A comparative study of enterotoxic and non-enterotoxic micrococci
by David R Morledge
A THESIS Submitted to the Graduate Committee in partial fulfillment of the requirements for the
degree of Master of Science in Bacteriology
Montana State University
© Copyright by David R Morledge (1949)
Abstract:
Eighteen strains of micrococci, ten of which had "been isolated from cases of food-ooiscning, were
studied as to their physiological reactions and the ability of their boiled filtrates to invoke
entero-toxlcity in cats uoon Intravenous injection.
All but one of the strains gave the same physiological reactions and morphological characteristics listed
for Micrococcus pyogenes var. aureus in the sixth edition of Sergey’s Manual, except that none of them
was observed to produce hydrogen sulfide.
Incubation at 30°C brought about a more rapid liquefaction of gelatin by the strains than incubation at
37° C. The length of time the tubes were cooled after each incubation Influenced the observable
liquefaction in some of the tubes.
Three of the strains were found enterotoxic to cats. In addition to having all the physiological
characteristics of Micrococcus pyogenes van aureus, these three strains were coagulytic, hemolytic,
fibrinolytic, and gave a positive Stone reaction in 48 hours. Pour strains not enterotoxic to cats gave
these same reactions. No visible serological reaction was observed between homologous enterotoxin
and rabbit serum.
The results of these experiments lead one to the same conclusion reached by most of the other workers
on food-poisoning micrococci, namely, that at present there is no physiological characteristic
successful in separating enterotoxic from non-enterotoxic micrococci. A COMPARATIVE STUDY O i
EWTEROTOXIC AED EOR-EETEROTOXIC MICROCOCCI
by
D av id R. M o rle d c e
A THESIS
S u b m itte d t o t h e G ra d u a te C om m ittee
in
p a r t i a l f u l f i l l m e n t o f th e r e q u ir e m e n ts
f o r th e d e g re e o f
M a s te r o f S c ie n c e i n B a c t e r i o l o g y
at
M ontana S t a t e C o lle g e
Auuro v e d :
Chairman, E x a m in in g C om m ittee
Bozem an, M ontana
J u n e 19^9
]\j 3 I *
(S-I)Ij- ^
ACnrOVLSDOMBHT
The a u t h o r w is h e s to ac k n o w led g e th e f i n e p e r s o n a l
I n t e r e s t a n d s u p e r v i s i o n o f Dean F . B. C o tn e r and
M r. W. G. W a lte r a n d t h e e n c o u ra g e m e n t o f D r. B. L . J o h n s o n
tlir o u g h o u t t h i s s tu d y .
9254^1
TABLE OF CONTENTS
P ag e
ABSTRACT ...........................................................................................
2
INTRODUCTION ..................................................................................
3
HISTORICAL BACKGROUND...............................................................
3
PHYSIOLOGICAL STUDY
...............................................................
8
M ethods and M a t e r i a l s ................................................
8
R e s u l t s .............................................................................*
12
BIOLOGICAL STUDY ........................................................................
19
M ethods an d M a t e r i a l s ................................................
19
R e s u l t s ....................................................................................... 21
SEROLOGICAL STUDY..............................................................................23
M ethods and M a t e r i a l s ......................................................24
R e s u l t s ........................................................................................25
GENERAL DISCUSSIO N......................................................................... 2$
SUMMARY......................................................................................................31
LITERATURE CITED AND CONSULTED............................................ 3 4
-2<
ABSTRACT
E ig h te e n s t r a i n s o f m ic r o c o c c i, t e n o f w hich had "been i s o l a t e d
fro m c a s e s o f f o o d - n o l s o n i n g , w e re s t u d i e d a s t o t h e i r p h y s i o l o g i c a l
r e a c t i o n s an d t h e a b i l i t y o f t h e i r b o i l e d f i l t r a t e s t o in v o k e e n t e r o t o x l c l t y in c a ts uoon in tra v e n o u s i n j e c t i o n .
A l l b u t one o f t h e s t r a i n s g a v e t h e same p h y s i o l o g i c a l r e a c t i o n s
a n d m o r p h o lo g ic a l c h a r a c t e r i s t i c s l i s t e d f o r M ic ro c o cc u s -pyogenes v a n .
a u r e u s i n t h e s i x t h e d i t i o n o f S e r g e y ’ s M a n u al, e x c e p t t h a t none o f
them was o b s e r v e d t o p ro d u c e h y d ro g e n s u l f i d e .
I n c u b a t i o n a t JO0 C b r o u g h t a b o u t a m ore r a p i d l i q u e f a c t i o n o f
g e l a t i n by t h e s t r a i n s t h a n i n c u b a t i o n a t 3 7 ° C.
The l e n g t h o f tim e
t h e t u b e s w e re c o o le d a f t e r e a c h i n c u b a t i o n i n f l u e n c e d t h e o b s e r v a b l e
l i q u e f a c t i o n i n some o f t h e t u b e s .
T h re e o f t h e s t r a i n s w e re f o u n d e n t e r o t o x i c t o c a t s .
In a d d itio n
t o h a v in g a l l t h e p h y s i o l o g i c a l c h a r a c t e r i s t i c s o f M ic ro c o c c u s p y o g e n e s
v a n a u r e u s , t h e s e t h r e e s t r a i n s w ere c o a g u l y t i c , h e m o l y ti c , f i b r i n o l y t i c ,
a n d g a v e a p o s i t i v e S to n e r e a c t i o n i n 48 h o u r s .
e n t e r o t o x i c t o c a t s g av e t h e s e same r e a c t i o n s .
Four s tr a in s n o t
No v i s i b l e s e r o l o g i c a l
r e a c t i o n was o b s e r v e d b e tw e e n hom ologous e n t e r o t o x i n a n d r a b b i t seru m .
The r e s u l t s o f t h e s e e x p e r im e n ts l e a d one t o t h e sam e c o n c lu s io n
r e a c h e d by m ost o f t h e o t h e r w o rk e rs on f o o d - p o i s o n in g m ic r o c o c c i, n a m e ly ,
t h a t a t p r e s e n t t h e r e i s no p h y s i o l o g i c a l c h a r a c t e r i s t i c s u c c e s s f u l i n
s e p a r a t i n g e n t e r o t o x i c fro m n o n - e n t e r o t o x i c m ic r o c o c c i.
-3 INTEODUCTIOB
E ig h te e n s t r a i n s o f m ic r o c o c c i, some o f w h ich h a d b e e n i s o l a t e d fro m
c a s e s o f f o o d - p o i s o n i n g , w e re s t u d i e d .
An a t t e m p t was made t o d e te r m in e
w h e th e r o r n o t t h o s e m ic r o c o c c i fo u n d t o e l a b o r a t e e n t e r o t o x i n c o u ld be
d i f f e r e n t i a t e d fro m o n e s fo u n d t o be n o n - e n t e r o t o x i g e n i c b y t h e i r s e r o ­
l o g i c a l a n d p h y s i o l o g i c a l p r o p e r t i e s a n d t h u s be p l a c e d i n a c a te g o r y
by t h e m s e lv e s .
HISTORICAL BACKGROUND
Food p o i s o n i n g by m ic r o c o c c i h a s come t o be r e c o g n i s e d a n d common­
l y a c c e p t e d a s s u c h o n ly w i t h i n th e l a s t tw e n ty y e a r s .
M ic ro c o c c u s fo o d
p o i s o n i n g i s p r o d u c e d b y an e n t e r o t o x i n fo rm e d by o r g a n is m s w h ic h w ere
f o r m e r l y c l a s s i f i e d a s S ta p h y lo c o c c u s a u r e u s a n d Si a l b u s (D o lo a n and
W ils o n , 1938) b u t a r e now c l a s s i f i e d a s M ic ro c o c c u s p y o g e n e s v a r . a u re u s
a n d Mi p y o g e n e s v a r . a l b u s i n t h e s i x t h e d i t i o n o f " B e r g e y 's M anual o f
D e t e r m in a tiv e B a c t e r i o l o g y " ( 1 9 4 8 ) .
S in c e i t s
common r e c o g n i t i o n , a l a r g e
num ber o f o u t b r e a k s o f m ic ro c o c c u s f o o d p o i s o n i n g h a v e b e e n r e p o r t e d i n
c u rre n t s c ie n tif ic lite r a tu r e .
A c c o rd in g t o Dack ( 1 9 4 3 ) , i t
i s p r o b a b ly
t h e m ost common o f a l l t y p e s o f f o o d p o i s o n i n g , an d t h e num ber o f c a s e s
c i t e d i s b u t a s m a ll p r o p o r tio n o f th e a tt a c k s o c c u rr in g .
T h is i s
p r o b a b l y b e c a u s e i t f o ll o w s a r a p i d an d n o n - f a t a l c o u r s e , u s u a l l y among
s u s c e p t i b l e mem bers o f a s i n g l e f a m i l y , a n d i s p a s s e d o f f a s a " s l i g h t
i n te s ti n a l d is tu r b a n c e ."
As t h i s s i c k n e s s i s o f s h o r t d u r a t i o n , i s
n o n - f a t a l , a n d a f f e c t s m ost i n d i v i d u a l s a t v a r i o u s tim e s d u r i n g t h e i r
liv e s ,
i t i s n o t g iv e n a t t e n t i o n and i s so o n f o r g o tte n .
I t i s when l a r g e
o u f b r e s k e o c c u r i n s c o m m n lty t h a t t h e c a u s a t i v e a g e n t i s t r a c k e d down
and th e c a se r e p o r te d .
The eynotO B a o f m ic r o c o c c u s fo o d p o i s o n i n g g e n e r a l l y a p p e a r i n
tw o t o f o u r h o u r s a f t e r th e i n g e s t i o n o f f o o d c o n t a i n i n g a l a r g e num ber
o f e n t e r o t o x i n - p r o d u c i n g m ic r o c o c c i ( J o r d a n a n d B u rro u g h s , 1 9 ^ 5 )•
U s u a l l y , C r e a a w f l l l e d b a k e r y g o o d s h ig h i n s t a r c h c o n t e n t a r e t h e r e s p o n ­
s i b l e I te m s , a l t h o u g h many o t h e r f o o d s s u c h a# c h e e s e , m il k , c h ic k e n
g r a v y , ham, to n g u e , c h ip p e d b e e f , f i s h s a u s a g e , and l i v e r s a u s a g e h a v e
b e e n fo u n d t o h a v e c a r r i e d t h e c o n ta m in a t in g o rg a n is m s i n t h e r e p o r t e d
eases.
I n s e v e r a l i n s t a n c e s , m il k fro m cows s u f f e r i n g , o r h a v in g
s u f f e r e d , w i t h m a s t i t i s h a s b e e n p r o v e d t o b e t h e f o o d in v o lv e d
( S h a u g h n e e e y and O ru b b , 1936;
M in e tt, 1 9 3 8 ).
H ow ever, D aek ( 19U3 )
p o i n t s o u t t h a t t h e m eet common mode o f I n f e c t i o n h a s r e s u l t e d fro m th e
h a n d l i n g o f t h e f o o d s by I n d i v i d u a l # i n f e c t e d w i t h m ic r o c o c c i a n d t h e
s u b s e q u e n t s t o r a g e o f t h e s e f e e d s u n d e r c o n d i t i o n s f a v o r a b l e f o r th e
g ro w th o f t h e e n t e r o t o x i n - o r o d u e i n g o r g a n is m s .
The tim e i n t e r v a l b e tw e e n
t h e i n g e s t i o n o f t h e c o n ta m in a te d f o o d a n d t h e a p p e a r a n c e o f th e f i r s t
symptom 1# i n f l u e n c e d b y t h e am ount o f t h e e n t e r o t o x i n consum ed a n d t h e
e u e c e o tib ility o f th e in d iv id u a l.
The f i r s t symptom i s t h a t o f s a l i v a ­
t i o n , a n d i s f o llo w e d b y n a u s e a , r e t c h i n g , a b d o m in a l c ra m p in g e f v a r y i n g
s e v e r i t y , and d ia r r h e a .
m ild e r c a s e s .
Some o f t h e s e sym ptom s may n o t b e p r e s e n t i n
I n s e v e r e c a s e s b lo o d and m ucus may be o b s e r v e d i n th e
• t o o l s and th e v o a it u e .
I n t h e l a t t e r came# t h e t e m p e r a t u r e o f t h e
i n d i v i d u a l may b e h i g h e r o r lo w e r t h a n t h e norm , a n d h i e b l o o d p r e s s u r e
-5 may l i k e w i s e v a r y .
R e c o v e ry u s u a l l y t a k e s p l a c e i n fro m one t o t h r e e
d a y s , a lt h o u g h so m e tim e s i t i s a s lo n g a s one o r two w eeks u n t i l t o t a l
re c o v e ry i s o b ta in e d .
T h is v a r i e s w ith t h e s u s c e p t i b i l i t y a n d p h y s i c a l
f i t n e s s o f t h e p e r s o n a t t a c k e d , a n d t h e am ount o f e n t e r o t o x i n I n g e s t e d
(D a c k , 19U3 ) .
The c a u s a t i v e a g e n t o f m ic ro c o c c u s f o o d p o i s o n i n g w as " d is c o v e r e d ”
f o u r tim e s b e f o r e i t was commonly r e c o g n i z e d a s s u c h .
T h e se f o u r
d i s c o v e r i e s w e re b y Denys i n 1 8 9 4 , by R. V. G. Owen i n 1 9 0 6 , by M. A.
B a r b e r i n 1 9 1 4 , a n d by D ack, C a r r y , W o o lp e r t, a n d W iggere i n 1 9 3 0 .
T h e se r e s u l t e d i n o u t b r e a k s o c c u r r i n g i n B e lg iu m ; K alam azoo, M ic h ig a n ;
t h e P h i l i p p i n e I s l a n d s ; an d C h ic a g o , r e s p e c t i v e l y .
The c o n ta m in a te d
f o o d w as f o u n d t o b e f r e s h b e e f , d r i e d b e e f , m ilk fro m a cow w h ic h h a d
m a s t i t i s , a n d a t h r e e - l a y e r sp o n g e c a k e .
S in c e 1930 a l a r g e num ber o f
s i m i l a r o u t b r e a k s h a v e "been r e p o r t e d i n t h e l i t e r a t u r e .
The d i a g n o s i s o f m ic r o c o c c u s f o o d p o i s o n i n g i s u s u a l l y r e a c h e d
b y f i r r - t f e e d i n g th e s u s p e c t e d f o o d t o human v o l u n t e e r s t o t e s t i t s
t o x i c i t y , h y e x a m in in g i t f o r l a r g e num bers o f m ic r o c o c c i, a n d by
c u l t u r i n g t h e p r e d o m in a n t o rg a n is m s a n d t h e n f e e d i n g t h e c u l t u r e f i l t r a t e s
t o human v o l u n t e e r s .
The a p p e a r a n c e o f c l i n i c a l symptoms i n d i c a t i v e o f
m ic ro c o c c u s f o o d p o i s o n i n g i s
(D ack jet £ l ,
c a r e f u ll y o b se rv e d .
The u s e o f m onkeys
1 9 3 0 ) e n d k i t t e n s (D olm an a n d W ils o n , 1938) i n p l a c e o f
hum an v o l u n t e e r s h a s b e e n g i v e n a g r e a t d e a l o f a t t e n t i o n .
t h e i r d is a d v a n ta g e s :
B o th have
t h e m onkeys a r e e x p e n s iv e t o k e e p a n d u s e , a n d th e
k i t t e n s a re d i f f i c u l t to o b ta in th e y e a r a ro u n d .
Hammon ( l 9 4 l )
in je c te d
b o t h k i t t e n s a n d c a t s I n t r a v e n o u s l y w i t h e n t e r o t o x i n a n d f o u n d t h a t by
-6 -
h ie method I e e e emounte were need ed .
I e c a te were found a s s u s c e p t ib le
as k lt t e n e and a re e a s i l y o b ta in e d , th e problem o f p ro cu rin g and k eep in g
n e c e ssa r y an im als was c o n s id e r a b ly le s s e n e d .
However, as Hamaon p o in ted
o u t, k it t e n s end c a t s lo s e t h e i r s u s c e p t i b i l i t y to th e e n t e r o t o x ln a f t e r
b a v in
had fo u r o r f i v e a tta c k s o f th e d is e a s e .
Other ex p erim en ta l
a n im a ls. In c lu d in g th e dog (Jordan and Burroughs, 1933i M in ett, 1938;
Higdon, 1 938) and ro d en ts (Jordan and McBroom1 1 9 3 1 ; C orpening end
F e x h a ll, 1935) have been t r i e d , but w ith o u t a comparable amount o f
su ccess.
Hopkins and Poland (B ack, 19%3) r e p o r te d th a t young p ig s are
good t e s t a n im a ls, but o th e r workers have n o t u sed them in o rd er to
con firm t h i s .
There have been numerous a ttem p ts to e f f e c t a la b o r a to r y d ia g n o s is
by fin d in g some c o n s is t e n t c h a r a c t e r is t ic s e t t i n g th e s e organism s apart
from th e r e s t o f th e m icr o co cci group.
S ton e (1 9 3 5 ) d ev elo p ed a c u lt u r a l
method f o r th e c l a s s i f i c a t i o n o f fo o d -p o is o n in g m icr o co cci by t h e ir
p ro d u ctio n o f li q u e f a c t io n in h i s medium c o n ta in in g g e l a t i n when in cu b ated
a t 37° C f o r 24 h o u rs.
Any d eg ree o f li q u e f a c t io n was c o n sid ered as
in d ic a t in g a fo o d -p o is o n in g s t r a in ; th e co n v erse b ein g tr u e i f no
li q u e f a c t io n o ccu rr ed .
Chin (1 9 3 6 ) and KPpchik (1937) c o u ld not c o r r e la te
t h e i r f in d in g s w ith th o se o f S ton e; whereas Chapman e t a l (1 9 3 7 ) found
tiv ;t th e Stone r e a c t io n ( g e l a t i n li q u e f a c t io n ) was p o s i t i v e i n 75*5 P®*
c e n t o f t y p ic a l fo o d p o is o n in g m icro co cci and p o s it iv e i n 2 7 .6 p er cen t
o f s t r a in s w ith s im ila r p r o p e r tie s but i s o l a t e d from n o n -fo o d -p o iso n in g
so u r c e s
-7 -
Chapman * t a l (19?4) found th a t a h ig h c o r r e l a t i o n e x is t# betw een
th e m ic ro c o c c i'# a b i l i t y to produce c o ag u la se and hem olysin and t h e i r
p a th o g e n ic ity .
C rulckehank (1937) su rm o rted th e r e l a t i o n s h i p between
p a th o g e n ic ity and p ro d u c tio n o f c o a g u la se , w hereas S t r a l t e r and Jo rd a n
(1935) f QTiud t h a t fo o d -p o iso n in g s t r a i n s d id not c o n s ti tu te a c l e a r l y
marked d iv is io n ^ 'ith any hom ogeneity In biochem ical* h e m o ly tic , o r
a g g lu tin a tiv e c h a r a c te r s .
Kupchik (1937) went a s te p f a r t h e r and
found in h is e x p erim en ts t h a t th e r e was no d i s t i n c t i v e r e a c tio n w hich
would s e p a ra te e n te r o to x lc from n o n -en t e r o t o x ic - p r oducing s t r a i n s by
pigm ent p ro d u c tio n , a c tio n on m ilk , l iq u e f a c t io n o f n u t r i e n t g e l a t i n ,
a s w e ll ae th e b io c h e m ic a l, a g g lu tin a tiv e , and o o a g u la tiv e c h a r a c te r s
s tu d ie d by C rulokshank, and S t r a l t e r and J o rd a n .
Dolman and W ilson
(1 9 3 8 ), Haynes and Bucker ( I 9W)) and Bueseman and Tanner (19^9) were
a le o o f th e o p in io n t h a t any c u l t u r a l c h a r a c t e r i s t i c I s u n s a t i s f a c t o r y
f o r d ia g n o s is .
Thygeson (1938) b e lie v e d t h a t th e m a n n ito l fe rm e n ta tio n
t e s t i s a r e l i a b l e I n d ic a to r o f th e to x ig e n ic powers o f m ic ro c o c c i.
T o x ig en ic ones are p o s i t i v e f o r th e most p a r t ; n o n -to x lg e n lc ones a re
n e g a tiv e .
Ohapman (1938) has rem ained c o n fid e n t th ro u g h o u t h is s tu d ie s
t h a t th e fo o d -p o iso n in g m icro co cci can be s e p a ra te d as a d i s t i n c t group
by d i f f e r e n t i a l c h a r a c t e r i s t i c s .
Thus, a c c o rd in g to him , th e y p o sses#
a l l o r a m a jo rity o f th e fo llo w in g c h a r a c t e r i s t i c s :
( I ) p ig m e n ta tio n ,
(2 ) c o a g u ly tlc , ( 9 ) g e la t in o l y t i c , (4 ) f i b r i n o l y t i c , ($ ) la c to s e
fe rm e n ta tiv e , (6 ) m a n n ito l fe rm e n ta tiv e , end (7 ) h e m o ly tic pow ers.
Through th e s e , he b e lie v e s t h a t th e organism can be th o ro u g h ly d iag n o sed
c u l t u r a l l y a n d h a s d e s ig n e d s e v e r a l c u l t u r e m e d ia f o r th e d i f f e r e n t i a t i o n
o f t h e f o o d o o l e o n l n g m ic r o c o c c i.
Dolman an d V lle o n (1 9 3 8 ) fo u n d t h a t e n t e r o t e x l n I s a s p e c i f i c
a n tig e n .
K i t t e n s g iv e n a s e r i e s o f i n j e c t i o n s o f f i l t r a t e s
e n t e r o t o x l n a c q u i r e a c t i v e im m u n ity .
c o n ta in in g
Immune r a b b i t e n d h o r s e serum
c o n f e r a a s s i v e p r o t e c t i o n a g a i n s t t h e e n t e r o t o x l n upon n o rm a l k i t t e n s .
By a r e c i p r o c a l a b s o r p t i o n m e th o d , i n v o l v i n g t o x i c f i l t r a t e s p r e p a r e d
fro m two s e l e c t e d s t r a i n s o f m ic r o c o c c i ( o n l y one o f them e n t e r o t o x i g e n i c ) ,
an d t h e i r r e s p e c t i v e a n t i e e r a , a s p e c i f i c f l o c u l a t i o n r e a c t i o n was d e m o n s tr a te d
b e tw e e n t h e e n t e r o t o x l n an d i t s
c o r r e s p o n d in g a n t i b o d y .
H ow ever, no o t h e r
w o r k e r i n t h i s f i e l d h a s b e e n a b l e t o o b t a i n t h e same r e s u l t s .
PHYSIOLOGICAL STUDY
M a t e r i a l s and m eth o d s
The e i g h t e e n s t r a i n s o f m ic r o c o c c i u s e d I n t h i s e x p e r im e n t a r e
l i s t e d i n T a b le I .
To a s s u r e t h e n u r l t y o f t h e c u l t u r e s u s e d i n t h i s
e x p e r im e n t, t h e s e s t r a i n s w e re s t r e e k e d on p l a t e s c o n t a i n i n g t h e ChapmanS to n e m edium, w h ic h Chapman (1 9 4 8 ) h a d d e v i s e d t o p ro m o te t h e m ore o u t ­
s ta n d in g q u a l i t i e s o f s t r a i n s b e lie v e d to o ro d u ce e n te r o to x ln .
T h is
medium lie s t h e f o l l o w i n g c o m p o s itio n :
W a t e r ...............................................
1 ,0 0 0 .0 ml
d - M a n n ito l ( D l f c o ) . . . . .
................................................
1 0 .0 g
B a c to t r y p t o n e ..................................................................................
1 0 .0 g
Bacto a g a r........................................................................................
Sodium c h lo r id e , Merck r e a g e n t......................
B a c to y e a s t e x t r a c t .................................................................
KgHPOU, a n h y d ro u s , M erck r e a g e n t .......................................
B a c to g e l a t i n ..................................................................................
Ammonium s u l f a t e , B a k e r a n a ly s e d ,
A .C .S . p y r ld in e - f r e e ........................................................
Sodium h y d ro x id e, 10 p er c e n t ..........................................
15.0
5 5 .0
2 .5
5 .0
3 0 .0
g
g
g
g
g
7 5 .0 g
6 .0 ml
-9 TABLE I .
SOURCE AHD HISTORY OF MICROCOCCUS STRAINS
S t r a i n Ho.
S o u rc e o f I s o l a t i o n and H i s t o r y
I 72 a
C h ip p e d b e e f , 1 9 3 8 .
17 Sa
Ham, 1 9 3 9 .
T o x ic t o m onkeys, 1 9 3 9 . 1944.
196s
Ham, 1 9 4 8 .
T o x ic t o m onkeys on num erous t e s t s .
s - l 6b
Ham, 1 9 3 4 .
C o a g u la s e 4, S to n e r e a c t i o n - ,
S-31B 15
E c l a i r , 1935•
C o a g u la e e 4* S to n e r e a c t i o n 4* 1 9 4 8 .
5.290%
T u rk e y , 1 9 4 2 .
C o a g u la s e / ,
3-295%
Ham, 1 9 4 5 .
C o a g u la e e
3 -2 9 6 ^
H e , 1945.
C o a g u la s e 4* S to n e r e a c t i o n </, 1 9 4 8 .
s - 325 %
Lamb, 1 9 4 8 .
9664
C ase o f f o o d —o o l s o n l n g , A m erican Type C u l t u r e C o l l e c t i o n
M -I
S to c k c u l t u r e , D e p a rtm e n t o f B o ta n y a n d B a c t e r i o l o g y ,
M ontana S t a t e C o lle g e
T o x ic t o m onkeys, 1 9 3 8 .
1948.
S to n e r e a c t i o n 4* 1 9 4 8 .
S to n e r e a c t i o n 4* 1 9 4 8 .
C o a g u la s e 4* S to n e r e a c t i o n •/, 1 9 4 8 .
M-2
n
M-3
n
M-4
H
m- 5
U r in e o f a d u l t w i t h u r o s t a t i c i n f e c t i o n , 1943 .
S t a te C o lle g e .
2220-18®
M ilk , 1 9 4 3 .
2 8 2 2 -2 °
M ilk o f m a e t i t i c cow, 1 9 4 6 .
2908 °
C om bined m il k o f m a e t i t i c h e r d o f cow s, 19 4 7 .
M ontana
H e m o ly tic , 19 4 6 .
C u l t u r e s o b t a i n e d fro m
a ) "Dr. G. M. D ack , D e p t, o f B a c t e r i o l o g y , U n i v e r s i t y o f C h ic a g o .
b ) D r. R. V. S to n e , D i r e c t o r o f P u b l i c H e a l th L a b o r a t o r i e s , Loe A n g e la s C o ., C a l i f ,
c
> . E . A. T u n n i c l i f f , D e p t, o f V e t e r i n a r y S c ie n c e , M ontana S t a t e C o lle g e .
10*»
An i s o l a t e d c o lo n y w as rem oved fro m e a c h o f t h e f i r s t s e t o f p l a t e s and
s t r e a k e d on a s e c o n d s e t .
From t h e s e c o n d s e t o f p l a t e s a c o lo n y was
rem oved fro m e a c h a n d s t r e a k e d on a t h i r d s e t .
A ll p l a t e s w e re in c u b a t e d
f o r 42 h o u r s a t 3 0 ° C "before c o l o n i e s w e re p i c k e d .
An i s o l a t e d c o lo n y
fro m e a c h o f t h e t h i r d s e t o f p l a t e s was p i c k e d i n t o n u t r i e n t b r o t h .
A ll
m e d ia u s e d i n t e s t i n g t h e p h y s i o l o g i c a l r e a c t i o n s o f t h e s e e i g h t e e n
s t r a i n s w e re i n o c u l a t e d from 2U h o u r n u t r i e n t b r o t h c u l t u r e s a n d w ere
i n c u b a t e d a t 3 0 ° C f o r a p e r i o d o f one w eek , u n l e s s o t h e r w i s e s p e c i f i e d .
The S to n e R e a c tio n a n d p i g m e n ta t io n w e re o b s e rv e d on e l a t e s
c o n t a i n i n g t h e C hapm an-S tone m edium , a f t e r 42 h o u r s i n c u b a t i o n .
The S to n e
R e a c ti o n i s e s s e n t i a l l y t h a t o f l i q u e f a c t i o n o f g e l a t i n w i t h i n t h e medium
a n d i s d e te r m in e d b y t h e c l e a r e d a r e a s im m e d ia te ly s u r r o u n d i n g t h e
c o lo n ie s .
The p ig m e n t c o l o r o f t h e s t r a i n s was com pared w i t h t h e c o l o r s
g iv e n on P l a t e IV o f " C o lo r S ta n d a r d s a n d N o m e n c la tu re " by R o b e rt
R idgw ay ( 1 9 1 2 ) .
F e r m e n ta tio n s t u d i e s w e re c a r r i e d o u t on t h e f o l l o w i n g c a rb o h y ­
d ra te s !
g lu c o s e , f r u c to s e , x y lo s e , g a la c to s e , 1 -a ra b in o s e , d -a ra b in o s e ,
su c ro se , tre h a lo s e ,
l a c t o s e , m a l to s e , e e l l o b i o e e , m e l l b i o s e ,
g l y c e r o l , m a n n i to l , d u l e i t o l ,
i n o s i t o l , and e a l i c i n .
w as made u p i n f i v e p e r c e n t a q u e o u s s o l u t i o n ,
ra ffin o s e ,
Sach c a r b o h y d r a te
s te r il iz e d by f i l t r a t i o n
th ro u g h p y re x b ran d f r i t t e d d i s c s o f u l t r a - f i n e p o r o s ity , and s to r e d a t
IOc C u n t i l u s e d .
F iv e d a y s b e f o r e u s e , one m l o f e a c h c a r b o h y d r a te was
t r a n s f e r r e d a e e p t i c a l l y t o t e s t tu b e s w h ic h c o n ta i n e d Durham f e r m e n t a t i o n
t u b e s a n d f o u r ml o f n u t r i e n t b r o t h w i t h brom e r e s o l p u r p l e a s an
♦
-11.
In d ic a to r.
The t u b e s w e re s u b s e q u e n t l y h e l d a t room t e m p e r a t u r e t o
c h e c k f o r a n y c o n ta m in a t io n .
They w e re t h e n i n o c u l a t e d a n d o b s e rv e d
a t v a r i o u s i n t e r v a l s f o r a c i d an d g a s p r o d u c t i o n .
K i t r a t e r e d u c t i o n , p r o d u c t i o n o f i n d o l , h y d ro g e n s u l f i d e , ammonia
fro m p e p to n e , m o t i l i t y , s t a r c h h y d r o l y s i s , g e l a t i n l i q u e f a c t i o n , g ro w th
on !'THltHpPOi4., a n d r e a c t i o n s on l i t m u s m ilk a n d brom c r e s o l p u r p l e m ilk
w e re s t u d i e d on s t a n d a r d m e d ia a n d t e s t e d a c c o r d in g t o t h e m eth o d s
p r e s e n t e d i n t h e "M anual o f M ethods f o r P u re C u ltu r e S tu d y o f B a c t e r i a "
(1 9 4 8 -1 9 4 9 ).
H e m o ly sis o f s h e e p e r y t h r o c y t e s was t e s t e d on p l a t e s u s i n g b a c to
b lo o d a g a r b a s e a n d f i v e p e r c e n t s t e r i l e , d e f l b r i n a t e d b l o o d .
The c o a g u la s e t e s t w as r u n a c c o r d in g t o C hapm an's p r o c e d u r e
(1 9 4 6 ), v i s . :
"Mix a l o o p f u l o f c u l t u r e , grow n e x a c t l y 42 h o u r s on th e
s p e c i a l medium ( C hapm an-S tone a g a r ) w i t h 0 . 1 m l o f b a c to b r a i n - h e a r t
i n f u s i o n ; s h a k e v i o l e n t l y a t f r e q u e n t i n t e r v a l s f o r e x a c t l y 15 m in u te s
a n d a d d 0 . 2 m l o f t e s t e d b lo o d ( p r e v i o u s l y 0T a i n t e d ) .
In c u b a te th e
m i x t u r e , p r e f e r a b l y i n a w a te r b a t h , a t 3 7 ° C an d l n s n e c t i n e x a c t l y
one h o u r ."
C o a g u la tio n o f t h e b lo o d i s i n d i c a t i v e o f a p o s i t i v e t e s t .
The t e s t f o r f l b r i n o l y s l n p r o d u c t i o n was r a n a c c o r d i n g t o th e
t e c h n i c o f T i l l e t t an d G a rn e r ( 1 9 3 3 ) .
The p la s m a u s e d f o r t h e d e te r m in a ­
t i o n was p r e p a r e d by m ix in g 20 ml o f human b lo o d w ith one m l o f a two
p e r c e n t p o ta s s iu m o x a l a t e s o l u t i o n .
and c e n tr if u g e d .
The b lo o d was s h a k e n th o r o u g h ly
O n e - h a lf o f t h e s u p e r n a t a n t o f e a c h c u l t u r e d s t r a i n ,
grow n i n n u t r i e n t b r o t h f o r t w e n t y - f o u r h o u r s , was m ixed w i t h one ml
-1 2 o f 1 :5 d i l u t i o n o f p la s m a ; t h e n 0.2 5 ” 1 o f a n 0 .2 5 Pe r c e n t c a lc iu m
c h l o r i d e s o l u t i o n I n n o rm a l s a l i n e was a d d e d ; th e tu b e s w e re s h a k e n
t h o r o u g h l y a n d k e p t a t 3 7 ° C t o h e o b s e r v e d a t t h e e n d o f 24 h o u r s f o r
l y s i s o f t h e p la s m a c l o t .
R esu l t s
F e r m e n t a t iv e a b i l i t i e s
a r e g iv e n i n T a b le I I .
o f t h e e i g h t e e n s t r a i n s o f m ic r o c o c c i
W ith t h e e x c e p t io n o f s t r a i n M -I, a l l o rg a n is m s
p r o d u c e d a c i d w i t h o u t g a s fro m g l u c o s e , f r u c t o s e , g a l a c t o s e , s u c r o s e ,
tre h a lo s e ,
l a c t o s e , m a l to s e , g l y c e r o l , and m a n n i to l , w h e re a s t h e y
p r o d u c e d n e i t h e r a c i d n o r g a s fro m x y l o s e , 1 - a r a b i n o s e , d - a r a h l n o s e ,
c e llo b io e e , m e lib io s e , r a f f i n o s e , d u l c i t o l ,
i n o s i t o l , and s a l i c i n .
S tra in
M -I d i f f e r e d fro m t h e r e s t i n n o t f e r m e n t in g s u c r o s e , g a l a c t o s e , l a c t o s e ,
o r m a n n ito l.
T h ese f e r m e n t a t i o n s w e re , f o r t h e m ost p a r t ,
t h e en d o f a 4 g - h o u r i n c u b a t i o n .
c o n ro le te a t
H ow ever, t h e tu b e s w e re r e t a i n e d f o r
t h r e e w eeks a n d o b s e r v a t i o n s w ere made p e r i o d i c a l l y .
T h e se r e s u l t s a r e
i n a g re e m e n t w i t h t h o s e o b t a i n e d Tyr Chapman (1 9 4 0 ) a n d o t h e r s i n t h e i r
s t u d i e s on t h e c a r b o h y d r a te f e r m e n t a t i o n r e a c t i o n s o f s t a o h y l o c o c c l .
T h y g eso n (1 9 3 8 ) fo u n d t h a t m a n n i to l f e r m e n t a t i o n i s a r e l i a b l e
i n d i c a t o r o f t h e t o x i g e n i c p o w e rs o f s t a p h y l o c o c c i .
S t r a l t e r (1 9 3 5 ) a n d
Chapman (1 9 4 0 ) b e l i e v e d th e f e r m e n t a t i o n o f l a c t o s e a n d m a n n i to l h a s
some d i f f e r e n t i a l v a lu e i n s e t t i n g a p a r t e n t e r o t o x i n ~ o r e d u c i n g m ic r o c o c c i.
S t r a i n 9664 f a i l e d t o f e r m e n t m a n n ito l w i t h i n 24 h o u rs ( t h e o t h e r s t r a i n s ,
w i t h t h e e x c e p t i o n o f s t r a i n M -I, d i d ) .
S t r a i n S - l6 p r o d u c e d much l e s s
a c i d i n l a c t o s e w i t h i n 24 h o u r s t h a n d i d th e r e s t o f t h e s t r a i n s .
-1 3 TXBLB I I .
yBBMEMTXTIVl! BBACTIONS 0 7 MICROCOCCI
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• F e r m e n t a t iv e c h a r a c t e r i s t i c * f o r M ic ro c o c c u s -ojogenes v e r . a u re u e ae
g iv e n I n t h e e l x t h e d i t i o n o f B eTgeyr S- M m u a l.
14O th e r p h y s i o l o g i c a l r e a c t i o n s , n i t r a t e r e d u c t i o n , i n d o l u re d u c ­
t i o n , h y d ro g e n s u l f i d e p r o d u c t i o n , p r o d u c t i o n o f ammonia fro m p e p to n e ,
m o tility , h y d ro ly s is o f s ta r c h ,
l i q u e f a c t i o n o f g e l a t i n , g ro w th on
r e a c t i o n s on l i t m u s m ilk a n d brom c r e s o l p u r p l e m il k , p ig m e n ta ­
t i o n , l i q u e f a c t i o n o f g e l a t i n a n d t h e S to n e r e a c t i o n a r e g i v e n i n T a b le
III.
I t w as o b s e r v e d a f t e r one w eek ’ s i n c u b a t i o n t h a t a l l s t r a i n s
r e d u c e d n i t r a t e t o n i t r i t e , p r o d u c e d ammonia fro m p e o to n e , p r o d u c e d a c i d
i n l i t m s a n d brom c r e s o l p u r p l e m ilk ( w i t h t h e e x c e p t io n o f s t r a i n M - l) ,
r e d u c e d l i t m u s m il k , a n d p r o d u c e d p ig m e n ts — fro m l i g h t y e ll o w t o d eep
o ran g e .
A l l s t r a i n s g a v e n e g a t i v e r e s u l t s when t e s t e d f o r h y d ro g e n
s u l f i d e , i n d o l , s t a r c h h y d r o l y s i s , g ro w th on ammonium a c i d p h o s p h a te
a g a r , and m o t i l i t y .
T h e se a r e , w i t h one e x c e p t i o n , t h e c h a r a c t e r i s t i c s
l i s t e d f o r Micrococcus?, p y o g e n e s v a r . a u r e u s a s g iv e n i n t h e s i x t h e d i t i o n
o f S e r g e y 's M an u al, i n w h ic h g ro u p e n t e r o t o x i n - p r o d u c l n g m ic r o c o c c i a r e
in c lu d e d .
The one g e n e r a l e x c e p t io n i s t h a t no n e o f t h e e i g h t e e n s t r a i n s
p r o d u c e d h y d ro g e n s u l f i d e on t h e medium c o n t a i n i n g p e p to n e , m eat e x t r a c t ,
so d iu m s u l f i t e ,
f e r r o u s s u l f a t e , a n d a g a r , w h e re a s S e r g e y 's M anual l i s t s
a e l i c i t am ount o f h y d ro g e n s u l f i d e a s a c h a r a c t e r i s t i c .
The l i q u e f a c t i o n o f g e l a t i n a n d t h e S to n e r e a c t i o n , a s o b s e rv e d
a f t e r v a r i o u s l e n g t h s o f i n c u b a t io n , a r e g i v e n i n T a b le I T .
T w elve o f
t h e e i g h t e e n s t r a i n s g av e a S to n e r e a c t i o n w i t h i n a 4 8 - h o u r i n c u b a t i o n
p e rio d .
T h re e s t r a i n s (S -3 1 B , 9 6 6 4 , and 2 8 0 9 ) l a t e r showed t h e S to n e
r e a c t i o n a t 96 h o u r s , a n d one s t r a i n (M -l) was c o n s id e r e d p o s i t i v e a t
t h e e n d o f n in e d a y s .
Two s t r a i n s ( S - l 6 , M -2) f a i l e d t o g i v e any
-1 5 TABIS I I I .
PHYSIOLOGICAL BEACTI ONS OF MICROCOCCI ON STANDARD MEDIA
ti
0
+5
S
1
t-i
r-t
S t r a i n No.
172
178
196
S - l6
S-31B
S -2 9 0
S -2 9 5
s -296
s -325
9664
M -I
M-2
M-3
M-U
M-5
2 2 2 0 -1 8
2 8 8 2 -2
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P ig m en t
l i g h t cp.dmium
a n i l i n e y e llo w
a n i l i n e y e llo w
l i g h t cadmium
m a iz e y e llo w
a p r i c o t y e llo w
M a rtu s y e llo w
a n i l i n e y e llo w
Martua y e llo w
lem o n y e llo w
m a iz e y e llo w
a n i l i n e y e llo w
p i n n a r d y e llo w
a p r i c o t y e llo w
M a rtu s y e llo w
a p r i c o t y e llo w
l i g h t cadmium
m a iz e y e llo w
y e llo w to orange
• P h y s i o l o g i c a l c h a r a c t e r i s t i c s f o r M ic ro c o c c u s p y o g e n e s v a r . a u re u s a s
g i v e n I n th e s i x t h e d i t i o n o f S e r g e y 's M an u al.
A, a c i d i c ; B1 b a s i c ; C, c u r d ; R, r e d u c t i o n ; W, whey.
TABLE IT,
LIQUEFACTION OF GELATIN AND THE STONE HBACTION
S t r a i n No.
172
178
196
S -l6
S-31B
S -2 9 0
S -2 9 5
S -2 9 6
S-325
9664
M-I
M-2
M-3
M-U
M-5
2220-18
2908
G e la tin L iq u e fa c tio n
Days
4 “
- S
+
+
+
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+
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The S to n e H e a c tio n
Days
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-1 7 -
ln d lc a t lo n o f th e Stone r e a c t io n .
t o a s c e r t a in fo r th e most p a r t .
The Stone r e a c tio n was v ery d i f f i c u l t
Many o f th e s t r a in s w hich were
c o n sid e r e d p o s i t i v e m erely showed a v ery narrow zone o f d ecrea sed
o p a c it y a d ja c e n t to growth on th e p l a t e s .
Two s t r a in s ( S - l 6 , S -31B ) d id n o t li q u e f y g e l a t i n a f t e r a f iv e - d a y
in c u b a tio n p e r io d a t 30° C.
Moat o f the s t r a in s l i q u e f i e d g e l a t i n w ith in
a 72-h o u r in c u b a tio n ; however, liq u e f a c t io n became obvious w ith f i v e o f
them (M- 2 , M~3, 9 6 6 4 , 2 9 0 8 , and S -2 9 0 ) o n ly a f t e r a f iv e - d a y in c u b a tio n .
I t was found th a t in c u b a tio n a t 30° C brought about a more ra p id liq u e ­
f a c t io n o f g e l a t i n by the organism s than in c u b a tio n a t 37° C.
The le n g th
o f tim e th e tu b es were h e ld i n the r e f r ig e r a t o r a f t e r each in cu b a tio n
( i n ord er to determ ine which s t r a in s a c t u a ll y li q u e f i e d th e g e l a t i n )
in flu e n c e d th e o b serv a b le li q u e f a c t io n in some o f th e tu b e s .
When h e ld
a t 8 ° C fo r a. p e r io d o f two h o u rs, e ig h t o f th e e ig h te e n tu b es became
s o l i d and any p o s s i b l e li q u e f a c t io n in them was u n d is c e r n ih le .
When
h e ld a t S0 C f o r a p e r io d o f 4$ m in u tes, o n ly two o f th e tu b es showed
no li q u e f a c t io n .
The u n in o c u la te d c o n tr o l tu b es were s o l i d a f t e r each o f
th e d if f e r e n t c o o lin g p e r io d s .
Table V g iv e s th e r e s u l t s o f th e c o a g u ly t ic , f i b r i n o l y t i c , and
h e m o ly tic a b i l i t i e s o f the c u lt u r e s .
A ll but fo u r s t r a in s (M -3, M-4,
2 o 8 2 -2 , S - l 6 ) show ed some f i b r i n o l y t i c a c t i o n on c o a g u la te d human b lo o d
p la s m a , f i v e s t r a i n s ( M - l, M -4, 2 9 0 8 , S - 1 6 , S-31B ) d id n o t h e m o ly z e s h e e p
e r y t h r o c y t e s , an d a l l e i g h t e e n s t r a i n s c o a g u l a t e d d e f i b r i n a t e d r a b b i t
w h o le b lo o d t o a c e r t a i n e x t e n t .
Chapman a t a l (1 9 3 4 ) f o u n d t h a t a h ig h
c o r r e l a t i o n e x i s t s b e tw e e n t h e m i c r o c o c c i 's a b i l i t y t o p r o d u c e c o a g u la s e
IS TABLB 7 .
MICROCOCCUS REACTIOITS WITH BLOOD
S t r a i n Ho.
172
178
196
H e m o ly tic
C o a g u l y ti c
F ib rin o ly tic
4+
1+
3+
5+
3+
3+
4+
2+
2+
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S -31B
s-290
-
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s-2 9 5
2+
3+
3+
1+
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4+
3+
3+
4+
s-296
s-325
9664
M-I
M-2
-
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-
3+
2+
5
M-5
2220-18
2882—2
2908
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-1 9 a n d h e m o ly s in find t h e i r p a t h o g e n i c i t y .
C r u lc k s h a n k (1 9 3 7 ) s u p p o r te d
t h e r e l a t i o n s h i p b e tw e e n p a t h o g e n i c i t y end p r o d u c t i o n o f c o a g u la s e ,
w h e re a s J o r d a n a n d B urrow s ( 1 9 3 5 ) . K u p c h ik ( 1 9 3 7 ) . Dolman a n d W ilso n
( 1 9 3 9 ) . «ucL H ussem ann and T a n n e r (1 9 ^ 9 ) c o u ld n o t f i n d t h e c o a g u la s e
t e s t o r a n y o t h e r p h y s i o l o g i c a l c h a r a c t e r i s t i c w h o lly s u c c e s s f u l i n
s e p a r a t i n g t h e e n t e r o t o x l n - f o r m i n g fro m t h e n o n - e n t e r o t o x i n - f o r m i n g
m ic r o c o c c i.
BIOLOGICAL STUDY
The b i o l o g i c a l s tu d y o f t h e e i g h t e e n s t r a i n s o f m ic r o c o c c i
c o n s is te d o f t e s t i n g c u ltu r e f i l t r a t e s f o r e n te r o to x ln th ro u g h c a t
in o c u la tio n .
M ethods and m a t e r i a l s
F a v o r i t e a n d Hammon (19% 1) d e v is e d t h e f o ll o w i n g medium f o r
e n t e r o t o x l n p r o d u c t i o n fro m m ic ro c o c c u s f o o d - p o i s o n in g s t r a i n s :
C a s e in h y d r o l y s a t e ................................................
D i s t i l l e d w a t e r ........................................................
P h e n o l r e d ( 0 . 2$ ) ................................................
T h ia m in c h l o r i d e ............................. . . . . .
H i c o t i n l c a c i d ..........................................................
15
9 OO
U
5
1200
g
ml
ml
gamma
gamma
The pH o f t h i s medium was a d j u s t e d t o 7*6 w i t h Uo p e r c e n t NaCH and th e
volum e b r o u g h t t o 990 m l.
S i x t y ml p o r t i o n s o f t h i s medium w e re p l a c e d
i n K o lle f l a s k s a n d s t e r i l i z e d .
The r a t i o o f s u r f a c e a r e a i n c o n ta c t
w i t h a tm o s p h e re t o th e volum e o f c u l t u r e medium i s h ig h i n K o lle f l a s k s ;
t h u s , more g ro w th a n d g r e a t e r am o u n ts o f t o x i c s u b s ta n c e s i n a g iv e n
am ount o f medium a r e o b t a i n e d .
c e n t aqueous s o lu tio n o f s t e r i l e
A f t e r a u t o c l a v i n g , one ml o f a 25 p e r
g lu c o s e w as a d d e d to th e medium o f e a c h
-2 0 fla s k .
They were then I n o c u la te d w ith one ml o f 24-hour "broth c u ltu r e s
and in cu b ated a t 30° C f o r 72 hours In a p y rex brand 5000 ml vacuum
d e e s ic a t o r j a r under a te n s io n o f 3 ° p er cen t carbon d io x id e and JO per
cen t oxygen.
At th e end o f t h i s p erio d the c u ltu r e s were f i l t e r e d
through p yrex brand f r i t t e d d is c s o f u l t r a f i n e p o r o s it y and th e f i l t r a t e s
tr a n s fe r r e d to v a c c in e b o t t l e s .
The b o t t l e s were then immersed in b o ilin g
w ater f o r 30 m inutes to d e str o y alp h a snd b e ta h em olysin s and any oth er
t h e n o l a b i l e t o x in s e la b o r a te d by th e organ ism s.
I t w as i m p o s s ib l e t o o b t a i n a s u f f i c i e n t num ber o f k i t t e n s upon
w h ic h t o t e s t t h e f i l t r a t e s ,
a s t h i s p a r t o f t h e e x p e rim e n t was c a r r i e d
o u t b e f o r e t h e k i t t e n i n g s e a s o n o c c u r r i n g i n l a t e s p r i n g a n d e a r l y summer.
T h e r e f o r e , i t was n e c e s s a r y t o t e s t t h e e n t e r o t o x i c i t y o f t h e c u l t u r e s by
c a t in o c u la tio n .
N ine c a t s , a n d two k i t t e n s o f one o f t h e c a t s , w ere u s e d
to t e s t th e p re s e n c e o f e n te r o to x in in th e c u ltu r e f i l t r a t e s .
was i n j e c t e d w i t h f o u r d i f f e r e n t f i l t r a t e s ,
b e tw e e n i n j e c t i o n s .
Sach c a t
t h r e e d a y s t o a w eek a llo w e d
T h re e ml o f e a c h f i l t r a t e
w ere f i r s t i n j e c t e d i n t r a ­
m u s c u l a r l y b e h in d t h e s h o u ld e r b l a d e s o f t h e c a t s i n o r d e r t o s e e w h e th e r
i n j e c t i o n s u s i n g t h i s r o u t e w o u ld e l i c i t f o o d - p o i s o n in g sym ptom s.
As no
e n t e r o t o x i c sym ptom s w e re o b s e r v e d u p o n i n t r a m u s c u l a r i n j e c t i o n s o f t h e
filtra te s ,
i n t r a v e n o u s i n j e c t i o n s , i n a c c o r d a n c e w ith th e Hammon im p ro v e d
c a t t e s t ( i g U l ) w e re u s e d .
c lip p e d o f h a i r .
The i n n e r s u r f a c e s o f t h e c a t ' s t h i g h s w ere
Then p r e s s u r e was a p p l i e d i n t h e i n g u i n a l r e g i o n t o make
t h e v e i n s " s t a n d o u t" a n d t h e n e e d le i n s e r t e d i n t o t h e s a o h e n o u s v e i n .
T h is v e i n , a l t h o u g h s m a l l , i s t h e e a s i e s t v e i n t o w ork w i t h i n t h e c a t .
I t was fo u n d n e c e s s a r y t o h a v e t h e h e l p o f a n o t h e r p e r s o n i n o r d e r t o h o l d
.21.
th e c a t p e r f e c t l y s t i l l w h ile in s e r t i n g the n eed le n r o o e r ly in t o th e
v e in to p rev en t i t s d e s tr u c tio n through sudden movement.
The two k it t e n s
were each g iv e n th r e e ml o f a f i l t r a t e p r e v io u s ly showing p o s i t i v e rea c­
t io n s on a c a t .
T his was in accordance w ith th e Dolman k i t t e n t e s t (1 9 3 8 ).
A ctu a l v o m itin g was c o n sid ered a p o s i t i v e r e a c t io n .
R e s u lts
None o f th e f i l t r a t e s from th e e ig h te e n s t r a in s was found to
in vok e fo o d -p o is o n in g symptoms upon in tra m u scu la r in j e c t i o n o f th e c a t s .
However, upon in tra v en o u s in o c u la t io n , c a ts in j e c t e d w ith th e f i l t r a t e s
o f th r e e s t r a in s (I J S , M-5, S -325) showed t y p ic a l fo o d -p o is o n in g symptoms
w ith v o m itin g .
The two k i t t e n s , each about th ree weeks o ld , were in o c u la te d
ln t r a p e r lt o n e a lly w ith th r e e ml amounts o f th e e n t e r o t o x ig e n ic s t r a in S-325
and a ls o showed t y p ic a l fo o d -p o is o n in g symptoms w ith v o m itin g .
The f o llo w in g are d e s c r ip t io n s o f th e symptoms o f fo o d -p o is o n in g
dem onstrated "by each c a t upon in tra v en o u s in o c u la t io n w ith th r e e ml o f
each f i l t r a t e p ro v in g e n t e r o t o x ig e n ic .
Male c a t , age one yea r:
UjOO p.m .
I n je c te d w ith f i l t r a t e from s t r a in S -3 2 5 .
appeared n a u sea ted and d e je c te d .
Uj20 vom ited .
UjlO
U j12 showed r e t c h in g m otion s.
U j 20-U j 30 vom ited tw ic e more.
T his c a t refu sed
t o e a t d u rin g th e n ex t two or th ree days and became much th in n e r .
At th e co m p letio n o f th e experim ent some J>0 days l a t e r , i t had
n ot y e t f u l l y r e c o v ered in w eigh t o r s p i r i t .
•22F em ale c a t , a g e tw o y e a r s :
4 :0 5 p .m .
I n j e c t e d w i t h f i l t r a t e fro m s t r a i n S -3 2 5 .
4 :2 0 show ed d e j e c t i o n , n a u s e a , an d s a l i v a t i o n , f o l l o w e d by
r e s t l e s s n e s s a n d r e t c h i n g m o tio n s .
v o m ite d .
4 : 4 0 v o m ite d .
4 ;4 l
D u rin g t h e n e x t 30 m in u te s i t d i d n o t v o m it a g a i n ,
b u t e x h i b i t e d d e j e c t i o n and l a s s i t u d e .
The n e x t d a y i t
a p p e a r e d t o be e a t i n g n o r m a lly a n d n o n e t h e w o rse f o r h a v in g
had an a t t a c k o f fo o d -p o is o n in g .
M ale c a t , a g e t h r e e y e a r s :
3 : 1 0 p .m .
I n je c te d w ith f i l t r a t e
fro m s t r a i n I J S .
3:30 show ed d e j e c t i o n a n d e x c e s s i v e s a l i v a t i o n .
m o tio n s o b s e r v e d .
4 : 0 5 , 4 : 0 8 , 4 ;1 0 v o m ite d .
3:35 r e t c h i n g
T h is w as f o llo w e d
b y d e j e c t i o n b u t no a d d i t i o n a l s i g n s o f n a u s e a o r r e t c h i n g .
The n e x t d a y i t a t e n o r m a lly and show ed no v i s i b l e s i g n s o f
l o s s o f w e ig h t o r w e a k n e s s .
F em ale c a t , a g e one y e a r :
3:05 p .m .
I n je c te d w ith f i l t r a t e
p e a r e d n a u s e a t e d and d e j e c t e d .
fro m s t r a i n M-5»
3$35 show ed h e a v y s w a llo w in g
m ovem ents, l i c k i n g o f l i n e , and u n r e s t .
v o m ite d .
3 $ 3 0 ap ­
3 $36 , 3 $3 7 , 3$^5
An h o u r a f t e r i n j e c t i o n o f t h e f i l t r a t e
it
s till
a p p e a r e d d e j e c t e d , b u t t h e n e x t d a y i t a t e n o r m a lly .
The tw o k i t t e n s show ed s i m i l a r eymotoms when i n j e c t e d i n t r a p e r i t o n e a l l y
w i t h t h r e e ml am o u n ts o f t h e f i l t r a t e
fro m s t r a i n S -3 2 5 , b u t n u r s e d
n o r m a l ly t h e n e x t d a y and a p p e a r e d to l o s e l i t t l e ,
i f a n y , w e i g h t.
•23'
Of the te n s t r a in s u sed in t h i s experim ent which were is o la t e d
from a c tu a l c a s e s o f m icr o co cci fo o d -p o is o n in g , on ly two, s t r a in 1J8
and s t r a in S - 325 . proved to invoke v o m itin g when in j e c t e d in tr a v e n o u sly
in t o c a t s .
E v id e n tly the o th er s t r a in s , i f th ey were a c t u a lly th e
sou rce o f th e fo o d -p o is o n in g c a s e s from which th ey were i s o l a t e d , had
l o s t t h e ir a b i l i t y to produce e n te r o to x in a f t e r b ein g c u ltu r e d on
a r t i f i c i a l media (some a s lo n g as f i f t e e n y e a r s ) , whereas th o s e in v o k in g
fo o d -p o is o n in g symptoms in th e c a te had n o t l o s t t h is a b i l i t y .
It is
p o s s i b l e th a t th e t e c h n ic fo llo w e d to o b ta in e n te r o to x in f i l t r a t e s was
a t f a u l t , but a s ea ch f i l t r a t e was o b ta in ed in th e same way, and a s two
o f them a c t u a lly produced v o m itin g in c a t s , t h is does n ot seem to be th e
c a u se .
S tr a in M-5, which a ls o invoked v o m itin g in a c a t , was not I s o la t e d
from a ca se o f fo o d -p o is o n in g but was o b ta in ed from th e u r in e o f an
in d iv id u a l s u f f e r in g w ith a p r o s t a t ic i n f e c t i o n .
The Hammon Cat T est and th e Dolman K itte n T est are c o n sid ered by
most workers in t h i s f i e l d a s b e in g th e most r e l i a b l e means o f d eterm in in g
th e e n t e r o t o x i c it y o f a m icrococcus s t r a in .
SEROLOGICAL STUDY
W oolpert and Dack (1 9 3 3 ) were th e f i r s t I n v e s t ig a t o r s to dem onstrate
th e a n t ig e n ic it y o f e n t e r o t o x in .
They accom plished a c t iv e im m unization
in monkeys w ith o u t d i f f i c u l t y , but were u n ab le to immunize them p a s s iv e ly .
A lthough Dolman, W ilson, and C ockcroft (1 9 3 6 ) rep o rted th a t p relim in a ry
exp erim en ts in d ic a t e d th a t e n te r o to x in was a n t ig e n ic , s u f f i c i e n t confirm a­
t io n o f t h is f a c t was n o t forth com in g u n t i l Dolman and W ilson (1 9 3 8 ) and
M inett (1938) p u b lish e d t h e i r o b s e r v a tio n s .
Dolman and W ilson dem onstrated
a f l o c c u l a t i o n r e a c t io n I n v o lv in g th e e n te r o to x in and th e s p e c i f i c a n tib o d y .
However, no o th er au th or has been a b le to confirm t h i s .
Methods and M a ter ia ls
In t h i s exp erim en t, two r a b b its were g iv e n a s e r i e s o f in j e c t io n s
f o llo w in g a sch ed u le su g g e ste d by Boyd ( 19U7 ) .
One ra b b it was in j e c t e d
w ith a f i l t r a t e from s t r a in S-325 which had showed a p o s i t i v e c a t t e s t ,
th e o th e r w ith a f i l t r a t e from s t r a in S-295 which had showed n e g a tiv e
r e s u l t s in th e c a t t e s t .
P reced in g each s e r i e s o f i n j e c t i o n s , blood
was o b ta in ed from the r a b b it s ' e a r s .
S e r o lo g ic a l t e s t s were run w ith
th e serum o f t h i s b lood in o rd er to d eterm ine whether th e r e were any
a n tib o d ie s p r e se n t d ir e c t e d a g a in s t the e n te r o to x in which would g iv e
a v i s i b l e s e r o l o g i c a l r e a c t io n .
The f o llo w in g im m unization sch ed u le
was u sed fo r both r a b b its :
Dgr
B led
Dosage
I
2
3
5 ml
1 .5 ml
1 .5 ml
1 .5 ml
Intravenous
Intraven ous
Intravenous
7
8
9
5 ml
4 .5 ml
1 .5 ml
1 .5 ml
ln t r a p e r lt o n e a l
Intravenous
Intravenous
13
lU
15
5 ml
4 .5 ml
1 .5 ml
1 .5 ml
ln t r a p e r lt o n e a l
Intravenous
Intravenous
23
5 ml
-
-
-
-
Mode o f I n o c u la tio n
The ln t r a p e r lt o n e a l in j e c t io n p reced in g th e second and t h ir d s e r ie s
o f in tra v en o u s in o c u la t io n s p rev en ted th e p o s s i b l e occu rren ce o f
a n a p h y la c tic sh ock .
—25*“
The se ru m o b t a i n e d fro m th e ab o v e r a b b i t b l e e d i n g s was
t e s t e d f o r th e p r e s e n c e o f p r e c i p i t a t i n g a n t i b o d i e s d i r e c t e d a g a i n s t
th e f i l t r a t e
a c c o r d in g t o th e p r o c e d u r e o u t l i n e d i n th e "M anual o f
M ethods f o r P u r e C u l t u r e S tu d y o f B a c t e r i a " ( 1 9 4 8 - 1 9 4 9 ) .
P ro g re s s iv e ly
d o u b le d s e r i a l d i l u t i o n s o f a n t i g e n w ere p r e p a r e d i n s a l i n e b e g in n in g
w ith . 1 :1 0 0 a n d e x te n d i n g th r o u g h 1 :2 5 ,0 0 0 .
O n e - te n th ml o f t h e serum
wav t r a n s f e r r e d t o th e b o tto m o f s m a ll t u b e s a n d an e q u a l am ount
( 0 . 1 m l) o f e a c h d i l u t i o n o f th e a n t i g e n ( f i l t r a t e ) wae l a y e r e d o n to
t h e s e ru m .
The t u b e s w ere I n c u b a t e d a t
37° 0
f o r two h o u r s an d
o b s e r v e d a t 3® m in u te i n t e r v a l e f o r p r e c i p i t a t e a t t h e j u n c t u r e o f
t h e seru m a n d a n t i g e n .
a t 4 ° C.
it
They w ere t h e n s h a k e n a n d in c u b a t e d o v e r n ig h t
Py t h i s tim e t h e p r e c i p i t a t e s h o u ld h a v e s e t t l e d o u t s o t h a t
c o u ld b e r e a d b y a g e n t l e s h a k in g o f t h e t u b e s .
C o n t r o ls o f a n t i g e n
w i t h s a l i n e a n d seru m w i t h s a l i n e w ere i n c l u d e d .
B e s u lts
A lth o u g h Dolm an and W ils o n (1 9 3 8 ) d e m o n s tr a te d a s p e c i f i c
f i o c u l a t i o n r e a c t i o n b e tw e e n e n t e r o t o x i n a n d i t s
o b t a i n e d fro m r a b b i t im m u n iz a tio n , no v i s i b l e
o b s e r v e d i n t h i s e x p e r im e n t.
c o r r e s p o n d in g a n tib o d y
s e r o l o g i c a l r e a c t i o n was
A f t e r a f o u r w e e k s ' im m u n iz a tio n p e r i o d ,
n o r e a c t i o n w h a ts o e v e r w as o b s e rv e d b e tw e e n v a r i o u s a m o u n ts o f a n a lo g o u s
e n t e r o t o x i n an d r a b b i t seru m r u n i n th e p r e c i p i t a t i o n t e s t .
G-ENSKAl DISCUSSION
A l l h u t one o f t h e e i g h t e e n s t r a i n s s t u d i e d g a v e t h e same
p h y s i o l o g i c a l r e a c t i o n s a n d m o r p h o lo g ic a l c h a r a c t e r i s t i c s a s t h o s e l i s t e d
f o r mic ro c o c c u s p y o g e n e s v a r . a u r e u s i n t h e s i x t h e d i t i o n o f S e r g e y 's
•26”
M a n u al.
S t r a i n M -I was th e i n t e r e s t i n g e x c e p tio n t o t h i s .
I t gave a
b a s i c r e a c t i o n i n l i t m u s a n d brom c r e e o l m r p l e m ilk , f a i l e d t o fe rm e n t
s u c r o s e , g a l a c t o s e , l a c t o s e , a n d m a n n i to l , and p r o v e d t o h a v e c e l l s o f
t w ic e t h e d i a m e te r o f th e o t h e r s t r a i n s .
No o t h e r o rg a n is m l i s t e d u n d e r
t h e g e n u s M ic ro c o c c n : i n S e r g e y 's M anual i s c h a r a c t e r i s e d by a b a s i c
r e a c t i o n i n l i t m u s m ilk .
S v en w i t h t h e s e d i s c r e p a n c i e s , s t r a i n M -I i s
c l o s e r t o t h e e n e c i e s o f M icro c o c c u s p y o g e n e s v a r . o u r eug t h a n i t i s t o
a n y o t h e r o rg a n is m c l a s s i f i e d i n S e r g e y 's M a n u al.
A p o s s i b l e e x p la n a ti o n
i s t h a t s t r a i n M -I i s a v a r i a n t o f t h e s p e c i e s t o w h ic h th e o t h e r s t r a i n s
b e lo n g .
V a r i a t i o n so m e tim e s o c c u r s among b a c t e r i a b e c a u s e o f t h e i r h ig h
g e n e r a t i o n r a t e a n d t h e tr e m e n d o u s ly d i f f e r e n t e n v ir o n m e n ta l c h a n g e s t o
w h ic h t h e y a r e c o n s t a n t l y b e in g s u b j e c t e d .
I t was f o u n d t h a t I n c u b a t i o n a t 3 0 ° C b r o u g h t a b o u t a more r a p i d
l i q u e f a c t i o n o f g e l a t i n by t h e o rg a n is m s t h a n i n c u b a t i o n a t 3 7 ° C.
The
l e n g t h o f tim e t h e t u b e s w ere h e l d i n t h e r e f r i g e r a t o r a f t e r e a c h in c u b a ,t i o n ( i n o r d e r t o d e te r m in e w h ic h s t r a i n s a c t u a l l y l i q u e f i e d th e g e l a t i n )
i n f l u e n c e d t h e o b s e r v a b l e l i q u e f a c t i o n i n some o f th e t u b e s .
When h e ld
a t S0 C f o r e p e r i o d o f tw o h o u r s , th e g e l a t i n i n e i g h t o f t h e e i g h t e e n
t u b e s becam e s o l i d and a n y p o s s i b l e l i q u e f a c t i o n i n them w as u n d i e c e r n i b l e .
When h e l d a t 8° C f o r a p e r i o d o f U5 m in u te s , o n ly two o f t h e tu b e s show ed
no l i q u e f a c t i o n .
The u n i n o c u l a t e d c o n t r o l t u b e s w ere s o l i d a f t e r e a c h o f
th e d i f f e r e n t c o o l i n g p e r i o d s .
Most o f t h e s t r a i n s l i q u e f i e d g e l a t i n w i t h i n
a 72 - h o u r i n c u b a t i o n ; h o w e v e r, l i q u e f a c t i o n becam e o b v io u s w i t h f i v e o f
them ( S - 29O, 966U , 2 9 0 8 , M -2, M -3) o n ly a f t e r a f i v e - d a y i n c u b a t i o n .
G e l a t i n i s g e n e r a l l y n o t l i q u e f i e d r a p i d l y i n t h e g ro u p t o w h ic h t h e s e
•27'
e t r # .i n e b e lo n g .
Two a t r a i n s ( S * l 6 , S-31B ) f a i l e d to l i q n e f y g e l a t i n
a t th e and o f f iv e day*.
The S to n e r e a c t i o n , t h e H o n e f a c t i o n o f g e l a t i n l a t h e C h a m a n S to n e m e d ia * , *fne n o e lfc lv e f o r tw e lv e o f t h e e i g h t e e n e t r a i n * w i t h i n
US h o u r * .
O R rt.
T h i s r e a c t i o n was v e r y d i f f i c u l t t o a s c e r t a i n f o r t h e m ost
Many o f th e a t r a l n e w h ic h w ere c o n s i d e r e d o o e l t l v e m e r e ly showed
a v e r y n a rro w e o n s o f d e c r e a s e d o p a c i t y a d j a c e n t t o g ro w th on t h e p l a t e s .
O n ly tw o o f t h e s t r a i n s ( H -5 , 3 -2 9 0 ) b r o u g h t a b o u t a o ro n o n n c e d c l e a r i n g
( t r a n s l u c e n c y ) o f th e m edium .
T h re e s t r a i n s C966U, 2 9 0 8 , M-1 ) o u t o f
t h e s i s w h ic h show ed no d i s c e r n i b l e S to n e r e a c t i o n w i t h i n UR h o u r s d id
s o w hen o b s e r v e d a t
96
h o u r s an d n i n e d a y s .
Of t h e t h r e e s t r a i n s w h ic h
f a i l e d t o g iv e t h e S to n e r e a c t i o n , S - l 6 , a n d S-31B d i d n o t l i q u e f y
g e l a t i n , mad M»2 v a e a s lo w l l q u e f l e r . 1
A l l e i g h t e e n s t r a i n s c o e ,g a le te d d e f i b r t n & t e d r a b b i t w h o le b lo o d
to a c e rta in e x te n t.
F o r t h e m o st p a r t i t c a n b e assum ed t h a t a l l
s t r a i n s w e re i s o l a t e d fro m p a t h o l o g i c a l s o u r c e s ( s t r a i n s M -I, M-P, M -3,
r n d M-U *m re o b t a i n e d fro m s t o c k c u l t u r e s w h ic h h ad no h i s t o r y o f i s o l a ­
tio n so u rc e ).
Chapman ( 193U) <md C ru lc k a h a n k (1 9 3 7 ) fo u n d t h a t a h i g h
c o r r e l a t i o n e x i s t s b e tw e e n t h e a b i l i t y o f m ic r o c o c c i t o p r o d u c e
o o a g u la e e and t h e i r p a t h o g e n i c i t y , b u t s u b s e q u e n t e x p e r im e n ts c a r r i e d
o u t b y o t h e r w o r k e rs ( J o r d a n a n d B u rro w s, 1935? Kvrochlk, 1937$ 1J o la a a
and b i l e o n , 1939? end H ussem nnn and T e n n e r, I 9U9 ) c o u ld n o t s u b s t a n t i a t e
th is
T a b le T I c o m p a res t h e e n t e r o t o r l c l t y on c a t s o f t h e e i g h t e e n
s t r a i n s w i t h t h e p h y s i o l o g i c a l r e a c t i o n s w h ic h v a r i o u s w o r k e r s h a v e
t h o u g h t a t one tim e o r a n o t h e r t o he s i g n i f i c a n t i n r e g a r d t o f o o d o o i s o n i n g m ic r o c o c c i.
( 172,
The t h r e e s t r a i n s fo u n d e n t e r o t o x i c t o c a t e
« 325. »4-5) l i o n e f l e d g e l a t i n , f e r m e n te d l a c t o s e a n d m a n n i to l ,
w e re c o r .g u l y t i c , f i b r i n o l y t i c ,
m d h e m o l y ti c , and p ro d u c e d a p o s i t i v e
S to a e r e a c t i o n w i t h i n 43 h o u r s .
To u r s t r a i n s ( 1 7 2 , 19*5, 3 - 2 9 5 , 2 2 2 0 -1 3 ) o f t h e r e m a in in g
f i f t e e n w h ic h w e re a o n - e n t e r o t o x i g e n i c t o c a t s p r o d u c e d t h e same
p h y s io lo g ic a l r e a c tio n s .
The r e m a in d e r o f t h e s t r a i n s d i f f e r e d by
b e in g n e g a t i v e i n o n e o r more o f th e u h y s i o l o g i c a l r e a c t i o n s .
If
th e s e s t r a i n s h a d o r i g i n a l l y b e e n a b l e t o p r o d u c e t h e s e r e a c t i o n s ,
th e y h ad a p p a r e n t l y l o s t t h i s a b i l i t y a f t e r b e i n g c u l t u r e d on a r t i f i c i a l
m e d ia f o r f a i r l y lo n g p e r i o d s o f tim e s i n c e t h e i r o r i g i n a l i s o l a t i o n .
I t i s t o b e n o t e d t h a t th e t h r e e s t r a i n s w h ic h p ro d u c e d f o o d - p o i s o n in g
sym ptom s i:i c a t s h a d b e e n c u l t u r e d on a r t i f i c i a l m ed ia f o r a s h o r t e r
tim e th a n m o st o f t h e r e s t o f t h o s e i s o l a t e d fro m f o o d - n o l e o n in g c a s e s .
The s i x e t r a i n s f u r n i s h e d by S to n e ( 1 9 ^ 9 ) w ere a l l f o u n d t o be
c o & g a la e e p o s i t i v e when t e s t e d i n h i e l a b o r a t o r y i n 1 9 4 2 .
c o n fir m e d t h i s r e a c t i o n .
O ur r e s u l t s
The S to n e r e a c t i o n h e f o u n d f o r t h e s i x s t r a i n s
c o rre sp o n d e d w ith o u rs w ith one e x c e p tio n .
Ee h a d fo u n d s t r a i n S - J lB t o
g i v e s. p o s i t i v e S to n e r e a c t i o n w h e re a s i t g a v e a n e g a t i v e S to n e r e a c t i o n
i n t h i s s tu d y .
B ack (1 9 4 8 ) had fo u n d M s t h r e e s t r a i n s ( 1 7 2 . 1 7 8 , I S 6)
t o b e t o x i c t o monkey® s h o r t l y a f t e r i s o l a t i o n .
e x o e rim e n t we fo u n d o n ly s t r a i n
H ow ever, i n th e o r e s e n t
1J8 t o b e e n t e r o t o x i c t o c a t e .
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-3 0 Cate are presum ably a s s e n s i t i v e to e n te r o to x ln am are aon keye, so the
e n t e r e to x lo a b i l i t y o f th e o th er two s t r a in e (w hich produced no r e a c tio n
i n c a t e ) m e t have been l o s t .
-he o n ly c o n c lu s iv e t e s t fo r e n te r o to x ln would have to be c a r r ie d
out u s in g human v o lu n t e e r s .
However, as Hueeemann and Tanner (19^9)
brought o u t, th e w hole f i l t r a t e s o f m icro co cci thought to be the source
o f fo o d -p o is o n in g sh ould be u se d to t e s t t h e i r p a th o g e n ic ity , an a l l the
m e ta b o lic product;; o f the m icr o co cci are in g e s te d w ith th e contam inated
fo o d .
Seme m icr o co cci produce l e t h a l to x in , derm atoxin, sad hom olysine
as w e ll as e n t e r o t o x ln , and th e s e co u ld be f a t a l i f in g e s t e d .
T h erefo re,
ex p erim en ta l an im als must of n e c e s s it y be u s e d , even i f the e l i c i t i n g o f
fo o d -o o ie o n in g symptoms w ith in them cannot be co n sid ered as c o n c lu s iv e to
e n te r o to x ln .
At the p r e se n t tim e c a t s are th e moot commonly u sed anim al
to t e s t for e n t e r o t o x ln .
I t would be o f advantage to f in d a more s u it a b le
and a l , however, f o r c a t s are d i f f i c u l t t o m ain tain end h a n d le, and they
d ev elo p an I n c r e a s in g to le r a n c e t o e n te r o to x ln a f t e r s e v e r a l a tta c k s o f
fo o d -p o is o n in g .
A lthough Bolinan and W ilson (1938) dem onstrated & s p e c i f i c f l e c u l a tio v r : a c tio n betw een e n te r o to x ln and i t s corresp on d in g a n tib o d y o b tain ed
from ra b b it im m unization, no v i s i b l e s e r o lo g i c a l r e a c tio n was observed in
t h i s exp erim en t.
A fte r a fo u r w eeks' im m unization p e r io d , no r e a c tio n
w h atsoever was ob served betw een v a r io u s amounts o f homologous e n te r o to x ln
and r a b b it serum run in th e p r e c ip it a t io n t e s t .
M. Hammon (19^1) was
a ls o u n ab le to o b ta in any v i s i b l e s e r o lo g i c a l r e a c tio n betw een homologous
-31e n te r o to x ln and e e r w .
Workers a t v a r io u s tim es have th o u ^ it t h a t some p h y s io lo g ic a l
r e a c tio n s c h a r a c te r is e d food—p o is o n in g m ic r o c o c c i.
However, subsequent
exp erim en ts by o th e r workers on th e s e have n o t le d to c o n fir m a tio n .
Jordan and Barrows (1 9 3 5 ) , Chinn (1 9 3 6 ), Kupchik (1 9 3 7 ), Dolman and Wil­
son (1 9 3 8 ), Hamaon (19%1), Haynes and Stacker ( I 9U6 ) , Stone (1 9 ^ 9 ). and
Huseemann and Tazmer (19^ 9) co u ld n o t fin d any p h y s io lo g ic a l c h a r a c t e r is t ic
w h o lly s u c c e s s f u l in s e p a r a tin g the e n te r o to x in -fo n a in g from th e nona n te r o to x in -fo r m in g m ic r o c o c c i.
The r e s u l t s o f the p r e s e n t experim ent
in d ic a t e th e same c o n c lu s io n .
SUMMARY
E ig h teen s t r a in s o f m ic r o c o c c i, te n o f which had been I s o la t e d
fr o 1 c a s e s o f fo o d -p o is o n in g , were s tu d ie d a s to t h e ir p h y s io lo g ic a l
r e a c t io n s and th e a b i l i t y o f t h e i r b o ile d f i l t r a t e s to in vok e e n te r o t o x i c i t y in c a t s upon in tra v en o u s i n j e c t i o n .
An attem pt was made to
o b ta in a v i s i b l e s e r o lo g i c a l r e a c tio n betw een one o f th e s t r a in s (S -3 ? 5 )
found to e l i c i t v o m itin g in c a t s , and i t s homologous serum o b ta in ed by
r a b b it im m unization.
A ll but one o f th e s t r a in s gave th e same p h y s io lo g ic a l r e a c tio n s
and m o rp h olog ica l c h a r a c t e r is t ic s a s l i s t e d f o r M icrococcus pyogenes
v a r . aureus in the s i x t h e d it i o n o f S e r g e y 's Manual, e x ce p t th a t none
o f them was ob served to produce hydrogen s u l f i d e .
The s t r a i n (M -l)
d i f f e r i n g from th e r e s t gave a b a s ic in s te a d o f a c id ic r e a c t io n on litm u s
and brom c r e s o l p u rp le m ilk , f a i l e d to ferm ent su c r o se , g a la c t o s e , la c t o s e ,
and m a n n ito l, and proved to have c e l l s o f tw ic e the diam eter o f the oth er
-3 2 e tra ia e .
B re n w i t h t h e s e d i s c r e p a n c i e s , s t r a i n M -I i s c l o s e r t o th e
s-o e c ie e o f M ic ro c o c c n s pyo--enee f a r . a u r e n s t h a n t o a n y o t h e r
o rg a n is m c l a s s i f i e d i n S e r g e y 's M an u al.
T h re e o f t h e s t r a i n s ( 1 7 8 , 8-325» M -5) w ere fo u n d t o h e e n t e r o t o r l c
to c a ts .
Two o f t h e s e ( 1 7 8 , S -3 2 5 ) w e re i s o l a t e d fro m c a s e s o f f o o d -
o o i s o n i n g , w h e re a s s t r a i n M-5 w as i s o l a t e d fro m th e u r i n e o f an a d u l t
w ith a p r o s t a t i c i n f e c t i o n .
T h ese t h r e e s t r a i n s , i n a d d i t i o n t o h a v in g
a l l t h e p h y s i o l o g i c a l c h a r a c t e r i s t i c s l i s t e d f o r M ic ro c o c c u s p y o g e n e s
v a r . a u r e u s , w e re c o a g u l y t i c , f i b r i n o l y t i c , h e m o l y ti c , and g a v e a
p o s i t i v e S to n e r e a c t i o n i n Hg h o u r s .
F o u r o t h e r s t r a i n s ( 1 7 2 , 196,
S - 296 , 2 2 2 0 -1 8 ) g a v e t h e s e sam e c h a r a c t e r i s t i c s h u t w e re n o t e n t e r o t o x l c
to c a ts .
The r e m a in in g e le v e n s t r a i n s d i f f e r e d hy n o t g i v i n g one o r
m ore o f t h e s e r e a c t i o n s .
No v i s i b l e
s e r o l o g i c a l r e a c t i o n was o b s e rv e d b e tw e e n v a r i o u s
a m o u n ts o f e n t e r o t o x l c f i l t r a t e a n d h o m o lo g o u s r a b b i t serum r u n i n th e
p re c ip ita tio n t e s t .
I t was f o u n d t h a t i n c u b a t i o n a t 3 0 ° C b r o u g h t a b o u t a more r a p i d
l i q u e f a c t i o n o f g e l a t i n by t h e o rg a n is m s t h a n i n c u b a t i o n a t 3 7 ° C.
It
was a l s o o b s e r v e d t h a t t h e l e n g t h o f tim e t h e t u b e s o f g e l a t i n w ere h e ld
i n t h e r e f r i g e r a t o r a f t e r e a c h i n c u b a t i o n i n f l u e n c e d th e o b s e r v a b l e
l i q u e f a c t i o n i n some o f t h e t u b e s .
The S to n e r e a c t i o n was v e r y d i f f i c u l t to a s c e r t a i n f o r th e m ost
p a rt.
Many o f t h e s t r a i n s w h ic h w ere c o n s i d e r e d p o s i t i v e m e r e ly showed
a v e r y n a rro w gone o f d e c r e a s e d o p a c i t y a d j a c e n t t o g ro w th on th e p l a t e s ,
o n l y tw o o f t h e s t r a i n s b r o u g h t a b o u t a p ro n o u n c e d c l e a r i n g .
Some o f t h e
s t r a i n s w h ic h show ed no d i s c e r n i b l e S to n e r e a c t i o n w i t h i n Ug h o u r s d id so
-33w hen o b s e r r s d a t 96 h o u r s and n i n e d ays.
The r e s u l t s o f t h e s e e x p e r im e n ts l e a d one to t h e sam e c o n c lu s i o n
r e a c h e d by m o st o f t h e o t h e r w o rk e rs on f o o d - p o i s o n in g m ic r o c o c c i,
n a m e ly , t h a t a t p r e s e n t t h e r e i s no p h y s i o l o g i c a l c h a r a c t e r i s t i c w h o lly
s u c c e s s f u l i n s e p a r a t i n g th e e n t e r o t o x i n - f o r m i n g from t h e n o n - e n t e r o t o x i n - f o r m i n g m ic r o c o c c i.
LITERATURE CITED AND CONSULTED
S e r g e y 's m an u al o f d e t e r m i n a t i v e b a c t e r i o l o g y .
a n d W ilk in s Company, B a l ti m o r e , M a ry la n d .
I 9U8 .
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________________
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w h o le b lo o d f o r t e s t i n g th e c o a g u l a t i n g mower o f e tam h y I o c o c c i .
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~
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