A study of corynebacteria isolated
by B Bayliss
A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
of Master of Science in Bacteriology
Montana State University
© Copyright by B Bayliss (1951)
Abstract:
The history of the disease, diphtheria, is discussed, especially in the light of recent epidemics. Throat
cultures from 200 Montana State College students were studied to determine the number carrying
diphtheria or diphtherialike organisms. Thirteen organisms were isolated and compared
morphologically and physiologically with 14 known cultures of Corynebacterium diphtherias,
submitted by Frobisher and Galbraith, that had been cultured from the throats of patients with
diphtheria infections. All these organisms varied in some characteristic, so no serious attempt to
classify them was made. Five strains appeared very similar, to known cultures of C. diphtherias type
minimus. The other eight differed in at least one character from any of the corynebacteria described in
Bergey's Manual. The advantages and disadvantages of Loeffler's and Pai's media for primary
culturing, of the tellurite medium employed and of "chocolate" agar for purification are presented. The
Schick reactions of the students from whom these cultures had been obtained were determined, but
being negative, no further work was done. Cultures were made also from the throats of students with
pharyngitis, but no diphtheria or diphtherialike organisms were found. Possible reasons for this are
discussed.
A STUDY.OF OORYNEBAOTERIA ISOLATED FROM
HUMAN THROATS
by
' BERENICE G, BAYLISS
A THESIS
S u bm itted t o th e G raduate F a c u lty .
in
p a r t i a l f u l f i l l m e n t o f th e re q u ire m e n ts
f o r th e d eg ree of
M aster o f S cience i n B a c te rio lo g y
at
Montana S ta te C o lleg e
Approved:
M ajor D epartm ent
Chairman, Examining Committee
Jfe/m , iGrafdudte
ix v isio n
■Bozqman,. Montana
lJu n e, 1951
A/37/
J? 34-3 s
-2 TABLE OF CONTENTS
ACKNO15JLEDGEMHtTS
3
ABSTRACT
U
INTRODUCTION
5
REVIEW OF LITERATURE
H i s t o r i c a l background
D e s c rip tio n of Coryneba c t erium d ip h th e r ia e
M alig n an t d ip h th e r ia
C. d ip h th e r ia e ty p e minimus
D ip h th e ria and im m unization
D ip h th e ria c a r r i e r su rv e y s
Types of m edia u sed
V iru le n c e t e s t i n g of C. d ip h th e ria e
6
7
10
11
12
13
111
16
METHODS AND MATERIALS
P r e p a ra tio n
Survey
P u rific a tio n
C u ltu ra l s tu d ie s
V iru le n c e
21
22
23
25
EXPERIMENTAL RESULTS
Number of c u ltu r e s o b ta in e d
Comparison o f th e P a i 's and L o e f f l e r 's media
G rouping o f th e organism s
C l a s s i f i c a t i o n of groups
R e s u lts o f th e S chick t e s t s
B a c te r ia l grow th on th e t e l l u r i t e medium used
26
27
27
30
35
35
DISCUSSION
36
SUMMARY
39
REFERENCES
hi
20
100893
ACKNOWLEDGEMENTS
The a u th o r w ishes to e x p re s s h e r g r a titu d e to D r. C. W. Hammer* Mra
•W. G. W alter* and Dr. R. H. McBee* under whose d i r e c t i o n t h i s i n v e s t ig a ti o n
was perform ed* f o r t h e i r a s s is ta n c e and encouragem ent d u rin g th e c o u rse o f
t h i s . s t u d y . She" a ls o w ish es t o th a n k th e n u rs in g p e rs o n n e l o f th e S tu d e n t
H e a lth S e rv ic e a t Montana S ta te C ollege* th e s t a f f o f t h e V e te rin a ry Re­
s e a rc h L aboratory* and th o s e who a s s i s t e d i n th e f i n a l ty p in g of t h i s
t h e s i s f o r t h e i r c o o p e ra tio n .
ABSTRACT
The h i s t oiy o f th e d i s e a s e , d ip h th e r ia , i s d is c u s s e d , e s p e c ia ll y in
th e l i g h t o f r e c e n t e p id e m ic s . T hroat c u ltu r e s from 200 Montana S ta te
C o lleg e s tu d e n ts Tivere s tu d ie d t o d eterm in e th e number c a r r y in g d ip h th e r ia
o r d i p h t h e r i a l i k e org an ism s. T h irte e n organism s w ere i s o l a t e d and com­
p ared m o rp h o lo g ic a lly and p h y s io lo g ic a lly w ith llr known c u ltu r e s o f
Co ry n eb acteriu m d i p h t h e r i a s , .su b m itte d by F r o b is h e r and G a lb r a ith , t h a t had
been c u ltu re d from th e t h r o a t s o f p a t i e n t s w ith d ip h th e r ia i n f e c t i o n s . A ll
th e s e organism s v a r ie d i n some c h a r a c t e r i s t i c , so .n o s e r io u s a tte m p t to
c l a s s i f y them was made. F iv e s t r a i n s a p p ea re d v e ry sim ilar, to known c u l­
tu r e s o f C. d ip h th e r ia e ty p e m inim us. The o th e r e ig h t d i f f e r e d i n a t
l e a s t one c h a r a c te r from any o f th e c o ry n e b a c te ria .d e sc rib e d i n B ergey1s
M anual. The ad v an tag es and d is a d v a n ta g e s o f L o e f f l e r 's and F a i 1s media
f o r prim ary c u ltu r in g , o f th e t e l l u r i t e medium employed and o f "c h o c o la te "
a g a r f o r p u r i f i c a t i o n a re p re s e n te d . The S ch ick r e a c tio n s of th e s tu d e n ts
from whom th e s e c u ltu r e s had been o b ta in e d w ere d e te rm in e d , b u t b ein g
n e g a tiv e , no f u r t h e r work was done. C u ltu re s w ere made a ls o from th e
t h r o a t s of s tu d e n ts w ith p h a r y n g itis , b u t no d ip h th e r ia o r d ip h th e r ia lik e
organism s w ere fo u n d . P o s s ib le re a so n s f o r t h i s a re d is c u s s e d .
-5 A STUDY OF CORYNEBACTERIA ISOLATED FROM HUMAN THROATS
INTRODUCTION
.
D ip h th e ria was once c o n sid e re d t o be a d is e a s e whose c h ie f s u f f e r e r s
Tfere c h ild r e n , p a r t i c u l a r l y th o se o f p re s c h o o l age.
I n some o f th e more
r e c e n t ep id em ics, how ever, a t t e n t i o n has been drawn to th e number of th o se
a f f l i c t e d who w ere o f c o lle g e age or o ld e r .
A lthough s e v e r a l surveys
have b een made t o determ in e w heth er c h ild r e n were c a r r i e r s o f d ip h th e r ia
o rg an ism s, v e ry few such i n v e s t ig a ti o n s have been conducted on c o lle g e
s tu d e n ts .
T h e re fo re , i t was f e l t t h a t i n view o f the s h i f t in age of
th o s e c o n tr a c tin g d ip h th e r ia , i t would be v a lu a b le t o d eterm ine th e per=
c e n ta g e o f an u n s e le c te d group o f fresh m en s tu d e n ts a t M ontana S t a t e ■
C o lle g e Titiio h a rb o re d d ip h th e r ia o r d i p h th e r ia lik e organism s.
As a p a r t o f t h e i r Freshman Week p h y s ic a l e x a m in a tio n , each s tu d e n t
i s g iv e n a S ch ick t e s t .
Those showing a p o s i t i v e r e a c tio n a re g iv e n th e
s ta n d a rd im m unization in o c u la tio n s a g a in s t d ip h th e r ia .
S in ce t h e inform a­
t i o n c o n ce rn in g th e r e s u l t s o f th e S ch ick r e a c tio n whs a v a il a b le , i t was
plan n ed t o r e c u l t u r e th e t h r o a t s o f th o s e s tu d e n ts showing a p o s i t i v e
■
S chick r e a c t i o n , and from whom c o ry n e b a c te ria had been i s o l a t e d to d e t e r mine th e e f f e c t o f t h e i r im m unization on th e f l o r a of th e t h r o a t .
T h is' was
to be done ap p ro x im a te ly s i x t y days a f t e r th e c o m p letio n o f th e immuniza­
tio n .
I n th e c u l t u r a l d ia g n o s is o f d i p h th e r ia , Loef i l e r ' s b lo o d serum has.
commonly been employed s in c e i t was f i r s t d e s c rib e d by L o e f f le r i n l8 8 iu
McGnigan and F r o b is h e r (1936) m entioned t h e u se o f P a i 's medium a^ a sub­
s titu te .
I n t h e i r o p in io n th e l a t t e r was as r e l i a b l e a medium- as L o e f f l e r 1
and s im p le r to p r e p a r e » I t w as, th e r e f e r e> d e cid e d to com pare th e s e media
when c u ltu r in g th e organism s from th e s tu d e n ts * t h r o a t s .
Some c o n tr a d ic to r y s ta te m e n ts a re c o n ta in e d i n th e l i t e r a t u r e as t o
w h eth er th e d ip h th e r o id s , organism s m o rp h o lo g ica lI y resem b lin g
Goryn e b ac te riu m d ip h th e r ia s (F lugg e) Lehmann and Neumann, b u t w ith d i f f e r ­
e n t p h y s io lo g ic a l p r o p e r t i e s , ace i n h i b i t e d by th e p o ta ssiu m t e l l u r i t e
employed i n th e m edia now w id e ly used f o r th e i s o l a t i o n and i d e n t i f i c a t i o n
o f G. d ip h th e r ia e .
There i s a ls o a q u e s tio n as to w hether th e C. d ip h th e -
r i a e p o p u la tio n o f a p p a r e n tly normal t h r o a t s d i f f e r s from t h a t of th r o a t s
showing p a th o lo g ic a l c o n d itio n s .
The p r e s e n t stu d y was u n d e rta k e n t o
o b ta in f u r t h e r in fo rm a tio n on t h e s e p o i n t s .
REVIEW OF LITERATURE
H i s t o r i c a l Background
'
D ip h th e ria was e s ta b lis h e d as a c l i n i c a l e n t i t y i n 1826 by B retonneau
o f T o u rs, F rance (Sm ith and M a rtin , 191+8).
I t .was n o t, how ever, u n t i l
1883 t h a t th e d ip h th e r ia organism was d e s c rib e d by K lebs i n sm ears from
th e membrane i n t h e th r o a t s o f p a t i e n t s w ith th e d is e a s e .
L o e f f le r i s o -
l a t e d th e organism i n a pure c u ltu r e i n t h e fo llo w in g y e a r .'
■
For t h i s
re a s o n G orynebacterium d ip h th e r ia e (F lugge) Lehmann and Neumann i s o fte n
r e f e r r e d t o a s t h e K le b s -L o e ffle r b a c i l l u s .
Form erly t h e r e was some doubt
as to th e r e l a t i o n s h i p o f th e s e organism s t o d ip h th e r ia , s in c e th e y were
a ls o o c c a s io n a lly found i n t h e th r o a ts o f norm al p eo p le and w ere n p t always
p r e s e n t i n pseudomembranes.
Some o f th e sy ste m ic m a n ife s ta tio n s - of th e
i n f e c t i o n w hich a r e now known t o be due t o th e acjtioh p f th e ,d ip h th e ria
-7 to x in w ere a ls o a cause o f c o n fu s io n .
The d is c o v e ry of. th e e x o to x in by
Roux and Y e rsin i n 1888 e lim in a te d th e s e d o u b ts and e s ta b lis h e d th e e t i ­
o lo g ic a l r e l a t i o n s h i p o f C, d ip h th e r ia s to d i p h th e r ia .
A m a lig n a n t form o f .d ip h th e ria , ap p eared i n F rance sometime betw een
1850 and i8 6 0 (Sm ith and M a rtin , 19U8) and sp re a d over th e w o rld , re a c h in g
a peak i n London i n l87lj..
This v i r u l e n t form was found i n B oston i n 1872,
where th e d e a th r a t e s in c re a s e d from 35 p e r 100,000 i n 1872 t o 218 p e r
100,000 i n 1881.
S m aller epidem ic waves r e c u r r in g a t f i v e to te n y e a r
i n t e r v a l s w ere re c o rd e d th ro u g h o u t t h e w o rld from 1880 t o 1930.
S ince
th e n th e d e a th r a t e from d ip h t h e r i a h as d e c lin e d , even i n t h e c o u n trie s
w here p r o p h y la c tic im m unization has n o t b e e n employed, as i n Norway.
In
New York t h e d e a th r a t e dropped from 1 ,1 7 5 and 1,005 p e r 100,000 i n 1881
and 1887, r e s p e c t i v e l y , t o a lm o st none i n 1935»
This d e c lin e i n th e num­
b e r o f d e a th s wad a t t r i b u t e d to d ip h th e r ia a n tito x in w hich was made a v a i l ­
a b le to e v e ry d ip h th e r ia p a t i e n t b y .1900 and t o mass im m unization w hich
was s t a r t e d i n 1915.
(A mord d e t a i l e d h i s t o r y o f d ip h th e r ia can be found
i n Smith and M a rtin , 19U8.)
D e s c rip tio n o f C orynebacterium d ip h th e r ia s
C orynebacterium d ip h th e r ia s i s a non-m oti l e , gram p o s itiv e r o d , o c c u rr i n g 's i n g l y , and v a ry in g g h e a tly i n s i z e (B rped, e t a l . , 19U8). - The a rra n g e ­
ment o f t h e c e l l s i s e s p e c i a l l y c h a r a c t e r i s t i c .
Ih e n th e c e ll s d iv id e , t h e
c e l l membrane g iv e s way on one s i d e . • T his r e s u l t s in , a sn ap p in g p o s tf i s s i o n movement and i n th e fo rm a tio n o f a p a lis a d e o r chine Se l e t t e r arrangem ent .
F r o b is h e r (19lt9) f e e l s t h i s to be "an e s p e c i a l l y u s e f u l
-6 d ia g n p s tic f e a t u r e " /
However, 'B is s e t (1950) r e f e r s t o t h i s p o s t - f i s s i o n
movement as b e in g "an i n t e r e s t i n g b u t q u ite u n im p o rtan t a r t i f a c t 11= U sual­
l y th e ro d s a r e s t r a i g h t , b u t o fte n th e y appear to be s l i g h t l y curved and
f r e q u e n tly a re sw o llen a t one o r b o th e n d s.
The ro d s do n o t, a s a r u l e ,
s t a i n e v e n ly w ith m ethy]ehe b lu e , b u t show a l t e r n a t e bands o f s ta in e d and
u n s ta in e d m a t e r i a l o
Very o fte n m etachrom atic g ra n u le s a p p e a r.
These a r e
r e f e r r e d t o a s p o la r b o d ie s when th e y a re lo c a te d a t th e ends o f th e
organism .
These g ra n u le s a re a ls o term ed a r t i f a c t s by B is s e t (19!?0) as
th e y a re found o n ly i n d r ie d and s ta i n e d p r e p a r a tio n s ,
He b e lie v e s them
t o be " d rie d a g g re g a te s o f n u c le a r and, p ro b a b ly , r e s e rv e fo o d m a te r ia l" .
J u s t as th e organism s v a ry g r e a tly i n m orphology, so t h e colony
form s v a ry .
These v a r i a n t s have b een d e s c rib e d as fo llo w s :
1.
Smooth (S) w ith a g l i s t e n i n g s u r f a c e and even e d g e s .
2.
Rough (R) h aving a d u l l s u r f a c e and uneven m a rg in s.
3.
I n te r m e d ia te s , w hich in c lu d e
a . Sr., which more n e a r ly re sem b le s th e pure S form .
b . r S , w hich a re v ery s im ila r to th e R.
c . SR c o lo n ie s w hich show p r o p e r t i e s t h a t a re n o t l i k e e i t h e r
S o r R.
Iu
•
Dwarf (D) w ith c o lo n ie s so sm a ll t h a t m a g n ific a tio n i s re q u ire d
t o make them v i s i b l e to t h e human ey e.
Based upon th e s e v a r i a t i o n s i n morphology o f th e organism i t s e l f
and ihe d i f f e r e n t colony fo rm s, s e v e ra l, ty p e s o f G. d ip h th e r ia s have
been d e s c rib e d and named.
The fo u r ty p e s m ost u n iv e r s a ll y re c o g n ize d a re
b r i e f l y d e s c rib e d i n t a b l e I ,
-9 -“
.
T able I
Types o f C orynebacterium d ip h th e r ia e
Type
:
Colony forms
Morphology
g ra v is
s h o r t ro d s
u n ifo rm ly s ta i h in g
SR
m itis
lo n g ro d s
m etachrom atic g ra n u le s
S
in te rm e d in s
lo n g ro d s
b a rre d forms
clubbed ends'
sR
minimus
lo n g ro d s
b a rre d forms
clubbed ends
D
S e r o lo g ic a l in v e s t i g a t i o n h as shown th a t- th e s e ty p e s a re a ls o a n t i ­
genic a l l y d i s t i n c t from one a n o th e r (Jo rd o n and Burrows, 19il7) .
Some
■
a u t h o r i t i e s re g a rd th e gravi'Sr, m i t i s , in te rm e d in s and m inim us, n o t as ty p e s
b u t r a t h e r a s s u b s p e c ie s o f Co d ip h th e r i a e »
Both -v iru le n t and a v i r u l e n t
s t r a i n s have b e e n i s o l a t e d i n a l l of th e ty p e s m entioned,
how ever, F ro b is h e r
e t a l . , (1951) r e p o r ts t t |a t s t r a i n s o f th e minimus ty p e produce only
f a i n t in tr a d e rm a l r e a c tio n s i n r a b b i t s -vixen t h e i r v iru le n c e i s t e s t e d by
s ta n d a rd p ro c e d u re s , b u t t h a t i n , t h e a g a r—p la te method o f t e s t i n g v ir u l e n c e ,
n o t one minimus s t r a i n h as e v er been found t o b e n o n - v ir u le n t.
The v iru le n c e o f th e d ip h th e r ia organism i s c lo s e ly c o r r e l a t e d w ith
th e p ro d u c tio n o f a p o te n t e x o to x in .
S tu d ie s by P o v its k y , E is n e r , and
Jack so n (1933) showed ttjab a l l d ip h th e r ia to x in s a re s i m i l a r .
I f th e y
-1 0 d i f f e r e d , i t was o n ly i n th e degree o f t o x i c i t y , n o t i n th e k in d .
The
g r a v is s t r a i n i s o l a t e d d u rin g th e d ip h th e r ia epidem ic a t H a lif a x , Nova
S c o tia i n 19Ul5 when grown i n a medium c o n ta in in g fo u r m icrogram s o f ir o n
p e r ml o f c u ltu r e f l u i d , s y n th e s iz e d te n tim e s a s much t o x i n ..as o th e r
s tra in s te s te d .
M alig n an t d ip h th e r ia
There i s a p o s s i b i l i t y t h a t in th e f u tu r e a ty p e o f d ip h th e r ia may
be e n co u n tered a g a in s t which our p re s e n t methods o f p r o te c tio n w i l l n o t
p r e v a i l . , T h is s e v e re ty p e o f d i p h th e r ia has a lr e a d y ap p eared 'on th e
European c o n tin e n t, and s p re a d to th e B r i t i s h I s l e s about 1931.
This
m a lig n a n t, h y p e rto x ic , o r grave d i p h th e r ia d i f f e r s from th e o rd in a ry
d ip h th e r ia by th e p re se n c e o f marked c e r v ic a l s w e llin g , extreme-' to x e m ia ,
and a lb u m in u ria .
There i s f r e q u e n tly a developm ent, o f n e u ro p a th ie s and a
v e ry h ig h d e a th r a t e , i n s p i t e o f e a r ly and la r g e doses o f a n t i t o x i n
(Updyke and F r o b is h e r , 19U7)«
The f i r s t o u tb re a k o f t h i s m a lig n a n t d ip h th e r ia t o a p p ea r on th e
N orth American c o n tin e n t, o c c u rre d i n H a lif a x in 19UO and 19U l.
I t has
been s u sp e c te d t h a t t h i s o u tb re a k was cau sed by a Norwegian s a i l o r from a
w haling v e s s e l .
W heeler and Morton (1912) r e p o r t t h a t i n t h i s epidem ic a
h ig h p e rc e n ta g e o f c a s e s were in th e o ld e r age group.
They su g g est th a t
t h i s m ight be th e r e s u l t o f t h e im m u n iz a tio n ' o f most o f th e sch o o l c h ild r e n
A somewhat s im ila r o u tb re a k o f d ip h th e r ia o ccu rred i n B a ltim o re ,
M aryland i n 1911.
D uring the f i r s t s ix months o f t h a t y e a r th e m o r t a lit y
r a t e from o rd in a ry d ip h th e r ia was s i x p e r c e n t, w h ile t h a t from th e
-1 1 m a lig n a n t d ip h th e r ia -was UU p e rc e n t.
I t was f e l t by some t h a t t h i s se ­
v e r i t y might be d u e, n o t t o the d ip h th e r ia organism a lo n e ? b u t to i t s
s y n e r g is ti c a c tio n w ith some o th e r organism , p o s s ib ly s tr e p to c o c c i.
E a rly
work by Updyke and F r o b is h e r (19UU) seemed t o p o in t tow ard h em o ly tic
s t r e p t o c o c c i 'o f th e L a ic e f ie ld Group B, a s s o c ia te d w ith d ip h th e r ia organ­
ism s o f th e m i t i s ty p e .
However, f u r t h e r w ork, w ith a n im a ls, a ls o done
by Updyke and F r o b is h e r (19U7) d id n o t su p p o rt t h i s s u p p o s itio n , and th e y
f e l t t h a t th e e tio lo g y o f t h i s m a lig n a n t ty p e o f d ip h th e r ia was s t i l l
u n c e r ta in .
A few i n v e s t i g a t o r s have t r i e d to d eterm in e w h eth er a c e r t a i n ty p e
o f C. d ip h th e r ia e m ight n o t be r e s p o n s ib le f o r t h i s grave o r m a lig n a n t
d i p h th e r ia .
A nderson, H appold, McLeod, and Thomson (1931) f e l t th a t
i n t h e epidem ic w hich o c cu rred i n L eed s, E ngland, C. d ip h th e r ia e ty p e
g ra v is was a s s o c ia te d w ith th e s e v e re and to x ic c a se s of th e d is e a s e and
th e m-i t i g ty p e w ith th e m ild e r c a s e s.
F ro b is h e r (19U3) has found, how­
e v e r , many s t r a i n s o f G. d ip h th e r ia e ty p e g ra v is in t h e U n ited S ta te s
w hich w ere c o m p letely a to x lg e n ic .
K ello g g -an d Wende (19U6) su g g este d th e p o s s i b i l i t y t h a t r e tu r n in g
tro o p s from Europe m ight b rin g b ack s t r a i n s o f th e s e more v i r u l e n t form s .
o f d ip h th e r ia organism s t h a t a re p r e v a le n t i n N o rth e rn E u ro p e, and t h a t
th e p o t e n t i a l t h r e a t s t i l l made d ip h th e r ia a problem t h a t w a rra n te d
a tte n tio n .
G o ry n eb acterium d ip h th e r ia e ty p e minimus
I n 19U7 and 19U8 an epidem ic o f d i p h th e r ia ap p eared i n U tah w ith
s e v e r a l u n u su a l f e a t u r e s .
The f i r s t was t h a t th e r a t h e r r a r e
-1 2 G. d ip h th e r ia e ty p e minimus was i s o l a t e d from 35 p e rc e n t of. th e cases
(J e n k in s , l5 1 |8 ),
-The second was th e la r g e number o f c a s e s am ong,school
c h ild r e n and o ld e r a d u lts .
I t was fo u n d t h a t th e c a r r i e r r a t e o f t h i s
minimus ty p e was. a s h ig h as 21.5 p e rc e n t i n a c o lle g e in Utah from which no
p re v io u s c a s e s had been r e p o r te d .
B ram hall, G a lb ra ith ,, and F r a s e r (19U8) i n t h e i r r e p o r t of th e Utah
ep id em ic, s t a t e t h a t C. d ip h th e r ia e ty p e minimus seems t o be o c c u rrin g
more commonly i n t h e U nited S t a t e s , h av in g been found a ls o i n Id a h o ,
G eo rg ia, M aryland, and Kentucky-.
A t h i r d f e a t u r e o f t h i s epidem ic was t h a t o f th e s e minimus cases r e ­
p o rte d , 58 p e rc e n t had been immunized a g a in s t d ip h th e r ia .
D ip h th e ria and im m unization
F r o b is h e r (19U0) p o in ts o u t t h a t d ip h th e r ia e p id em ics o f a fu lm in ­
a tin g n a tu r e , which seem to p ro g re s s r e g a r d le s s of im m unity, a re n o t un­
common.
T his may be because d ip h th e r ia a n t i t o x i n im m unity, r e g a r d le s s of
the- immunizing p ro c e d u re , i s n o t perm anent a s re p o rte d t y V olk e t a l. (19U2) s
and t h a t a s tim e goes on, th e a n t i t o x i n c o n te n t of th e b lo o d i s red u ced .
<l
Volk adv o cated th e re im m u n iz a tio n of c h ild r e n f i v e to s ix y e a r s a f t e r th e
im m unization o f in f a n c y .
-This lo s s o f immunity could b e, i n p a r t , th e
cause o f th e high' p e rc e n ta g e o f c a s e s i n th e o ld e r age gro u p i n th e Utah
ep id em ic.
The p e rc e n ta g e o f th o s e p re v io u s ly immunized, whose d ip h th e r ia
was caused by C .■d ip h th e r ia e ty p e g ra v is was much lo w er.
-1 3 -
•
D ip h th e ria c a r r i e r su rv ey s
Grossmann (19U0) d id a S chick t e s t and d i p h t h e r i a - c a r r i e r su rv e y o f
w h ite sc h o o l c h ild re n in V ir g in ia ,
He fo u n d th a t i n th e age group from
f iv e t o f o u r te e n y e ars# Ch d ip h th e r ia s C a r r ie r s were f i v e tim e s a s g r e a t
i n th e S ch ick n e g a tiv e c h ild r e n SS i n th o s e who were S ch ick p o s i t i v e .
G i l l (19U0) conducted a c a r r i e r su rv e y on w h ite c h ild r e n i n Alabama
d u rin g th e y e a r s 1 93? t o 1939 and found a c a r r i e r in c id e n c e o f to x ig e n ic
C. d ip h th e r ia e o f 0 .8 ? p e rc e n t fo r c h ild r e n from th e ag es o f f i v e t o
f o u r te e n y e a r s .
Schuman and D o u ll (19U0) i n t h e i r Study o f c a r r i e r s i n
C lev elan d d u rin g th e same tim e# found a low er ra te # 0 .9 6 p e rc e n t# f o r th e
same age group.
The l a t t e r e s tim a te d t h e r a t i o of c a r r i e r in f e c tio n s t o
c l i n i c a l a tta c k s as b e in g 9 3 3 :1 .
They a t t r i b u t e d t h i s h ig h r a t i o to th e
lo s s in p a th o g e n ic ity Of th e C. d ip h th e r ia e o f t h a t p e rio d .
From a study o f th e su rv e y s made i n th e U nited S ta te s # i t ap p ears
t h a t th e d i p h t h e r i a p ic tu r e as re g a rd s, in cid en ce# s u s c e p t i b i l i t y # and
c a r r i e r ' s t a t u s i s not u n iform th ro u g h o u t th e c o u n try 6 F ro b is h e r (19h2)
s t a t e s t h a t c a r r i e r su rv e y s have shown t h a t many ty p e s o f d ip h th e r ia
organism s# e . g . g ra v is o r m i t i s # V ir u le n t o r a v ir u le n t# may predom inate
and a t tim es may be th e on ly ty p e p re s e n t i n some com m unities b u t not in
o th e r s .
T his phenomenon may be lo c a l iz e d or g e n e r a l and c o n c lu sio n s in
re g a rd t o p re v a le n c e o r r a t e s a re n o t v a l i d beyond th e c o n fin e s of a co u n ty
or s ta te .
F r o b is h e r (I9I4O) su g g e s ts th a t th e s tu d y o f t h e ty p e o f C. d ip h th e r ia e
found i n p a r r i e r su rv e y s and th e number o f p o s itiv e c u ltu r e s o b ta in e d
-illm ight be u s e f u l g u id e s i n d i p h th e r ia c o n tr o l by in d ic a tin g the tre n d s
and changes.
Types o f m edia used f o r th e c u l t i v a t i o n
and i s o l a t i o n o f Ge d ip h th e r ia s
A lthough L o e f f le r 's b lo o d serum medium has been th e s ta n d a rd u sed f o r
th e i s o l a t i o n o f C, d ip h th e r ia s s in c e i t was f i r s t in tro d u c e d i n IBBli5 i t
h as been r e s p o n s ib le f or many e r r o r s i n th e d ia g n o s is o f d ip h th e r ia .
These m ista k e s have been caused by th e s c a r c i t y of grow th of C, d ip h th e r ia s
and th e overgrow th o f o th e r organism s o f th e nose and t h r o a t ,
M edalia
a t a l . (1931) d e s c rib e d a m o d ific a tio n o f L o e f f l e r 's medium t h a t made i t
more s e l e c t i v e .
T his was done by adding W /l NaOH t o g iv e a pH o f 7«6 to
th e f i n a l m ix tu re „ However5 because o f i t s o p a c ity i t i s d i f f i c u l t t o
m easure th e pH o f t h i s medium w ith o u t u sin g a pH m eter,
Laypourn (1935)
p roposed a m ethod by which t h e pH c o u ld b e d e term in e d by u s in g a verysm a ll amount of the w a te r o f h y d ra tio n o f a c u ltu r e .
I n a p ap er p u b lis h e d i n th e C hinese M edical J o u rn a l in 193S5 P a i de­
s c r ib e d an egg medium f o r th e c u l t i v a t i o n and i s o l a t i o n o f £» d ip h th e r ia s ,
McGuigan and F r o b is h e r (1936) found th a t t h i s medium p ro d u ced C, d ip h ­
t h e r i a s m o rp h o lo g ic a lly and to x i g e n i c a l l y s im ila r to th o s e c u ltiv a te d on
L o e f f l e r 1S5 and i n a d d itio n was e a s i e r t o p re p a re ,
T in s d a le (I9li7) h as fo rm u la te d a medium which u se s th e p ro d u c tio n o f
hydrogen s u l f i d e as a d i f f e r e n t i a t i n g c h a r a c t e r i s t i c .
D if f e r e n t organism s
produce d e f i n i t e ty p e s o f c o lo n ie s and c e r t a i n organism s form c h a r a c t e r i s t i c
h a lo e s .
The medium h a s the adv an tag e o f g iv in g d e f i n i t e r e s u l t s i n a
—If?—
c o m p a ra tiv e ly s h o r t p e rio d o f tim e . On t h e o th e r hand, t h e com p o sitio n o f
■
t h i s medium i s v e ry d e t a i l e d and c o m p lic a te d and i t s keeping q u a l i t i e s 'a r e
p o o r.
The u se o f p o ta ssiu m t e l l u r i t e a s a means o f i n h i b i t i n g th e growth o f
o th e r organism s and f o r d i f f e r e n t i a t i o n i n th e c u ltu r in g and i s o l a t i o n o f
Go d ip h th e r ia s has been w id e ly in v e s t ig a te d and u sed
of su ccess.
w ith v a ry in g d e g re es
I t was f i r s t u sed by Conrade and Troch i n 1912.
I t had been
d is c o v e re d a s e a r ly a s 1885 t h a t some members o f th e p l a n t kingdom, among
them th e " i n f u s o r ia " , could re d u c e ' th e t e l l u r i t e s i n sm a ll amounts t o
m e ta llic te llu r iu m "without i t s becoming to x ic , to them .
About 1900 K le tt
and o th e rs found th a t some b a c t e r i a w ere c o lo re d by th e red u ced m e ta l.
In
K l e t t 's s tu d y , u s in g numerous molds and b a c t e r i a , i t was found t h a t some
organism s c o u ld t o l e r a t e t h e t e l l u r i t e s much b e tte r, th a n o th e r s .
I t was
a ls o n o tic e d t h a t some organism s were not a f f e c t e d a t a l l by c o n c e n tra tio n s
t h a t would c o m p le te ly i n h i b i t o th e r o rg an ism s.
B a c illu s d ip h th e r ia s
(G. d ip h th e ria s )w a s m entioned a s one o f t h e ' organism s used i n !th is stu d y
t h a t was a b le to t o l e r a t e th e t e l l u r i t e s and was c o lo re d b y them .
Jo ach im u g le' and H iro se i n s tu d ie s t o d e term in e th e c o n c e n tr a tio n o f t e l l u ­
r i t e s t o l e r a t e d by C. d ip h th e r ia e fo u n d , f o r exam ple, t h a t C. d ip h th e r ia s
could t o l e r a t e a s o lu tio n c o n ta in in g U5OOO tim e s a s much of th e m e ta l a s
could S alm o n ella ty p h o sa ( G ilb e r t and Humphreys, 1 9 2 6 ).
Many w orkers have ex p erim en ted w ith m edia c o n ta in in g p o ta ssiu m t e l l u ­
rite .
Among them a re McGuigan. and F r o b is h e r (1 9 3 6 ), H a ll (1 9 3 9 ), Levin
(19U3), and K ellogg and Wende (19U 6).
A com parative s tu d y _of th e t e l l u r i t e
-1 6 p l a tin g m edia was c a r r i e d out by F r o b is h e r 5 P a rs o n s 5 Y e a te s 5 and bsy (l9it-8)
u s in g F r o b is h e r 1s own c y s tin e —te l l u r i t e medium (1937) as a b a s is f o r com­
p a r is o n , s in c e he had u sed i t f o r many y e a rs and knew i t s p r o p e r t i e s .
The
r e s u l t s o f t h i s stu d y showed t h a t th e - t e l l u r i t e medium o f K ellogg and Wende
(19)4.6) gave a s h ig h or h ig h e r p e rc e n ta g e o f p o s i t i v e s .
I e l l p g g and Wende
(19^6) a ls o claim ed a very h ig h p e rc e n t of p o s itiv e s w ith i t ^ having check­
ed i t c a r e f u l l y and. f in d in g t h a t i t seldom f a i l e d to d e m o n strate C. d ip h ­
t h e r i a s i f p r e s e n t.
H a ll5 whose medium d i f f e r s v e ry l i t t l e from t h a t o f
K ellogg and Wende5 l i s t s th e ad v an tag es o f h e r medium a s th e a b i l i t y to
i n h i b i t organsim s such a s m ic ro c o c c i, s tr e p to c o c c i, N e is s e r ip c a t a r r h a l l s ,
pneum ococci, e t c . , "while th e CL d ip h th e r ia s grows w ith t y p i c a l c o lo n ie s
ra n g in g from g ra y is h -w h ite t o b la c k i n c o lo r .
She a lso c laim s t h a t th e
p l a t e s can be p re p a re d and kept f o r two or th r e e weeks in th e r e f r i g e r a t o r .
T his i s a d e c id e d advantage a s m ost of "the t e l l u r i t e m edia m ust be p re p a re d
o n ly a day or so b e fo re t h e i r u s e .
V iru le n c e t e s t i n g ■o f G. d ip h th e r ia s
There has b een some do u b t a s to w h eth er anim al in o c u la tio n s r e v e a l
a l l th e p a th o g en ic p o t e n t i a l i t i e s o f G. d ip h th e r ia s m a n ife ste d i n human
b e in g s . F r o b i s h e r e t ^ ( W ) found t h a t n o n to x ig e n ic s t r a i n s w i l l produce
p a r t i a l or com plete p r o te c tio n a g a in s t v i r u l e n t organism s i n r a b b i t s , and
a ls o t h a t th e s o - c a lle d a v i r u l e n t d ip h th e r ia organism s c o n ta in e n d o to x in s.
He f u r t h e r s t a t e s t h a t numerous c a s e s o f c l i n i c a l l y t y p i c a l d ip h th e r ia
have been e n c o u n te re d i n w hich th e e t i o l o g i c a l ag en t has p roved to b e only
s t r a i n s o f C. d ip h th e r ia e n o n to x ig e n ic to g u in ea p ig s .
T h is i s p a r t i c u l a r l y
-1 7 t r u e -with th e minimus s t r a i n s o f th e organism , a s th e s e te n d to produce
o n ly f a i n t in tr a d e r m a l r e a c tio n s , i n .r a b b its when t h e i r v ir u le n c e i s t e s t ­
ed by s ta n d a rd p ro c e d u re s (F ro b is h e r s t . &!,, 1951) •
G uinea p ig s a re f r e q u e n tly employed f o r t e s t i n g the v ir u le n c e o f ■'
C. d i p h t h e r i a s .
Two an im als a r e u s u a lly u s e d , th e f i r s t b e in g in o c u la te d
s u b c u ta n e o u sly w ith a su sp e n sio n o f th e organism to be t e s t e d .
The second
s e rv e s as a c o n tr o l and i s g iv e n d ip h th e r ia a n t i t o x i n p lu s th e same amount
o f b a c t e r i a l s u sp e n sio n i n th e same m anner.
I f the organism s a re v i r u l e n t ,
th e t e s t anim al w i l l d ie i n th r e e t o f i v e d a y s, b u t th e c o n tr o l anim al
w i l l s u r v iv e .
Schaub and F o ley ( 19h 7) d e s c rib e a method employing o n ly one g uinea
p ig w hich s e rv e s b o th a s te s tin g ' anim al and c o n tr o l.
This h as th e advan­
ta g e s o f b e in g more econom ical and e lim in a tin g the v a r i a t i o n in s e n s i t i v i t y
o f in d iv id u a l a n im a ls.
A heavy s u s p e n s io n o f th e c u ltu r e i s in o c u la te d
in tr a d e im a lly i n th e shaven s id e o f th e g u in ea p i g .
A t a l a t e r s p e c if ie d
tim e an i n t r a p e r i t o n e a l i n j e c t i o n o f d i p h t h e r i a a n t i t o x i n i s g iv e n .
A
s h o r t tim e l a t e r th e o th e r s id e o f th e abdomen i s i n j e c t e d w ith t h e same
amount of th e c u ltu r e p r e v io u s ly u s e d .
A. r e a c tio n o c c u rrin g on th e -sid e
in o c u la te d f i r s t , b u t n o t on th e o p p o s ite s id e i s c o n s id e r e d .p o s itiv e ,
and d e n o te s a v i r u l e n t s t r a i n of C. d i p h th e r ia e .
s id e s i g n i f i e s a n o n to x ig e n ic s t r a i n .
A r e a c tio n on n e ith e r
A r e a c t i o n on b o th s id e s i s s a id to
be due to some o th e r f a c t o r th a n d ip h th e r ia to x i n .
A nother im p o rta n t
advantage of t h i s method i s th a t s e v e r a l c u ltu r e s can be t e s t e d on th e
same a n im a l, depending upon i t s s i z e .
F ro b is h e r e t aL. (1942) d e s c r ib e s a method i n which he u sed seven to tw enty
day o ld c h ic k s .
He f e l t t h a t th e s e c o u ld be recommended as a s u b s t i t u t e
f o r r a b b i t s or g u in e a p ig s i n t e s t i n g th e v ir u l m c e of 0. d ip h th e r ia s ^
e s p e c i a l l y when c o n d u c tin g a s m a ll number o f t e s t s , i f ro d e n ts tubr e n o t
r e a d i l y a v a il a b le .
A lthough th e need f o r an i n v i t r o method f o r t e s t i n g C. d ip h th e r ia s
f o r v ir u le n c e h as lo n g been re c o g n iz e d , i t was n o t u n t i l r e c e n tly t h a t
such a t e s t h as been d e s c rib e d (K ing, F ro b ish e r- and P a rs o n s, 1 9 4 9 ).
The
p r in c ip le of the t e s t i s b a se d on th e t o x i n - a n t i t o x i n f lo c c u l a t i o n a s de­
v is e d by N ic o lle i n 1920.
The t e s t as d e v ise d by E lek i n 1948 c o n s is ts of
th e p r e p a r a tio n o f a n a g a r b ase w h ic h m ust be c l e a r and w hich m ust c o n ta in
0*3 gram o f m a lto s e and 0*07 ml o f l a c t i c a c id p er 100 m l.
c o o led base, i s added 20 p e rc e n t of norm al h o rs e serum .
th e n poured i n t o p e t r i d i s h e s .
To t h i s m e lte d ,
The m ix tu re i s
A s t e r i l e f i l t e r p ^per s t r i p i s dipped, in
d ip h th e r ia a n t i t o x i n c o n ta in in g 100 u n i t A p e r ml, d ra in e d , and. p la c e d i n th e
c e n te r of tiie d is h w here i t i s allowed...to s in k in to th e warm a g a r .
A fte r
s o l i d i f i c a t i o n , t h e p l a t e s a re d rie d i n th e in c u b a to r and in o c u la te d o n •th e
same day.
Four organism s may be t e s t e d on each p l a t e i
s t r a i n sh o u ld be in c lu d e d .
A known v i r u l e n t
A w h ite l in e d e v elo p in g a t An a n g le on each
s id e o f the inoculum n e a r th e f i l t e r s t r i p d e n o tes a to x ig e n ic a g e n t.
K ing, e t a l , (1949) n o te d th e so u rc e s o f e r r o r i n t h i s i n v i t r o t e s t
and o u tlin e d m ethods by which th e s e e r r o r s could be m inim ized.
The most
im p o rta n t f a c t o r n e c e s s a ry t o a ssu re , c o r r e c t r e s u l t s , b e s id e s th e c o r r e c t
a d ju stm e n t o f th e pH, th e amount of a n t i t o x i n , and o th e rs a s o b v io u s, i s
th e c o l l e c t i o n and p r e p a r a tio n of th e serum u sed i n t h e a g a r b a s e . -Many
—19”
l o t s o f serum are n o t s a t i s f a c t o r y f o r th e minimus ty p e o f C. d ip h th e r ia s .
The r e a c tio n i n minimus s t r a i n s i s i n h i b i t e d by serum o n ly f a i n t l y c o lo re d
by th e l y s i s of th e e r y th r o c y te s .
a f f e c te d b y t h i s .
O ther s t r a i n s a re n o t so r e a d i l y
I t h as a l s o been found t h a t some unknown su b sta n c e or
c o n d itio n w hich i n t e r f e r e s w ith th e i n v i t r o v iru le n c e t e s t ap p ears in th e
serum when i t rem ains i n c o n ta c t w ith th e c l o t f o r a day or lo n g e r.
U sing t h i s m ethod. King e t a l . (19U9) ra n a lo n g s e r i e s o f t e s t s
em ploying v ir u l e n t and a v ir u le n t c u ltu r e s of C. d ip h th e r ia s i n com parison
w ith anim al in o c u la tio n s . ' Complete agreem ent w ith th e i n v i t r o and anim al
in o c u la tio n s was o b ta in e d i n 290 c a s e s , of w hich 123 were a v ir u le n t and
l6 ? w ere v i r u l e n t .
A p o s i t i v e r e a c tio n w ith the i n v i t r o t e s t seem s to be an e n t i r e l y
r e l i a b l e in d i c a t i o n o f v ir u le n c e o f d ip h th e r ia organism s.
However-, a
n e g a tiv e r e a c tio n can be f a l s e l y o b ta in e d u n le s s th e c o n d itio n s fo r t h a t
s t r a i n a re p ro v id e d .
They su g g est t h a t a l l n e g a tiv e r e a c t i o n s be checked
by anim al in o c u la tio n s .
They a ls o concluded t h a t th e t e s t needed f u r t h e r
s tu d y .
However, i n a more r e c e n t p a p er th e s e same i n v e s t i g a t o r s , a f t e r
f u r t h e r stu d y of t h i s m ethod, recommend i t to be 1,w holly ! r e l i a b l e , sim p le ,
r a p i d , and in e x p e n s iv e , i f p ro p e rly and c a r e f u l l y p e rfo rm ed ".
o th e r d i f f i c u l t i e s and so u rc e s o f e r r o r .
They m ention.,
For exam ple, c e r t a i n commercial
a n t i t o x i n s a r e u n s a t i s f a c t o r y -for t h i s u s e , a lth o u g h p e r f e c t l y cap ab le of
fu n c tio n in g as p r o p h y la c tic and th e r a p e u ti c a g e n ts .
s tre s s
Once a g a in th e y
th e c a re t h a t must be ta k e n i n p re p a rin g th e serum u sed a s n u t r i ­
ment en rich m en t o f th e a g a r when t e s t i n g s t r a i n s o f th e minimus ty p e .
-2 0 They su g g e s t u sin g a s a c o n tr o l a known p o s i t i v e l y r e a c t i n g s t r a i n o f
minimus when p l a t i n g unknown minimus s t r a i n s (F ro b is h e r e t a l .j 19f?l)«
METHODS MD MATEETALS
P r e p a ra tio n
B efore th e a c t u a l su rv e y on t h e s tu d e n ts f o r .the d e te rm in a tio n o f
d i p h th e r ia c a r r i e r s was begun, known v i r u l e n t and a v i r u l e n t c u ltu r e s o f
G orynebacterium d ip h t h e r i a s were o b ta in e d from Frobis„her and from
——
*
'
' T1 “
.
_
G a lb ra ith t h a t in c lu d e d s t r a i n s of t h e g r a v is , m i t i s , in te r m e d iu s , and
minimus- ty p e s .
These w ere used t o become f a m ilia r w ith th e m orphology,
s ta i n in g r e a c tio n s , and 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 th e o rg an ism s.
The gram s t a i n was employed only t p be c e r t a i n t h a t a l l of th e o r ■ganism s w ere gram p o s i t i v e .
A ll m o rp h o lo g ica l s tu d ie s w ere niade on s l i d e s
-
s ta in e d w ith m ethylene blue*
•
L o e f f l e r 1s a lk a l in e m ethylene b lu e and a
m ethylene b lu e s t a i n made w ith o u t th e a d d itio n of th e a l k a l i gave s im ila r
s ta in in g r e s u l t s .
S ince t h e l a t t e r was sim p le r to p re p a re , i t was used
th ro u g h th e r em ainder of th e s tu d y .
A com parison o f th e m orphology o f 0 . d ip h th e .ria e on P a i 's egg medium
and L o e f f l e r 's serum medium was made.
The g ra n u le s and b a r r in g of th e
organism s were b e t t e r d e m o n strated on P a i 's medium a lth o u g h th e L o e f f l e r 1s
medium produced a more l u x u r i a n t g ro w th .
T h e re fo re , i t was d ecid ed to
in o c u la te h a l f of the th r o a t c u ltu r e s o b ta in e d d u rin g th e su rv e y onto
P a i 's medium and the- o th e r h a l f onto L o e f f l e r ' s medium i n o rd e r to o b ta in
in fo rm a tio n c o n cern in g th e u s e f u ln e s s o f t h e form er a s a s u b s t i t u t e i n
r o u tin e th r o a t c u ltu r e s of s u sp e c te d cad es o f
d ip h th e r ia .
-2 1 The L o e f f l e r ’s medium used th ro u g h o u t t h i s s tu d y was p re p a re d
from
B a c to -L o e ffle r b lo o d serum .
P a i 1s medium was p re p a re d a c c o rd in g t o th e f orm ula g iv e n by McGuigan
and F ro b is h e r (1 936).
Both th e L o e f f l e r 1s' and P a i 's m edia w ere c o a g u la te d and s t e r i l i z e d as
fo llo w s :
a . The b a s k e ts w ere wrapped- in ab so rb en t c o tto n and s la n te d by le a n in g
on a wooden su p p o rt i n th e c e n te r of th e a u to c la v e cham ber.
b . A ll th e e x h au st v alv es- "were t i g h t l y c lo s e d and the steam was adm it­
te d a t a f a i r l y slow b u t even r a t e .
c . A t th e end o f 90 m in u te s, th e steam v alv e.w as c lo se d , and th e p re s ­
s u re allow ed t o go down w ith o u t opening th e ex h au st v a lv e s .
d . A f te r th e p re s s u re re a c h e d z e r o , th e e x h au st v a lv e s w ere opened
f o r a few m in u te s b e fo re th e s t e r i l i z e r d o o r was opened.
Smooth s l a n t s f r e e o f gas b u b b les were o b ta in e d .
These were s to r e d at,
£ C u n t i l used.
K ellogg an d 'Wende1s t e l l u r i t e medium was used fo r th e i s o l a t i o n and
i d e n t i f i c a t i o n 'o f t h e organism s o b ta in e d on th e P a i ’s and L o e f f l e r ’s m edia.
Human b lo o d was s u b s t i t u t e d f o r th e recommended r a b b i t b lo o d .
I
.
:
was n o t s to r e d f o r more th a n a day or two b e fo re u s e .
T his medium
' Survey
The survey was c a r r ie d o u t a s su g g este d by F ro b is h e r (19U2 and 19U9)«
T h ro at c u ltu r e s were made from 200 a p p a re n tly h e a lth y s tu d e n ts , 100
men and 100 women, d u rin g th e Montana S ta te C o lleg e FreShman Week,
-
Septem ber, ±9b9o.
22 - .
One hundred o f. th e s e , s e le c te d a t random , were c u ltu re d
on P a i* s medium and one hundred on Loef i l e r ' s medium.. The d i s t r i b u t i o n
o f sex e s c u ltu r e d on each medium was ab o u t e q u a l.
A f te r 2U h o u r s '
in c u b a tio n a t 35 C th e c u ltu r e s w ere t r a n s f e r r e d t o t h e t e l l u r i t e p la te s
p re v io u s ly p re p a re d and a g a in in c u b a te d a t 35 C.
I
A t th e en d o f 1+8 h o u rs th e p la te 's were' o b serv ed f o r c o lo n ie s .
grow th ap p eared from 78 o f t h e t r a n s f e r s .
No '
From th e 122 p l a t e s on which
growth a p p e a re d , r e p r e s e n ta tiv e c o lo n ie s were p ic k e d and t r a n s f e r r e d to .
L o e f f le r o r P a i s l a n t s .
Smears w ere made ,from th e s e s l a n t s a t th e end of
21+ h o u rs and s ta in e d by Gram 's method.
A ll c u ltu r e s
showing gram p o s i t i v e
ro d s i n th e t y p i c a l p a lis a d e and C hinese l e t t e r fo rm a tio n w ere sav ed f o r
p u r i f i c a t i o n and i d e n t i f i c a t i o n .
D uring th e w in te r q u a r te r o f 1950 th e t h r o a t s o f a l l s tu d e n ts coming
t o th e H e a lth S e rv ic e w ith s e v e re s o re t h r o a t s were swabbed and su b seq u en t­
l y h a n d led in t h e same manner a s th e f i r s t 200 c u l t u r e s .
I
s tu d e n ts w ere in c lu d e d i n th e su rv e y .
Thus, 35 o th e r
P u rific a tio n
A g r e a t d e a l of d i f f i c u l t y was e n co u n te re d i n p u r ity in g th e mixed
c u ltu r e s ,.
The c o n tam in atin g organism s were m ic ro c o c c i, e x ce p t f o r two
c u ltu r e s w hich w ere co ntam inated w ith s tr e p to c o c c i ,
The m ic ro c o c c i grew
w e ll on th e t e l l u r i t e medium, prod u cin g c o lo n ie s which w ere more sh in y and
mucoid in ap p earan ce th a n th o s e of C. d ip h th e r ia e .
The s tr e p to c o c c i grew
"very p o o rly on the t e l l u r i t e medium, p ro d u cin g a t b e s t a v e ry sm all g ia y
colony t h a t was d i f f i c u l t t o s e e .
For t h e m ost p a r t , th e y j u s t rem ained
-2 3 dorm ant b u t v ia b le , and re a d y t o grow i f th e p ro p e r environm ent was a g a in
p re s e n te d .
The t e l l u r i t e i n h i b i t e d th e d i p h t h e r i a l i k e organism s to th e
e x te n t th a t a very heavy in o c u la tio n was n e c e s s a ry t o o b ta in grow th.
th e n , made i t d i f f i c u l t to f i n d i s o l a t e d c o lo n ie s .
T h is ,
Even, th o u g h a colony
d id appear t o c o n s i s t of on ly one s p e c ie s , upon t r a n s f e r to L o e f f l e r ’s
medium, or e s p e c i a l l y t o a l i q u i d medium, i t w as' o fte n found to be im pure.
A fte r much e x p erim en ta t i on i t was fo u n d t h a t a " c h o c o la te " medium
made a c c o rd in g t o the fo rm u la f o r th e t e l l u r i t e but, w ith o u t th e p otassium
t e l l u r i t e , made a b e t t e r s u b s tr a te f o r i s o l a t i o n .
This medium gave good
growth of a l l o f t h e organism s, p e r m ittin g th e u se o f a s m a lle r inoculum .
The d i p h t h e r i a l i k e b a c t e r i a produced sm all tra n s p a r e n t to g ra y is h c o lo n ie s ,
th e m ic ro c o c c i formed la r g e w h ite c o lo n ie s , and th e c o lo n ie s o f t h e s t r e p ­
to c o c c i w e re • surrounded by d e f i n i t e y e llo w zo n es.
A fte r s tr e a k in g th e c u ltu r e s and p ic k in g i s o l a t e d c o lo n ie s on s e v e r a l
o c c a s io n s , 13 c u l t u r e s w ere f i n a l l y o b ta in e d i n a pure s t a t e .
These w ere
m a in ta in e d on L o e f f l e r ’ s o r P a i 1s m edia and s to r e d a t 5 0,
C u ltu r a l s tu d ie s
The morphology o f t he organism s was a s c e r ta in e d by s tu d y in g s l i d e s
made from 2li-hour c u ltu r e s on L o e f f l e r ’s o r P a i 1s m edia and s ta in e d w ith
m ethylene b lu e .
M easurem ents w ere made em ploying a p re v io u s ly c a li b r a t e d
o c u la r m ic ro m e te r.
M o tility was checked by the hanging d ro p method, u s in g
a 2U-hour b ro th c u l t u r e .
A ll p h y s io lo g ic a l t e s t s were ru n i n d u p lic a te and i f any d o u b t a ro s e
a s t o t h e i r agreem ent, f u r t h e r checks w ere made.
S ev eral, d i f f e r e n t d y p e s - -
o f b r o th w ere t r i e d , i n a n e f f o r t t o f in d one t h a t would g iv e a s u f f i c i e n t l y
!
—2i|.~
good grow th f o r th e p h y s io lo g ic a l t e s t s .
D ifc o 1s v e a l in f u s io n medium
c o n ta in in g 0 .1 p e rc e n t a g a r was found to su p p o rt th e b e s t grow th.
This
medium was a ls o u sed fo r making th e a g a r p l a t e s and s l a n t s by in c re a s in g
th e a g a r to 1 .5 p e rc e n t.
The t e s t s f o r n i t r a t e r e d u c tio n and in d o l p ro d u c tio n w ere made on th e
same tu b e of v e a l in f u s io n medium to Vnich had been added enough t r y p t one
(B acto) and p o ta ssiu m n i t r a t e t o g iv e a 0 .1 p e rc e n t c o n c e n tra tio n of each .
The organism s w ere t e s t e d f o r t h e i r a b i l i t y t o fe rm e n t g lu c o se ,
g a la c to s e , f r u c to s e , s u c r o s e , m a lto s e , l a c t o s e , d e x tr in , m a n n ito l, and
g ly c e r o l.
A ll th e s e s u b s tr a te s w ere s t e r i l i z e d ty f i l t e r i n g through a
s t e r i l e f r i t t e d g la s s f i l t e r , and a s u f f i c i e n t q u a n tity o f th e s t e r i l e
s o lu tio n added to .th e b a s a l medium to g iv e a f i n a l c o n c e n tra tio n of one
p e r c e n t.
Brom c r e s o l p u rp le was u sed a s an i n d i c a t o r .
E very p o s s ib le
p re c a u t ion was ta k e n t o p re v e n t c o n ta m in a tio n and a l l tu b e s were in c u b a te d
f o r 2h h o u rs a t 35 C b e fo re b e in g . examined and s to re d a t 5 C u n t i l u se d .
Less th a n one p e rc e n t of th e s e tu b e s of m edia was d is c a rd e d because of
c o n ta m in a tio n .
G e la tin liq u e f a c t i o n was d e term in e d in -v eal in f u s io n medium t p which
had been added 12 p e rc e n t g e l a t i n .
S ta b in o c u la tio n s were made and th e
c u ltu r e s in c u b a te d a t 35 C f o r a t l e a s t two w e e k s .'
C a ta la s e p ro d u c tio n was d e m o n strated by a d d in g a few drops of th r e e
p e rc e n t com m ercial hydrogen p e ro x id e to c o lo n ie s on an a g a r p l a t e .
C u ltu re s i n th e v e a l in f u s io n
b ro th w ere s tu d ie d i n 2k and it8 h o u rs
f c r ty p e o f g row th, p e l l i c l e fo rm a tio n , sedim ent a n d a re a of h e a v ie s t
grow th.
-2 5 C o lo n ies on some cf th e p l a t e s and s l a n t s could n o t be observed
•without a Ipc hand le n s .
The medium f o r d e te rm in in g h y d ro ly s is o f u re a was made ty ad d in g
a s e p tic a l l y 10 ml of a c o n c e n tra te d u re a s o lu tio n (D ifco ) to a s o lu tio n
c o n s is tin g o f 90 ml of w a te r and 1 .5 p e rc e n t a g a r.
T his medium was th e n
poured i n t o s t e r i l e tu b e s and s la n t e d b e fo re s o l i d i f i c a t i o n to o k p la c e .
These s l a n t s w ere s tr e a k e d w ith a heavy inoculum , in c u b a te d and w atch ed
f o r th e developm ent o f a p in k c o lo r t h a t d e n o te d u re a h y d r o ly s is .
V iru le n c e
V iru le n c e of t h e organism s i s o l a t e d and p u r i f i e d was d eterm in ed by
in tr a c u ta n e o u s s k in t e s t s on g u in e a p ig s as d e s c rib e d b y Schaub and F o le y
(19U7).
A heavy su sp e n sio n of t h e organism s i n p h y s io lo g ic a l s a l i n e was
made by w ashing th e growth from a. 2l|.—h o u r c u ltu r e on L o e f f l e r 1s s l a n t s .
O n e -te n th m i l l i l i t e r of t h i s su sp e n sio n was i n je c te d in tr a c u ta n e o u s I y i n t o
th e shaven s id e of th e abdomen of a g u in e a p i g .
By s ta g g e rin g th e s i t e s
of th e i n j e c t i o n s , sev en c u ltu r e s w ere i n j e c t e d i n t o one s id e o f a la rg e
g u in ea p i g .
A f te r in o c u la tio n , t h e re m a in in g su sp e n sio n s were p la c e d in
th e r e f r i g e r a t o r .
F iv e h o u rs l a t e r th e anim al was given 100 u n i t s of
d ip h th e r ia a n t i t o x i n i n t r a p e r i t o n e a l l y , c a re b e in g ta k en n o t to p u n ctu re
th e i n t e s t i n e s .
T h irty m in u tes l a t e r a n o th e r 0 .1 ml of t h e su sp e n sio n
was i n j e c t e d in tr a c u ta n e c u s ly i n t o th e o p p o s ite shaven s id e o f th e g u in e a
p i g ’ s abdomen. • P re c a u tio n was ta k e n t o b e su re th a t each organism was
i n j e c t e d e x a c tly o p p o s ite th e f i r s t i n j e c t i o n .
s e rv e d as a c o n tr o l.
This second i n j e c t i o n
A c h a r t of th e s i t e s o f th e i n j e c t i o n s was made s o
t h a t t h e r e co u ld be no m ista k e as to th e p o s itio n o f each organism .
—26—
A re c o rd , was made of. th e r e a c tio n s o f each a t 2li)
and 72 h o u r i n t e r v a l s .
E rythem a, edema, and n e c r o s is a t th e s i t e of the t e s t i n j e c t i o n and the
absence of such a r e a c tio n on' th e c o n tr o l s id e meant a p o s itiv e s k in t e s t ,
and in d ic a te d t h e p ro d u c tio n of to x in by the organism i n q u e s tio n .
-
■
Known
.
v i r u l e n t and a v ir u le n t s t r a i n s w ere a ls o t e s t e d so a s t o check th e .
te c h n iq u e ,
EXPERIMENTAL RESULTS
Number o f c u ltu r e s o b ta in e d
From t h e 200 c u ltu r e s ta k e n from seem in g ly h e a lth y s tu d a i t s d u rin g
Freshm an Week , 122 were found to g iv e b la c k c o lo n ie s on th e t e l l u r i t e
medium.
A lthough many o f them w ere much t o o s h in y and mucoid, i n appearance
to be C orynebacterium d ip h th e r ia e , L o e f f le r and P a i s l a n t s were in o c u la te d
w ith r e p r e s e n ta tiv e c o lo n ie s from a l l p l a t e s .
A fte r 2k h o u rs each c u ltu r e
was examined by gram s t a i n in g .
Most o f them w ere found t o c o n s is t o f
m icro q o cci and were d is c a rd e d .
Some of th e c u ltu r e s were a mixbure of
gram p o s i t i v e r o d s ■and o th e r org an ism s, p r i n c i p a l l y m ic ro c o c c i.
A few
a p p ea re d t o be seem ingly p u re c u ltu r e s o f gram p o s itiv e ro d s .
No c u ltu r e s c o n ta in in g gram p o s itiv e ro d s TBr e o b ta in e d from th e
35> s tu d e h ts r e p o r tin g to t h e H e a lth S e rv ic e w ith so re t h r o a t s d u rin g th e
w in te r and s p rin g q u a r te r s of 195)0.
A v e ry r a p id ly grow ing s tre p to c o c c u s
g e n e r a lly p red o m in ated .. U n su c c e ssfu l a tte m p ts were made t o o b ta in gram
p o s i t i v e ro d s by t r a n s f e r r i n g t h r o a t c u l t u r e s in o c u la te d on L o e f f le r
s l a n t s , t o t h e t e l l u r i t e p l a t e s a f t e r an in c u b a tio n p e rio d of lU t o 18
h o u rs as w e ll as a t th e end o f 2k h o u rs .....S in ce no d i p h th e r ia lik e organism s.
-2 7 w ere o b ta in e d from th e s e 35 s tu d e n ts , th e c u ltu r in g o f p a th o lo g ic a l t h r o a t s
was d is c o n tin u e d .
• Comparison o f th e P a i t S and L o e f f l e r 1s m edia
A r e c o rd was k ep t as t o th e medium on which the o r i g i n a l t h r o a t c u l­
tu r e s were in o c u la te d .
Of th e 13 s t r a i n s o b ta in e d i n p u re c u ltu r e , e ig h t
were o r i g i n a l l y p la c e d on P a l ’ s medium and f i v e on L o e f f l e r t s medium.
How­
e v e r, i t was n o te d t h a t o f t h e f iv e c u ltu r e s w hich w ere i s o l a t e d from th e
t e l l u r i t e a g ar p l a t e s ' i n an a p p a r e n tly p u re s t a t e , fo u r w ere o r i g i n a l l y
c u ltu r e d on L o e f f le r s l a n t s and only one on a
Pai s la n t.
I
G rouping of th e organism s
A ll of th e 13 organism s o b ta in e d had s e v e r a l m o rp h o lo g ic a l and
p h y s io lo g ic a l c h a r a c te r s i n common.
They were a l l gram p o s i t i v e ro d s show- '
in g C hinese l e t t e r and p a lis a d e g ro u p in g , th e y a l l produced c a ta la s e , b u t
f a i l e d t o form in d o l from try p to p h a n e , or to liq u e f y g e l a t i n .
With b u t
one e x c e p tio n , c u ltu r e 113, a l l g u in e a p ig t e s t s fo r to x in p ro d u c tio n
w ere n e g a tiv e .
By p la c in g to g e th e r th o s e which re sem b le d one a n o th e r i n t h e o th e r
m o rp h o lo g ica l and p h y s io lo g ic a l c h a r a c te r s fo r which th e y w ere t e s t e d ,
f o u r groups were e s ta b lis h e d .
Group f o u r , o r th e l a s t g ro u p , c o n s is ts o f
th r e e m is c e lla n e o u s organism s w hich do n o t show th e s i m i l a r i t y th a t i s
found i n th e o th e r g ro u p s.
The m o rp h o lo g ica l c h a r a c t e r i s t i c s o f th e
organism s i n th e s e groups a re l i s t e d i n t a b l e I I and th e p h y s io lo g ic a l i n
ta b le I I I .
T able I I
M o rp h o lo g ical c h a r a c t e r i s t i c s o f d ip h th e r ia lik e organism s i s o l a t e d from s tu d e n t th r o a t s
Groun C u ltu re
101, 102,
I
103, IOti.,
Ill
2 ' 107, 109,
HO
3
h
108, IlU
(1 #
(
(
(
(
( 112
(
(
(
( 113
S iz e
(m icrons)
I . 2 -3 .8 x
0 .6 - 1 .2
B ro th
C e l lu la r appearance.
C o lo n ie s
D i s t i n c t b a r r in g , oc­
c a s io n a l c lu b sh a p e s,
some p o la r b o d ie s .
M inute, tr a n s l u ­
c e n t, s h in y , con­
vex and sm ooth.
1 . 2 -3 .6 x
' O .U-1.2
B a rrin g and p o la r
b o d ie s p r e v a le n t, mo­
d e r a te number of club
shaped c e l l s .
1-2 mm, w h ite ,
opaque and sh in y ,
s o lu b le brown
pigm ent produced
a f t e r 2i| h o u rs .
P e l l i c l e form ed.
T u r b id ity , upper
h a l f in c h ,
s l i g h t sed im en t.
0
Very s m a ll r o d . V a ria ­
b i l i t y of s t a i n i n g .
1-2 mm, smooth,
s h in y , convex and
opaque.
Uniform t u r b i ­
d i t y upper
h a l f in c h .
1 .2 -2 x
0 .6 - 1 .2
Some b a r r in g .
1-2 mm, smooth,
s h in y , opaque,
o ra n g e -p in k p ig ­
m ent.
Uniform t u r b i ­
d i t y u p p er h a lf
in c h , p in k
sed im en t.
1 . 2 -1 .9 %
0 .U -0 .7
P o la r b o d ie s b u t no
b a rrin g .
1-2 mm, w h i t e , '
opaque, d ry and
g r a n u la r .
G ran u lar t u r b i ­
d i t y u p p er h a lf
in c h .
I . 2 -3 .0 x
0 .6 - 0 .9
Some b a r r in g .
1-2 mm, w h ite ,
s h in y , sm ooth,
convex.
Y
0 .2 - 0 .6
Uniform t u r b i ­
d i t y u p p er
h a l f in c h of
. c u ltu re .
Uniform t u r b i ■ d i t y upper
h a l f in c h .
T ab le I I I
P h y s io lo g ic a l c h a r a c t e r i s t i c s of d ip h th e r ia lik e organism s i s o l a t e d from s tu d e n t t h r o a t s
Group
I
2
O
G u ltu r e
(
(
J
(
101
102
103 .
io l
h
/
( H l
( 107
( 109
( HO
/
/'
/
/
/
( io5
( 112
( 113
/
/
Mai—
to s e
—
.
/
/
/
-
7
/
/
/
-
-
/
-
—
/
/
-
.
.
Suero s e
/
/
/
/
/
/
/
-
( 108
G alacto s e
. F ru c to s e
/
/
/
-
( 111
'
G luco se
/
.
—
6 .
-
—
/ .
7
—
-
/
.
-
a
—
/
-
/
"
.
.
•
NO^ r e d u c tio n
/
-
-
/
-
/
/
/
-
/
C
/
/
/
—
-
-
-
-
-
—
—
_
_
—
/
-
-
-
-
■»
—
-
—
U rease
-
-
■
/
7
M annito l
_
/
—
-
-
—
—
/
DexG lyt r i n . cero l
Lbc—
to s e
-
—
—
—
—
-
-
-
.
. /
-
—
—
—3 0 C l a s s i f i c a t i o n o f groups
A- com parison o f th e m o rp h o lo g ica l and p h y s io lo g ic a l c h a r a c t e r i s t i c s
o f th e organism s i s o l a t e d w ith thos.e o f C» d ip hthe r i a e a s g iv en i n
B grgeyt S Manual o f D e te rm in a tiv e B a c te rio lo g y (B reed e t a l , 19U8) i s
Cf
p re s e n te d i n t a b l e IV ,
The m o rp h o lo g ica l and p h y s io lo g ic a l c h a r a c t e r i s t i c s o f th e organism s
o b ta in e d from F ro b is h e r and from G a lb ra ith were a l s o d e term in e d .
A ll
w ere gram p o s i t i v e ro d s w ith fr e q u e n t a n g u la r and p a lis a d e fo rm a tio n s.
L ik e w ise , a l l produced c a ta la s e and re d u c ed n i t r a t e s to n i t r i t e s .
how ever, produced in d o l from try p to p h a n e o r l i q u e f i e d g e la t in ..
None,
The o th e r '
c h a r a c t e r i s t i c s d eterm in ed a re p re s e n te d i n t a b l e s V and V I.
I t w i l l be n o te d i n ta b le IV t h a t Group I and Group 2 p r e s e n t a
p ic tu r e v e ry s im ila r t o t h a t of C. d ip h th e r ia s as d e s c rib e d i n B ergsgJs
.M anual.
Group 2 organism s were s e p a ra te d from Group I o n ly on the b a s is
o f t h e p ro d u c tio n o f th e brown s o lu b le p ig m en t i n s o l i d m edia.
This
a b i l i t y i s n o t m entioned i n c o n n e c tio n w ith any of th e c o ry n e b a c te ria and
seems t o p la c e th e s e th r e e organism s i n a group a p a r t.
Group 3 d i f f e r s
from C. d ip h th e r ia s in th a t n i t r a t e s a re n o t red u ced t o n i t r i t e s .
The
m is c e lla n e o u s organism s i n Group U show s l i g h t v a r ia tio n s i n n i t r a t e
r e d u c tio n , u re a h y d r o ly s is , and sugar fe rm e n ta tio n .
C u ltu re 1G£ r a th e r
c lo s e ly resem b les G orynebacterium pseudod ip h th e ritic u m e x c e p t t h a t i t .
_
' -T
produces a v e ry d e c id e d ly o ra n g e -p in k pigm ent in s te a d o f b ein g " g ra y is h to
.c re a m -c o lo re d " a s d e s c rib e d i n B erg e y 's M anual.
I t i s i n t e r e s t i n g t o n o te t h a t ’ th e organism s o f Group I very c lo s e ly
resem ble two s t r a i n s o f C. d ip h th e r ia s ty p e minimus o b ta in e d from
T ab le IV
M o rp h o lo g ical and p h y s io lo g ic a l c h a r a c te r s of organism s i s o l a t e d from th r o a t s of
s tu d e n ts compared w ith C. d ip h th e r ia as d e s c rib e d in B ergeyi S Manual
C h a ra c te r
Shape
S iz e (m icrons)
Appearance
Group I
■R ods, few
c lu b b e d .
I o 2 -3 o8 x
0 .5 - 1 .2
B a rrin g , m etach ro - B a rrin g , a
few p o la r
m atic g ra n u le s .
b o d ie s .
A ngular and p a l i Same,
sade.
C o d ip h th e ria e *
Rodss sometimes
c lu b s «
I . 0 -9 .0 x 0 .3 -0 .8
Group form atio n
Gram r e a c tio n
• /
Og re q u ire m e n t A e ro b ic ,
M o tility
C a ta la s e
—
G e la tin liq u e fa c tio n
In d o l
N itr a te re d u c tio n
/
Urea
Sugar fe rm e n ta tio n .
G lucose
/
F ru c to s e
/
G a lac to se
V a ria b le .
M altose
V a ria b le .
V a ria b le .
S ucrose
D e x trin
V a r ia b le .
G ly c e ro l
V a r ia b le .
^A ccording to B ergeyi s Manual
Group. 2
Rods.
Group 3
Sm all r o d s .
I , 2 -3 ,6 x
0 .6 - 1 .7 x
0 .2 - 0 .6
Unevenly
s ta in in g .
0. 4- 1.2
B a rrin g and
m etach ran at i c g ra n u le s .
Same.
Same,
/
A e ro b ic .
-
/
A e ro b ic ,
-
-
-
-
/
A e ro b ic .
.
/
-
/
-
—
/
V a ria b le .
—
V a ria b le .
-
/
/
V a ria b le .
V a ria b le ..
V a ria b le .
V a r ia b le .
/
y
V a ria b le .
V a ria b le .
V a ria b le .
V a r ia b le .
—
Group h
Rodsi . '
I o 2—3 »0 x
0 .4 - 1 .2
B ar- G ranr in g u le s
Same.
/
/
A ero b ic.
-
-
“
/
7
/
—
-
—
—
—
••
—
—
—
-»
T able 7
M orphological c h a r a c t e r i s t i c s of G_o_ d ip h th e r ia s c u ltu r e s o b ta in e d
from F ro b is h e r (F) and from G a lb ra ith (G)
C u ltu re S iz e (m icrons)
F I I «0-2 0)4. X OoU-dOoS
.
F 2
0 .6 - 2 .2
X
0 .4 —0 .6
F 3
1 .2 - 3 .0
X
0 .4 - 0 .5
F h
1 .8 —3 .6
X
0 .5 - 0 .6
X
0 ,4” 0 e6
X
0 .4 - 0 .7
F £ • 0 .U -1 .8
F 6 0 .9 - 2 .I
-
I
X
On
O
0 .6 - 3 .8
O
F 7
F 8 O6S-Ile 2
F 9 • 1 .0 - 2 .U
X
0 .5 - 1 .0
0 , 4™o.6
GI
0 .6 -1 .8
X
0 .4 - 0 .5
G2
I . 2 -1 .8
X
o .4 - o .7
G '3
0 .4 -1 .8
X
0 .4 -0 .8
G U 0 .4 - 1 .4
X
0 .3 - 0 .5
0 .5 - 1 .2
X
0 .3 -0 .5
G.5
X
C e l lu la r appearance
L i t t l e v a r i a t i o n in s ta i n in g ,
few g ra n u le s .
Go lo n ie s
W h ite, opaque.
smooth, s h in y ,
c o n v e x .'
Il
B roth
T u r b id ity upper ?
in c h , s l i g h t p e l l i c l e fo rm a tio n .
U
Uniform s t a i n i n g , o c c a s io n a l
g r a n u le .
H
II
O ccasio n al b a r r e d ro d , m etachrom atic g r a n u le s .
O ccasio n al b a rre d r o d , m etaW hite, opaque, ro u g h ,
"
chrom atic g ra n u le s .
irre g u la r, d u ll.
U niform ly s ta i n in g r o d , club
W h ite, opaque, smooth,
!l
forms p r e s e n t.
s h in y , convex.
Il
Il
V a ria tio n i n s t a i n i n g , o ccas io n a l clu b b ed fo rm .
B arred r o d s , club form s p r e M inute, tr a n s l u c e n t . Uniform t u r b i d i t y
s e n t.
smooth, s h in y .
upper I' in c h .
It
B arred ro d s , c lu b form s p r e s e n t,
"
B arred r o d s , m etachrom atic
W hite, opaque,
T u rbid upper ^
g ra n u le s .
smooth, sh in y )
in c h , s l i g h t p e l convex.
l i c l e fo rm a tio n .
B arred r o d s , o c c a s io n a l m eta- M inute, t r a n s l u c e n t , Uniform t u r b i d i t y
■ch ro m atic g ra n u le s .
s h in y , convex.
upper I in c h .
11:
Il
B arred ro d s and m etachrom atic
g ra n u le s .
Il
it
U niform ly s ta i n in g ro d s of
v a ry in g s i z e s .
11
Il
B arred r o d s , o c c a s io n a l clu b
sh ap e.
ii
11
U niform ly s ta in in g ro d s , occas lo n a l c lu b sh a p e „
T ab le VI
P h y s io lo g ic a l c h a r a c t e r i s t i c s of C_c_ d ip h th e r ia s c u ltu r e s
o b ta in e d from F ro b is h e r (F) and from .G a lb ra ith (G)
Glu­
cose
C u ltu re
F ru c­
to s e
G alac­
to s e
Suc­
ro s e
Lac­
to s e
Mcil™
to s e
Dex­
trin
Manni­
to l
F I '
/
A
/
-
-
/
/
F 2
7
/
/
—•
-
-
/
-
F 3
/
/
/
-
/ .
-
/
-
F U
/
/
/
-
-
/
/
F $
/
/
/
-
-
-
-
F 6
/
-
/
-
-
/
F 7
/
-
7
-
-
F 8
/
-
-
-
-
F 9
/
-•
/
-
• G 2
/
-
G.3
/
GU
G3
• G l
.*
Gly­
cero l
U rease
/
-
--
-
/
-
-
-
-
-
-
-
■’ /
-
-
/
-
-
-
7
-
A
-
-
-
—
/
-
/
-
-
/
-
.
■
/
-
-
-
-
-
-
-
-
—
-
-
-
-
-
-
—
—
- •
—
-
«■
—
—
-
' «
-
)
■
-3U '
'
T ab le V II
Com parison of- Group I organism s i s o l a t e d from
s tu d e n t s ' th r o a ts w ith Co d ip h th e r ia s ty p e
.minimus f u r n is h e d by F ro b is h e r
Known Minimus
Group I Organisms
S iz e : (m icrons) ■
0 .6 - 3 .8 x 0 . 3 - 1 . O
'1 .2 -3 .8 x 0 .5 - 1 .2
A ppearance on
s ta i n e d smear
B arred fo rm s, c lu b
s h a p e s , few p o la r
b o d ie s »
B arred form 's, clu b
s h a p e s, few g ra n u le s
and p o la r b o d ie s .
T ra n s p a re n t, beaded,
d u ll o
T ra n s p a re n t, beaded,
d u ll.
M in u te, sm ooth, convex,
s h in y , t r a n s l u c e n t .
M in u te, sm ooth, convex,
s h in y , t r a n s l u c e n t .
Gray, m in u te , sm ooth,
s h in y , tr a n s l u c e n t .
G ray, m in u te , sm ooth,
s h in y , t r a n s lu c e n t.
S m all, c o l o r l e s s .
S m all, c o l o r l e s s .
Growth in . b r o th
Uniform t u r b i d i t y upper
^ in c h .
Uniform t u r b i d i t y upper
g in c h .
Urea
No h y d r o ly s is .
No h y d r o ly s is .
/
/
C a ta la s e
/
/
G e la tin
No l i q u e f a c t i o n .
No l i q u e f a c t i o n .
C olony c h a r a c t e r i s t i c s
a . Agar s l a n t
b 0 Agar p l a t e
Ce
B lood a g a r p l a t e s
do L o e f f le r s l a n t s
N i t r a t e r e d u c tio n
In d o l p ro d u c tio n
-35F ro b is h e r and s tu d ie d s im u lta n e o u s ly .
This s i m i l a r i t y i s shown in
t a b le VIIo
R e s u lts o f S ch ick t e s t s
The S chick r e a c t i o n of t h e s tu d e n ts from whom th e s e 13 c u ltu r e s were
i s o l a t e d w ere a l l fo u n d to b e n e g a tiv e , hence no fo llo w -u p work was done.
I t had o r i g i n a l l y been p la n n ed t h a t th r o a t c u ltu r e s w ould a g a in b e s tu d ie d
from th o s e who showed p o s itiv e r e a c tio n s t o see i f th e same ty p e o f organ­
ism s c o u ld be r e i s o l a t e d from th e t h r o a t a f t e r im m unization.
B a c te r ia l grow th on th e t e l l u r i t e medium used
As was s t a t e d e a r l i e r , th e most f r e q u e n tly e n c o u n te re d organism on th e
t e l l u r i t e medium u se d , was a gram p o s itiv e m icrococcus grow ing a p p a re n tly
u n in h ib ite d .
The c o lo n ie s w ere v e ry s h in y , sm ooth, and convex.
When
to u ch ed w ith t h e in o c u la tin g n e e d le , th e grow th was found t o be r a th e r
s tic k y and mucoid,
'
S tr e p to c o c c i, i f they grew a t a l l , produced a v ery s m a ll tr a n s p a r e n t
co lo n y .
These w ere d i f f i c u l t to s e e and i t was ‘easy to p ic k them up w ith .
th e o th e r c o lo n ie s .
However, c o n ta m in a tio n w ith s tr e p to c o c c i p re s e n te d a
problem i n o nly a few of th e c u ltu r e s o b ta in e d .
■ One p l a t e showing f l a t g ra y c o lo n ie s proved when sub c u ltu r e d on a n o th e r
medium, to be an organism w ith th e c h a r a c t e r i s t i c ap p earan ce o f th e y e a s ts .
T his coIbny co u ld n o t be c o n fu sed w ith C. d ip h th e r.ia e , b u t d i d grow w e ll
enough and r a p id ly enough t o overgrow the slo w er growing c o ry n e b a c te ria .
A s m a ll gram n e g a tiv e hod was e n c o u n te re d o c c a s io n a lly , b u t t h e .
c o lo n ia l c h a r a c t e r i s t i c s were n o t o b serv ed .
-3 6 —
. Co d ip h th e ria e produced v e ry b la c k , d u l l c o lo n ie s on th e t e l l u r i t e
medium.
I f a p la te w ere h e a v ily in o c u la te d w ith t h i s organism , i t grew
v ery w e ll, b u t when fe w e r organism s w ere p r e s e n t and i n a mixed c u ltu r e ,
th e y w ere o fte n overgrown by th e gram p o s i t i v e c o c c i.
Nd organism s p re s e n tin g Ifche t y p i c a l c h a r a c t e r i s t i c s o r appearance o f
C. p s e u d o d ip h th e r iticum a s d e s c rib e d i n B ergey1s Manual were o b ta in e d .
As th e s e a re l i s t e d a s being p re s e n t i n 26 o u t of
c o n tr o l c a s e s , th e y
must have been e i t h e r i n h i b i t e d by th e t e l l u r i t e i n th e medium o r o f a
colony s i z e n o t v i s i b l e .
.
.
DISCUSSION
Of th e 200 c u ltu r e s ta k e n from th e t h r o a t s o f a p p a re n tly h e a lth y
■young a d u lts on th e Montana S ta te C o lleg e, campus, 13 c u ltu r e s o f d ip h th e r ia
o r d i p h t h e r i a l i k e organism s w ere i s o l a t e d .
F iv e of th e se (Group I ) a re
v e ry s im ila r t o th e C. d ip h th e r ia e ty p e minimus found by o th e r w o rk ers.
T h is i s of i n t e r e s t because o f th e epidem ic of d ip h th e r ia t h a t o ccu red
i n Utah i n 19U7 and 19U8, and i n w hich 39 out .of 73 o f th e d ip h th e r ia
c u ltu r e s i s o l a t e d , o r 3 3 .3 p e r c e n t, -were of ihe minimus ty p e (G a lb ra ith
e t ali, 1 9 ^ 8 ).
Je n k in s (19U8) s t a t e s t h a t th e h ig h e s t c a r r i e r r a t e o f
m-in-imn.c! found by him i n U tah a t t h a t tim e was among c o lle g e s tu d e n ts .
The
in c id e n c e o f d ip h th e r ia c a s e s from w hich minimus ty p e organism s were
i s o l a t e d was a ls o in t h i s age group, b ein g 60 p e rc e n t.
The c a r r i e r r a t e
o f organism s re s e m b lin g th is ty p e b ased on f i v e out of th e 13 o b ta in e d in
t h i s stu d y a t Montana S ta te C o lleg e w ould b e 3 8 .3 p e r c e n t.
This seems
v e ry h ig h , b u t when we ta k e th e p e rc e n t t h a t t h i s r e p r e s e n ts o f th e t o t a l
-3 7 c u ltu r e s made5 w hich was 200, i t i s r e a l l y v e ry sm a ll, b ein g o n ly 2 ,5 p e r­
c e n t of th e t o t a l .
The t o t a l c a r r i e r r a t e w ith 13 p o s itiv e - c u ltu r e s o u t
of th e 200 i s 6.5> p e rc e n t.
Of t h e o th e r t h r e e g ro u p s, Group 2 d i f f e r e d from C. d ip h th e r ia e only
i n t h a t th e s e organism s produced a s o lu b le brown pigm ent i n s o l i d m edia.
■This fo rm atio n o f w a ter s o lu b le , brown pigm ent by coryneb a c t e r i a was n o t
d e s c rib e d I n any o f th e l i t e r a t u r e re v ie w e d .
Hence, i t i s n o t p o s s ib le to
a tte m p t t h e i r c l a s s i f i c a t i o n .
Group 3 resem b les G. d i p h th e ria e w ith th e e x c e p tio n t h a t n i t r a t e s
w ere n o t re d u c e d .
Group h c o n ta in s th re e organism s t h a t d i f f e r e d from
c o ry n e b a c te ria in a number of r e a c tio n s ( t a b l e IV ).
A U th e c u l t u r e s w ere o b ta in e d from S c h ic k n e g a tiv e i n d iv id u a ls .
In
th e Utah ep id em ic, Je n k in s (19US) found t h a t o f t h e .c a s e s o f d ip h th e r ia
caused by th e minimus ty p e s t r a i n s o f G. d ip h th e r i a e , 58,33 p e rc e n t were
p e rso n s who had been immunized, w h ile o n ly 37.03 perdejnt o f a l l o th e r
c a se s r e p o r te d had been immunized.
The f a c t t h a t our g u in e a p ig in o c u la tio n s were n e g a tiv e i s not of
much s ig n if ic a n c e s in c e G a lb ra ith e t a l , (19U8) found t h i s minimus ty p e
d ip h th e r ia organism t o be r e l a t i v e l y a v i r u l e n t t o guinea p ig s , even though
some o f th e s t r a i n s were i s o l a t e d from p a t i e n t s w ith se v e re d ip h th e r ia
in fe c tio n s .
F r o b is h e r e t £ 1 .(1 9 5 1 ) a ls o r e p o r t s th e minimus ty p e organism s
to be on ly s l i g h t l y v i r u l e n t t o r a b b i t s when t e s t e d b y s ta n d a rd i n t r a derm al p ro c e d u re s .
I t was hoped t h a t some in fo rm a tio n w ould be o b ta in e d a s to t h e m e rits
o f P a i ’s egg medium f o r .use i n r o u tin e t h r o a t c u ltu r e s .
While th e number
“ 3 8o f c u ltu r e s ta k e n was n o t la r g e enough t o prove th e s u p e r i o r i t y o f e i t h e r
medium f o r th e i s o l a t i o n o f C0 d i p h t h e r i a s , P a i 1s medium d id prove to have
a d v a n ta g e s,
A t l e a s t , a s many d ip h th e r ia lik e organism s -were o b ta in e d on
th e P ai s l a n t s a s on t h e L o e f f le r s l a n t s .
The e a se o f p r e p a r a tio n o f P a i ’s
medium and t h e e x c e l l e n t d e m o n stratio n o f th e ty p ic a l co ry n eb acteriu m
m orphology, such as b a r r in g , c lu b sh a p e s, and m etachrom atic g ra n u le s , a re
fa c to rs in i t s fa v o r.
O ther b a c t e r i a seem t o develop a s r a p id ly a s th e
d i p h t h e r i a l i k e o rganism s, e s p e c i a l l y t h e m ic ro c o c c i.
On L o e f f l e r 1s
medium th e c o ry n e b a c te ria t e n d to outgrow th e o th e r t h r o a t b a c t e r i a , so
t h a t i n e a r ly c u l t u r e s t h i s organism w i l l p red o m in ate.
This does n o t seem
t o be t r u e of th e P a i medium, f o r of th e f i v e c u ltu r e s show ing p re d o m in a te ly
d ip h th e r ia lik e organism s on th e t e l l u r i t e p l a t e s , fo u r of them w ere t r a n s ­
f e r r e d from L o e f f le r s l a n t s t o th e t e l l u r i t e , and o n ly one from a P a i
s la n t.
The re a s o n s f o r o b ta in in g no d i p h th e r ialike.' b a c t e r i a i n the. th r o a t s
o f th e 33 s tu d e n ts r e p o r tin g to th e Montana S ta te C o lleg e H ealth. S e rv ic e
w ith severs s o re t h r o a t s , i s nob c le a r .
nant Iy s tr e p t o c o c c i .
These c u ltu re s ' were a l l predom i- •
The i n f e c t i o n seemed t o be se v e re i n m ost c a s e s .
c u ltu r e showed a good grow th of th e s e c o c c i i n s i x h o u rs ,
One
A s e r ie s of
c u ltu r e s from p a th o lo g ic a l th r o a ts sh o u ld be done d u rin g s e v e ra l d i f f e r e n t
sea so n s and over a p e rio d o f y e a rs b e fo re any c o n c lu sio n s i n t h i s m a tte r
can be made.
The c u l t u r e s o b ta in e d from F ro b is h e r and G a lb ra ith showed a c o n sid e r­
a b le amount o f v a r i a t i o n ( t a b l e s V and V I).
This m ig h t b e -ex p ected , f o r .
—39”
a s p o in te d o u t by F r o b is h e r (I9 lt0 ), th e r e i s a v a r ia tio n i n th e c u ltu r e s
o b ta in e d from d i f f e r e n t g e o g ra p h ic aL Io c a b io n s 0- C o n seq u en tly , no a tte m p t
h as been made a t d e t a i l e d c o m p arativ e s t u d i e s «
On th e t e l l u r i t e medium u se d , th e m ic ro c o cc i grew v e ry w e ll.
S tr e p ­
to c o c c i ap p eared b u t r a r e l y and w ere th e n few i n number and d i f f i c u l t t o
d e te c t.
On two o c c a sio n s gram n e g a tiv e ro d s -were o b ta in e d from th e
t e l l u r i t e p la te s .
On t h i s t e l l u r i t e medium t h e ad v an tag es a re n o t a s •'
a p p a re n t as some w o rk ers i n th e f i e l d have in d ic a te d .
The c o ry n e b a c te ria
a re e a s i l y re c o g n iz e d , b u t t h e lu x u r i a n t growth o f th e m ic ro c o c c i m ight be
a d is a d v a n ta g e ,
SUMMARY
■ T hroat c u ltu r e s were, o b ta in e d from 200 h e a lth y c o lle g e s tu d e n ts a t
Montana S ta te C o lleg e d u rin g Freshman- -Week o f 19U9, i n an e f f o r t to d e te r ­
mine th e number h a rb o rin g d ip h th e r ia o r d i p h t h e r i a l i k e o rg an ism s,
L o e f f l e r ’s and P a i 1s m edia were compared f o r prim ary c u ltu r in g by in o c u la t­
in g .equal numbers o f t h r o a t c u ltu r e s ' on each medium.
T e l l u r i t e medium and
" c h o c o la te " a g ar were employed f o r i s o l a t i o n and p u r i f i c a t i o n .
T h irte e n
p u re c u ltu r e s of d ip h th e r ia o r d i p h t h e r i a l i k e o rg a n is e s w ere i s o l a t e d and
s tu d ie d .
-Five s t r a i n s ap p eared t o be very s im ila r to known c u ltu r e s o f
C orynebacterium d ip h th e r ia e ty p e m inim us.
The o th e r e i g h t c u ltu r e s d i f f e r ­
ed in a t l e a s t one c h a r a c te r from any o f th e c o ry n e b a c te ria d e s c rib e d i n
B ergey1S M anual.
A ll th e s tu d e n ts from whom th e s e organism s were i s o l a t e d gave h e g a tiv e
S ch ick r e a c t i o n s , hence no com parisons were made as to th e change in c a r r i e r
s t a t u s a f t e r im m unization.
—Uo—
L o e f f l e r 1s and P a d 's m edia w ere found t o be q u ite com parable,
L o e f f l e r 's b e in g p r e f e r r e d f o r i s o l a t i o n and P a i 1S fo r m o rp h o lo g ical
s tu d ie s .
No d ip h t h e r i a l i k e organism s w ere o b ta in e d from any o f th e 3E> p a th ­
o lo g ic a l th r o a t s from which c u ltu r e s were made.
S tr e p to c o c c i ap p eared t o
be th e p red o m in atin g organism .M o rp hological and p h y s io lo g ic a l s tu d ie s made on th e lU c u ltu r e s o f
C. d ip h th e r ia e o b ta in e d from F ro b is h e r and from G a lb r a ith , showed some
v a ria tio n .
T his was a l s o found t o be tr u e of t h e organism s i s o l a t e d
d u rin g th e s e s t u d i e s .
-Ui- .
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“
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