The utilization of carbohydrate intermediates and amino acids by Vibrio Fetus
by James K Alexander
A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
of Master of Science in Bacteriology at Montana State College
Montana State University
© Copyright by James K Alexander (1954)
Abstract:
A suitable basal medium was developed to determine the utilization of various energy sources by
Vibrio fetus. This basal medium consisted of 0.575 per cent Difco's dehydrated brain liver heart
medium, 0.5 per cent sodium chloride, 0.1 per cent disodium phosphate, and 0.05 per cent agar.
Under the conditions of these experiments lactate, pyruvate, acetate, oxaloacetate, alpha-ketoglutarate,
succinate, fumarate, malate, aspartate, asparagine, glutamate, and proline were found to be suitable
energy sources. Serine supported growth of only one of the two strains used in this study. Glucoae,
hexosediphosphate, glycerol, glycerophosphate, citrate, isocitrate, oxalosuccinate, tartrate, glycine,
alanine, leucine, lysine, ornithine, arginine, histidine, phenylalanine, threonine, cysteine and
methionine all were unable to support appreciable growth.
Resting cell suspensions oxidized lactate, pyruvate, isocitrate, and fumarate. Hexosediphosphate may
have been oxidized slightly. Glucose and glycerophosphate were not oxidized. THE UTILIZATION OF CARBOHYDRATE INTERMEDIATES AND AMINO ACIDS
BY VIBRIO FETUS
• by
JAJIES K= ALEXANDER '
A THESIS
S u b m itte d t o t h e G ra d u a te F a c u l t y
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TABLE OF CONTENTS
ABSTRACT
3
INTRODUCTION
h
HISTORICAL REVIEW
5
MATERIALS AND METHODS
P r e l i m i n a r y w ork
B a s a l medium
M an o m etric s t u d i e s
7
9
Il
RESULTS
G row th s t u d i e s
M an o m etric s t u d i e s
12
17
DISCUSSION
20
SUMMARY
21
REFERENCES
23
110376
ABSTRACT
A s u i t a b l e b a s a l m edium was. d e v e lo p e d t o d e te r m in e t h e u t i l i z a t i o n o f
v a r i o u s e n e r g y s o u r c e s b y V i b r io f e t u s .
T h is b a s a l medium c o n s i s t e d o f
Oo575 p e r c e n t D i f c o 's d e h y d r a t e d b r a i n l i v e r h e a r t m edium , 0 . 5 p e r c e n t
so d iu m c h l o r i d e , 0 . 1 p e r c e n t d is o d iu m p h o s p h a te , an d 0 .0 5 p e r c e n t a g a r .
U nder t h e c o n d i t i o n s o f t h e s e e x p e r im e n ts l a c t a t e , p y r u v a t e , a c e t a t e ,
o x a lo a c e ta te , a lp h a - k e to g l u ta r a te , s u c c in a te , fu m a ra te , m a la te , a s p a r t a t e ,
a s p a r a g i n e , g l u t a m a t e , a n d p r o l i n e w e re f o u n d t o b e s u i t a b l e e n e r g y s o u r c e s
S e r i n e s u p p o r te d g ro w th o f o n l y one o f t h e tw o s t r a i n s u s e d i n t h i s s tu d y .
G lu c o a e 3 h e x o s e d i p h o s p h a te , g l y c e r o l , g l y c e r o p h o s p h a t e , c i t r a t e , i s o c i t r a t e
o x a lo s u c c in a te , t a r t r a t e , g ly c in e , a la n in e , le u c in e , l y s i n e , o r n ith in e ,
a r g i n i n e , h i s t i d i n e , p h e n y l a l a n i n e , t h r e o n i n e , c y s t e i n e a n d " m e th io n in e a l l
w e re u n a b le t o s u p p o r t a p p r e c i a b l e g ro w th .
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R e s tin g c e l l s u s p e n s io n s o x id iz e d l a c t a t e , p y r u v a te , i s o c i t r a t e , and
f u m a r a t e . H e x o s e d ip h o s p h a te may h a v e b e e n o x i d i z e d s l i g h t l y .
G lu c o se a n d
g ly c e r o p h o s p h a t e w e re n o t o x i d i z e d .
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-1
-I).INTRODUCTION
:
:
1
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A lth o u g h V i b r i o f e t u s w as r e c o g n i z e d a s t h e p r o b a b l e c a u s a t i v e a g e n t
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I
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o f c a s e s o f i n f e c t i o u s a b o r t i o n o v e r IiO y e a r s a g o , l i t t l e i s known o f this
c u ltu r a ,! c h a r a c t e r i s t i c s e x c e p t t h a t i t i s a n o b lig a te m ic r o a e r o p h ile and
d i f f ic u l t to c u ltiv a te .
C o n s e q u e n tly , t h e c u l t u r a l m eth o d s p r e s e n t l y em­
p lo y e d a r e u n s a t i s f a c t o r y f o r t h e r e i s n o w ay t o d i s t i n g u i s h TL f e t u s fro m
s im ila r v ib r io s .
M o re o v e r, i t i s d i f f i c u l t t o i s o l a t e t h i s o r g a n is m .
The in a d e q u a c y o f t h e c u l t u r a l m eth o d s h a s d i s c o u r a g e d s t u d i e s o f t h e
n a t u r a l h a b i t a t o f t h e o rg a n is m a n d o f t r a n s m i s s i o n f a c t o r s i n v o lv e d i n t h e
d is e a s e .
-
I n v ie w o f t h i s l a c k of_ k n o w le d g e i t a p p e a r s t h a t a m ore c o m p le te
u n d e r s t a n d i n g o f t h e m e t a b o l i c c h a r a c t e r i s t i c s p f t h i s o r g a n is m m ig h t a i d
i n d e v e lo p i n g m ore s u i t a b l e c u l t u r a l m e th o d s ,
f T p e tte r u n d e r s t a n d i n g o f
t h e m e ta b o lis m m ig h t make i«t p o s s i b l e t o d i s t i p g u i s h TL1 f e t p s fro m s i m i l a r
v ib rio s .
M o re o v e r, a k n o w le d g e o f t h e n u t r i t i o n a l r e q u i r e m e n ts , c o u ld p o s ­
s i b l y l e a d t o t h e d e v e lo p m e n t o f a p a r t i a l l y s e l e c t i v e m edium f o r t h e i s o ­
l a t i o n o f t h i s o r g a n is m .
,
The d e t e r m i n a t i o n o f t h e u t i ^ i z a b l e e n p rg y s o u r c e s w as u n d e r t a k e n a s
t h e f i r s t s t e p i n s tu d y i n g t h e m e ta b o lis m o f t h i s o r g a n is m .
V a r io u s com­
p o u n d s h a v e b e e n i n v e s t i g a t e d t o s e e o f t h e y w o u ld s e r v e a s s o u r c e s o f
e n e r g y f o r t h e g ro w th o f t h i s o r g a n is m .
In a d d itio n to
t h is , re s tin g c e ll
s u s p e n s i o n s w e re u s e d t o d e te r m in e t h e a b i l i t y o f t h i s o rg a n is m t o o x i d i z e
s e v e r a l com p o u n d s.
The g ro w th f a c t o r a n d am ino a c i d r e q u i r e m e n t s h a v e b e e n
l e f t f o r fu tu re s tu d ie s .
HISTORICAL R E V IEF.
The f i r s t r e p o r t o f v i b r i o s b e i n g a s s o c i a t e d w i t h i n f e c t i o u s a b o r t i o n
i n s h e e p a n d c a t t l e w as made b y M cFadyean a n d Stockm an i n 1913 ( c i t e d b y
S m ith , 1 9 1 8 ) .
S m ith a n d T a y lo r ( 1 9 ^ 9 ) s t u d i e d t h e c u l t u r a l c h a r a c t e r i s t i c s o f a n
o rg a n is m w h ic h w as p r e s u m a b ly t h e same a s t h e v i b r i o i s o l a t e d b y M cFadyean
a n d Stockm an an d named t h e o rg a n is m V i b r i o f e t u s .
A lth o u g h t h e o rg a n is m
i s f o u n d b o t h i n t h e fo rm o f a s p i r i l l u m a n d i n t h e fo rm o f a v i b r i o , t h e
l a t t e r fo rm p r e d o m in a te s i n y o u n g c u l t u r e s , h e n c e , t h e g e n e r i c name o f
V i b r i o w as c h o s e n .
The o r g a n is m w as d e s c r i b e d a s r e l a t i v e l y d i f f i c u l t t o ■
c u l t i v a t e im m e d ia te ly a f t e r i s o l a t i o n .
A f te r r e p e a te d t r a n s f e r s th e o rg an ­
is m so m e tim e s becam e s a p r o p h i t i z e d a n d c u l t i v a t i o n becam e l e s s d i f f i c u l t .
I n d o le w as n o t fo rm e d i n b o u i l l o n , a n d n o g ro w th w as a t t a i n e d i n m ilk o r
n u t r i e n t g e l a t i n o r on p o t a t o ' m edium .
The o r g a n is m p r o d u c e d n o g a s an d 'th e
r e a c t i o n r e m a in e d n e u t r a l o r s l i g h t l y a c i d i n f e r m e n te d b o u i l l o n w i t h I p e r
c e n t d e x t r o s e , l a c t o s e , op s a c c h a r o s e .
A redh-ded oxygep t e n s i o n 'w a s r e p o r t e d
a s n e c e s s a r y f o r g ro w th qij1 t h e ‘o r g a n is m .
.
S tockm an (19& 9) r e p o r t e d thaib n o g ro w th p f' V. f e t u s o c c u r r e d e i t h e r i n
a fre e
s u p p ly o f p i r p r u n d e r s t r i c t l y a n a e r o b i c c o n d i t i o n s .
B aP ger (1 9 2 8 )
g rew t h e o rg a n is m i n an a tm o s p h e re i n w h ic h a b o u t 10 p e r c e n t o f th e a i r w as
r e p l a c e d b y c a rb o n d i o x i d e .
The c o n c e n t r a t i o n o f a tm o s p h e r ic c a r b o n d i o x i d e m o st s u i t a b l e f o r g ro w th
'
w as d e te r m in e d b y P l a s t r i d g e a n d W illia m s (1 9 U 3 ). W ith a s e m i s o l i d medium
•
•
:
o f l i v e r i n f u s i o n a n d p e p to n e a d j u s t e d to , pH 7 .U t h e m o st a b u n d a n t g ro w th
w as o b t a i n e d when 10 p e r c e n t o f t h e a i r 'w a s . r e p l a c e d b y c a rb o n d i o x i d e .
No g ro w th w as o b t a i n e d w hen 30 p e r c e n t o f t h e a i r w as r e p l a c e d b y c a rb o n
d io x id e .
T hese W p rk e rs a l s o i n v e s t i g a t e d t h e u t i l i z a t i o n o f c a r b o h y d r a te s
b y t h i s o r g a n is m .
No e v id e n c e f o r a c i d p r o d u c t i o n w as f o u n d w i t h I p e r
c e n t a ra b in o s e , d e x tro s e , d p x tr in , d u l c i t o l , g e la c to s e , i n u li n , la c to s e ,
I e v u l o s e 3 m a l t o s e , p a n n i t o l , r a f f i n o s e , s a l i c i n , s u c r o s e , s 0jrt> ito l o r
tre h e lo s e .
The b a s a l m e d iu p .u s e d i n t h i s s t u d y c o n s i s t e d o f b e e f i n f u s i o n ,
1 .0 p e r c e n t p e p t o n e , 0 .3 p e r c e n t a g a r , a n d 1 . 0 p e r c e n t A n d r a d e 's
in d ic a to r.
The n u t r i t i o n o f V_. f e t u s w as s t u d i e d b y B a t l i n an d W ils o n ( 1 9 3 0 ) .
A
medium c o n s i s t i n g o f h y d r o ly s e d c A s e in , s a l t s , a n d g ro w th f a c t o r s w as s u f f i
c i e n t t o m a i n ta i n g ro w th th r o u g h s e r i a l t r a n s f e r s .
L ik e w is e a medium
com posed o f 16 am ino a c i d s , s a l t s , a n d g ro w th f a c t o r s w as a l s o s u f f i c i e n t
I
t o m a i n ta i n g r o w th .
T hese m ed ia w e re p r e p a r e d witfcj I p e p c e n t m e th o c e l a s
a t h i c k e n i n g a g e n t a n d i n c u b a t i o n w as u n d e r 10 p e r c e n t a d d e d c a rb o n
d io x id e .
A c c o rd in g t o P l a s t r i d g e e t . a l . . ,
(1 9 5 1 ) i n c r e a s e d c a rb o n d i o x id e
t e n s i o n i s r e q u i r e d f o r 'grow th o f r e c e n t l y i s o l a t e d s t r a i n s o f V. f e t u s
a n d e n h a n c e s t h e g ro w th o f o t h p r s t r a i n s .
May (195.3) fo u n d t h a t t h e
o rg a n is m a p p a r e n t l y d i d n o t r e q u i r e i n c r e a s e d c a rb o n d i o x i d e i n t h e a tm o s­
p h e re .
The m o st a b u n d a n t g ro w th w as o b t a i n e d w hen a b o u t o n e - h a l f o f th e
a i r w as re m o v e d .
No s u r f a c e g ro w th w as a t t a i n e d i n a f r e e s u p p ly o f a i r .
■The a tm o s p h e r ic r e q u i r e m e n t s o f
f e t u s a r e som ew hat p u z z l i n g .
From
a r e v i e w o f t h e l i t e r a t u r e i t a p p e a r s t h a t t h i s o rg a n is m i s a n o b l i g a t e
a e r o b e , b u t r e q u i r e s e i t h e r a r e d u c e d oxygen t e n s i o n o r a n i n c r e a s e d c a rb o n
—7—
d i o x i d e t e n s i o n f o r g r o w th .
A p p a r e n tl y i t i s m ore d i f f i c u l t tcj p r o v id e
f a v o r a b l e a tm o s p h e r ic c o n d i t i o n ^ f o r s u r f a c e g ro w th t h a n f o r g ro w th i n a
s e m i s o l i d medium o r i n a b r o t h njedium .
The e f f e c t o f a d d i t i o n a l c a rb o n
d i o x i d e i n t h e a tm o s p h e re d o e s n o t a p p e a r t o b e u n d e r s t o o d ,
•MATERIALS AND METHODS
Two s t r a i n s o f
f e t u s , b o t h o f o v in e o r i g i n , w e re u s e d i n t h i s s t u d y .
T h e se s t r a i n s , o b t a i n e d fro m t h e V e t e r i n a r y R e s e a r c h L a b o r a t o r y , M ontana
S t a t e C o l l e g e , B ozem an, M ontanp,, hpd b e e n a s s i g n e d a c c e s s i o n n u m b ers 2035
a n d UhbO b y t h a t l a b o r a t o r y .
S to c k c u l t u r e s w e re m a i n ta i n e d i n tu b e s ' c o n t a i n i n g a b o u t 7 m l o f D i f c o 1s
b r a i n l i v e r h e a r t se m i s o l i d m edium .
b ra in liv e r h e a rt p er l i t e r .
T h is medium c o n t a i n e d ij.6 g o f d e h y d r a te d
The d e h y d r a t e d b r a i n l i v e r h e a r t w as S t o r e ^ i n
t h e c q ld a s a p r e c a u t i o n a g a i n s t d e t e r i o r a t i o n f o r May (1 9 5 3 ) fo u n d t h a t
t h i s medium so m e tim e s u n d e rw e n t p r o g r e s s i v e d e t e r i o r a t i o n i f
s t o r e d a t room
te m p e ra tu re .
P r e l i m i n a r y W ork.
g ro w th e x p e r i m e n t s .
-A s e a r c h w as made f o r a s u i t a b l y b a s a l medium fo p t h e
T h is b a s a l m edium w as d e s ig n e d t o ^ u p p ly a n e x c e s s '
am o u n t o f 't h e g ro w th f a c t o r s ; a n d am ino, a e f d s n e e e ^ fc a ry ' f o p ‘t h e g ro u fth o f
t h i s o rg a n is m b u t t o Ipupply1O nly a l i m i t e d am b u n t o f u t i l i z a b l e e n e rg y
so u rc e s.
S in c e t h e g ro w th f a c t o r pnd am ino a c i d r e q u i r e m e n t s o f t h i s o r g a n ­
is m a r e unknow n, n o a t t e m p t w as made t o d e v e lo p a c h e m i c a l ly d e f i n e d m edium .
Two b a s a l m ed ia w e re t r i e d .
One c o n t a i n e d y e a s t e y t h a c t , p e p t o n e ,
s o l u b l e s t a r c h , so d iu m c h l o r i d e a n d a g a r .
fa c to rs
The o t h e r c o n t a i n e d . s i x g ro w th
( t h i a m i n e , p y r i d o x a l , r i b o f l a v i n , n i c o t i n a m i d e , c a l c i u m ;p a n t o t h e n a t e ,
a n d b i o t i n ) - c a sa m in o a c i d s , a d d i t i o n a l t r y p t o p h a n e a n d c y s t e i n e , s o l u b l e
/
—8—
S t a r c h 5 so d iu m c h l o r i d e a n d a g a r .
N o .a p p r e c i a b l e i n c r e a s e i n g ro w th r e ­
s u l t e d w hen a s u i t a b l e e n e r g y s o u r c e w as a d d e d t o t h e s e m e d ia .
I n th e c o u rs e o f t h i s p r e lim in a r y w o rk , a v a r i a n t o f
e n c o u n te r e d that!, g re w w e l l on t h e s e b a s a l m e d ia .
f e t u s w as
H ow ever5 s i n c e i t o b v i o u s l y
w as l e s s d e m a n d in g i n i t s c u l t u r a l r e q u i r e m e n t s th a n w as t h e p a r e n t s t r a i n ,
i t w as n o t u s e d f o r t h e r e m a in d e r o f t h e s t u d y .
A m edium c o n t a i n i n g a b o u t 0 .3 p e r c e n t d e h y d r a te d b r a i n l i v e r h e a r t a n d
a d d i t i o n a l so d iu m c h l o r i d e a n d a g a r w as f o u n d t o s u p p o r t a s l i g h t am ount o f
g r o w th .
No g ro w th w as a t t a i n e d u n l e s s so d iu m c h l o r i d e w as a d d e d t o th e m edium .
A p p a r e n tly t h e o r g a n is m r e q u i r e s a c e r t a i n , osmotiLsc p r e s s u re ': f&s*: g r o w th '
b e c a u s e t h e a d d i t i o n o f 3 p e r c e n t y e a s t a u t o l y s a t e o r h o r s e seru m o r 0 . 1
.
p e r c e n t c a sa m in o a c i d s w o u ld n o t r e p l a c e so d iu m c h l o r i d e .
The o r g a n is m s w e re grow n bn a g a r s l a n t s a t 35 C i n B re w e r a n a e r o b e j a r s
w i t h a b o u t o n e - h a l f . o f t h e a i r re m o v e d .
A m ark ed i n c r e a s e i n t h e am ount o f
g ro w th w as n o t e d when a s u i t a b l e e n e r g y s o u r c e w as a d d e d t o t h i s b a s a l m edium .
H ow ever5 a t t e m p t s t o m e a s u re t h e am ount <pf g ro w th a t t a i n e d on t h e a g a r s l a n t s '
w e re u n s u c c e s s f u l b e c aq .se i t w as d i f f i c u l t t o o b t a i n u n if o r m c e l l s u s p e n s i o n s .
A l s o , When w a s h in g t h e c e l l s o f f t h e a g a r s u r f a c e , a s m a l l am ount o f a g a r w qs
o f te n in tro d u c e d i n to th e c e l l s u s p e n s io n .
I t w as t h e r e f o r e n e c e s s a r y t o
f i n d a m eth o d w h e re b y q u a n t i t a t i v e m e a s u re m e n ts c o u ld b e m ade.
'
S e v e r a l u n s u c c e s s f u l a t t e m p t s w e re made t o d e v e lo p a b r o t h medium u s i n g
i
a s m a ll c o n c e n tr a tio n
( a b o u t 0 .3 p e r c e n t ) o f d e h y d r a t e d b r a i n l i v e r h e a r t
w i t h a d d i t i o n a l so d iu m c h l o r i d e .
u n l e s s a r e d u c i n g a g e n t w as a d d e d .
No g ro w th w as a t t a i n e d i n t h i s medium
A s l i g h t am ount o f .g r o w th w as a t t a i n e d
■=9~
w hen so d iu m t h i o g l y c o l l a t e o r so d iu m s u l f i d e w e re a d d e d t o t h e m edium .
How­
e v e r 5 n o g ro w th r e s u l t e d w hen sodium f o rm a ld e h y d e s u l f o x y l a t e w as a d d e d t o
t h e m edium .
B a s a l m edium .
A s a t i s f a c t o r y b a s a l medium was, d e v e lo p e d b y i n c r e a s i n g
t h e c o n c e n t r a t i o n o f d e h y d r a t e d b r a i n l i v e r h e a r t fro m a b o u t 0 .3 p e r c e n t t o
a b o u t 0 . 6 p e r c e n t a n d u s i n g a s m a ll am ount o f a g a r , p r e s u m a b ly t o m a i n ta i n
a r e d u c e d oxy g en t e n s i o n .
The b a s a l m edium u s e d i n t h e g ro w th s t u d i e s co n ­
t a i n e d 0 .5 7 p e r c e n t d e h y d r a t e d b r a i n l i v e r h e a r t m edium , 0 . 5 p e r c e n t
so d iu m c h l o r i d e , 0 . 1 p e r c e n t d is o d iu m p h o s p h a te a n d 0 .0 5 p e r c e n t a g a r*
The c o n c e n t r a t i o n o f t h e e n e r g y s o u r c e s w as 0 .2 p e r c e n t .
The pH o f t h e medium w as a d j u s t e d e l e c t r o m e t r i c a l l y t o 7 .0 a f t e r th e
a d d it io n o f th e e n e rg y s o u rc e s .
The p h o s p h a te b u f f e r w as a d d e d t o p r e v e n t
t h e -pH o f t h e medium fro m c h a n g in g d u r in g a u t o c l a v i n g .
The b u f f e r k e p t t h e
pH o f t h e m edium w i t h i n a r a n g e o f 6 .8 t o 7 .3 $ w h e re a s w i t h o u t t h e b u f f e r t h e
pH c h a n g e d fro m 7 .0 t o a b o u t 8 .0 when c e r t a i n e n e r g y s o u rc e is w e re a d d e d .
A p p a r e n tl y t h i s c h a n g e ini.p& X as d u e t o ^ h e p a r t i a l d e c o m p o s itio n o f some o f
t h e a d d e d e n e r g y s o u r c e s w hen h e a t e d .
f o r 15 f t i n u t e s ,
The medium! wa,s a u t o c l a v e d a t 121 G
,-
G lu c o s e , h e x o s e d i p h o s p h a t e , o x a l o a c e t a t e , o x a l o s u c c i n a t e , a n d i s o c i t r a t e
s o l u t i o n s w e re a d j u s t e d t o n e u t r a l i t y , s t e r i l i z e d b y f i l t r a t i o n , , an d t h e n
a d d e d t o t h e s t e r i l e b a s a l medium a s e p t i c a l l y .
The o x a l o a c e t a t e w as p r e p a r e d
b y t h e m eth o d d e s c r i b e d b y K ra m p itz an d W erkm an. ( l 9 i j .l ) . ' The r e m a in in g e n e r g y
s o u r c e s a n d a l s o g l u c o s e w e re a d d e d t o t h e medium a n d s t e r i l i z e d by a u to -r
c la v in g ,
A s e r i e s w i t h n o a d d i t i o n a l e n e r g y s o u r c e i n t h e b a s a l medium a n d
a s e r i e s w i t h l a c t a t e a d d e d t o th e b a s a l medium w e re i n c l u d e d i n e a c h
-IO-"
e x p e rim e n t.
E a c h compound w as r u n a t l e a s t i n t r i p l i c a t e .
T h is b a s a l medium s u p p o r t e d o n l y m in im a l g r o w th .
H ow evetj when' a s u i t - '
a b l e e n e r g y s o u r c e w as a d d e d a m arked i n c r e a s e i n gtOW th r e s u l t e d .
This,
i n c r e a s e w as much g r e a t e r t h a n a n y d i f f e r e n c e w h ic h ' o c c u r r e d i n r e p l i c a t e s ,
o f t h e same com pound.
The s m a ll am o u n t o f a g a r a d d e d t o t h e b a s a l medium d i d n o t - i n c r e a s e
t h e v i s c o s i t y o f t h e medium a p p r e c i a b l y .
s u s p e n s i o n t h e medium h a d t o b e s h a k e n .
I n o r d e r t o o b t a i n a u n if o r m c e l l
A lth o u g h t h i s s h a k in g c a u s e d t h e
f o r m a ti o n o f a i r b u b b l e s i n t h e m edium , t h e s e b u b b l e s so o n d i s a p p e a r e d , a n d
t h e am o u n t o f g ro w th c o u ld b e e s t i m a t e d t u r b i d i m e t r i c a l l y b y d e te r m i n in g
th e o p t i c a l d e n s it y o f th e s u s p e n s io n .
The q e e d c u l t u r e s f o r t h e g ro w th e x p e r im e n ts w e re grow n i n 1$Q m l p y r e x
m ilk d i l u t i o n b o t t l e s p lu g g e d w i t h 1 r u b b e r s t o p p e r s .
Each b o t t l e c o n ta i n e d
20 m l o f D i f c o 's b r a i n l i v e r h e a r t s e m i s o l i d medium an d w as i n c u b a t e d on i t s
s id e .
O n e - d a y -o ld c u l t u r e s o f s t r a i n 2035 w e re u s e d f o r in o c u lu m .
S tra in
IdUjD a p p e a r e d t o grow s lo w e r th a n d i d s t r a i n 2 0 3 5 , h e n c e , tw o - 4 a y - o M
c u l t u r e s w e re u s e d a s in o c u lu m .
H ow ever, i t w as l a t e r o b s e r v e d t h a t a f t e r
s e v e r a l r a p i d t r a n s f e r 's s t r a i n ItUijO a p p a r e n t l y c o u ld grow a t a b o u t t h e same
r a t e a s s t r a i n 2035«
E a c h b o t t l e r e c e i v e d 0 . 1 m l o f in o c u lu m .
The medium
w as i n o c u l a t e d w i t h i n a fe w h o u r s a f t e r a u t o c l a v i n g a s a p r e c a u t i o n a g a i n s t
o x i d a t i o n o f t h e medium b e f o r e g ro w th c o u ld b e . e s t a b l i s h e d .
The o r g a n is m s w e re grow n i n 25 m l o f t h e b a s a l medium a n d t h e a d d e d
■
e n e r g y s o u r c e s c o n ta i n e d i n m ilk d i l u t i o n - b o t t l e s .
-
T h ese b o t t l e s w e re
p lu g g e d w i t h r u b b e r s t o p p e r s a n d w ere i n c u b a t e d a t 35 C on t h e i r s i d e s .
'
V - : ;,:
V.
.
.
'
.
S t r a i n 2035 w as i n c u b a t e d tw o d a y s | s t r a i n UUUO w as i n c u b a t e d f i v e d a y s . ■
The am o u n t o f g ro w th w as m e a su re d w i t h a K lh tt-S u m m e rs o n p h o t o e l e c t r i c
c o lo r im e te r u s in g a b lu e f i l t e r .
E ach c u l t u r e w as e x a m in ed m i c r o s c o p i c a l l y
f o r c o n ta m in a tio n .
-
M a n o m e tric s t u d i e s .
A fe w manomet r i e
e x p e r im e n ts w e re c o n d u c te d i n
o r d e r t o com pare t h e am ount o f g ro w th a t t a i n e d w i t h v a r i o u s com pounds w i t h
t h e a b i l i t y o f t h e o rg a n is m t o o x i d i z e t h e s e com pounds.
The a c t i v i t y o f
r e s t i n g c e l l s u s p e n s i o n s w as d e te r m in e d b y c o n v e n t i o n a l m a n o m e tric m eth o d s
(U m b re it e t a l . , ± 9 h 7 )•
The c e l l s w e re grow n i n a b r o t h medium c o n t a i n i n g 1 .0 p e r c e n t t r y p t o s e
0.5> p e r c e n t so d iu m c h l o r i d e , 0 . 1 p e r c e n t y e a s t e x t r a c t , 0 . 1 p e r c e n t
s o lu b le s ta r c h .
The c u l t u r e s w e re i n c u b a t e d one d a y a t 35 0 .
I t w as f o u n d
t h a t a l a r g e s u r f a c e a r e a i n r e l a t i o n t o t h e am ount o f medium w as n e c e s s a r y
f o r t h e .o r g a n i s m s t o grow w e l l .
A p p a r e n tl y t h e am ount o f g ro w th i n t h i s
medium w a s 'l i m i t e d b y t h e am ount o f oxygen a v a i l a b l e . . H e n c e , th e d e p th o f ■
t h e b r o t h w as n o t m ore th a n a b o u t 0 .7 cm.
The am ount o f g ro w th a t t a i n e d i n
a l i t e r o f t h i s m edium p r o v id e d enough c e l l s f o r one e x p e r im e n t.
The s e e d c u l t u r e s w e re grow n i n 25 m l o f t h e
same medium c o n ta i n e d i n
125 m l e r le n m e y e r f l a s k s a n d w e re i n o c u l a t e d fro m one d a y - o l d c u l t u r e s .
The
c o n t e n t s o f one f l a s k s e r v e d a s in o c u lu m f o r a b o u t 350 m l o f m edium,
Th£ c e l l s w e re c e n t r i f u g e d a n d s u s p e n d e d i n M/50 d i p o ta s s iu m p h o s p h a te
b u f f e r , pH 7 . 0 .
The c e l l s w e re n o t w a sh e d b e c a u s e t h e e n d o g e n o u s r e s p i r a ­
t i o n o f t h e u n w ash ed c e l l s w as n o t g r e a t a n d w a s h in g a p p e a r e d t o r e n d e r t h e
c e lls le s s a c tiv e .
The o x y g en u p ta k e w i t h u n w ash ed c e l l s w as a b o u t 20 p e r
c e n t g r e a t e r t h a n w i t h c e l l s w ash ed i n I p e r " c e n t s o l u t i o n s o f y e a s t e x t r a c t
o r p e p to n e .
The c o n c e n t r a t i o n o f t h e c e l l s u s p e n s i o n s w as a d j u s t e d t u r b i d i -
”12m e tric a lly o
C e l l s u s p e n s i o n s o f a b o u t t h e same t u r b i d i t y w e re u s e d i n e a c h
e x p e rim e n t.
One m l o f c e l l s u s p e n s i o n w as u s e d i n e a c h v e s s e l j t h e t o t a l v e s s e l
c o n t e n t s w e re 2 .3 m l.
a t 37 C.
The oxygen u p ta k e w as m e a su re d i n an a i r a tm o s p h e re
The c a rb o n d i o x i d e w as a b s o r b e d b y a l k a l i ' i n t h e c e n t e r p e l l .
The c o n c e n t r a t i o n o f s u b s t r a t e i n t h e v e s s e l s w as 0 .0 0 3 M.
The s u b s t r a t e s
w e re d i s s o l v e d i n l / 3 0 p h o s p h a te b u f f e r a n d t h e pH w as a d j u s t e d t o 7 . 0 ,
The s i d e arm o f t h e e n d o g e n o u s v e s s e l c o n ta i n e d 0 . 3 m l o f M/30 p h o s p h a te
b u f f e r w h ic h w as a d d e d t o t h e v e s s e l a t t h e same tim e t h e s u b s t r a t e s w e re
added.
L a c t a t e w as u s e d a s a p o s i t i v e c o n t r o l i n e a c h e x p e r im e n t.
E x c e p t w i t h f u m a r a t e 5 t h e e x p e r im e n ts w e re t e r m i n a t e d a f t e r t h e r a t e
o f oxygen u p ta k e i n t h e v e s s e l s c o n t a i n i n g s u b s t r a t e becam e a b o u t t h e same
a s t h e r a t e o f oxygen u p t a k e i n t h e e n d o g e n o u s v e s s e l .
A ll d e te rm in a tio n s
w e re made a t l e a s t i n d u p l i c a t e .
RESULTS
G row th s t u d i e s .
The am o u n t o f g ro w th a t t a i n e d by tw o s t r a i n s o f
V. f e t u s w i t h v a r i o u s n o n - n i t r o g e n c o n t a i n i n g com pounds a n d am ino a c i d s
w as d e te r m in e d i n t h e g ro w th s t u d i e s .
The r e s u l t s sh o w in g t h e am ount o f g ro w th o b t a i n e d w i t h t h e n o n - n i t r o g e n
c o n t a i n i n g com pounds a r e su m m arized i n T a b le I .
L a c ta te 3 p y ru v a te 3 a lp h a -
k e t o g l u t a r a t e 3 S u c c i n a t e 3 f u m a r a te 3 a n d m a la te a l l s e r v e d a s e x c e l l e n t
e n e rg y s o u rc e s .
A c e t a t e s u p p o r t e d . e x c e l l e n t g ro w th o f s t r a i n UUhO b u t o n ly
s l i g h t g ro w th o f s t r a i n 2 0 3 3 .
G lu c o s e 3 w h e th e r s t e r i l i z e d by f i l t e r i n g
s u p p o r t a p p r e c i a b l e g r o w th .
o r a u to c la v in g 3 d id n o t
Of t h e c a r b o h y d r a te i n t e r m e d i a t e s 3 "■
-1 3 T able I
G row th o f V i b r i o f e t u s w i t h 0 .2 p e r 'c b n t o f t h e
v a r i o u s n o n - n i t r o g e n c o n t a i n i n g com ^o^nds a d d e d
t o t h e b a s a l m edium .
A v erag e t u r b i d i t y o f a t
l e a s t ,'3 d e t e r m i n a t i o n s . ,
Compound a d d e d
S t r a i n 2033
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N one*.
Gl u c o s e . . . . .
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G lu c o s e * * . . . . © »
H e x o s e d ip h o s p h a te * * .
G ly c e ro l . . . .
© e
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L a c ta te * . . . .
» »
P y r u v a te . . . .
o o
A c e ta te .
O x a lo a c e ta te * *
C itra te ,
Is o c itra te * *
O x a ld s u c e in a te * *
A lp h a - k e t o g I u t a r a t e
S u c c in a te .
F u m a ra te . . . .
M a la te . . . . .
T a rtra te . . . .
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* R e p r e s e n t s t h e a v e r a g e o f a t l e a s t 21 d e t e r m i n a t i o n s ,
* * Compound s t e r i l i z e d b y f i l t r a t i o n a n d a d d e d a s e p t i c a l l y t o t h e b a s a l
m edium .
Compounds n o t so m ark ed w e re s t e r i l i z e d b y a u t o c l a v i n g .
h e x o s e d i p h o s p h a te , g l y c e r o l a g l y c e r o p h o s p h a t e , c i t r a t e , i s o c i t r a t e a n d
o x a l o s u c c i n a t e d i d n o t s u p p o r t a p p r e c i a b l e g r o w th .
L ik e w ise , n o in c r e a s e d
g ro w th w as a t t a i n e d w i t h t a r t p a t e .
!
T a r t r a t e w as t r i e d a s a p o s s i b l e e n e r g y s o u r c e b e c a u s e o f t h e s im i­
l a r i t y b e tw e e n i t s m o le c u la r s t r u c t u r e a n d t h e s t r u c t u r e o f s u c c i n a t e ,
f u m a r a t e , arid m a l a t e .
Ih e i n a b i l i t y o f t h e o rg a n is m t o u t i l i z e
ta rtra te
m ig h t b e d u e t o t h e s p e c i f i c i t y o f o n e o r m ore enzym es i n v o l v e d .
M ost o f th e com pounds o f t h e K re b s c y c l e s e r v e d a s e x c e l l e n t e n e rg y
so u rc e s.
I t m ig h t p o s s i b l y b e t h a t c i t r a t e w as n o t u t i l i z e d b e c a u s e o f
t h e l a c k o f p e r m e a b i l i t y o f t h e c e l l m em brane f o r t h i s com pound.
th e p o o r u t i l i z a t i o n
L i k e w is e ,
o f o x a l o a c e t a t e an d o x a l o s u c c i n a t e may h a v e b e e n due
t o t h e l i m i t e d p e r m e a b i l i t y o f t h e c e l l mem brane f o r t h e s e com pounds.
No i n c r e a s e d g ro w th w as a t t a i n e d w i t h a n y o f th e com pounds o f t h e
g l y c o l y t i c schem e w h ic h w e re i n v e s t i g a t e d , e x c e p t p y r u v a t e .
T h is may i n ­
d i c a t e t h ,a t t h e o r g a n is m d o e p n o t h a v e t h e a b i l i t y t o r e s p i r e , a n a e r o b i ­
c a lly .
I f t h i s i s t r u e , t h e n i t may p o s s i b l y b e a n e x p l a n a t i o n o f t h e
n e e d f o r oxygen b y t h i s o r g a n is m .
,T^e r e s u l t s sh o w in g t h e am ount o f g ro w th o b t a i n e d when d i f f e r e n t am ino
a c i d s w e re a d d e d t o t h e b a s a l medium a r e su m m arized i n T a b le I I . ' A s p a r t a t e
a s p a r a g in e , .f u m a r a te , and p r o lin e
V. f e t u s .
s u p p o r te d g ro w th o f b o t h s t r a i n s o f
S e r i n e s u p p o r te d e x c e l l e n t g ro w th o f s t r a i n 2035 b u t s u p p o r te d
n o a p p r e c i a b l e g ro w th o f s t r a i n liiUlO.
A s p a r t a t e a n d a s p a r a g i n e w o u ld be
e x p e c te d t o be c o n v e rte d i n to o x a lo a c e ta te by d e a m in a tio n .
u t i l i z e d t h e s e l a t t e r com pounds b e t t e r t h a n d i d s t r a i n 2035«
S t r a i n )i)|)|0
The g r e a t e r
u t i l i z a t i o n o f t h e s e com pounds b y s t r a i n hLW may be due t o a m ore a c t i v e
•15'
. Table. I I
G row th o f V i b r i o f e t u s w i t h 0 .2 p e r c e n t o f t h e
v a r i o u s am ino a c i d s a d d e d t o t h e b a s a l m edium .
A v e ra g e t u r b i d i t y o f a t
l e a s t 3 d e te rm in a tio n s .
Compound a d d e d
None* . . . . . .
G ly c in e . . . . .
D L -A la n in e . . . .
1 - A l a n i n e I, . . .
D L -S e rin e . . . .
L -S e rin e .
.
.
D L - A s p a r ta t e . . .
L -A s p a r t a t e . . .
A s p a ra g in e . . » .
L -G lu ta m a te . . .
L - R r o lin e . . . .
L e u c in e . . . . .
L - Q r n it h i n e . . .
L -A rg in in e . . . . .
H is tid in e . . . .
D L -T h re o n x n e . . .
L - L y s in e . . . . .
D L - P h e h y la la n in e .
C y s te in e * * . . . .
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-SHS- Some o f t h e t u r b i d i t y p r o b a b l y due t o t h e f o r m a ti o n o f c y s t i n e i n t h e
m e d iu m .'
- 16 enzym e o r enzyme s y s te m i n t h i s s t r a i n t h a n i n s t r a i n 2 0 3 5 .
1
S t r a i n Itl^O u t i l i z e d g lu ta m a te a n d p r o l i n e b e t t e r t h a n d i d s t r a i n
2035«
B o th o f t h e s § com pounds w o u ld b e e x p e c te d t o b e c o n v e r t e d i n t o
a lp h a - k e t o g l u t a r a t e .
However 9 s t r a i n 2035 u t i l i z e d a l p h a - k e t o g l u t a r a t e
b e t t e r t h a n d i d s t r a i n WjltO«
.W ith t h e e x c e p t io n o f s e r i n e t h e am ino a c i d s w h ic h s u p p o r t e d a p p r e ­
c i a b l e g ro w th w e re e i t h e r t r a n s a m i n a t i n g am ino a c i d s o r com pounds w h ic h
m ig h t be e x p e c t e d t o fo rm t r a n s a m i n a t i n g am ino a c i d s i n t h e p r o c e s s o f d i s ­
s im ila tio n ,
O b v io u s ly t h e t r a n s a m i n a t i n g am ino a c i d s w ould b e e x p e c te d t o
b e m o st r e a d i l y i n t r o d u c e d i n t o t h e K re b s c y c l e ,
L - a s p a r t a t e s u p p o r te d b e t t e r g ro w th o f b o t h s t r a i n s t h a n d i d DL-
1
a s p a rta te .
More g ro w th o f s t r a i n 2035 w as a t t a i n e d w i t h L - s e r i n e t h a n w i t h
D L -s e rin e ,
H ow ever, o n ly s l i g h t g ro w th o f s t r a i n W4.I4.O r e s u l t e d w ith e i t h e r
L - s e r in e o r D L -s e rin e L
little
The c o n f i g u r a t i o n o f t h e a l a n i n e m o le c u le made .
d i f f e r e n c e i n t h e am ount o f g ro w th a t t a i n e d w i t h e i t h e r s t r a 'i n .
W it h 'e a c h am ino a c i d w itty whf.ch g ro w th yras a t t a i n e d w i t h t h e D L - c o h f ig u r a t i o n a n i n c r e a s e d am o u n t o f g ro w th w as a t t a i n e d w i t h t h e L - c o n f i g u r a t i o n «
A p o s s i b l e e x p l a n a t i o n f o r t h e m ore a b u n d a n t g ro w th w i t h t h e L -is o m e r m ig h t
b e t h a t t h e D-isomer h a d a b l o c k i n g e f f e c t on t h e enzym e i n v o l v e d .
A n o th e r
p o s s i b i l i t y i s t h a t o n ly t h e L - is o m e r w as u t i l i z e d and t h a t t h e i n c r e a s e d
g ro w th w as due t o a g r e a t e r s u p p ly o f t h e u t i l i z a b l e f o rm .
G l y c in e , a l a n i n e , t h r e o n i n e , p h e n y l a l a n i n e , h i s t i d i n e , l y s i n e , a r g i ­
n i n e , c y s t e i n e , a n d m e th io n in e w e re a l l u n a b le t o s u p p o r t a p p r e c i a b l e
g r o w th .
tytytyO,
S l i g h t g ro w th w as a f f o r d e d s t r a i n . 2.035 b y l e u c i n e b u t n o t s t r a i n
O r n i t h i n e s u p p o r te d o n ly s l i g h t g ro w th o f b o th s t r a i n s .
I t is
-1 7 p o s s i b l e t h a t p h e n y l a l a n i n e a n d m e th io n in e e n t i r e l y i n h i b i t e d g ro w th o f
s t r a i n if.lj.l|.0<>
W ith t h e e x c e p t i o n o f a l a n i n e <, n o n e o f t h e s e am ino a c i d s
w o p ld b e e x p e c te d t o b e e a s i l y i n t r o d u c e d i n t o th e K re b s c y c l e .
M a n o m e tric s t u d i e s .
r i z e d i n T a b le I I I .
The r e s u l t s o f t h e manomet r i e
s t u d i e s a r e summa­
In g e n e r a l th e s e r e s u l t s a re in l i n e w ith th e r e s u l t s
o b t a i n e d fro m t h e g ro w th e x p e r i m e n t s .
The a c t i v e o x i d a t i o n o f i s o c i t r a t e
a n d t h e s l i g h t o x i d a t i o n o f h e x o s e d i p h o s p h a te 5 h o w e v e r, g r e n o t i n 'a c c o r d
w i t h t h e r e s u l t s o f th e ' g ro w th s t u d i e s .
The l a r g e am ount o f oxygen u p ta k e w i t h i s o c i t r a t e i s d i f f i c u l t t o
u n d e r s t a n d i n v ie w o f t h e s l i g h t am o u n t o f g ro w th o b t a i n e d w i t h t h i s com"
pound.
1
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,
The s l i g h t o x i d a t i o n o f h e x o s e d ip h o s p h a te may i n d i c a t e t h a t t h i s com­
p o u n d c a n b e o x i d iz e d b y V. f e t u s .
On t h e o t h e r h a n d , t h e l a c k o f i n c r e a s e d
g ro w th w i t h t h i s compound a n d o t h e r com pounds i n t h e g l y c o l y t i c sch em e, a n d
t h e i n a b i l i t y o f t h e o rg a n is m t o o x i d i z e g lu c o s e a n d g l y c e r o p h o s p h a t e ,
w o u ld s#em t o b e r e a s o n n o t t o e x p e c t h e x o s e d ip h o s p h a te t o b e o x i d i z e d .
The o x y g e n u p ta k e w i t h h e x o s e d ip h o s p h a te m ig h t p o s s i b l y h a v e b e e n due t o
t h e o x i d a t i o n o f an i m p u r i t y i n t h e p r e p a r a t i o n a n d n o t d u e t o t h e o x id a ­
t i o n o f h e x o s e d i p h o s p h a te .
F i g u r e I show s t h e r a t e o f oxygen u p t a k e i n one e x p e r i m e n t .
A sh o rt
l a g a t f i r s t w as e v i d e n t w i t h l a c t a t e , f o ll o w e d by a r a p i d r a t e o f oxygen
u p ta k e .
The r a t e o f o x i d a t i o n t h e n g r a d u a l l y d i m i n is h e d , p r e s u m a b ly b e c a u s e
o f t h e l a c k o f s u b s t r a t e , u n t i l t h e r a t e o f o x y g e n u p ta k e becam e a b o u t th e
same a s i n t h e e n d o g e n o u s v e s s e l s .
I t i s e v i d e n t fro m f i g u r e I t h a t t h e
r a t e o f o x i d a t i o n o f f u m a r a te w as r e l a t i v e l y c o n s t a n t up t o t h e tim e t h a t
-1 8 T a b le I I I
O x i d a ti v e d i s s i m i l a t i o n o f e n e r g y s o u r c e s
b y s t r a i n 2035 o f V i b r io f e t u s .
S u b s tra te
(0.005M )
G lu c o s e . . . . .
H e x o s e d ip h o s p h a te
G ly c e r o p h o s p h a te .
L a c ta te * * * . . . .
p y ru v a te . . . . .
Is o c itra te . . . .
Fum arateiHC* . . .
Oxygen u p t a k e i n m i c r o l i t e r s *
(A v e ra g e o f a t l e a s t 2 d e t e r m i n a t i o n s )
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* E n d o g en o u s r e s p i r a t i o n h a s b e e n s u b t r a c t e d fro m t h e t o t a l o x y g e n u p t a k e »
* * R e p re s e n ts th e a v e ra g e o f 5 d e te r m in a tio n s .
* * * E x p e r im e n t t e r m i n a t e d b e f o r e o x i d a t i o n w as c o m p le te .
—19—
OXYGEN UPTAKE IN MICROLITERS
LACTATE
FUMARATE
ENDOGENOUS
MINUTES
F ig u re I .
R a te o f o x i d a t i o n o f l a c t a t e an d f u m a r a te b y s t r a i n 2035 o f
V i b r io f e t u s .
-2 0 t h e e x p e r im e n t w as t e r m i n a t e d .
Ih e o x i d a t i o n o f f u m a r a t e may b e l i m i t e d b y
t h e p e rn ie a b i l i t y o f t h e c e l l membrane f o r t h is ; com pound.
F u m a ra te w as o x i d i z e d a t a s lo w e r r a t e t h a n w e re l a c t a t e , p y r u v a t e ,
and i s o c i t r a t e ,
DISCUSSION
P r e v io u s t o t h i s s t u d y t h e m ed ia u s e d f o r t h e c u l t i v a t i o n o f t h i s o r g ­
a n is m h a v e b e e n e x tr e m e l y c o m p le x .
As a r e s u l t , i s o l a t i o n h a s b e e n d i f f i ­
c u l t b e c a u s e , c o n ta m in a n ts o f t e n o v e rg re w V. f e t u s .
The i n f o r m a t i o n
o b t a i n e d i n t h i s s tu d y m ig h t b e u s e d i n d e v e lo p i n g s p e c i a l m ed ia f o r t h e
i s o l a t i o n o f V. f e t u s .
L a c t a t e , f o r i n s t a n c e , -prould p r o b a b l y s e r v e a s a
s u i t a b l e e n e r g y s o u r c e i n su c h m ed ia f o r i t c a n b e r e a d i l y u t i l i z e d a n d
c a n b e a u t o c l a v e d w i t h o u t d e c o m p o s in g .
I n t h e p a s t , t h e r e h a s n o t been- e n o u g h known t o i d e n t i f y V. f e t u s b y
its
c u ltu ra l c h a ra c te r is tic s .
The i n a b i l i t y o f V. f e t u s t o e i t h e r o x i d iz e
o r f e r m e n t g l u c o s e w o u ld a p p e a r t o b e a n e x c e l l e n t c r i t e r i o n f o r d i s t i n - '
g u i s h i n g t h i s o r g a n is m fro m m o st o t h e r v i b r i o s .
The r e s u l t s o f t h i s s t u d y i n d i c a t e t h a t t h i s o rg a n is m c a n n o t u t i l i z e
a n y o f t h e com pounds i n t h e g l y c o l y t i c schem e e x c e p t p y r u v a t e .
From w h a t
i s known o n ly com pounds a s s o c i a t e d w i t h a e r o b i c m e ta b o lis m ' c a n b e r e a d i l y
u tiliz e d .
I t a p p e a r s t h a t t h i s o rg a n is m c a n o n ly o b t a i n e n e r g y fro m com­
p o u n d s i n t h e K re b s c y c le o r com pounds w h ic h c a n b e e a s i l y i n t r o d u c e d
i n t o t h e K re b s c y c l e .
I n many r e s p e c t s V. f e t u s a n d B r u c e l l a a b o r t u s a p p e a r t o r e s e m b le
each o th e r .
Th^ s i m i l a r i t y o f t h e s e o r g a n is m s a p p e a r s t o e x te n d bey o n d
t h e i r p a th o g e n i c e f f e c t i n c a t t l e .
B o th o r g a n is m s seem t o h a v e s t r i c t
-2 1 a tm o s p h e r i c r e q u i r e m e n t s , a r e r e l a t i v e l y d i f f i c u l t t o c u l t i v a t e , a n d
a p p a r e n t l y r e q u i r e 6 c e r t a i n o s m o tic p r e s s u r e f o r g r o w th ,
In a d d itio n
t o t h e s e s i m i l a r i t i e s t h e r e a p p e a r s t o b e a m ark ed r e s e m b la n c e b e tw e e n
t h e a b i l i t y o f B, a b o r t u s t o o x i d i z e com pounds i n t h e K re b s c y c le and
am ino a c i d s . a n d t h e u t i l i z a t i o n
o f t h e s e -compounds, b y V. f e t u s .
B.
' a b o r t u s h a s b e e n f o u n d t o o x i d i z e m o st o f t h e com pounds i n t h e K re b s c y c le
{G e r h a r d t , L e v i n e , a n d W ils o n , 1 9 5 0 ) I V, f e t u s w as a f f o r d e d e x c e l l e n t
g ro w th b y m o st o f t h e s e com pounds.
A s p a ra g in e , g lu ta m a te , and p r o lin e
w e re o x i d i z e d b y B, a b o r t u s ; t h e s e com pounds s u p p o r te d c o n s i d e r a b l e g ro w th
o f V, f e t u s ,.
T h is r e s e m b la n c e w as n o t a b s o l u t e , h o w e v e r, f o r a l a n i n e w as
o x id iz e d , b y B, a b o r t u s b u t w as n o t u t i l i z e d b y V. f e t u s .
M o re o v e r, s e r i n e
w as n o t o x i d i z e d a t an a p p r e c i a b l e e x t e n t b y B. a b o r t u s b u t s u p p o r te d
g ro w th o f one s t r a i n o f V. f e t u s .
A s p a rta te , a rg in in e , o r n ith in e , h i s t i ­
d i n e ,' l e u c i n e , l y s i n e , p h e n y l a l a n i n e , m e t h io n i n e , a n d t h r e o n i n e w e re n o t
o x i d i z e d a t a n a p p r e c i a b l e r a t e by B. a b o r t u s .
W ith t h e e x c e p t io n o f
a s p a r t a t e , n o n e o f t h e s e am ino a c i d s s u p p o r te d a p p r e c i a b l e g ro w th o f
V. f e t u s .
■SUMMARY
The u t i l i z a t i o n
been in v e s tig a te d .
o f e n e r g y s o u r c e s f o r t h e g ro w th o f V i b r i o f e t u s h a s .
I t a p p e a r s t h a t o n ly com pounds i n t h e K re b s c y c le o r
com pounds w h ic h c a n e a s i l y b e i n t r o d u c e d i n t o t h e K reb s c y c l e w i l l s e r v e
a s s u i t a b l e e n e r g y s o u r c e s f o r t h i s o r g a n is m .
.
L a c ta te , p y ru v a te , a c e t a te , o x a lo a c e ta te , a lp h a -k e to g lu ta r a te , suc­
c i n a t e , f u m a r a t e , m a l a t e , a s p a r t a t e , a s p a r a g i n e , g lu ta m a te a n d p r .o lin e a l l
s e rv e d a s s u it a b le e n e rg y s o u rc e s u n d e r th e c o n d itio n s o f th e s e
. =22™'
e x p e rim e n ts .
G lu c o s e , h e x o s e d i p h o s p h a te , g l y c e r o l , g l y c e r o p h o s p h a t e ,
c i t r a t e , i s o c i t r a t e , o x a lo s u c c in a te , t a r t r a t e , g ly c in e , a la n in e , th re o n in e ,
c y s t e i n e , m e t h io n i n e , h i s t i d i n e , p h e n y l a l a n i n e , l e u c i n e , l y s i n e , o r n i t h i n e
a n d a r g i n i n e d i d n o t s u p p o r t a p p r e c i a b l e g r o w th .
B e s t i n g c e l l s u s p e n s i o n s o x i d i z e d l a c t a t e , p y r u v a t e , i s o c i t r a t e , an d
fu m a ra te .
H e x o s e d ip h o s p h a te may h a v e been, o x i d i z e d s l i g h t l y .
g l y c e r o p h o s p h a t e w e re n o t o x i d i z e d .
G lu c o se an d
-2 3 REFERENCES
B a r g e r , E-. He 1928 R p p o r t o f a c a s e o f a b o r t i o n in d u c e d b y V i b r i o f e t u s .
J . Am. V e t. -Med. A s s n . , 2 £ , 1^6S-ItTlt.
B a t l l n ? A l e x a n d e r , a n d W ils o n , J . B. 1950 N u t r i t i o n a n d a n t i g e n
p r o d u c t i o n o f V i b r i o f e t u s . B a c t . P r o c , , I tl ( A b s t r a c t ) .
G e r h a r d t , P h i l i p p , L e v i n e , H. B . , a n d W ils o n , J . B. 1950 The' o x i d a t i v e
d i s s i m i l a t i o n o f am ino a c i d s a n d r e l a t e d com pounds b y B r u c e l l a
a b o rtu s .
J . B a c t . , 6 0 , It5 9 -lt6 7 .
K r a m p itz , L . 0 . , a n d W erkman, C. H. 1 9 ltl
a c i d . B io ch em .' J . , 3 5 , 595«
P re p a ra tio n of o x a lo a c e te tic
• May, L . K. .1 9 5 3 The m i c r o a e r o p h i l i c n a t u r e o f V i b r io f e t u s .
T h e s i s . M ontana S t a t e C o l l e g e , B ozem an, M o n tan a.
M a s t e r 's
P l a s t r i d g e , W. N . , a n d W il li a m s , L . F , 19lt3 O b s e r v a tio n s on V i b r io f o e t u s
in f e c tio n in c a t t l e .
J . Am. V e t. Med. A s s n ., 1 0 2 , 89-95>.
P l a s t r i d g e , W. N ., W il li a m s , L . F . , E a s t e r b r o o k s , H. L . , W a lk e r, E . C ., a n d
B e c c i a , R . N. 1951 V i b r i o s i s i n c a t t l e . . B u l l e t i n 2 8 l . , S t o r r s
A g r i c u l t u r a l E x p e r im e n t S t a t i o n , C o lle g e o f A g r i c u l t u r e ,
U n i v e r s i t y o f C o n n e c t i c u t , S t o r r s , C o n n e c t ic u t.
S m ith , T0
1 9 1 8 . S p i r i l l a a s s o c i a t e d w i t h d i s e a s e o f t h e f e t a l m em branes
in c a t t l e ( I n f e c tio u s A b o rtio n ).
J . E x p t l . M e d ., 2 8 , 7 0 1 -7 1 9 .
S m ith , T . , a n d T a y l o r , M. S . 1919 Some m o r p h o lo g ic a l a n d b i o l o g i c a l
c h a r a c t e r s o f t h e ' s p i r i l l a ( V i b r io f e t u s N. s p . ) a s s o c i a t e d
w i t h d i s e a s e o f t h e f e t a l m em branes i n c a t t l e .
J . E x p t l . M ed .,
3 0 , 299 - 312 .
S to c k m a n , S . 1919
U 99-50li.
V ib rio n ic a b o r tio n .
J . Am. V e t. Med. A s s n . , 8 ,
Umbrei t , W. W ., B u r r i s , R . H ., a n d S t a u f f e r , ■J . F . 19U7 M an o m etric
t e c h n i q u e s a n d r e l a t e d m eth o d s f o r t h e s tu d y o f t i s s u e
m e ta b o lis m . B urge1s s P u b l i s h i n g Co.-, M in n e a p o lis .
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