l t u r e s from s o i l

th esis

is

composed

d e sc rib in g th e and

of

o b tain in g of

the s c re e n in g of th e s e

second b e in g concerned w ith th e

sep aratio n

5 of an a n t i b i o t i c and t h e b io tic

th ird

from a s t r a i n

d iv isio n

As t h e r e s e a r c h w i t h

to

it

polym yxa

the

sep aratio n

co v ering

from a M arburg ty p e

progressed,

o f B.

o f B.

su b tilis

the a n ti b i o ti c

became a p p a r e n t t h a t

it

g raphic

the i d e n t i t y o f the

77

som e r e s e m b l a n c e

it

a n tib io tic

c o m p a r is o n s c o u ld be made.

37)•

from c u l t u r e

bore

77),

o f an a n t i ­

(cu ltu re

^ a e r o s p o r i n ” and " p o ly m y x in ” ; h o w e v e r,

to determ in e

(cu ltu re

was n o t p o s s i b l e u n til

M oreover,

chrom ato­

u n til

the June,

1 9 4 9 , A n n a l s o f t h e New Y o r k A c a d e m y o f S c i e n c e s a p p e a r e d , i t w a s n o t k n o w n how much o f t h e

c h e m is try o f th e polym yxins

had b e en w orked o u t .

1949,

fied h y d ro ch lo rid e

In A ugu st,

of th e c u l t u r e

a sam ple

of th e p u r i ­

77 a n t i b i o t i c was s e n t

t o D r . H a r o l d N a sh o f t h e P itm a n - M o o r e Company.

U sing

p a p e r c h ro m a to g ra p h y . D r. N ash com pared t h e m a t e r i a l w i t h known p o l y m y x i n s .

He r e p o r t e d t h a t t h e s a m p l e a p p e a r e d

c o n t a i n m ore t h a n one p o ly m y x in w i t h a zone o f a d v a n c e t h a t m ost o f th e c arried

chem ical r e s e a r c h

out,

a n tib io tic

corresponded

it

was d e c i d e d t o

from c u l t u r e

organism s in

th e

to

the m ajo r p a r t having t o p o l y m y x i n D.

S ince

on t h e p o l y m y x i n s h a d b e e n term in a te re s e a rc h

on t h e

77 i n f a v o r o f i n v e s t i g a t i n g

o th er

c o llec tio n .

S e v e r a l o f t h e s e w e re e x a m in e d i n a p r e l i m i n a r y way before

i t was d e c id e d

c u ltu re ism .

37.

I t had

c u ltu re

N. S .

by r a t s

the s t r a i n

o f B.

C r e e a r had begun r e s e a r c h

sh o w n p r o m i s e

b r o t h s had

to le rate d

to stu d y

in t h a t

su b tilis.

on t h i s

organ­

c o n c e n tr a te s from the

in h ib ite d B ru cella upon i n t r a p e r i t o n e a l

su is

and had been

in je ctio n .

H ow ever,

6 th e

cu ltu re

lo g ic ally ,

re s e m b le d B. and s i n c e

from s t r a i n s p o ssib ility b io tic .

o f B.

a n tib io tic s

c u l t u r a l l y and m orpho­

so many a n t i b i o t i c s h a d b e e n r e p o r t e d

s u b t i l i s , th ere

ex iste d

a fe a r of

of o b tain in g an o th er a lre ad y -d isc o v ered

C om parison t e s t s

c o n ce n tra te s

su b tilis

in d icated to w arrant

on s m a l l q u a n t i t i e s

the a n ti­

of a c tiv e

enough d if f e r e n c e s from re p o rte d co n tin u ed

in v estig atio n s.

7 EXPERIMENTAL I.

E stab lish m en t

of a C o llectio n M icroorganism s

T w e n ty -s e v e n sam ples th e

iso latio n

of about

o f Indiana

of A n tag o n istic

s o i l s w ere used f o r

one h u n d r e d m i c r o o r g a n i s m s w h ic h

showed a n t a g o n i s m i n one o f t h e t h r e e

i s o l a t i o n m ethods

em ployed--the

crow ded p l a t e m e th o d , t h e b a c t e r i a l a g a r

p l a t e m ethod,

and t h e b a c t e r i a l

A . Crowded P l a t e M ethod

(19).

O r d i n a r y s o i l was p l a t e d th e

ag ar of ch o ice,

sp ra y m ethod.

in

stan d ard P e t r i d ish e s

using d i l u t i o n s

of s o il

o f 1:100 to

1 :1 0 0 ,0 0 0 .

P l a t e s w e re i n c u b a t e d a t room t e m p e r a t u r e

(2 1 -2 4 ° C.)

or at

seven d a y s .

3 0 ° C.

D ifferen t

w ere loo k ed

f o r p e r i o d s r a n g i n g fro m one to

so ils

and c r o w d in g a t d i f f e r e n t

gave v a ry in g ty p e s o f grow th

d ilu tio n s.

perio d ,

and a n t a g o n i s t s w e r e p i c k e d

ste rile

agar p la te s .

In g e n e ra l,

F o r t h i s m ethod used w i t h th e

and t r a n s f e r r e d

d ilu tio n s

crow ded p l a t e m eth o d e x c e p t in to

the

so il

h ig h e r tem p eratu re

o f 1 :1 0 0 0 and

o rg a n is m s to to fa v o r th e

same a s t h a t th at a te s t

agar to g e th e r w ith th e

and p l a t e s w e re i n c u b a t e d f i r s t th e

to

(20).

th e p r o c e d u r e was t h e

o rg a n is m was i n c o r p o r a t e d

to allo w

in cu b atio n

best re s u lts .

B. B a c t e r i a l A g a r P l a t e M e t h o d

so il,

Zones o f i n h i b i t i o n

f o r from day to day th ro u g h o u t the

1 : 1 0,00 0 gave th e

in

a t a low er te m p e ra tu re

grow b e f o r e t r a n s f e r grow th o f th e t e s t

to a o rganism .

8 C_. B a c t e r i a l S p r a y M e t h o d A co n sid erab le

(21).

amount o f r e s e a r c h w as d e v o te d t o

developm ent o f a s a t i s f a c t o r y te c h n iq u e s p r a y m ethod.

The m eth o d p r o v e d

to

v alu ab le a f t e r proper c o n d itio n s e s t a b l i s h e d , th e m ost of th e r ig h t

A ir p ressu re

used

it

T he f o l l o w i n g

u se had b e e n se lec tio n

The m o s t s a t i s f a c t o r y

b ein g n e c e ssa ry to

test

sp ra y the

r o t a t in g the

p late

o rg a n is m s w ere found

s p r a y m ethod:

b ro n ch ise p t ic a , B a cillu s

E sch erich ia

type

th ro u g h

c o l i , B ru cella

c o u ld be used

p r o p e r m edium and s p r a y e d

same

to be e a s i l y

s u b t i l i s » an d B a c i l l u s

M ycobacterium t u b e r c u l o s i s in th e

for its

and

p r o v i d e d b y h an d b u l b s was s u p e r i o r t o p r e s s u r e

a number o f tim e s w h ile

in th e

be q u i t e w o r k a b l e

f o u n d t o b e a s m a l l s i z e D e V i l b i s s A'1 2 7 .

from th e a i r l i n e ,

360°.

th e b a c t e r i a l

f a c t o r b ein g th e

type o f a to m iz e r.

f o r t h i s w ork was

p late

im p o rtan t

in

the

if

it

cereus. w e re grown

onto a s u i t a b l e

agar.

D. A n t i b a c t e r i a l S p e c t r a . The a n t i b a c t e r i a l s p e c t r a o b ta in e d by an a g a r

s t r e a k m ethod

c u ltu res

of the

su b tilis

(Ford t y p e ) ,

b ro n ch isep tica

fo llo w in g t e s t

(ATCC 6 0 7 ) .

1 show s t h e

blood

ly sis

(2 0 ),

T able

c u l t u r e s was

u sin g 24-h o u r b r o th

organism s:

(P e o ria B 140), S alm o n ella B r.

and v i a b l e .

iso lated

B.

c e r e u s » B.

S tap h y lo co ccu s a u re u s , E.

V e te r in a r y 30628),

sev en ty -th ree

of the

su is

c o li » B r.

p u llo ru m

(Purdue

(A 1 9 ), Myco. t u b e r c u l o s i s a n tib ac terial

sp ectra

o f t h e s e m ic ro o rg an ism s t h a t rem ained The t a b l e

by th e

a lso

in d icates

in d iv id u a l

the

c u ltu res.

of a ctiv e

ten d en cy tow ards

9 E.

B lood L y s i s S u r v e y . A Jfo b l o o d

fib rin a te d ployed

in

allo w ed

a g a r m ad e b y i n c o r p o r a t i n g s t e r i l e ,

c a l f blood th is

to

in to

screenin g

procedure.

and p l a t e s w e re r e a d

In T able 1 a z e ro

in d ic ate s

a n u m b e r f r o m one t o f i v e first, II.

o f B.

s e p t i c a , S.

t h a t hem olysis

fo u rth ,

or f i f t h

77 (B a c i l l u s

T a b l e 1 o r g a n i s m 77 i n h i b i t e d s u b t i l i s . S t.

a u r e u s , E.

occurred

day.

th e grow th

c o l i , B r.

bro n ch i-

p u l l o r u m a n d M yc o. t u b e r c u l o s i s w h e n t e s t e d of b lo o d .

C olony I s o l a t e s .

The m i c r o o r g a n i s m w as t a k e n t h r o u g h c o lo n y t r a n s f e r s , and s l a n t s a n tib io tic

and

polym yxa)

b y t h e a g a r s t r e a k m ethod and showed n o l y s i s A. S ingle

d a y s a t room

f o r hem olysis each day.

in d ic ate s

The S t u d y o f C u l t u r e

c e r e u s , B.

fo r fiv e

t h a t no h e m o l y s i s o c c u r r e d ,

second, th ir d ,

As s h o w n i n

a g a r w a s em­

E ach c u l t u r e was

g r o w on a b l o o d a g a r p l a t e

tem perature

on t h e

o rd in ary n u tr ie n t

de-

p ro d u ctio n .

so

a series

of sin g le

o b ta in e d w ere com pared f o r

No d i f f e r e n c e s

among s l a n t s

could

be d e t e c t e d . B.

S electio n In

o f T e st O rganism .

order to

ing th e a c t i v i t y

select

in

only th e

organism f o r m easur­

shaker c u ltu re at

cup a s s a y p l a t e s

a u r e u s , E . c o l i , and B r. th at

test

p ro d u ced by o rg a n is m 7 7 , u n f i l t e r e d

o f o r g a n i s m 7 7 , grown i n teste d

a su itab le

o f B.

28° C. , w ere

c e r e u s , B.

b ro n ch isep tic a .

g ram -n eg ativ e organism s,

E.

beers

su b tilis, S t.

T a b le 2 shows co li

a nd B r .

10 b r o n c h i s e p t i c a , w ould have a n y v a l u e a s cup d i f f u s i o n B r. C.

assay.

b ro n ch isep tic a D eterm in atio n

B140 i n t h i s

and s u b s e q u e n t t a b l e s

o f B e s t M e d iu m f o r A n t i b i o t i c in

th e stu d y was th e

g r o w th m edium w h ic h w ould y i e l d

is

P ro d u c tio n .

se lec tio n

of a

t h e maximum a m o u n t o f t h e

The e f f e c t

of v a ria tio n

i n a n NZ a m i n e m e d i u m o n a n t i b i o t i c E rlen m ey er f l a s k s

o f 500 m l.

m l.

flask

o f m edium p e r 28° C .

try p tica se

in

c o n cen tratio n

p r o d u c t i o n was s t u d i e d .

c a p a c i t y w e re u se d w i t h 100

s h a k e r and

sta tio n a ry

c u ltu re a 24-hour

soy

medium

b ro th

of

7 .1

before

to

sodium h y d r o x id e .

c u ltu re

T able

zone in

77.

The pH o f t h e

in o cu latio n

3 shows t h e

(m m.) a s m e a s u r e d f r o m t h e

sep tica .

of glucose

E a c h f l a s k was i n o c u l a t e d w i t h 1 m l. o f

was a d j u s t e d

the

a

p rin c ip le .

1.

at

organism s in

(B 14 0 - P e o r i a ) .

The n e x t s t e p

a ctiv e

test

T he c o m p o s i t i o n

of E.

o f 10%

zones of in h ib itio n

edge of t h e

cup a s s a y p l a t e s

by a d d i t i o n

cup to

the

c o l i and B r.

edge of

b ro n ch i­

o f t h e NZ a m i n e m e d i u m i s

liste d

b e l o w T a b l e 3• For the

rest

t i o n £ 500 m l . m edium p e r In o cu latio n try p tica se

of th e experim ents re p o rte d

E rlen m ey er f l a s k s

f l a s k w ere used of soy

c o n t a i n i n g 100 m l.

shake c u ltu r e

b ro th

of cu ltu re

c u ltu re .

Cup a s s a y p l a t e s

A lso, in a l l

7 7 , v/hich b r o t h

teste d

of

a 24-hour

a ro s e from

s l a n t s made f r o m s i n g l e

a l l beers

sec­

a t 2 8 ° C.

e a c h f l a s k was a l w a y s w i t h 1 m l. o f

a t r a n s f e r f r o m one o f t h e of th e

in

in th is

c o lo n ies

w ere u n f i l t e r e d .

t h e s e ru n s w ere h e ld

in the

co ld

11 room f o r for

4 hours b efore b ein g placed

at

3 7 ° C.

in cu b atio n

ov ern ig h t. 2.

F iv e m edia whose c o m p o s it io n s a r e l i s t e d

w ere d i s t r i b u t e d flask s.

in

100 m l .

p o rtio n s

to

500 m l.

These w ere i n o c u l a t e d w ith 1 .0 m l.

try p tica se

soy b ro th

colony s l a n t

of cu ltu re

of c u ltu re

77,

below

E rlen m ey er

of a 24-hour

7 7 made f r o m $3 s i n g l e

and w ere

g r o w n on a s h a k e r a t

2 8 ° C. T a b le 4 shows t h e by u n f i l t e r e d

beers

zones

of th ese

of in h ib itio n sam ples i n

i n mm. g i v e n

cup a s s a y p l a t e s

B r. b ro n c h ise p tic a . M edium

C o m p o s i t i o n o f M edium Peptone Y east e x tr a c t 1C2HP04

0.75% 0.25% 0 . 20%

P eptone Y east e x tr a c t G lucose k 2h p o 4

0.75% 0.25% 0 . 5 0% 0 . 20%

NZ a m i n e G lucose S a lt m ix tu re pH a d j u s t e d t o 7 . 1

1 . 00% 0 . 5 0% 150 m l . / L

NZ a m i n e G lucose S a lt m ix tu re pH a d j u s t e d t o 7 . 1

2 . 00 % 0 . 5 0% 150 m l . / L

"T ry p ticase T ry p ticase P h y t one NaCl K2HP04 G lucose *

S o y B r o t h f t «t* 1 . 70% 0 . 30% 0 . 5 0% 0 . 2 5% 0 . 2 5%

B altim o re B io lo g ic a l L ab o ra to ry

of

12 3.

Upon c o n s i d e r a t i o n

m edia s t u d y i t 28° 0 . in th e Medium

was d e c i d e d fo llo w in g

of th e

re su lts

to grow c u l t u r e

o f th e p re c e d in g

77 on s h a k e r a t

te n m edia: C o m p o s i t i o n o f Medium

6

Soya f l o u r G lucose (NH4 ) 2S0, KHoPO/, M gSO,.7H2 0 N aCl* F e (NH4 ) 2S 0 4 • 6H2 0

1.5% 1.0% 2 .0 % 0.2% 0.2% 0.005% 0.0014%

7

P eptone Y east e x tr a c t

0.75% 0.25%

8

Same a s 7 ,

p l u s 0.5% g l u c o s e

9

Same a s 7>

p l u s 2.0% g l u c o s e

10

Same a s 8 , w i t h 0 .2 5 g . CaCOV 100 m l. added a f t e r a u to c la v i n g .

11

Same a s 8 , w i t h 1 . 0 g . CaC O^/ 100 m l. added a f t e r a u t o c l a v i n g .

12

P eptone Y east e x tra c t G lucose KH2 P 0 4

0.75% 0 . 25% 0.5% 0.

13

P eptone Y east e x tr a c t G lucose Speakman s a l t s

14

Same a s 6 , b u t w i t h 0 . 5 % y e a s t e x t r a c t i n s t e a d o f 1 . 5% s o y f l o u r a n d w i t h 1 . 0 g . CaCOo a d d e d a f t e r au to c lav in g .

15

C a s e i n h y d r o l y s a t e 1.0% G lucose 0.5% Speakman s a l t s 0 . 5 m l . A) 0 .5 m l. B )P e r 100

T h e pH v a l u e s o f m e d i a 7 ,

8 , 9,

0.75% 0.25% 0 .5 % 0 . 5 m l . A) 0 :5 m l: B )p e r 100 m l -

12,

13 a n d 15 w e r e

_

13 a d j u s t e d w here n e c e s s a r y t o 6 .8 w ith

10% NaOH.

The g l u c o s e

was added a f t e r a u t o c l a v i n g i n m edia 6 and 14•

E a c h m edium

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

and e a c h o f t h e s e

in t r i p l i c a t e

in

se p tica .

The a v e r a g e z o n e

th erefo re

the

the a c t i v i t y at

average

in d iv id u a l

cup a s s a y a g a i n s t B r . size

(mm.)

liste d

of n in e r e a d in g s .

of th e b e e rs a f t e r

flask s b ro n ch i­

in T able 5 i s

The t a b l e

shows

2 , 4 , and 6 d a y s i n c u b a t i o n

28° 0 . 4 .U s i n g w h a t

appeared

p r e c e d i n g r u n (M edium 6 ) t h e

t o be t h e b e s t medium fo llo w in g

in th e

v a r i a t i o n s w ere

prepared : Medium 6

C om position

o f M e d iu m

Soya f l o u r 1.5% G lucose 1 . 0% ( N H i ) pSO j 2. 0 % KH2P 0 f * 0 . 2% M g S O ,. 7 H 2 0 0.2% NaCl 0.005% E e ( N E , ) 2 S 0 , . 6Ho0 0.0014% (G lucôse a u to c la v e d s e p a r a t e l y )

16

Same a s 6 , b u t

w i t h 1.0%

(NH^J^SO^

17

Same a s 6 , b u t

w i t h 0 .5 %

( N E ^ ) 2 GO^

18

Same a s 6 , b u t

w i t h no

19

Same a s 6 , b u t

w i t h 1% CaCO^ a d d e d

(NH^)2S 0^

20

Same a s 6 , b u t w i t h 2% NHaNOo i n s t e a d o f (N H ^ ) 2 S 0 ^

21

Same a s 6 , b u t w i t h 1% NH.NOo i n s t e a d o f (NH^)2S 0^

22

Same a s 6 , b u t w i t h 2% ( N E , )E2 P0, i n s t e a d o f ( N E ^ ) 2 SO^ 4 ^

23

Same a s 6 , b u t w i t h 1% (NE, ) E 2 P0. i n s t e a d of (N H /^S O ^ 4 *

C o m p o s i t i o n o f Medium

M e d iu m 24

T able 6 l i s t s beers

Same a s 6 , b u t w i t h g l u c o s e a u t o clav ed to g e th e r w ith r e s t o f c o m p o n e n t s a f t e r a d j u s t m e n t o f pH to 6 .7 5 • the

re su lts

from s h a k e r c u l t u r e 5•

at

ob tain ed

2 8 ° C. a f t e r 1 ,

A g a i n u s i n g M e d iu m 6 a s t h e

fo llo w in g v a r ia tio n s M e d ium 6

of

from th e

u n filte re d

3 and 6 d a y s .

b a s i c medium , t h e

i t w ere p r e p a r e d , e ac h in

trip lic a te

C o m p o s i t i o n o f M e d ium Soya f l o u r G lucose (NH4 ) 2 S 0 4 KH2P 0 4

1.5% 1.0% 2 .0 % 0.2%

MgS04.7H20

0.2%

NaCl 0.005% F e (NH4 ) 2 s 0 4 #6H2 0 0.0014% (G lucose a u to c la v e d s e p a r a t e l y ) 25

Same a s 6 ,

b u t w i t h 0.5% so y a

flo u r

26

Sam e a s 6 , b u t w i t h 1 .5 % y e a s t e x t r a c t i n s t e a d o f soya f l o u r

27

Same a s 6 , b u t w i t h 0 . 5 % y e a s t e x tr a c t in ste a d of soy f lo u r

28

Same a s 6 , b u t w i t h 1 . 5 % f e r m e n t s o l u b l e s i n s t e a d of so y f l o u r

29

Same a s 6 , b u t w i t h 0 .5 % f e r m e n t s o l u b l e s i n s t e a d o f soy f l o u r

30

Same a s 6 , b u t w i t h 1 . 5 % p e p t o n e in s te a d of soy f l o u r

31

Same a s 6 , b u t w i t h 0 .5 % p e p t o n e in s te a d o f soy f l o u r

32

Same a s 6 , b u t w i t h 0 . 5 % l i v e r e x t r a c t i n s t e a d o f soy f l o u r

33

Same a s 6 , b u t w i t h 1 . 5 % c o r n s t e e p s o l i d s in s te a d o f soy f l o u r

15 M e d iu m

C o m p o s i t i o n o f M e d iu m

34

Same a s 6 , b u t w i t h 0 . 5 % c o r n s t e e p s o lid s in ste a d of soy f lo u r

35

Same a s in stead

36

Same a s 6 , b u t w i t h t h e 1 .0 % g l u c o s e a u t o c l a v e d w i t h r e s t of medium

Each o f th e tio n s in

to

th ree

13 m e d i a w a s d i s t r i b u t e d

at

2 8 ° C.

i n cup a s s a y p l a t e s

and 5 d a y s ,

the average

As t h e t a b l e 1 . 5% s o y a

ferm en tatio n

Each f l a s k w as of B r.

show s,

of n in e

no a c t i v i t y

f l o u r was r e p l a c e d so lu b les,

o r b y C u r b a y BG-80.

rep laced

flo u r,

soya

in 3 d ay s,

one f l a s k

two f l a s k s

same a s g i v e n ste rile It

in

i s no ted e ith er

out

1,

3,

zones. a t a l l was g iv e n w h e re

liv e r ex tra ct,

of th re e

showed a c t i v i t y

by

by c o r n

in

sho wed a c t i v i t y

5 days.

and i n a c t i v e

cup a s s a y p l a t e s ,

b e e r s w ere

t h e e f f e c t was th e b e e r a nd

w ater. t h a t m edia w h ic h gave a c t i v i t y soya f l o u r ,

yeast e x tra c t,

e a r l y r u n s m edia w hich gave a c t i v e 77 c o n t a i n e d e i t h e r s o y a p ep to n e

at

tri­

i n h i b i t i o n a s g iven

by m i x i n g e q u a l v o lu m e s o f a c t i v e

d istille d

co n tain ed

por­

I n M e d ium 3 0 , w h e r e p e p t o n e

When e q u a l v o l u m e s o f a c t i v e m i x e d a nd t e s t e d

in

b y 0 . 5% s o y a f l o u r ,

by s o lu b le

steep s o lid s ,

tested

b ro n ch isep tica

zones of the

in T able 7 b e in g th e a v e ra g e

th e

i n 100 m l.

500 m l. E r le n m e y e r f l a s k s w h ic h w e re p l a c e d

shaker c u ltu re

p lic ate

6 , b u t w i t h 0 . 5 % C u r b a y BG-80 o f soy f lo u r

flo u r,

beers

yeast

(alw ays w ith y e a s t e x t r a c t ) ,

(w hich gave s m a ll z o n e s ) .

in th is

run

or p ep to n e.

In

from t h i s

c u ltu re

e x t r a c t , NZ a m i n e , or case in h y d ro ly zate

16 6• found

The p r e c e d i n g

th at

th e

of th e f i r s t

experim ent

resu lts

of th e

except in

the

(5) was r e p e a t e d .

second t r i a l

fo llo w in g :

I t was

d u p licated

those

(a ) A c t i v i t y was ob ­

t a i n e d w i t h t h e m edium c o n t a i n i n g

0.5% so y a f l o u r .

zones

f r o m M e d iu m 35 ( C u r b a y B G ) .

( 3 . 0 - 4 . 5 mm.) w e r e

D. R e l a t i o n

o btained

a c tiv ity

1. in

It

developed

was found

sta tio n a ry

c u ltu re

a t 28° C. f o r

in c u b a ti o n used w ith 2.

I t was

washed c e l l s a n tib io tic cells

o n ly in

th at

c e r t a i n m edia.

tu b e s w i t h 5 m l.

the tu b e s the

of cu ltu re

pro d u ctio n

flask s

in stead in

i n o c u l a t i o n w i t h washed o r un­

in y east

any d if f e r e n c e

e x t r a c t medium ,

in a n t i b i o t i c u n til of the

of yeast e x tra ct lev el

the

T a b le 8 shows t h e

a n tib io tic . showed a d e c r e a s e

o f y e a s t was r e d u c e d ,

o f 0 . 10% y e a s t e x t r a c t

organism in

b u t washed

s m a ll z o n es w hich la c k e d t h e

p ro d u ctio n as th e

at a lev el

in

o f C u r b a y BG m e d i u m g a v e a b e e r

ex h ib ited

lev e ls

in cu ­

shake c u l t u r e .

s h a rp n e s s o f edge u s u a l l y found w ith t h i s D ifferen t

o f 500 m l.

o f t h e 24 h o u r

77 d i d n o t g i v e

used f o r i n o c u l a t i o n

3.

o f m edia

o f m ed ia a l l o w i n g 48 h o u r s

found t h a t

w hich when t e s t e d ,

te st

t o e x p l a i n why a n t i ­

c o u ld be u sed i n s t e a d

E rle n m e y e rs w ith 100 m l. b a tio n

S m all

of N itro g e n Source to A n t i b i o t i c P ro d u c tio n .

Now e x p e r i m e n t s w e r e d e s i g n e d b io tic

(b)

t h e r e w a s no g r o w t h

3 day in c u b a tio n p e r io d . zones of i n h ib i t io n

produced

at

v ario u s y east le v e ls . 4.

I t was f o u n d t h a t

th eir a b ility

to

cause

com m ercial p e p to n e s d i f f e r e d

(or in h ib it? )

a n tib io tic

in

p ro d u ctio n .

17 T able 9 g iv e s grown i n at

zones of i n h i b i t i o n

5 m l.

28° C.

volum e

in t e s t

of beers

tubes in

P e p to n e s w ere t e s t e d

of

c u ltu re

sta tio n ary

at le v e ls

of 0 .5

77

c u ltu re and 1 . 0 $ ,

e ac h sam ple i n d u p l i c a t e . 5. re la tiv e

U s i n g m i x t u r e s o f 18 a m i n o a c i d s a t p e r c e n t a g e s to each o th e r

as

found i n c a s e i n ,

d i f f e r e n t m ix tu re s gave d i f f e r e n t no c o n c l u s i o n s n oted

is

th an in than did g iv es

th at

c o u ld be

a n tib io tic

the g ro w th in

1$ y e a s t

p ro d u ctio n ,

draw n fro m t h e r e s u l t s .

e x tra ct

th e average p e r cen t

A lso

to

but be

t h e am ino a c i d m i x t u r e w as l e s s

c a s e i n h y d r o ly z a te w hich in the

i t was found t h a t

t u r n had l e s s g ro w th

c o n t r o l medium.

T a b l e 10

p r o d u c t i o n w i t h 1$ y e a s t e x t r a c t

t a k e n a s 100$ . E.

The O b t a i n i n g o f a n A n t i b i o t i c . JL. C a r b o n A d s o r p t i o n .

was c l a r i f i e d H alf o f th is was l e f t

A ctiv e

1 .5 $ Y east E x tr a c t B eer

by s t i r r i n g w ith "C e lite " filtra te

was t a k e n

and f i l t r a t i o n .

t o pH 2 . 5 w i t h HC1 a n d h a l f

a s pH 6 . 5 .

D u p licate

tubes

o f 10 m l .

s h a k i n g 30 m i n u t e s w i t h 1$ , (Darco G --60).

1 / 2$ ,

The f i l t r a t e s

a s c e r t a i n what p e rc e n ta g e

o f e ach w ere t r e a t e d 1 / 4$ ,

from t h e

o r 1 / 10$ o f

by carbon

c a rb o n w ere t e s t e d

to

o f a c t i v i t y was rem oved b y th e

carbon.

T a b le 11 show s t h a t

a c tiv ity

and t h a t

1 $ c a r b o n a t pH 6 . 5

removed a l l

c a r b o n a t pH 6 . 5 w a s m o r e e f f e c t i v e

than

a t pH 2 . 5 . 2.

D eterm in atio n

o f s o l v e n t s was u se d to

o f B est E lu tio n P ro c ed u re .

A v a rie ty

f in d w h ic h w ould rem ove t h e a n t i b i o t i c

18 from th e

carbon.

ity

of the

a te .

It

T able

12 s h o w s t h e p e r c e n t a g e

o rig in a l u n treated

is

seen th a t

beer th at

a cid ifie d

eth a n o l

at d iffe re n t

a c id itie s per

th ree

gave

the

From t h i s

7 5% a c e t o n e

resu lts

liste d

on a b a t c h

experim ent

it

com plete rem oval o f th e

did

th at

m ethan­

e ffe ct.

m e t h a n o l a nd

under d i f f e r e n t

in T able curve

elu -

13 w h i c h

of th e th at

an a c i d i t y

shows

u n treated

50% a c e t o n e ,

of 0 .0 1 N o r 0 .0 5

0 . 0 5 N HC1 w o u l d g i v e

a c t i v i t y from th e carb o n .

a c e t o n e rem oved m ore c o l o r fro m th e

the m ost I t was

carbon th an

e t h a n o l and m e t h a n o l . J.

F ive

a ce to n e ,

appeared

and 1 0 0 % a c e t o n e a t

N i n HC1 , o r 75% e t h a n o l a t

noted

any a p p r e c ia b le

c o n c e n t r a t i o n s and

cent e lu t i o n based

beer.

so lv en ts,

a c tiv ­

appeared in th e

a c e t o n e and a c i d i f i e d

o l w ere t h e o n l y e l u a n t s w h ich had E lu tio n w ith

of the

A p p l i c a t i o n o f A d s o r p t i o n and E l u t i o n T e c h n i q u e s .

c a r b o n a d s o r p t i o n and e l u t i o n

sem i-larg e

s c a l e w ere c a r r i e d

experim ents

o u t.

at

2 8 ° C.

or

The f e r m e n t a t i o n l i q u o r s

u s e d w e r e p r o d u c e d b y g r o w i n g o r g a n i s m 77 i n m eyer f l a s k s

on a l a r g e

in shake c u lt u r e .

500 m l . E r l e n ­

These r u n s m ig h t

be s u m m a r iz e d : Run V o l u m e o f L i q u o r

Type

E lu an t 90% a c e t o n e 90% a c e t o n e 50% Me OH 100% Me OH 90% a c e t o n e 100% MeOH

1. 2. 3.

1 2 .0 l i t e r s 1 .2 l i t e r s 1 .0 l i t e r

1 . 0% Y e a s t e x t . 1.5% Soy f l o u r 1.0% Y e a s t e x t .

4. 5.

1 6 .0 l i t e r s 13-5 l i t e r s

1.5% Soy f l o u r 1.0% Y e a s t e x t .

The e x p e r i m e n t a l p r o c e d u r e F ourteen l i t e r s

of h arv ested

used

f o r Run 5 i s

l i q u o r had

R ecovery 50% 60% 19% 32% 23% 46% as fo llo w s:

a pH o f 5 . 5 .

It

19 was c l a r i f i e d M an v ille).

by s t i r r i n g

F iltra tio n

w i t h 280 g .

through a c lo th

"C e lite"

gave 1 3 .5

pH 5 • 7 .

The pH w a s r a i s e d

o f 2 .5 % NaOH.

lite rs

At t h i s

of "0e lite "

of c lear, to 6 .2

pH t h e

coated

lig h t

(Johns-

w i t h 200 g .

brown f i l t r a t e

by slow a d d i t i o n

filtra te

of of

o f 160 m l.

was s t i r r e d

for 2

h o u r s w i t h 54 g* o f D a r c o G - 6 0 . To t h e

carbon s u s p e n s io n was added

a n d a n e q u a l w e i g h t was s p r e a d m ix tu r e was f i l t e r e d lite rs th ree

of d is tille d

filte r

paper.

of " C e lite "

filte r

c lo th .

The

an d t h e c a r b o n c a k e w a s w a s h e d w i t h 4 w ater.

s u c c e s s i v e 1200 m l.

10 m i n u t e s w i t h

over the

54 g .

The c a r b o n w a s t h e n w a s h e d w i t h p o rtio n s

o f 50 % e t h a n o l b y s t i r r i n g

each p o rtio n fo llo w ed F in a lly ,

the

by f i l t r a t i o n

carb o n was r i n s e d

w ith

through 300 m l .

of a b so lu te m ethanol. The c a r b o n w as so lu te m eth an o l.

subjected

The f i r s t

to th re e

acid ,

in th e e lu a n t.

the

h y d ro ch lo ric

The s e c o n d a n d t h i r d

acid

A fte r each e lu tio n 100 m l.

except

in stead the

of c o n ce n tra te d

carbon b e in g s t i r r e d

e x a c t l y t h e same f a s h i o n

for

t h a t 2 .0 m l.

o f 3 .8 m l. was used

of co n cen trated in th e m eth an o l.

out and w ashed w i t h

o f a b s o l u t e m e t h a n o l c o n t a i n i n g no a c i d .

The t h r e e d ilu tio n s

Each such

co rresp o n d in g e lu a te .

e l u a t e s w ere t e s t e d

of each,

30 m i n u t e s

e l u t i o n s w e r e m ad e i n

c a r b o n was f i l t e r e d

w ash was com bined w i t h th e

ab­

e l u t i o n w a s m ad e w i t h 4 9 0 m l .

o f a b s o l u t e m e t h a n o l c o n t a i n i n g 3 «& m l . hy d ro ch lo ric

e lu tio n s w ith

corresponding

s t r e n g t h and 1 /3 b e e r s t r e n g t h .

f o r a c t i v i t y by m aking

to 3 tim es o r i g in a l b e e r A f t e r pH a d j u s t m e n t

th ese

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

tested

w ere d i l u t i o n s

of th e o rig in a l

tain ed by p lo ttin g o rig in al beer

zone

to

those the

size

of the

It

beer.

(mm.)

is

seen

th at

A b atch

T able

a ctiv ity

the

first

of

the

the t h i r d

a ctiv ity . fa ile d to

W ashes o f t h e

and

(w ater,

and

in d ic a te d above,

second

elu a te s

were f i l t e r e d

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

f o r m a t io n of

to

p articles*

bulk o f th e c le a r th e

so lid

on t h e

clear,

size

p a le yellow

of c a rb o n

9 » 270 m l.

had

due t o

the

was c o n t i n u e d

se ttled

ou t,

su p ern atan t. the

s u p e r n a t a n t was rem oved w it h o u t c o n ta in e r.

f u g a t i o n , m ore s u p e r n a t a n t was rem oved

of

increased to a

and a s i n t e r e d - g l a s s f i l t e r

b o tto m o f the

as

of 1030 m l.

S tirrin g

p o in t w here a f l o c c u l e n t , w h ite p r e c i p i t a t e

U sin g s u c t io n

ob tain ed

co lo r.

u n t i l i n a b o u t 20 m i n u t e s p a r t i c l e

lea v in g a p e rf e c tly

assay.

i m m e d i a t e l y became t u r b i d ,

sm all, w h ite

to ta l

a s the H y d r o c h lo r i d e .

filtra te

T h i s v o lu m e was a d d e d w i t h s t i r r i n g The m i x t u r e

o rig in a l

o rig in a l

t o rem ove t r a c e s The

to ­

The f i l t r a t e

(acid m ethanol),

v olum e was c l e a r and o f a y e l l o w

a ceto n e.

o f the

ob­

e th a n o l, m ethanol)

S e p a ra tio n of th e A n tib io tic first

is

second e l u a t e s

1 2% o f t h e

carbon

beer

By r e f e r r i n g

in each e lu a te

show a n y e v i d e n c e o f a c t i v i t y i n th e

4. P ooled

c o n tain ed

of

o rig in al

e l u a t e h a v i n g a b o u t 4%*

from th e c a rb o n s t i l l

c u rv e was ob­

e lu a te s.

g e t h e r c o n t a i n e d a b o u t 42% o f t h e a c t i v i t y beer, w ith

as

14 i n d i c a t e s t h e

of the th re e

percen tag e

assay p lates

of the d i l u t i o n s

d ilu tio n s

o f the d i l u t i o n s

curve th e

tain ed .

b ro n ch isen tio a

ag ain st d ilu tio n .

zones of in h ib i t io n and

in B r.

great d istu rb in g

F ollow ing c e n t r i ­

in a lik e

m anner.

21 The s o l i d w a s w a s h e d t w i c e w i t h a c e t o n e e th e r.

The e t h e r w a s h w a s r e m o v e d

ing

so lid .

the

p laced

S till

low ered

to

th at

b o ttle s

from t h i s

sm a ll-sc ale

been allo w ed

to

7 1 9 m g.

the p re s s u re

g rad u ally The l i g h t

d r y i n g , on r e m o v a l f r o m

o r to change a p p ea ra n c e

(In e a r l i e r ,

so lid

s o l i d was

f o r w e i g h i n g , sh o w ed n o t e n d e n c y w h a t s o ­

e v e r t o be h y g r o s c o p i c ,

the

the

o b t a i n a b l e w i t h a w a t e r pump.

grey m a te ria l r e s u ltin g c en trifu g e

except fo r a la y e r cover­

covered w ith e t h e r ,

i n a vacuum d e s i c c a t o r and

and o n c e w ith

e x p e rim e n ts w here th e

in

a n y way.

s o l v e n t had

e v a p o r a te o f f from th e p r e c i p i t a t e

had d a rk e n e d On t h e b a s i s

a nd l i q u i f i e d . ) o f 13*5 l i t e r s

The s o l i d

of sta rtin g

in a i r , w eighed

c la rific a te

t h i s w ould mean t h e o r i g i n a l f e r m e n t a t i o n l i q u o r c o n t a i n e d 0.053 m g ./m l. The

so lid

a c tiv ity

at

a d ilu tio n

corresponding to

mm. i n B r .

only a tr a c e

S ince aceto n e only under a c id

assay p la te s .

from w hich th e

of a c tiv ity

io n ic

the

p rec ip ita tio n

c o n d itio n s

so lid

c h lo r i n e , the

referred c u ltu re

so lid

resu ltin g so lid

resu lted

so lu tio n

showed

c o n cen tratio n .

in a so lid

product

formed u n d e r n e u t r a l of the

a n tib io tic ),

from p r e c i p i t a t i o n

co n tain ed

w h ic h w as o b t a i n e d w i l l be

to a s the h y d ro ch lo rid e 77.

o f a b o u t 13

p recip itated

(an o i l was

an

The a c e t o n e -

even a f t e r a 50- f o l d

c o n d itio n s w ith a c o n ce n tra te d and s i n c e

ex h ib ited

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

b ro n ch isep tica

m ethanol m ix tu re

o f 1 m g./m l.

of th e

an tib io tic

from

22 P u rifica tio n F o rm atio n . d isso lv ed 0 .045 g. w ater.

of p i c r i c

of d is tille d acid

On c o o l i n g ,

from w hich t h e To t h i s

a te

in

b rig h t

through P ic r a te

A s a m p l e o f 2 50 mg. o f t h e h y d r o c h l o r i d e w a s i n 32 m l .

d isso lv ed

of th e H y d ro ch lo rid e

w ater.

d isso lv ed

To t h i s w a s a d d e d

in 5 m l.

d istille d

a s m a l l amount o f o i l y m a t e r i a l form ed

s u p e r n a t a n t was rem oved a f t e r c e n t r i f u g a t i o n .

s u p e r n a ta n t was added 0 .2 2 8 g. 3 m l.

yello w

w hich s e t t l e d

of hot

of b o ilin g

of p ic ric

a b so lu te m eth an o l.

p rec ip ita tio n

occurred.

acid

An i m m e d i ­

The p i c r a t e ,

o u t a s a p l a s t i c m a s s on c o o l i n g a n d s t i r r i n g ,

was w ashed t w i c e w i t h

satu rated

aqueous p i c r i c a c id .

rem oval o f t h e l a s t wash s o l u t i o n ,

the

p icra te

A fter

was d r i e d

in

a vacuum d e s i c c a t o r . The d r i e d m e t h a n o l and

so lid

was d i s s o l v e d

the p i c r a t e

p rec ip ita te d

so lu tio n

in to

a fin ely

d ispersed

dried

a vacuum d e s i c c a t o r .

in

50 m l .

in

of e th e r.

a b o u t 6 m l.

of ab so lu te

by p o u rin g th e m eth an o l

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

s o l i d was washed t w i c e w i t h

form ed a s

eth er

and

I t w e i g h e d 2 6 6 mg. By t w i c e

d i s s o l v i n g and r e p r e c i p i t a t i n g

the

p o sitiv e

o r i g i n a l h y d r o c h l o r i d e was

reactio n

e lim in a te d to

from t h e

p icra te .

the h y d ro c h lo rid e ,

w hich p o s s e s s e d did

found i n th e

the

a S akaguchi-

On r e c o n v e r s i o n o f t h e

a w h ite am orphous s o l i d

6 l% m o r e a c t i v i t y

per

u n it

p icra te

was o b t a i n e d

of w eight than

o rig in a l h y d ro ch lo rid e.

F. P ro p e rtie s !•

p icra te ,

of th e A n tib io tic .

S ta b ility .

The s t a b i l i t y

of the a n t i b i o t i c

room t e m p e r a t u r e and a t 1 0 0 ° 0 . u n d e r

at

sev eral c o n d itio n s of

23 a c i d i t y and a l k a l i n i t y was i n v e s t i g a t e d . th e

p ercentage

m in u tes

in

a c t i v i t y r e m a i n i n g a f t e r 15 a nd a f t e r

0 .1 N h y d ro c h lo ric

a t pH 7 . 0 ,

T a b l e 15 r e p o r t s

a t pH 9 . 5 ,

and i n

acid ,

in 0 .1 N a c e ti c

1 20 acid ,

0 .1 N sodium h y d r o x id e

so lu tio n . It ployed

s h o u ld be p o i n t e d i n t h i s w ork

out th a t

in the ty p e

( t h e a g a r cup d i f f u s i o n

assay),

of any s in g le d e te rm in a tio n h as been e stim ate d W ith t h i s th at

the

i n m ind,

the r e s u l t s

a n tib io tic

0 .1 N h y d ro c h lo ric

is

stab le

th at

it

but not a t u n stab le

is

fairly

i n 0 .1 N sodium h y d r o x id e

tem p eratu re

(22,

23).

of th e

I t has

in h ib itio n

by th e p r e s e n c e calcium

T herefore, h y d roxide to

th e

been r e p o r te d zone

b io tic

in

and t h a t

it

a t 100° C.

rep o rted th at

sodium c h l o r i d e , zone

size

sta b le

a t pH

at 100°

is ap p aren tly The " i n c r e a s e a t room m ay

by o th e r w o rk ers

an i n c r e a s e

in

the

size

such as n o tassiu m p h o sp h a te , an d ammonium s u l f a t e . found w i t h th e

sodium

o f t h e h y d r o c h l o r i d e m ig h t be a t t r i b u t e d effect.

S p e c if ic ity of A c tio n . o b tained

is

in a g a r a s s a y p l a t e s c a n be cau se d

in creased

so lu tio n

it

in

subsequent ex perim ents)

o f an e f f e c t

a sodium c h l o r i d e 2.

in

of v a rio u s s a l t s ,

c h lo rid e,

th at

sodium h y d ro x id e s o l u t i o n

(and a l s o n o te d

be a d e m o n s t r a t i o n

i n T a b l e 15 i n d i c a t e

a t pH 9 . 5 a t r o o m t e m p e r a t u r e

th e h ig h er tem p eratu re,

in a c t i v i t y ” seen in

t o b e - 1 5 %.

but ra p id ly lo ses a c tiv ity

stab le

the e r r o r

f o r 2 h o u r s a t 1 0 0 ° C.

or a c e tic a c id ,

7 . 0 a t room t e m p e r a t u r e C.,

liste d

o f a s s a y em­

the

first

A c o n cen trate

larg e

scale

of th e

a n ti­

carbon ex p erim ent

24 was used

to

o b tain the v a lu e s rep o rte d

c o n c e n t r a t e w as made i n t o in co rp o rated

in to

A p ath o g en ic

stra in

a se ries

t r y p t ose a g a r o f B r.

below *

T h is w a te r

o f d i l u t i o n s w hich w ere

(D ifco)

s u i s was

in P e tri p la te s .

tested

on t h i s a g a r b y

th e V e t e r i n a r y D epartm ent of Purdue U n i v e r s i t y by a llo w in g a stan d ard surface

su sp en sio n o f th e

of th e a g a r .

o f e a c h upon th e

The f o l l o w i n g l i s t

o rig in a l beer stre n g th of the

test

th at

d ilu tio n

i s p o ssib le

n eg ativ e)

J.

of the term s of th e grow th

of a n tib io tic

2 x beer stre n g th beer stren g th beer stre n g th 1/80 b e e r stre n g th 1/80 b e e r stre n g th 1/80 b e e r stren g th

th a t th e l a s t

In th e

th ree at

0 .10 m g ./m l. 0 .05 m g ./m l. 0 .05 m g ./m l. 0 .0 0 0 6 m g ./m l. 0.0006 m g ./m l. 0 .0 0 0 6 m g ./m l.

organism s

( a l l gram -

a low er c o n c e n tr a ti o n ,

only t o x i c i t y experim ent

perform ed,

o n e w e i g h i n g 2 0 0 g . and o n e 3 0 0 g . , w e r e i n j e c t e d o f an im pure w a t e r c o n c e n t r a t e

of th e

a n t i b i o t i c w hich

co n tain ed

to th a t

found i n 8 m l.

of o rig in a l

estim ated

a n d 0 . 5 mg. pH o f 5 • 5

by

pro b ab ly corresponds:

i n t r a p e r i t o n e a l l y w i t h 1 m l.

is

the

o f 1/80 b e in g the low est t r i e d .

T o x ic ity .

tw o r a t s ,

c o n cen tratio n

m ight have been i n h i b i t e d

a d ilu tio n

in

co m p letely in h ib ite d

B ru cella s u is S tap h y lo co ccu s aureus B acillu s s u b tilis B ru cella b ro n c h ise p tic a E sch erich ia c o li S alm o n ella p u llo ru m It

in to

surface

g iv e s the d i l u t i o n s

o r g a n is m and t h e

to w hich t h i s

d iffu se

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

s tre a k in g 24-hour b ro th s agar.

organism to

th a t the

an a c t i v i t y

ferm en tatio n liq u o r .

volume i n j e c t e d

of the a n t i b i o t i c .

co rresponding

c o n ta in e d betw een 0 .4

S o lu tio n

i n j e c t e d was a t

( r e s u l t i n g from d i l u t i o n w ith p h o sp h ate b u f f e r )

and had a m a r k e d ly r e d

co lo r.

It

a

25 The r a t s sig n s

im m ed iately fo llo w in g i n j e c t i o n

of d isco m fo rt,

d istress.

b u t no c o n v u l s i o n s o r s i g n s

no g r o s s

in te rn al

of m ajor

sig n s

U p on a u t o p s y a f t e r

of a b n o rm a lity w ere

apparent in

the

R eactio n s.

The

organs.

4.

S o lu b ility

h y d ro ch lo rid e

and C l a s s i f i c a t i o n

of the a n t i b i o t i c

in w a te r and m e th a n o l, tia lly

in so lu b le

eth er,

ch lo ro fo rm ,

It

some

The n e x t m o r n i n g and f o r 4 s u b s e q u e n t d a y s t h e

a n i m a l s a p p e a r e d n o r m a l and w e re a c t i v e . 4 days,

sh o w e d

less

so lu b le

in n -b u tan o l. aceto n e,

g iv es p o s itiv e

of c u ltu re

It

in eth an o l, is

dioxane,

b iu ret

a lso

in

reactio n ,

the

first

but th is reactio n

and e s s e n ­

in so lu b le

and x a n t h o p r o t e i c

p rec ip ita tio n

v ery so lu b le

in

and p e t r o l e u m e t h e r .

and n e g a t i v e M i1 1 on and B e n e d i c t t e s t s . obtained

77 i s

reactio n s

The h y d r o c h l o r i d e

gave a p o s itiv e Sakaguchi

is n e g ativ e

in

th e m ore p u r e

h y d ro ch lo rid e. Gr. C o m p a r i s o n o f t h e A n t i b i o t i c Because o f

sim ila ritie s

o f C u l t u r e 77 w i t h P o l y m y x i n s .

in s p e c if ic ity ,

so lu b ility

ch aracteristics

to Polym yxin

A ero sp o rin

(Polym yxin A ),

a sam ple o f th e

ch lo rid e

of the

a n t i b i o t i c was

t h e P itm a n -M o o re Company. Nash com pared p o rted vance

th at

th e m ajor p a r t

developed

th at

and

( P o l y m y x i n D) a n d t o p u rified

hydro­

s e n t to D r. H a ro ld N ash o f

U sing p a p e r

ch ro m atography.

t h e m a t e r i a l w i t h know n p o l y m y x i n s .

corresp o n d in g to

a n tib io tic

sta b ility ,

D r.

He r e ­

of t h e m a t e r i a l had a zone o f a d ­ o f P o l y m y x i n D.

Polym yxin D i s

by th e S tam ford L a b o r a t o r i e s

A m e r i c a n C yanam id Company and i t s

of the

co m p lete c h e m ic a l m ake-up

26 was r e p o r t e d

in

t h e J u n e , 1 9 4 9 , A n n a l s o f t h e New Y o r k

Academy o f S c i e n c e s . from c u l t u r e

77 w a s t e r m i n a t e d

o th e r organism s III.

in

th e

cu ltu re

C u ltu re

o f the

37 i s

th at

c o lle c tio n . 37

(B.

s u b t i l i s -Mar b u r g )

O rganism .

a g ram -p o sitiv e,

grows r a p i d l y

aero b ic,

b e t w e e n 2 0 o - 5 2 ° C.

of a stu rd y ,

s l i g h t l y w rinkled p e l l i c l e

hours in

the

usual tem p eratu re

pH 5 . 9 .

It

p o sitiv e

t e s t f o r hydrogen s u l f i d e ,

It

range,

the

first

and a n e g a t i v e

glu co se,

but not m alto se o r m a n n ito l in

sla n ts,

th e

and g l y c e r o l .

cu ltu re

c o r r e s p o n d s t o B. teriu m w ere sen t P lan t In d u stry ,

are

A ccording to su b tilis

starch . fru cto se,

the

th is

O ther

(M arburg).

compounds

sucrose,

Burdon k e y ,

the

m annose,

c u ltu re

S lan ts

o f the

o f the

o rg a n ism could

f r o m t h e M a r b u r g t y p e o f B.

perh ap s by the

for

brom cresol pu rp le

U. S . D e p a r t m e n t o f A g r i c u l t u r e , th at

test

id e n tif ic a tio n key

t o D r . N a t h a n G-. S m i t h ,

M a r y l a n d , who s t a t e d

a

o rg a n is m was fo u n d t o f e r m e n t

and t o h y d r o l y z e

fe rm e n te d by the

24

and g ro w s r e a d i l y a t

by B u rd o n

S ince

w ith in

U s i n g an a b b r e v i a t e d (24),

at

the fo rm a tio n

gives a p o s itiv e V oges-P roskauer r e a c tio n ,

fo rm atio n .

tin g u ish ed

o f m edia

in itia te s

proposed

bu tt

spore-fo rm in g

on a w i d e v a r i e t y

tem p eratu res

in d o le

a n tib io tic

in fav o r o f in v e s tig a tin g

T he S t u d y o f C u l t u r e

A. I d e n t i f i c a t i o n

b a cillu s

T h e r e f o r e , r e s e a r c h on t h e

37 bac­

B ureau of B e ltsv ille ,

n o t be d i s ­

s u b t i l i s , except

a n ti b i o ti c produced. a n tib io tic

of cu ltu re

37 s e e m e d

to h o ld

some

27 p ro m ise, th e

i t was d e c i d e d

to

a n tib io tic s rep o rted

ob tain

enough f o r

com p ariso n w ith

from g r a m - p o s i t i v e b a c i l l i .

B. S e l e c t i o n

o f M e d iu m f o r A n t i b i o t i c P r o d u c t i o n .

ly

It

short

C om parison o f ^ e d i a .

was d e s i r e d

t i m e a m edium w h i c h w o u ld g i v e

of the

a n tib io tic

p rin cip le ,

and a t

fairly the

s e l f to m an ip u latio n fo r e lim in a tio n The i d e a of the

b eh in d

av o id in g th e

tain ed

su b tilin ,

F iv e m l.

stu d y ,

of the b a c te r ia l

sta tio n a ry

c u ltu re a t

c ells. chance

c ells;

and t y r o c i d i n e

th eir

it­

are

ob­

so lu b le,

u sefu ln ess. 2 8 ° C. w a s u s e d . in t r i p l i ­

tu b es.

Inoculum c o n s is te d try p tica se

th e

the

p o r t i o h s o f s i x m edia w ere d i s t r i b u t e d

c ate to t e s t

6 m edia

sa m e t i m e l e n d

and a r e d i f f i c u l t l y

or i n s o l u b l e , i n w a t e r w hich l e s s e n s

in a

good p r o d u c t i o n

from w i t h i n

g ram icid in ,

from th e b a c t e r i a l c e l l

For th is

fin d

c e l l s was t o l e s s e n

o b ta in in g an " e n d o - a n tib io tic " a n tib io tics

to

soy b r o th

o f one d r o p p e r t u b e o f a 2 4 - h o u r

of c u ltu re

37-

The c o m p o s i t i o n s o f t h e

fo llo w : M e d iu m

C o m p o s i t i o n o f M e d ium 1-A sp arag in e G lucose KH2 P0 , NaCl MnS0^-4H2 0

1 .5 % 1.0% 0.2% 0.005% 0.002%

Yeast e x tr a c t G lucose ( N H , ) 9 S0,

1.5% 1 .0 % 2 .0 %

KH2PO4

0.2%

4

M gS0,.7H 20 NaCl F e (NH4 ) 2 S0j • 6Hp 0 MnS04 -4H2 0

0.2% 0.005% 0 . 0 014% 0 .0 0 2 %

28 C o m p o s i t i o n o f M edium

M e d iu m 3

Same a s M e d iu m 2 b u t w i t h 0 . 5 % y e a s t e x t r a c t i n s t e a d o f 1 . 0%

4

Same a s M e d i u m 3 b u t w i t h 1 . 0 % a s p a r a g i n e added

5

" T r y p t i c a s e Soy B r o th " * T ry p ticase 1 ..77 % P hytone 0.3% G lucose 0.25% 0 . 50% NaCl k 2h p o 4 0.25%

6

Same a s M e d iu m 5 b u t w i t h 0 . 0 0 2 % M n S O ^ '4N^O i n c l u d e d

*B aItim o re B i o lo g i c a l L a b o ra to ry The t u b e s w e r e a s s a y e d 3 , 6 , and 9 d a y s i n c u b a t i o n . same a c t i v i t y a t a ctiv e .

ag ain st

B. c e r e u s p l a t e s

M edia

1 and 2 had a b o u t

3 d a y s w i t h M edia 4 ,

How ever,

at

grow th c o n s is te d

6 d a y s M e d ium 5 h a d t h e m o s t a c t i v i t y ,

o th er

of a com plete p e l l i c l e

from w hich th e

co n tain in g

e x p e r i m e n t s , m edia composed

lev e ls

of soy f l o u r

failed

to

a c tiv ity .

seemed c o n v e n i e n t f o r u s e b e c a u s e t h e

c o u ld be p o u red a s a c l e a r l i q u i d In

the

5 and 6 b e in g l e s s

a n d a t 9 d a y s M e d i a 1 a n d 5 h a d e q u a l and g r e a t e s t M edia 1 and 5 a l s o

a fter

or y e a s t

e x tra ct,

g i v e a s good a n t i b i o t i c

liq u o r

few c e l l s .

of d i f f e r e n t

g lu co se,

and s a l t s

p ro d u ctio n as did

try p ti­

case soy b r o th . T h e a g a r c u p d i f f u s i o n m e t h o d o f a s s a y w i t h B. as te s t a c tiv ity

o r g a n is m h a s b e e n r o u t i n e l y em ployed of ferm en tatio n liq u o rs ,

and o f t h e

sep arated

a n tib io tic.

of a c tiv e

cereus

to m easure

th e

co n cen trates,

29 E ffect

2.

some a n t i b i o t i c s

of M e ta llic

is

Ions.

The o p t i m u m p r o d u c t i o n

d e p e n d e n t upon th e p r e s e n c e

in

of

the

g r o w t h m edium o f s m a l l a m o u n ts o f one o r more m e t a l l i c

io n s.

The m e t a l s w h i c h h a v e b e e n a s s o c i a t e d w i t h s u c h s t i m u l a t i o n are

z in c,

effect

m anganese,

on a n t i b i o t i c

of d iffe re n t io n .

copper,

le v e ls

and i r o n .

p ro d u ctio n

T a b l e 16 show s t h e

in a tr y p tic a s e

of th ese m etals,

iro n b ein g as f e r r i c

The o n l y v e r y p r o n o u n c e d e f f e c t w a s

in a c t i v i t y

a f t e r 4 days th a t

m anganese.

Sam ples

w ere s t r o n g e r

th an th e

and c l e a r

the sharp d e c re a s e

in sam ples c o n ta in in g in a d d itio n

co n tro ls,

c u ltu re

O ther e x p e rim e n ts in d ic a te d lev e ls

occurred

c o n ta in in g copper,

s l i g h t l y m ore a c t i v e

s o y medium

to b ein g

had p e l l i c l e s w h i c h

liq u o r ben eath th e p e l l i c l e s .

th at

copper a t s l i g h t l y h ig h er

(up t o 2 . 0 p . p . m . ) had a b o u t th e

sa m e e f f e c t a s c o p p e r

a t 0 .5 p .p .m . À* E f f e c t

ad d itio n

of Yeast E x t r a c t .

o f a low c o n c e n t r a t i o n

to the t r y p ti c a s e

cupric

soy,

in ch t e s t

cu ltu re tu b e,

at

o r 1 00 m l .

f o r p r e l i m i n a r y w ork, b o ttle

for

28° C.,

larg e sc ale

and

a n tib io tic

(D ifco ),

(D ifco ) p ro d u ctio n .

0.4% ,

and

I n c u b a t i o n was c a r r i e d u s in g 5 m l.

o f medium p e r

out six -

p e r 500 m l. E r l e n m e y e r f l a s k 300 m l .

p e r tw o q u a r t ,

rectan g u lar

pro d u ctio n .

Çï. S t u d i e s P r e l i m i n a r y t o S e l e c t i o n _1. S t a b i l i t y

e x tra c t

p r o d u c t i o n was composed o f

3.0%, y e a s t e x t r a c t

s u l f a t e • 5H2 O, 0 . 0 0 0 0 7 5 % »

in su rfa c e

of y east

s o y medium i n c r e a s e d

T he m e d i u m c h o s e n f o r r o u t i n e try p ticase

I t was fo u n d t h a t t h e

o f a_ S e p a r a t i o n P r o c e d u r e .

of the A n t i b i o t i c ( s ) in F erm en tatio n L iq u o rs.

30 The s t a b i l i t y and 100° C. g ated .

o f the a c t i v i t y

under d i f f e r e n t

T a b l e 17 l i s t s

C e lite -filte re d

m ad e f r o m d i l u t i o n s the

th ere

is

a c tiv ity a rapid

is

( - 15% ),

How ever,

pH 6 . 5 ,

of the

lo ss

at

25°

o f th e a c t i v i t y of

It

is

It

and

A b a tc h

curve

l i q u o r was u se d t o

se e n from th e t a b l e

of a c t i v i t y at

present at

the

a n d pH 9 * 0 .

filte re d

pH 2 . 0 .

a f t e r 15 m i n u t e s , m ethod

flu id

c o n d i t i o n s o f pH w a s i n v e s t i ­

the p e rc e n ta g e

lo sses.

but e s p e c ia lly at much a c t i v i t y

c u ltu re

l i q u o r r e m a i n i n g a f t e r 15 m i n u t e s a n d a f t e r

6 0 m i n u t e s a t pH 2 . 0 ,

tain

of th e

100° a t a l l

appears th a t a t

ob­

th at

pH v a l u e s

2 5 ° C. a s

pH 9 . 0 a f t e r 6 0 m i n u t e s a s

co n sid erin g the

sa m e m ay b e t r u e

some a c t i v i t y i s d e s t r o y e d

accuracy o f the at

assay

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

a t pH 2 . 0

a n d pH 9 . 0 e v e n

a t room t e m p e r a t u r e . 2.* C a r b o n A d s o r p t i o n a n d E l u t i o n .

carbon

(D arco 0 -6 0 )

tio n liq u o rs

I t was found t h a t

w ould rem ove t h e a c t i v i t y fro m f e r m e n t a ­

but a tte m p ts to e lu te

the

a c t i v i t y w ere u n s u c ­

c essfu l. P r e c ip ita tio n E x p erim en ts. ferm en tatio n

l i q u o r of

produced a f l o c c u le n t m ost o f th e so lid

a c tiv ity

so o b ta in e d

ra isin g

c u ltu re

p rec ip ita te

w hich was found to

of th e liq u o r .

to

The t a n

pH 7 . 0 o r h i g h e r .

p o ssib le

to e ff e c t

in activ e

by p r e c i p i t a t i o n

of th e

3 7 t o a b o u t pH 5 - 0 a n d l o w e r

d i s s o l v e d when su sp e n d e d

th e re a c tio n

On d i l u t i o n

A cid ifica tio n

any s e p a ra tio n

or g rey -tan

i n w a t e r upon It

was n o t

of a c tiv e m a te ria l

at d iffe re n t

co n tain

pH l e v e l s .

of th e w ater c o n c e n tra te of th e acid

found from

31 p r e c i p i t a t e w ith, l a r g e e th e r m ix tu res, occurred

except w ith b u tan o l,

p re c ip ita tio n

th e

of a w h ite

c ip ita te d w ith o u t

0, the

if

th e

co lo r,

from s o l u t i o n s

10,

25,

su p ern atan t.

50,

60,

a ctiv e

B utanol caused th e

th e

a c t i v i t y b ein g in

p rin cip le

co u ld be p r e ­

p r e c i p i t a t i o n b y a c i d i f i c a t i o n was

78%*

W i t h no e t h a n o l p r e s e n t m o s t o f but

it

c a r r i e d th e c o lo r w ith

f r o m 78% e t h a n o l w a s a l m o s t f r e e

i t had v e ry l i t t l e

4* E x t r a c t i o n of th e

o f the

c o n t a i n i n g v a r y i n g am ounts o f e t h a n o l ,

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

co lo r b u t

c a s e s most

s o l i d w hich had o n ly a s l i g h t

a c t i v i t y was p r e c i p i t a t e d

it.

aceto n e—

su p ern atan t.

To d e t e r m i n e

trie d

in the

and i n a l l

a g a i n s t B. c e r e u s , m o st o f

colo red

aceto n e,

o r b u t a n o l , no a p p r e c i a b l e p r e c i p i t a t i o n

a c t i v i t y rem ained

a c tiv ity

excesses of e th a n o l,

a ctiv ity .

o f t h e A cid P r e c i p i t a t e .

acid p r e c ip ita te ,

it

of

In e x tr a c tio n s

was s e e n t h a t d i f f e r e n t

v e n t s rem oved v a r y i n g am o u n ts o f t h e a c t i v i t y ,

e .g .,

so l­ 30%

e t h a n o l rem oved a l l

a c t i v i t y w h i l e 95% e t h a n o l and b u t a n o l

rem oved m ost

but not a l l .

of i t ,

It

was a l s o

found t h a t

b u t a n o l and o t h e r a l c o h o l s e x t r a c t e d more a c t i v i t y , m ore t o t a l a tu re

it

so lid s,

was n o t e d

than d id

appeared c ip ita te ,

by e t h e r .

e th e r.

t h a t p r a c t i c a l l y a l l B.

b i o t i c s w ere r e p o r te d tra c ta b le

d ieth y l

From th e l i t e r ­ su b tilis

to be e t h e r - i n s o l u b l e Because o f

th is,

e t h e r was s e l e c t e d

in th e

th ere

acid -p re­

f o r e x t r a c t i o n w ith the

o f o b t a i n i n g a m ore p r e f e r e n t i a l e x t r a c t i o n ,

a n ti­

or not ex-

and b e c a u s e

to be a m ix tu re of a n t i b i o t i c s

and

idea

and p e r h a p s

32 an e x t r a c t

t h a t would c o n t a i n

In v estig a tio n In d ica tio n s suggested by acid

of lo s s

the

of P o ssib le

of a c tiv ity

p o ssib ility

eth an o l,

p o ssib le

sources

aliq u o ts

of the

o n l y one a n t i b i o t i c . I n a c tiv a tin g A g en ts.

in e x tr a c tio n

of in ac tiv a tio n

o r perhaps,

by l i g h t .

of in a c tiv a tio n ,

experim ents

b y 9 5% e t h a n o l ,

To c h e c k t h e s e

id en tica l

e x tractio n s

of

same a c i d - p r e c i p i t a t e w e r e made w i t h d i l u t e

e t h a n o l and w i t h 95% e t h a n o l i n t h e d a r k and i n o r d i n a r y incandescent l i g h t . th at

i n a c t i v a t i o n was n o t pro d u ced

e th a n o l, lo ss

T h i s and o t h e r e x p e r i m e n t s

but

th a t h ig h ly acid

by l i g h t

e t h a n o l could

in d icated

o r by 95% brin g about

of a c tiv ity .

D. The O b t a i n i n g Form .

o f t h e Ant i b i o t i c

For the d e te rm in a tio n the a n t i b i o ti c

of c u ltu re

has been p ro p o sed ,

of C u ltu re

37 i n a_ S t a b l e

of the a n t i b a c t e r i a l

spectrum of

3 7 , f o r w h i c h t h e n am e " x a n t h e l l i n "

e th e r e x tra c ts

of th e acid p r e c i p it a te s

o f f e r m e n ta ti o n l i q u o r s w ere u se d .

F ollow ing a 4 -d ay i n ­

c u b a t i o n p e r i o d , t h e s e l i q u o r s h a d a pH o f 7 * 6 - 8 . 0 . d é c a n ta tio n through the

flu id

lay ers

was s e p a r a t e d

On l o w e r i n g t h e

o f c o t t o n g a u z e and g l a s s w o o l,

from m ost o f th e

pH t o 2 . 7

by t h e

w ith

stirrin g ,

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

th r o u g h a S h a r p i e s c e n t r i f u g e , was

d isso lv e d by su sp en sio n to

p e llicle.

p r e c i p i t a t e w hich con­

t a i n e d m o st o f th e a c t i v i t y was f o r m e d . by p a s s a g e

b acterial

slow a d d i t i o n ,

o f 15% p h o s p h o r i c a c i d , a f l o c c u l e n t

co llec ted

By

i n w a t e r and a d j u s t m e n t o f

7*0 o r s l i g h t l y h i g h e r w i t h d i l u t e

a c t i v i t y was t h e n r e p r e c i p i t a t e d

t h e pH

sodium h y d r o x id e .

by a c i d i f i c a t i o n

to

The

pH 2 . 7

33 and c e n t r i f u g a t i o n . n atan t

liq u id

m ained

in

its

w ith a p ip e tte ,

th e 200 m l.

of th e

x a n th e llin

the m o is t,

c en trifu g e

w a te r under reduced

trac tio n

th e

F o llo w in g th e rem oval o f the s u p e r ­

pressure

a t 4 0 - 4 5 ° G.

cake w ith so d iu m -tre a te d

as the

free

com bined y e llo w

acid .

eth e r e x tra c ts

at

from th e

sodium c h l o r i d e ,

ag ain st

once in

5% s o d i u m b i c a r b o n a t e s o l u t i o n

by e v a p o r a t i o n o f e t h e r e x t r a c t s

th at

these

kept

in

the

the

so lid

It

from a l k a l i n e d id . as th e

Based salt

iso latin g

so lu tio n s

on t h i s

ju st d escrib ed

eth er

of the

d ifferen ce

sodium s a l t for

ob tain ed

acid .

I t was found

a c t i v i t y when

an o th er,

A t­

more

co u ld be o b t a i n e d .

th at

o r as th e f r e e

the

agar for

e v en i n a vacuum d e s i c c a t o r .

form o f x a n t h e l l i n had been n o ted

in to

w ere c ru d e p r e p a r a t i o n s of

form of th e f r e e

sta te ,

to a v isc o u s,

The r e s i d u e s

t e m p t s w e r e t h e r e f o r e made t o d e t e r m i n e i f sta b le ,

e th e r e x tra c ts

a n d t h e pH a d j u s t e d

p r e p a r a t i o n s l o s t m ost o f t h e i r

the

F ollow ing

t h i s r e s i d u e was d i s s o l v e d a t

to 7 .0 w ith 0 .1 N h y d ro c h lo ric a c id .

in

o f about 6 g.

e th e r.

For in co rp o ratio n

testin g

a n tib io tic

ex­

e t h e r removed

under dim inished p re s s u re

b acteria,

E x h au stiv e

the r a t e

d écan tatio n

red d ish -o ran g e re s id u e .

o f m ost of

Sodium c h l o r i d e was added t o

t o e l i m i n a t e w a t e r from th e

w ere c o n c e n tr a te d

cake w hich r e ­

b o t t l e was f r e e d

p e r 100 m l.

the

tan

did n o t e x t r a c t

a n tib io tic,

w hile b u ta n o l

in th e s o l u b i l i t y o f x a n t h e l l i n

acid ,

a p r o c e d u r e was d e v is e d

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

o b tain in g

th e

Upon e v a p o r a t i o n o f t h e e t h e r ,

a c tiv ity

free

The m e t h o d

a c i d was em p lo y ed .

the r e s i d u e was a t

once

for

34 d isso lv ed trac tio n

i n 5% s o d i u m b i c a r b o n a t e

so lu tio n .

o f t h i s deep y ello w s o lu tio n w ith

A sin g le

o n e -te n th

its

volum e o f b u t a n o l c o m p l e t e l y rem oved th e a n t i b i o t i c , e v id e n c e d by th e aqueous phase.

absence

o f c o l o r and a c t i v i t y

The b u t a n o l s o l u t i o n

o b t a i n e d was w ashed once w i t h w ater,

and sodium c h l o r i d e ,

was ad d ed t o

the

as

the

o f t h e sodium s a l t

o n e-tw en tieth

at

in

ex­

the r a t e

its

volum e o f

o f 10 g .

b u t a n o l t o rem ove w a t e r .

so

p e r 100 m l . ,

The b u t a n o l

so lu ­

t i o n was rem oved by m eans o f a p i p e t t e f r o m t h e so d iu m c h lo rid e-w ater la y e r, C.

and c o n c e n t r a t e d

under dim inished p re s s u re .

was d i s s o l v e d obtained

( 0 . 5- 1 .5 m l.

o f m ed ium )

f o r t h e amount

of a b s o lu te m ethanol,

th e m ethanol s o lu tio n

c en trifu g e d

c l e a r yellow s o lu tio n

added d ro p w ise to t h i r t y

volum e o f e t h e r ,

whereupon th e

appeared as a p a le w ashing th r e e su sp en sio n ,

y ello w ,

45° - 50o

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

i n a s m a l l volume

fro m 10 l i t e r s

to d ryness a t

i f necessary,

sodium s a l t

flo ccu len t

and t h e tim es

its

of x a n th e llin

p rec ip ita te .

t i m e s w i t h e t h e r by c e n t r i f u g a t i o n

A fter

and r e ­

t h e a m o rp h o u s p r o d u c t was t h o r o u g h l y d r i e d

in

a vacuum d e s i c c a t o r . The y i e l d sodium s a l t ,

is

of a n t i b i o t i c ,

a b o u t 1 0 mg. p e r l i t e r

ex tractio n

of th e

c en trifu g e

y i e l d s a red

su p ern atan t liq u id

amount o f x a n t h e l l i n c a n terized

b a s e d on t h e w e i g h t o f

by a s t r o n g ,

o f medium .

the

E th er

from th e S h a r p i e s

o i l from w hich a s m a ll a d d i t i o n a l be o b t a i n e d .

u n p leasan t

odor.

The o i l

is

charac­

35 E.

S o lu b ility

and S t a b i l i t y .

T h e r e m ay be some c o n n e c t i o n b e t w e e n t h e x a n th e llin tain ed

and i t s

so lu b ility

in c e r ta in

in th e absence of m o istu re,

h ib ited

so lu b ility

form ,

dry d ie th y l e th e r,

benzene,

ever,

on c o n t a c t w i t h w a t e r

so lv en ts.

th e fre e

in the a lc o h o ls ,

sta b ility

of

When o b ­

acid h as ex ­

aceto n e,

dio x an e,

ch lo ro ­

and p e t r o l e u m e t h e r .

or m o ist a i r ,

it

How­

changes in

p h y s i c a l a p p e a r a n c e and w i l l n o t d i s s o l v e , o r w i l l d i s s o l v e only s l i g h t l y , eth e r. is

in e th e r,

A ccom panying th e

a m arked d e c r e a s e

in th e s o li d

X an th ellin ,

as

or p etro leu m

c h a n g e i n a p p e a r a n c e and

in a n ti b i o ti c

been m entioned t h a t th e u n stab le

c h lo ro fo rm , benzene,

free

acid ,

a c tiv ity . as

It

o btained

sodium s a l t , i s

in e th e r ,

ch lo ro fo rm ,

lik e

the f r e e

sta b ility

a c e t o n e , and b u t a n o l ; i t

so lid

a t room te m p e r a t u r e

s a l t h a s sh o w n t h e sta te :

it

a n d c a n be h a n d l e d

sco p icity

or a c t i v i t y d ecrease.

tio n s

IB d a t a

pH 9 . 5

show ing t h e

has been sto re d

of a c t i v i t y

t h e r e was ra p id

lo ss

sta b ility

from th e

are p resen te d .

t h e r e w a s no l o s s

Un­

ch aracteristic

in a i r w ithout

of x a n th e llin prepared

1 . 0 mg. p e r m l .

in so lu b le

of aqueous

so lu tio n

b u t a t pH 2 . 0 th is

so lu ­

to c o n ta in

At 25° C. in n e u t r a l

of a c t i v i t y a t

of

show ing h y g r o -

sodium s a l t

in 2 h o u rs,

of

f o r m onths

i n a vacuum d e s i c c a t o r w i t h no l o s s

a ctiv ity ,

In T able

is

in w a te r,

b e n z e n e , and p e t r o l e u m e t h e r .

a c i d , the

in th e

to d a te , is

an am orphous powder

t h a t d i s s o l v e s r e a d i l y to give y ello w s o l u t io n s eth an o l,

has a lre ad y

sta te .

th e

m ethanol,

so lu b ility

or

tem p eratu re;

36 at

100° C . i n a c t i v a t i o n was r a p i d

even in n e u t r a l s o l u t io n .

B u t a n o l and e t h a n o l s o l u t i o n s o r the

s a l t have r e ta in e d

of e i t h e r the

fre e acid

a c t i v i t y d u r in g m onths o f s t o r a g e

a t 4 ° C. F.

The A n t i b a c t e r i a l S p e c t r u m o f X a n t h e l l i n . An a g a r s t r e a k m e t h o d w a s e m p l o y e d f o r t h e

of the a n t i b a c t e r i a l th e a c id - f a s t p lates

spectrum

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

o rg a n is m s and f o r B r.

fo r use in

eth er

of ferm en tatio n liq u o r s .

a g a r s t r e a k m ethod w ere s t r e a k e d soy b r o th s .

su is.

t h e a g a r s t r e a k m ethod

am ounts o f th e r e s i d u e from th e c ip ita te s

d eterm in a tio n

A ll t e s t s w ith B r.

except fo r

T ryptose ag ar co n tain ed

ex tra cts

v ary in g

of acid

O rganism s t e s t e d

s u i s w ere p e rfo rm ed by M iss

Purdue V e t e r i n a r y S c ien c e D epartm ent.

of try p to se

of x a n th e llin . tem p eratu re

s u i s c e l l s w ere used to

agar

(D ifco) p l a t e s

Standard flood

the

surfaces

c o n tain in g d if f e r e n t

These p l a t e s w ere allo w ed

For the a c id - f a s t

in th e

su sp en sio n s

to

dry a t

f o r one day b e f o r e b e i n g i n c u b a t e d a t

fo r 2 days.

by t h e

from 2 4 - h o u r t r y p t i c a s e

D o r i s E . B u n n e l l o f D r . L . M. H u t c h i n g s l a b o r a t o r y

of v ir u le n t B r.

pre­

lev e ls room

37° C.

organism s a m o d ifie d K irc h -

n e r ’ s m edium ( 2 5 ) was u se d i n a b r o t h d i l u t i o n a s s a y . T a b l e 19 su m m a riz e s t h e i n h i b i t i o n s p e c ie s by x a n th e llin . fo r B r. ism :

su is.

S arcin a

tim e w as 24 h o u r s e x c e p t

T h e r e a p p e a r s t o be a s p e c i f i c i t y lu tea

w h ile m ost o f th e at

In cu b atio n

of v ario u s b a c te ria l

and B r .

s u i s w ere m ost

g ram -n eg ativ e

th e h ig h e r c o n c e n tra tio n s.

of an tag o n ­

stro n g ly in h ib ite d ,

s p e c i e s w ere i n h i b i t e d In t e s t s

only

ag ain st B r. su is

37 d e fin ite

p o in ts

of in h ib itio n

com pared f a v o r a b l y , h ib itio n

e .g .,

and g r o w t h w e r e

o b ta in e d w hich

w i t h one p r e p a r a t i o n

(no c o l o n i e s v i s i b l e )

was o b t a i n e d

com plete

at

in ­

0 . 0 1 1 mg. p e r

m l . w i t h g r o w t h a t 0 . 0 0 7 mg. p e r m l . ; w i t h a n o t h e r p r e p a r a t i o n t h e r e was c o m p le te grow th a t £•

in h ib itio n

0 . 0 0 9 mg.

d e la tio n

in

of I n h i b i t i o n

to

I n c u b a t i o n T im e.

c o n c e n tra tio n o f the

t h a t gave com plete

try p to se b ro th

0 . 0 2 2 mg. p e r m l . w i t h some

p e r m l.

T a b l e 20 shows t h e x an th e llin

at

(D ifco ) a t

in h ib itio n su ccessiv e

S m all t u b e s

each c o n t a i n i n g 1 .0 m l.

in o cu latio n

purposes 20-hour try p to s e

g ive

the

of lig h t

at

lo o p fu l

b ro n ch isep tica

( 2 x 3 . 5 mm.)

to each tu b e .

B r. b ro n ch isep tica H. The E f f e c t

the p e r c e n t

B. c e r e u s - 80%, St_. a u r e u s

- 88%, S a r c i n a

of the d ilu te d

lu te a

- 80%.

One

b r o t h s was t r a n s f e r r e d S arcin a lu te a

grow th in th e c o n t r o l s a t 4 h o u r s , did n o t have v i s i b l e

and

grow th a t 8 h o u r s .

o f h o g s e r u m on t h e

a ctiv ity

of x a n th e llin

s u i s w as d e te r m i n e d by a b r o t h d i l u t i o n method

w ith

try p to se

salt

o f x a n t h e l l i n b e t w e e n 0 . 0 0 5 mg. p e r m l .

per m l..

For

o f S e r u m on A n t i b i o t i c A c t i v i t y .

The e f f e c t a g a in st B r.

used.

try p to se b ro th to

expressed as

I n c u b a t i o n was a t 37° C.

sh o w e d i n s u f f i c i e n t

o f tim e.

a w a v e l e n g t h o f 6 1 0 mjn o n a W i l -

k i n s - A n d e r s o n p h o t o m e t e r KWSZ: - 90%, B r .

in te rv als

b ro th c u ltu re s of th e

o rg a n is m s w ere d i l u t e d w i t h s t e r i l e

tran sm issio n

of

of s e v e r a l organism s

o f medium w e r e

test

fo llo w in g t u r b i d i t i e s

sodium s a l t

b ro th co n tain in g

T hree s e t s

c o n c e n tra tio n s of the

o f tu b e s w ere p r e p a r e d ,

sodium

a n d 0 . 1 5 mg. on e s e t

38 c o n t a i n i n g no serum , serum . 1 .0

one c o n t a i n i n g 10% s e r u m ,

one 20%

E a c h s m a l l t u b e , h a v i n g a t o t a l volum e o f medium o f

m l . , was i n o c u l a t e d w i t h

su is

and

c ells

one l o o p f u l

of v i r u le n t B r.

from a su sp e n s io n h av in g a t u r b i d i t y

in g t o M cFarland

stan d ard

#2 .

correspond­

A fte r in cu b atio n at

f o r 24 h o u r s ,

B r.

s e r u m down t o

a l e v e l o f 0 . 0 1 0 mg. p e r m l . , w i t h g r o w t h a t

t h e 0 . 0 0 5 m g.

p e r m l.

in a l l

s u i s was i n h i b i t e d

37° 0 .

lev el;

B r.

su is

I.

T o x ic ity of X a n th e llin .

u sing

ou t.

p rep aratio n

of x a n th e llin

ly ,

ad ju stm en t

each.

survive,

o f pH t o

lim ited

the a n t i b i o t i c

7 .0 ,

25 mg. e a c h ,

in tra p erito n ea 1-

and t h r e e r e c e i v i n g

the h ig h e r le v e l f a i l e d cage-m ate w ith in

test

in d icates a r e la tiv e ly

In v estig a tio n s T ech n iq u e.

low t o x i c i t y ,

9 0 0 mg. p e r k g .

t o D eterm in e

t h e y m ig h t be o f v a l u e

graphic

te c h n iq u e s to th e

(D arco G -6 0 ),

th e f i r s t T his w ith a

an A p p l i c a b l e C h r o m a t o g r a p h i c

in the

a p p lica tio n

a n tib io tic.

L loyd*s r e a g e n t ,

to

of body w e ig h t.

A num ber o f s u b s ta n c e s w ere i n v e s t i g a t e d if

of a crude

from e t h e r e x t r a c t s ) ,

was i n j e c t e d

by i t s

o f 36 g .

and a n o t h e r d y i n g a t t h e end o f 5 d a y s .

p o s s i b l e LD(jq o f a b o u t J.

(obtained

Two o f t h e m i c e a t

one b e i n g k i l l e d

24 h o u r s ,

of th e t o x i c i t y of

A sodium b i c a r b o n a t e s o l u t i o n

t h r e e m ice r e c e i v i n g

30 m g .

t h e r e was g r o w th of

s i x w h i t e m ice w i t h an a v e r a g e w e ig h t

was c a r r i e d

after

how ever,

tu b e s c o n t a i n i n g serum .

To o b t a i n some i d e a a test

i n t h e t u b e s w i t h no

silic a

to d e te rm in e of ch ro m ato ­

These in c lu d e d g e l,

D ecalso,

carbon

talc,

39 P erm u tit

( F o l i a ) , A m b e r l i t e I R - 4 B , and A m b e r l i t e I R - 4 0 0 .

X a n t h e l l i n was t o o t e n a c i o u s l y h e ld

by c arb o n ,

r e a g e n t was u n s a t i s f a c t o r y

sin ce

d isso lv ed

ab le

o rg a n ic s o l v e n t s w h ich w ere i n ­

ex te n t

ten d ed f o r so lu tio n

i n som e o f t h e use w ith

lite

W ith A m b e r l i t e

o f th e sodium s a l t

ly through i t tio n of

it.

w ith l i t t l e ,

the s a l t

it

any,

d id n o t w ork a t a l l

so lu tio n

a c tiv ity e ith e r t o rem ove i t

but

th ere

on t h e

from th e

D ecalso o r s i l i c a g rap h ic

an io n o f x a n t h e l l i n

from

be d e s t r u c t i o n

of the

or in th e procedure

appeared

and P e r m u t i t

sy stem w ith th e a n t i b i o t i c . free

could be e f f e c t e d

th at

i f the

(F o lin ),

acid

P erm u tit

proper

and p o s s i b l y

ch ro m ato g rap h y i n d i c a t e d

by d i l u t i n g

th at

and t a l c

from a b en zene s o l u t i o n

and

th e benzene w ith a

s m a ll amount o f a p o l a r o r g a n i c s o l v e n t .

p a rtitio n

and a w a t e r s o l u ­

g e l , m i g h t h a v e som e w o r t h i n a c h r o m a t o ­

r e a d i ly adsorbed th e e lu tio n

talc

passed d ir e c t­

Amber­

exchange r e s i n It

a b u tan o l

sa tisfa c to rily .

seemed t o

colum n.

s o lv e n t w ere em ployed,

IR -4B ,

change,

IR -4 0 0 a p p a r e n t l y rem oved t h e

b u tano l

to a c o n s id e r ­

o f the a n t i b i o t i c if

and L l o y d f s

cellu lo se ,

How ever, em ployed

s y s te m , m ig h t be o f g r e a t e r v a lu e

paper in a

th a n chrom ato­

graphy w ith any o f th e s e m a t e r i a l s . K. P a p e r C h r o m a t o g r a p h y a n d P a r t i t i o n

on C e l l u l o s e .

A sce n d in g p a p e r chro m ato g rap h y u s in g s t r i p s man # 1 f i l t e r

paper w ith d if f e r e n t

the fo llo w in g

resu lts:

a c id developed w ith (50,

2 5 , 2 5 ) sy stem ,

(1)

so lv en t

system s produced

a b u tan o l s o lu tio n

a bu tan o l,

g lac ia l

a co llid in e ,

w ater

of W hat­

a ce tic (75,

of the

free

acid , w ater 2 5 ) sy stem ,

or

40 a p y r id in e , b u tan o l, w ater

(3,

2,

one zo n e o f i n h i b i t i o n when the cereus assay p la te s , vent advance; m ade v i s i b l e the

(2)

of the

w ater (40,

(3, 10,

spray ing

zone;

(3)

a n tib io tic

2, 49,

sa lt,

(4)

h o u r s and n e u t r a l i z e d ,

bove th e spot

the

In an a tte m p t

so l­

sa m e p o i n t a s

in

of th e

a p y rid in e,

sodium

b u tan o l,

eth an o l, w ater,

the a p p e a ra n c e

acid

so lu tio n

a sealed

tube

one a t

of th e

at

b u tan o l, of th e

so lv en t r i s e

but in a d d itio n

the sta rtin g

sodium

1 1 0 ° C.

e th an o l, w ater,

amm onia

o f two

an d o n e n o t f a r a b o v e t h e

in

p o in t,

c o u l d be s e e n ,

of t h e

a l s o g a v e two g r e e n - y e l l o w

the h e ig h t

sta rtin g

the

a f t e r n in h y d rin trea tm e n t,

a f t e r b ein g h eated

a g a i n one a t

spot at

o r a b u tan o l,

a 6 N h y d ro ch lo ric

developm ent w ith

to p

an a q u eo u s s o l u t i o n

peak of the so lv e n t f r o n t p o in t;

at the

o n B.

s i m i l a r s t r i p s w ith n in h y d r in

sy stem r e s u l t e d

g reen -y ello w sp o ts

only

s t r i p s w ere in c u b a te d

developed w ith

2) s y s t e m , 1)

system produced

zone b e in g

a g reen ish -y ello w

in h ib itio n

salt

th is

2)

f o r 43

sp o ts a f te r

ammonia s y s t e m ,

an d one j u s t

a f a in t p u rp le

a b o u t h a l f way up t h e s t r i p . to r e p r o d u c e t h e phenomenon o c c u r r i n g

on p a p e r o n a l a r g e r

scale,

a colum n o f p o w d e red

c e llu lo se ,

"S o lk a-flo c"* ,

was e m p lo y e d .

The m a t e r i a l w a s p r e p a r e d

use by w ash in g

several

by s t i r r i n g

w ith

tim es,

l a r g e volum es o f w a te r ,

bu tan o l, 5 5 ° C. sa lt,

and a c e t o n e ,

fo r 2 hours. a colum n o f 1 . 3

*

a-

fo llo w ed

and d r y i n g a t

and

o v ernight

F o r u s e w i t h 3 5 . 3 mg. of cm.

in sid e

B ro w n Com pany M i l l s ,

filtra tio n ,

by w ashes w it h

3 2 ° C.

th e

New H a m p s h i r e

eth an o l, and a t

sodium

d i a m e t e r was f i l l e d

B e rlin ,

for

to a

41 d e p t h o f 9 * 5 cm. w i t h 4 .5 it

g. to

th e

c ellu lo se

in w a te r-sa tu ra te d se ttle

ing

su c tio n .

a ll

tim es,

use

was p l a c e d

frac tio n

and a band

and t h e

was f o u n d t h a t

(II)

(I)

passed d i r e c t l y

of the

b u t n o t from F r a c t i o n of F ra c tio n

I in

th is

b u tan o l,

and d i s s o l v i n g

I.

p re c ip ita te d

o f 7 * 2 mg.

through

fo llo w ed .

so lid

How ever,

by d i s s o l v i n g

resid u e

from

it

a yellow

Frac­

so lu tio n ,

in m ethanol,

F ractio n

a c tiv ity

and f o r

I w as m ore t h a n

In the a b s o r p tio n

sp ectra

a yello w

so lid

from F r a c t i o n

u ltra v io le t

tw ice a s a c t i v e 1)

I.

s o l i d was o b t a i n e d

from F r a c t i o n

(F ig .

ex­

i t was fo u n d

II.

The p r o d u c t s o f t h e t w o f r a c t i o n s w e r e c o m p a r e d f o r a n tib io tic

the

e v a p o r a tin g o f f the

of e th e r-p re c ip ita te d

a n d 1 1 . 7 mg.

the

in m eth an o l,

5% s o d i u m b i c a r b o n a t e

the

a

b u t a n o l from F r a c t i o n s I

s o lu tio n w ith b u ta n o l,

from F r a c t i o n I

fo llo w ed

shade of y e llo w

of the r e s id u e s

e th e r p re c ip ita te d

e th e r also

colum n,

in a

A s e p a r a t io n o f the

of a deeper

d isso lv in g

tra c tin g

A sam ple

c o lle c t­

i n t o two f r a c t i o n s w as a p p a r e n t :

F ollow ing e v a p o ra tio n

th at

s u c t i o n w ere

of the c e llu lo s e

on t h e

of the so lv e n t m ix tu re .

g reen ish -y ello w

resid u e

c o v ered by s o lv e n t a t

A f te r thorough e q u il i b r a t io n

o r ig in a l su b stan ce

II

th en slo w ly a p p ly ­

of a th re e -w a y s to o c o c k above th e

of b u ta n o l,

of

allo w in g

b u t a n o l - w a t e r m i x t u r e , the s a m p le , d i s s o l v e d

by p o r t i o n s

tio n

colum n,

and su dden c h an g e s in th e a p p li e d

s m a l l volum e

and I I ,

and

The c e l l u l o s e w a s k e p t

in g v e s s e l .

colum n,

in to th e

by g r a v i t y f o r 1 h o u r,

avoided by th e

w ith the

b u tan o l

by p o u r in g a s l u r r y

lig h t

ab so rp tio n .

as F ra c tio n

II.

no d i f f e r e n c e s w ere

42 e v id e n t in

th e p o s itio n

o f t h e m axim a,

was n o te d i n t h e r e l a t i v e c ip itatio n

of F ractio n

h eig h ts

I I did n o t

alth o u g h

a d ifferen ce

o f the m axim a. change i t s

A repre­

ab so rp tio n

spectrum . A nother c e l lu l o s e in sid e

diam eter)

satu rated c h lo ric

was p r e p a r e d

( 7 0 mm. i n h e i g h t a n d o f 6 mm. and e q u i l i b r a t e d

A sam ple

0 .6 m l.

o f 5 • 5 mg. o f F r a c t i o n

acid .

fo llo w ed

so lv en t m ix tu re,

by the

passed

no y e l l o w c o l o r

b ein g h eld

through the

it

was s e e n t h a t

d i r e c t l y th ro u g h the back.

the

usual

b utanol,

so lu tio n

resid u e

e th e r-p re c ip itab le The c e l l u l o s e w ater-satu rated satu rated

cellu lo se

of yellow s o l id

But by t a k i n g

the

so lid

ev ap o ratio n i n m ethanol,

of the

b u tan o l,

so lu tio n , and

t h e r e was o b t a i n e d

an

h a v i n g a w e i g h t o f 3 . 2 mg.

c o l umn w a s w a s h e d w i t h

about

50 m l .

of

t h a t h a d b e e n m ad e 0 . 0 1 8 N w i t h a m m o n i a .

A s a m p l e o f 6 . 0 m g. o f F r a c t i o n I w a s d i s s o l v e d of w a ter-sa tu ra ted

a lk a lin e

from

b u t a n o l , a nd t h e n e q u i l i b r a t e d w i t h w a t e r -

b utan o l

A fte r p lac in g

w ith

sam ple t h r o u g h

o f d i s s o l v i n g i n sodium b i c a r b o n a t e

e x tra c tin g w ith o f the

the y e llo w

o f the m a t e r i a l w hich had p a sse d

c ellu lo se .

step s

the co lu m n ,

F u r t h e r m o r e , i t was s e e n

e t h e r p r o d u c e d no p r e c i p i t a t i o n so lu tio n

wa s 0 . 0 8 N

U po n p l a c i n g t h e s a m p l e o n

sam ple s o l u t i o n

the m eth an o l

I I was d i s ­

o f w a te r-sa tu ra te d b u tan o l th a t

in h y d ro ch lo ric

th at

w ith w a te r-

b u t a n o l t h a t h a d b e e n m ad e 0 . 0 0 7 N w i t h h y d r o ­

acid .

solved in

colum n

the

i n 1 .0 m l.

b u t a n o l t h a t was 0 .0 2 5 N i n am m onia. s a m p l e on t h e

colum n,

fo llo w e d by the

s o lv e n t-m ix tu re , a very d e fin ite

y e l l o w b a n d was

43 f o r m e d w h i c h m o v e d g r a d u a l l y d own t h r o u g h t h e How ever, ly

a s m a ll amount o f th e y e llo w

th ro u g h the

from t h i s fra c tio n parent

colum n.

la tte r

it

N e ith e r product

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

w e i g h i n g 2 . 2 mg. w a s o b t a i n e d .

of the a cid

showed a n y a c t i v i t y

u ltra v io le t

a l t h o u g h i t was a p ­

A f te r ta k in g the re s id u e s

throug h th e so lid

a b so rp tio n

or a l k a l i n e

i n B.

tin u ed

of the

to r is e

sp ectra

pro­

of the p ro d u c ts s t i l l

u s u a l m ax im a a t

g rad u a lly in stead

p a rtitio n

c e r e u s cup a s s a y p l a t e s ;

p o s s e s s e d m ax im a a t 2 8 0 0 A a n d m i n i m a a t the p o s itio n s

any s o lid

b u t fro m t h e m ain slo w e r-m o v in g

was i n c o m p l e t e .

eth e r-p re c ip itab le

th e

E th e r did n ot p r e c i p i t a t e

frac tio n ,

from b o th f r a c t i o n s

cedures

sam ple p a s s e d d i r e c t ­

e th e r did c a u se p r e c i p i t a t i o n ,

th at

c ellu lo se .

2540 A , b u t fro m

2320 A t h e

cu rv es con­

of a p p ro a c h in g m inim a.

L. L i g h t A b s o r p t i o n C h a r a c t e r i s t i c s . X an th ellin e x h ib its v isib le

part

u ltra v io le t m ax im a a t

of the

lig h t ab so rp tio n

sta rtin g

in the

s p e c t r u m a n d r e a c h i n g a maximum i n t h e

reg io n a t

2800 A ( E i g . 2 ) .

2 8 0 0 A a n d 2 3 2 0 A,

th ere are

In a d d itio n to in fle ctio n s

a t 6500

A and 3200 A . To d e t e r m i n e a n tib io tic e x tra cts

if

fre e acid

of aqueous s o lu tio n s

photom eter.

form s o f

The

sp ectra

3 0 0 0 - 3 3 0 0 A.

f o r th e in

the

b u tan o l

o f x a n t h e l l i n a t pH 3 . 0 and

and com pared i n

for a d ifferen ce

th e re g io n

and s a l t

showed d i f f e r e n t a b s o r p t i o n s p e c t r a ,

pH 1 0 . 5 w e r e o b t a i n e d

except

the

t h e B e ck m an s p e c t r o ­

two f o r m s w e re t h e

th e appearance

same

of in f le c t io n s

in

44 When a n a q u e o u s s o l u t i o n

c o n t a i n i n g 0 . 0 3 mg. p e r m l .

o f so d iu m s a l t was t r e a t e d w i t h p e ro x id e p e r 4 .0 m l.

of so lu tio n ,

a c t i v i t y w ere d e s t r o y e d . oxide by b o i l i n g ,

o n e d r o p o f yfo h y d r o g e n

the

the

c o l o r and a n t i b i o t i c

A f t e r rem oving th e e x c e s s p e r ­

so lu tio n

sh ow ed a s h a r p d r o p i n l i g h t

a b s o r p t i o n a t 2800 A and a c o r r e s p o n d i n g i n c r e a s e A (F ig .

3).

observed preted ated

A s i m i l a r though l e s s

in a so lu tio n

as ev id en ce

pronounced s h i f t w hich was

exposed to a i r

f o r 4 d a y s was i n t e r ­

of a u to x id a tiv e d e te rio ra tio n

of u n satu r­

lin k ag es.

M. P r o p e r t i e s

of the A n tib io tic .

Th e s o l u b i l i t y the

a t 2320

a n tib io tic

an d s t a b i l i t y

in th e s a l t

have been d e s c r ib e d ,

c h aracteristics

form and

as have i t s

of

in th e f r e e a c id

lig h t

ab so rp tio n

form

charac­

teristics. A 1 4 m g. d isso lv ed m l. ic

i n 20 m l .

of th is acid ,

sam ple o f t h e sodium s a l t

of a c id .

pH v a l u e s , itse lf it

the

The r e m a i n i n g v o l u m e o f t h e

curve

ob tain ed

presen ted

in F ig .

c a r b o n d i o x i d e - f r e e w a t e r t o t h e same

and t h e amount

t o pH 9 . 0 w a s d e t e r m i n e d .

tra liz a tio n

is

amount o f a c id n e ed e d t o t a k e

t o pH 5 . 0 ,

change in

w i t h 0 .0 1 2 0 6 N sodium h y d ro x id e s o l u ­

The t i t r a t i o n

By t i t r a t i n g

T en

w i t h 0 .0 1 2 0 6 N h y d r o c h l o r ­

u s i n g a B e c k m a n pH m e t e r t o m e a s u r e t h e

sam ple was t i t r a t e d

4.

of carbon d io x id e - f r e e w a te r.

sam p le was t i t r a t e d

pH w i t h a d d i t i o n

tio n .

o f x a n t h e l l i n was

e q u iv ale n t

o f 1319 was o b t a i n e d ;

sam ple

of a l k a l i needed to ta k e

From a c a l c u l a t i o n

b a s e d on t h e a l k a l i based

the

on t h e

used,

amount o f a c i d

of neu­ a v alu e used, a

45 v alu e

o f 936 was o b t a i n e d .

A calcu latio n

b in atio n

o f t h e a m o u n ts o f a c i d and a l k a l i

pH r a n g e

6 .0

of u n s a tu r a te d

is

in d icated

in

c h l o r o f o r m o r w a t e r and t h e

is

on t h e

com­

used t h r o u g h

the

to 8 .0 gave a v a lu e o f 1350.

The p r e s e n c e

ate.

based

by th e ra p id

uptake

lin k a g es

o f brom ine fro m s o l u t i o n s

r e d u c t i o n o f co ld perm angan­

The c h e m i c a l b e h a v i o r o f x a n t h e l l i n

a r e a d i l y o x id ized

in th e m olecule

su b stan ce.

in d ic ate s

th at

it

I t h a s a l r e a d y b e e n m en ­

tio n ed

t h a t hydrogen p e ro x id e q u ic k ly d e s tro y s b o th th e a n t i ­

b io tic

a ctiv ity

and t h e

of fo rm in g a p i c r a t e , p icric test

acid

c o lo r of w a ter s o lu tio n s .

the

to p icram ic acid

fo r hydroxyl groups

n itra te

reagent

ox id ized

a n tib io tic

occurred,

compounds.

(27),

at

th e fr e e

a r a th e r rap id

and t h a t

least

The a n t i b i o t i c

(28),

a ro m atic

of a c t iv i t y

from

a t room

g a llic

on t h e

acid ,

at a lev el

at

its

in itia l

5 d a y s a t room t e m p e r a t u r e . has given a n e g a tiv e resp o n se

a n eg ativ e

or a li p h a ti c

p h en y lh y d razin e

of x a n th e llin

acid a s th e r e s i d u e

in k eep in g the a c t i v i t y

number o f c l a s s i f i c a t i o n test

of e a s ily

i t was d i s c o v e r e d t h a t a s c o r b i c

the a n tio x id a n t,

o f 0 .5 % w a s e f f e c t i v e for at

of the e e r ie

ch aracteristic

lo ss

of

eerie n itr a te

a l e v e l o f 1.0% , h ad a s p a r i n g a c t i o n

a c tiv ity ,

v alu e

In the

a d e co lo riz atio n

w hich i s

te m p e r a tu r e was e n c o u n te r e d ; acid ,

causes a red u c tio n

W i t h some a q u e o u s s o l u t i o n s

( t h o s e m ad e b y d i s s o l v i n g an e t h e r e x t r a c t )

(26).

In stead

te sts.

It

"p ero x id ase"

in a

gave a n e g a tiv e test

aldehyde group,

(28)

enol

f o r e i t h e r an

n eg ativ e

and 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e

t e s t s w ith (27),

a

n e g ativ e

test

for n itro

aqueous or a lc o h o lic n eg ativ e

p erio d ic

n eg ativ e

p ro tein

hyd rin

( 2 7 ) , no c o l o r change w ith

so lu tio n s

acid t e s t

of f e r r i c

(27),

and am in o a c i d

on f i l t e r

b u tan ol s o lu tio n or tw o,

paper,

(27),

a

te sts,

alth o u g h w ith n in ­

A n eu tral

so lu tio n

of

when s p r a y e d w i t h a d i l u t e

o f n i n h y d r i n a n d h e a t e d a t 1 1 0 ° G.

shows a f a i n t

c h arac teristic

c h lo rid e

a n e g a tiv e M olisch t e s t ,

t h e r e was c o l o r d e v e lo p m e n t.

x an th e llin

m in u te

groups

peach c o lo r in ste a d

for a

of the

b lue o r p u rp le .

A d d i t i o n o f an e q u a l volum e o f c o n c e n t r a t e d h y d r o c h l o r i c acid

to an aqueous s o lu tio n

b io tic

resu lted

y ello w

c o lo r,

S olid p resen t

w hich g r a d u a l l y changed to in the

acid

a ctiv ity ,

the

and c h a r a c t e r i s t i c

th ro u g h c e l lo p h a n e m em branes,

its

evidenced by a n t i b i o t i c

upon a c i d i f i c a t i o n ,

yellow c o lo r ,

a b s o rp tio n spectrum .

W i t h c o p p e r , m e r c u r y a nd

A nalyses

a l i g h t brow n.

so lu tio n .

d ia ly z a te b ein g

p re c ip ita tio n

w ater-in so lu b le

of th e a n t i ­

s o lu tio n d id n o t co m p letely d i s ­

of th e

X a n th ellin d ialy zes in

sodium s a l t

i n c l o u d i n g and a m arked d e e p e n in g o f t h e

s o l v e on n e u t r a l i z a t i o n

presence

of the

silv er,

th e s u b s ta n c e form s

sa lts.

of th e sodium s a l t

o f 7.8% and a n a b s e n c e

showed a n i t r o g e n c o n t e n t

o f s u l f u r and h a l o g e n .

47 DISCUSSION From t h e x a n th e llin for it

p r o p e r t i e s w h ich have been d e te rm in e d f o r

and fro m t h e p r o p e r t i e s w h ic h have b e e n r e p o r t e d

o th e r a n t i b i o t i c s d e riv e d from s t r a i n s is p o ssib le

to d i f f e r e n t i a t e

o f B.

sn b tilis,

x a n t h e l l i n from th e s e a n t i ­

b io tic s. S a b tilin . e n t. in pH;

S a b tilin

F o r ex am p le,

o rg an ic

and x a n t h e l l i n a r e e n t i r e l y d i f f e r ­

sa b tilin

so lv en ts,

has a very lim ite d

and i n w a t e r i s more s o l u b l e a t

and i n a q u e o u s s y s te m s

B a citra cin .

B acitracin

is p re c ip ita te d

is not p re c ip ita te d

o r i g i n a l h a r v e s t b y pH m a n i p u l a t i o n a s i s a l s o m uch m o r e s t a b l e

so lv en ts

th at

or 6 .5 ,

a g a in st E.

and i s n o t

B a cillin

c o li,

soluble

and a f f e c t s

It

b y e t h e r a t pH

g ram -p o sitiv e

has a c t iv i t y and g r a m - n e g a t i v e

sim ila r co n cen tratio n s.

on x a n t h e l l i n

p ro d u ctio n ,

A lso , m anganese, has a d e trim e n ta l

pro du ctio n.

I n f o r m a t i o n a b o u t eum ycin i s

lim ite d but

d iffe re n c e s are ap p aren t.

Eum ycin i s

so lu b le

in so lu b le

free acid ,

x a n th e llin

in e th e r;

i n w a t e r an d

from th e

i n m an y o f t h e

th an x a n th e ll i n ,

w hich i s n e c e s s a r y f o r b a c i l l i n

E nm ycin.

l o w pH.

x a n th e llin .

is not e x tra ctab le

i s more s t a b l e

organism s a t f a i r l y

effect

at

d isso lv e x a n th e llin .

B a c illin . 2 .0

low

x a n t h e l l i n h a s a w ide ran g e of s o l u b i l i t y i n o r g a n ic

so lv en ts,

is

so lu b ility

as th e

so lu b le

acid w h ile x a n t h e l l i n

in is

eth e r.

i n w a t e r and is

in so lu b le

E um ycin i s h e a t - s t a b l e

co m p letely u n sta b le

some

under such

in

48 c o n d itio n s. x an th e llin p e r m l.

E um ycin o n l y s l i g h t l y i n h i b i t s St..

It

is

aureus a t

p o ssib le

of B.

a n tib io tic,

one o f w h ic h c o u ld

in

or a lk a lin e

cello p h an e. sta b le

o f 0 . 0 5 mg. by a M ar­

o f more t h a n

correspond

B aciH om ycin

a n tib a c te ria l

acid

elab o rated

s u b t i l i s , may c o n s i s t

B acillo m y cin .

sta p h y lo c o cc i;

a co n cen tratio n

t h a t eum ycin,

burg s t r a i n

g ib le

in h ib its

to x a n th e llin .

is rep o rted

to have n e g l i ­

a ctiv ity ,

t o be s t a b l e

so lu tio n ,

and t o n o t d i a l y z e

to au to c la v in g through

X a n th e llin has a n ti b a c t e r i a l a c t i v i t y ,

under th e aforem en tio n ed

one

co n d itio n s,

is

not

and w i l l d i a ­

ly z e th ro u g h c e llo p h a n e m em branes. S u b te n o lin . in

W hile s u b t e n o l i n r e s e m b le s x a n t h e l l i n

some r e s p e c t s ,

th ese

it

h a s m any d i f f e r e n t

d ifferen c e s are :

acid s,

is

very h eat

i n 95?» e t h a n o l ,

su b te n o lin

s t a b l e , and i s

and i n e t h e r .

su b te n o lin g iv es a p o s itiv e te st,

a test

resem ble x a n th e ll i n

in

its

and b o t h show s t r o n g l i g h t reg io n ,

S u b tily sin . is

d ifferen t

and d o e s n o t

an tag o n ism s.

u n satu ratio n

in

S u b ten o lin

t h e i r m o lecu les,

in the

u ltra v io le t

2700 A and x a n t h e l l i n

2 8 0 0 A a n d 2 3 2 0 A. S u b tily sin

c o lo rle s s , p ro tein

h ib its

are th a t

a p o s i t iv e M olisch

aldehyde g ro u p ,

a b so rp tio n

by

in a c e to n e ,

O ther d if f e r e n c e s

s u b t e n o l i n h a v i n g a maximum a t

h a v i n g m axim a a t

it

in so lu b le

b acterial

and x a n t h e l l i n b o t h c o n t a i n

Some o f

is not p re c ip ita te d

enol te s t,

f o r an a r o m a t i c

p ro p erties.

d iffers

from x a n t h e l l i n

and e n z y m a t i c

in th a t

i n n a t u r e , and e x ­

a n tib a c te ria l sp e cificitie s*

Ant i f u n g a l F a c t o r s

o f M ic h en e r and S n e l l .

B esid es

49 b ein g r e l a t i v e l y

in ac tiv e

a re b o th in s o lu b le does n o t d ialy ze

ag ain st b a c te ria ,

in e t h e r .

One, t h e a s p e r i g i l l u s

thro u g h c e llo p h a n e ,

f a c t o r , w hich does d i a l y z e ,

th ese f a c to r s

is

facto r,

and t h e r h i z o c t o n i a

in so lu b le

in aceto n e,

b u tan o l,

and a b s o l u t e e t h a n o l . In sta te d

the

sep aratio n

th at

im purity)

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

by e t h e r e x t r a c t i o n

b a cillo m y cin .

It

is p o ssib le

same a s x a n t h e l l i n . to w h e t h e r th e B. m ycin i s

procedure fo r b acillo m y cin ,

th at

stra in

su itab le

so lu tio n ,

o f two f o r m s ,

u n d isso ciated

acid

tie s

sa lt,

the

makes no s t a t e m e n t a s

th at

produces b a c i l l o ­

it

is

suggested

one o f w h ic h c o r r e s p o n d s t o t h e

fo rm and th e

and t h a t

colum n,

betw een

th ese

o th e r to the

a n io n ic p a rt

form s e x h i b i t d i f f e r e n t m o b ili­

on p a p e r o r c e l l u l o s e . From t h e

changes th a t

ab so rp tio n

sp ectra

x a n th e llin

so lu tio n s

and

su b stan ce is

x a n t h e l l i n e x i s t s a s an e q u i l i ­

brium m ix tu re

of the

th is

o b tain ed w ith p a r t i t i o n

b u t a n o l a n d w a t e r on a c e l l u l o s e in

( a s an

th e M arburg ty p e .

From t h e r e s u l t s

th at

is

o f the a c i d - p r e c ip i t a t e d

The l i t e r a t u r e

su b tilis

i s rem oved

it

on s t a n d i n g i n

take p lac e

and i n t h e a n t i b a c t e r i a l

provide

a ir,

it

is

x a n th e llin m o lecu le. a useful

u ltra v io le t a c tiv ity of

on t r e a t m e n t w i t h h y d r o g e n p e r o x i d e suggested t h a t

p o ssib ly causes a d e te r io r a tio n in th e

in th e

index to

of th e

ox id atio n

u n satu rated

lin k ag es

L i g h t a b s o r p t i o n d a t a m ay

the a c t i v i t y

of a p re p a ra tio n .

50 SUMMARY 1.

M ethods a r e

fica tio n

of a n t i b i o t i c s

sam ples o f In d ia n a stra in

o f B.

2.

d escrib ed

fro m two b a c t e r i a

so ils,

su b tilis

f o r t h e p r o d u c t i o n and p u r i ­

a stra in

o f B.

o b tain ed

from

polymyxa and a

(M arburg).

The a n t i b a c t e r i a l p r o d u c t stra in

o b ta in e d from th e

o f B. polym yxa h a s b e e n

cu ltu re

filtra te s

of th e

found to be

a m ix tu re

o f p o ly m y x in s, w ith th e m a jo r component b e in g

p o l y m y x i n D. 3. len t

An a n t i b i o t i c w i t h i n v i t r o

stra in s

o f B r.

w ater-so lu b le stra in

o f B.

so lid

s u is h as been o b tain ed a s a s t a b l e , from th e c u l t u r e

su b tilis

(M arburg).

we h a v e n a m e d x a n t h e l l i n , a g a in st g ram -p o sitiv e Of t h e

filtra te s

of the

T h is a n t a g o n i s t , w hich

i n g e n e r a l e x h i b i t s more a c t i v i t y

organism s th a n a g a i n s t

organism s t e s t e d ,

m ost s t r o n g l y

a c tiv ity ag ain st v iru ­

S arcina lu te a

in h ib ited .

P ro tectio n

g ram -n eg ativ e.

and B r .

te sts

s u i s w ere

a g a in s t B r.

w ith g u in ea p ig s have n o t y e t been a tte m p te d ;

su is

a p relim in ary

t o x i c i t y t e s t w i t h m ic e i n d i c a t e s a r e l a t i v e l y low t o x i c i t y . The p r e s e n c e

o f blood serum a t

been found t o on B r .

the

A d ifferen tiatio n

su b tilis

a n tib io tics

of th e

in h ib ito ry e ffe c t

of x a n th e llin

su b stan ce

is

o f x a n t h e l l i n f r o m o t h e r B.

p resen ted .

in d icate

th at

an e q u iv a le n t w eig h t o f about 1000, d ized ,

o f 10% a n d 20% h a s

su is.

4.

tie s

decrease

lev e ls

and t h a t

u n satu ratio n

T he c h e m i c a l p r o p e r ­ it

is

th at

is present

an a c id w ith it

is

re a d ily o x i­

in th e m o lecu le.

51 The b e h a v i o r o f t h e a n t i b i o t i c colum n i n d i c a t e s stance form ,

a d ifferen ce

in th e form o f th e

in

so lu tio n .

on a c e l l u l o s e

in the m o b ility o f th e

free

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