A COMPARATIVE STUDY OF ADAPTED BACTERIOPHAGE
WITH SHIGELLA DYSENTERY ANTIGENS
AND THE PURIFICATION OF
BAGTERIOPHA GE- TOXINS
AND EXOTOXIN

Thesis

for

the Degree o f Ph.D.

C h a r l e s K.

Larwood

MlChlGAisi STATE COLLEGE
of
AGRICULTURE AND ^PPLlED SCIENCE

A COMPARATIVE STUDY OF ADAPTED BACTERIOPHAGE
Wi t h SHIGELL^ DYSENTERY ANTIGENS
«ND THE PURIFICATION OF
BAGTERIOPHXGE-TOXINS
a ND EXOTOXIN,
By
C h a r l e s H.

Larwood

A
TRESIS
IN
BACTERIOLOGY

SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOE THE DEGREE OF DOCTOR
OF PHILOSOPHY TO THE GRADUATE FACULTY
OF THE DEPARTMENT OF BACTERIOLOGY
AND HYGIENE

East

Lansing,

Michigan,

1939

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AOKN0 GLEDCHEN X

The a u t h o r a c k n o w l e d g e s w i t h g r a t i ­
t u d e h i s i n d e b t e d n e s s t o Dr. ‘V. L.
Malltnann u n d e r w h o s e d i r e c t i o n and
g u i d a n c e t h i s work was c a r r i e d o u t ,
and t o t h a n k Dr. K. Vi/. Larkurn and
Dr. 1. F o r e s t H u d d l e s o n f o r t h e i r
many h e l p f u l s u g g e s t i o n s .
This
r e s e a r c h was made p o s s i b l e by a
g r a n t f rom t h e U p j o h n Company.

125808

TABLE OF CONTENTS
I n t r o d u c t i on
Ex per i m e n t a l
Mediums u s e d and t h e i r
Source

preparations

of c u lt u r e s

Preparation

of exotoxins

Preparation

of endotoxins

Preparation

of

Preparation

of bacteriophage

Preparation

of bacteriophage-exotoxins

Preparation

of b a c t e r i o p h a g e - e x o t o x o i d s

Preparation

of p u rified

bacteriris

bacteriophage

a.

Purified

agar

b.

Purified

bacteriophage

Preparation

of purified

Preparation

of antisera

exotoxin

Bacteriophage a n tisera
Exotoxin a n tisera
Endotoxin a n tisera
Bacterin antisera
Bacteriophage-exotoxin
Bacteriophage-toxoid
Purified
D iscussion:

Summary:

antisera

antisera

bacteriophage a n tis e r a

INTRODUCTION
The d y s e n t e r i e s
are s e v e r e
that

or m i l d

are at

least

evacuations
present
eric

in

have a f f l i c t e d

inflammations
p artially

man f c r

of the

lower i n t e s t i n a l

characterized

by f r e q u e n t

of b l o o d y m u c o p u r u l e n t s t o o l s .
the

glands,

in testin al

b u t do n o t

tract,

centuries.

tract,

painful

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

and o c c a s i o n a l l y

invade the u r in a r y

They

tract

in

the mesent­

or t h e

blood

stream.
Th e r e a r e two mai n t y p e s
1*.

S h i g e 11a d v s e n t e r i a e w h i c h

produces acid

and w h i c h p r o d u c e s no a c i d

in mannite, l a c t o s e ,

and d u l c i t e ,
2*

Shigella

nite
The S h i g a

and d o e s n o t

paradvsenteriae

and o t h e r s )

type

differentiated

immune

sera.

f orm i n d o l e .
(Flexner,

which produce a c i d

is

type-specific

in

I t produces

( enterotoxin)»

( Y ) , Strong,

in g lu c o se

and man­

the an tig en

content

and

an e x o t o x i n
The p r e s e n c e

( n e u r o t o x i n ) as w e l l as
o f t h e s e two t o x i n s
investigators

an

have

during

years.
In 1898,

Shiga

(1)

s u f f e r i n g from d y s e n t e r y
his

Hiss

f r o m t h e p a r a d y s e n t e r y g r o u p by s p e c i f i c

b e e n d e t e r m i n e d by a number o f i n d e p e n d e n t
recent

in glucose

and f o rm i n d o l e .

may be

endotoxin

of dysentery b a c i l l i ;

name.

and F l e x n e r

discovered

i n Japan,

F o l l o w i n g S h i g a ' s wo r k ,
in t h e P h i l i p p i n e s

in

the s t o o l s

of

patients,

the m icro-organism which bears
discoveries

further

by K r u s e

i n Jermany

c o n f i r m e d S h i g a ' s T^ork

that
of

bacteria

cause dysentery.

paradysentery,

the Shiga
lated
iae

type,

ha ve been s t u d i e d

The g e n e r a l

it

is

true

for the Shiga

impression

t o be found

is

that

of S h ig e lla

Michigan,

tained

through the co u rtesy
The t r a n s m i s s i b l e

was d i s c o v e r e d
1917.

of

lytic

independently

d ' H e r e l l e has s t a t e d

in Mexico,

in

cells

of S h i g e l l a

search

of

this

of this

substance,

tnat

to d e lv e

further

study

the a n tig e n ic

Holm,

Anderson,

This s tu d y w i l l
and t h e i r

into

is

(2) w i l l
It
the

characters

and L e o n a r d a s

sporadic

products.

para­

out­

A t Owo s s o,

type i s

brought to

d y s e n t e r y was

called

ob­

is

still

in

t h e p h e n o me n o n ,

disease

a subject

of

locusts.

of d isp u te,

the absence of b a c t e r i a l

lead,

not

bacteriophage,

observed

the

no d o u b t ,

to

purpose o f

further

this

nature of b a cteriop hage,

designated

by Larkum ( 3 )

re­

but to

and

later

" S tap h ylococcus B a c t e r io p h a g e Toxoid.

e m p h a s i z e r e s e a r c h on l y s e d S h i g .

bacterial

by t h e

1915 and by d ' H e r e l l e

he f i r s t

substance in

point.

and

the Michigan Department of H e a l t h .

by Twort i n

by K r u e g e r and B a l d w i n

clarification

are caused u s u a l l y

19C9, w h i l e w o r k i n g on a b a c t e r i a l

production

severest

countries,

are o c c a s i o n a l

The n a t u r e o f b a c t e r i o p h a g e
bu t t h e

in i t s

occurrence of th is

The Owosso s t r a i n

differences

type for r e s e a r c h .

most o u t b r e a k s

of the

of these re­

These b a s i c

or s e m i t r o p i c a l

there

to

of S h ig. dvsenter-

dysentery in the United S t a t e s .

an i n s t a n c e

our a t t e n t i o n .

that

that dysentery

in t r o p ic a l

d y s e n t e r y and r e l a t e d g r o u p s
breaks

t han

produce n e u r o t o x i n .

w e r e t h e mai n f a c t o r s

sim ilarities

but t h e v i r u l e n c e

t y p e s h a s b e e n shown t o be l e s s

form i s

a number o f s t r a i n s

w i t h some c u l t u r a l and a n t i g e n i c

and t h e y do n o t

while

In r e c e n t y e a r s

dvsenter iae c e l l s

The i n v e s t i g a t i o n s
this

subject

ficities.

indicate

Animals

of b a c t e r i o l o g i s t s

that

b a c t e r io p h a g e s have a n t i g e n i c

injected

with bacteriophage

corresponding

antibacterial

antibodies.

bacteriophage

is

of t o x o id

not

an a n t i g e n

ties

that

endo-and

toxin.

staphylococcus

since

it

that

does

to r a b b i t s ,

the to x ic

proper­

Holm, A n d e r s o n and Le o n a r d ( 4 )

bacteriophage

exotoxin p r in c ip le s

develop

states

innocuous

produce a n t i t o x i n which n e u t r a l i z e s

of s ta p h y lo c o c c u s

state

Larkum ( 3 )

is

speci­

filtrates

character,

p r o d u c e h e m o l y s i s and d e r m o n e c r o s i s ,

and w i l l

who h a v e s t u d i e d

in s o l u t i o n

toxoid

contains

both

which they d e s i g n a t e

" A m b o - t o x i d ".
It

is

the purpose

bacteriophage antigens
Shig.

are toxoid

or t o x i n

to determine

if

in character

for

d v s e n t e r i a e and t o c ompa r e b a c t e r i o p h a g e and p u r i f i e d

teriophage with S h i g e ll a
toxin,

endotoxin

cedure

bacterin,

exotoxins

b a c t e r i o p h a g e No.

led th e

purification

bac­

bacteriophage-exo-

of b a c t e r io p h a g e accord in g

o f K r u e g e r and Tamada ( 1 1 )

Shigella

exotoxin,

these

and b a c t e r i o p h a g e - e x o t o x o i d .

The p u r i f i c a t i o n

that

of t h is research

author

of S h i g e l l a

and t h e s u b s e q u e n t d i s c o v e r y

18 c o n t a i n e d

to attempt
exotoxin.

to th e pro­

a sim ilar

b o t h e n d o - and
procedure for

the

EXPERIMENTAL

A review

of the

medi a r e q u i r e d

for

indicates

s e v e r a l media

(7)

that

have c a r r i e d

toxin.

production

isms f o r f i v e
was p r o d u c e d

of

definitely

dysen ter i a e

between th e e x o t o x i n
by g r o w i n g t h e

w hile

their

endotoxin

f rom a g a r g r o w t h s

The f i l t r a t e from t h e

organ­

latter

in
med­

the endotoxin.

Subsequent
t wo t y p e s o f me d i a

data w i l l
for

experiments

show t h a t

the production

two t y p e s

prepare b a cterio p h a g ic
and e n d o t o x i n s

the organisms

two d a y s .

of

and e n d o t o x i n

the S h ig e lla

T h e i r e x o t o x i n was o b t a i n e d

for

to types

u s e d . Olifcsky and K l i g l e r

study of

quite

by i n c u b a t i n g

i um c o n t a i n e d

antigens

on n u t r i e n t

b r o t h and n u t r i e n t
ferm entation

agar.

it

is

u n n ecessary to use

of both t o x i n s

o f media were used
i n b r o t h as w e l l

in

but in t h e
order to

as e x o t o x i n s

agar.

C u l t u r e me d i a f o r g r o w t h o f

for

exotoxin

days in a l k a l i n e - e g g b r o th ,

solution

author's

in r e f e r e n c e

must be

out e x t e n s i v e

They d i f f e r e n t i a t e

and e n d o t o x i n .

salt

the

literatuie

Each wa s

r e a c t i o n s were

the

organi sms w e r e n u t r i e n t

adjusted

1 per

cent

t o pH 7 . 6 .
peptone water

Medi a
con­

t a in i n g Andrade's

i n d i c a t o r w i t h 0 * 5 per c e n t e a c h o f g l u c o s e ,

mannite,

and d u l c i t e .

The t e s t

in

p eptone water

lactose,

7 day o l d c u l t u r e
t wo d r o p s

1 per c e n t

of E h r lic h 's

(6)

for

r e a g e n t was a d d e d ;

i n d o l e was made on
i n w h i c h one or

preparation

Strains
b ility

if

of m e d i a

of S h ig . dvsenteriae rapidly

frequent

transplants

are

lose

n o t made.

medium u s e d o n c e a mont h m a i n t a i n e d

their

via­

sem i-solid

a

the t o x ic it y

of th e or­

ganism.
The medium was p r e p a r e d a s f o l l o w s ;
1.

Seai-solid
Whites

Medium

of 2 eggs

Yolk of

1 egg

200 cc d i s t i l l e d
Mix t h e e g g s
ing the w a ter.
first
i z e at

water-

t h o r o u g h l y w i t h an e g g b e a t e r

B o il 2 minutes,

through a w ire s t r a i n e r ,
15 l b s .

pressure for

To a s t e r i l e
0 . 5 p Na Cl )

and a l l o w t o

solid ify.

Mi x ,

a gar

(1 .5 $ agar,

S teril­

soft

1% p e p t o n e ,

o f 5 cc o f

t u b e under a s e p t i c

Stab c u ltu r e s
quite

Filter

15 m i n u t e s .

e g g mi x i n t h e p r o p o r t i o n

of agar.

The medium i s

constantly.

and t h e n t h r o u g h c o t t o n .

veal infusion

add s t e r i l e

m ix t u r e t o 2 cc

stirring

b e f o r e add­

egg

conditions,

a r e made.

but w i l l

solidify

s omewhat

on

c ooling.
This
viability

o f medium w i l l

of organisms,

transplanted.
"seed ings"

type

I t was

in n u t r i e n t

not s u f f i c e

to m aintain the

e x c e p t when t h e s t r a i n s

are f r e q u e n t l y

found t h a t r a p i d t r a n s f e r s
broth net only rendered

of ten

or more

the organisms

viable

but enhanced t h e p r o d u c t i o n
2.

Nutrient
A.

o f b o t h e x o - and e n d o t o x i n s

Broth

Formula;
Ground v e a l ,
Peptone,

fat

free

Proteose,

Sodium c h l o r i d e ,

(Infusion)

5 0 0 gms
15 gms

Bacto

5 gms

G. P .

10

cc.

1000

cc.

S t e r i l e 25$ d e x t r o s e s o l u t i o n
D i s t i l l e d water
B.

Procedure;
5 0 0 gms .
in

of chopped,

1 liter

night

in

of d i s t i l l e d

v e a l were placed

w a t e r and i n f u s e d

the r e f r i g e r a t o r .

f o r an h o u r ,

20 m i n u t e s a t

ed o v e r n i g h t

and c o t t o n ,

autoclaved

in th e r e f r i g e r a t o r .

was t h e n skimmed f r o m t h e s u r f a c e

salt
to
for
to

filtered

15 pounds p r e s s u r e

ume was made up t o

over

I t was t h e n b o i l e d

passed through a p r e s s ,

through c h e e s e c l o t h
for

defatted

1 liter.

and c o o l ­
The f a t

and t h e v o l ­

The p e p t o n e and

we r e ad d e d and t h e r e a c t i o n was a d j u s t e d

pH 7 . 6 .

The me d i a w e r e h e a t e d

10 m i n u t e s .
pH 7 . 6 .

Erlenmeyer
One c c .

t o 100°C.

The r e a c t i o n was r e a d j u s t e d

lOOcc p o r t i o n s w e r e p l a c e d
flasks

portions

and s t e r l i z e d
of

25/a s t e r i l e

i o n was added a s e p t i c a l l y

to

under

in
pressure.

dextrose

each f l a s k

solut­
prior

to use.

All flasks

were in c u b a te d

at 35-36°C.

f o r 48 hours t o c h eck s t e r i l i t y .
3.

N u t r i e n t Agar.
a.

Formula;
Na C l ( G . P . )

5 gms.

Neo-Peptone

15 gms.

V e a l I n f u s i o n ( p r e p a r e d a s i n 2b 5 0 0
b u t made d o u b l e s t r e n g t h )

cc.

D istilled

cc.

water

500

Agar
b.

4 0 g ms .

The medium-was p r e p a r e d a s

i n 2 b.

SOURCE OF CULTURES
S h ig . dvsenteriae
number o f u n i v e r s i t i e s
tity

c u l t u r e s were k i n d l y

and b i o l o g i c a l

o f e a c h c u l t u r e was c h e c k e d

glucose,

mannite,

lactose

laboratories.

and d u l c i t e ,

by i n d o l e

and by a g g l u t i n a t i o n

dysenteriae antisera

obtained

listed

f r om t h e N a t i o n a l

The c u l t u r e s w i t h t h e i r
i n T a b l e 1.

tests

by a

The i d e n ­

by f e r m e n t a t i o n t e s t s

i n Dunham' s s o l u t i o n

Health.

supplied

on

production
with S h ig .
Institute

biochemical rea ctio n s

are

of

TABLE I
THE DIFFERENTIAL CULTURAL CHARACTERISTICS
OF SHIGELLA

w

co
o
o

p>
o
*
/

Organism
#1 - # 1 — N o r t h w e s t e r n U n i v e r s i t y
2 =

#0.35

"

"

/

3 =

#2

"

"

/

4 = #K7A W a s h i n g t o n U n i v e r s i t y

/

5 = D e tr o it Dept,

of Health

/

6 = # 1 3 3 9 P a r k e D a v i s & Co.

/

7 =

/

8

t

#0.1675

*

M

# 2 0 2 L e d e r l e B i o l o g i c a l Labor'
atories

9 = U niversity
10 ”

/

of W isconsin

"

"

/

"

/

11 = S t a n f o r d U n i v e r s i t y

i

12 - N a t i o n a l Type C u l t u r e
(London) #4837

/

13 = U n i v e r s i t y

/

of C in c in n a ti

14 = N a t i o n a l Type C u l t u r e
(London) # 3 5 1 0

/

15 = H ^ - S h i g a , I m p e r i a l U n i v e r ­
s i t y , Japan

/

16 = j 2

M

n

«

-/■

17 = N

"

"

"

4

18 =

ii

n

»

/

«

n

/

of Health

/

19 =
20

=

q

h

— Michigan Dept,

= acid

formation

H

n
S
IS
3

w
cc

o

Eh
o
<q

Pi

W
l-H
O
hJ

p>
Q

W
■J
o

PI
s
n

Remarks

PREPARATION OF EAOTQa INS

S t r a i n Mo.

18 was t r a n s f e r r e d

dextrose nutrient broth.
included

four lo o p f u l s

four hours
ferred

or l e s s ,

t o a 100 c c

of t h i s
ture

which p e r c e p t i b l y

flask

cc o f

the

24 h o u r s .

containing

of organisms

o f a 4 0 cm.

a layer

last

of

the

layer

coarse

thread.

silk

Erlenmeyer

flask

of

c u l t u r e was t r a n s ­

at

absorbent co tto n

physiological
for

Even d i s t r i ­

or s l i g h t l y

c u r v i n g about

the curved area

w h i c h was s e c u r e d w i t h

solution

i n a 500 cc

p r e s s u r e for 30 minutes#

loosened with
salt

transplant

to K o lle c u l ­

" s p r e a d e r " was s t e r i l i z e d

15 l b s .

bro th which

o r t h e a g a r was o b t a i n e d by

The o r g a n i s m s w e r e g r o wn 20 h o u r s
t h e gr o wt h was

agar.

i r o n w i r e and c o v e r i n g

This

«

b r o t h in

of n u t r i e n t

nutrient

in

of the broth

One c u b i c c e n t i m e t e r

over t h e s u r f a c e

stiff

with a thin

ate

10 c c

clouded

c o n t a i n i n g 2b c c

m a x i n g a " s p r e a d e r rt t h r o u g h a p r o c e s s
10 cm.

in

24 h o u r b r o t h c u l t u r e was made a s e p t i c a l l y

flasks

bution

Each t r a n s p l a n t

then 0 .1

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

f o u r or more t i m e s

a "spreader"

on n u t r i e n t
after

agar

and

5 t o 10 c c

had b e e n a d d e d and a l l o w e d

to

of
macer­

two h o u r s .
PROCEDURE FOR SEPARATION OF Ea QIQa IN
FROM ORGa MlGMS
1.

60c c for

the

of S h i g e l l a

1 h o u r and c h e c k e d f o r
2.

by r a p i d

Suspensions

Supernatant

e x o t c x i n was f i l t e r e d

at

sterility.

f l u i d was

centrifugation.

organism s were neated

separated

The s u p e r n a t a n t

f rom t h e
fluid

suspension

which c o n t a in s

through a Pasteur-Chamber land

L3.

c a n d l e and c h e c x e d
3.
venous

M.

L. D.

injection

paration

for

sterility.

of

t h e e x o t o x i n was d e t e r m i n e d

in the m arginal vein

4.
The p r e c i p i t a t e d
of e n d o t o x in .

cells

of

the

by i n t r a ­

ear o f a r a b b i t .

were r e t a in e d

for

future

pre­

PREPARATION OF ENDOTOXIN

Boivin

and M e s r o b e a n u ( 3 )

h a v e s hown t h a t
mostable

in

acteristic

l e t h a l amounts,
This

paralysis

me t h o d o f B o i v i n
The c e l l s
NagGOg f o r s i x t y

from S h i g .

s u b s ta n c e does not

for

dvsenteriae
into

susceptible

followed

by d e a t h

produce the char­

t e c h n i q u e , w h i c h was a m o d i f i c a t i o n

and M e s r o b e a n u ,
were d i g e s t e d

was c a r r i e d

an a d d i t i o n a l

added u n t i l

the c e l l s

ed t o s t a n d

th r e e hours,

sixty

m i x t u r e was now c e n t r i f u g e d .

hours.

w it h a change of d i s t i l l e d

and d i g e s t i o n was c o n Trichloracetic

a c i d was

and t h e m i x t u r e was a l l o w ­

The s u p e r n a t a n t
d istilled

water every

contained

and 0 . 5 ; o

n e u t r a l i s e d w i t h N / 1 NaOH.

by means o f a c e l l o p h a n e s a c a g a i n s t

the

the

t r y p s i n was r e p l e n i s h e d ,

g a v e a pH o f 3 . 5
then

of

out as f o l l o w s ;

with 0.5^ trypsin

h o u r s and t h e n t h e

the d i a l y s i s - s a c

a ther­

exotoxin.

no more s o d i u m c a r b o n a t e was a d d e d ,

tinu§d

technique,

produces diarrhea

of the

The a u t h o r ' s

in

acid

b o d i e s o f S f o r m w h i c h , when i n j e c t e d

i n 24 t o 4 8 h o u r s .

but

c o lla b o r a tio n w ith Cglalb)

trichloracetic

s u b s t a n c e c a n be e x t r a c t e d

bacterial
animals

by t h e

(in

l i q u i d was d i a l y z e d
water

12 h o u r s .

endotoxin.

The

f o r 36 h ou rs

The m a t e r i a l

The d i a l y s a t e

was

filtered

t h r o u g h a P a s t e u r - C h a m b e r l a n d L . 3 c a n d l e and g r a d

u a t e d amo u n t s w e r e i n j e c t e d

intravenously

d e t e r m i n e t h e minimum l e t h a l d o s e .

into rabbits

to

preparation

sh ig e lla bactsein

of

The same p r o c e d u r e f o r p r e p a r a t i o n
used

for S h ig ella

bacterin.

e x u d a t e s we r e l e f t
were i n j e c t e d
imum l e t h a l

intact

The b a c t e r a l c e l l s

after

intravenously

rabbits

w ith the adaptation

injected

duced p a r a l y s i s

the d iff e r e n t

of bacteriophage

intravenously.

and d i a r r h e a

D e a t h o c c u r r e d when l a r g e r
Shigella
successive

times

transfer

loops

clouding

in

through c o t t o n ,

This b a c t e r io p h a g e a l s o
injected

followed

bacteria

in

subcutaneously.

at

least

four

w e r e u s e d when

four

l C c c di d n o t p r o d u c e d e f i n i t e

less

incubation.
sewage.

by f i l t r a t i o n

e a r t h and f i n a l l y

free.

pro­

d o se s were g i v e n .

of organisms
or

8.

a rab b it weighing 2

and a d d i t i o n a l t r a n s p l a n t s

coated with f u l l e r ' s

activity

for

No.

began

N o . 18 b a c t e r i o p h a g e

d v s e n t e r i a e was t r a n s p l a n t e d

four hours

antigens

t© S h i g e l l a

in r a b b i t s

o b t a in e d from f r e s h u n t r e a t e d

it

t h e min­

BAGTERIOPHa GE

of

had a minimum l e t h a l d o s e o f 4 c c

render

to determ ine

c o m p a r i s o n was made w i t h s t r a i n

kilograms

a mo u nt s

dose.

The p r e l i m i n a r y wor k f o r

that

and b a c t e r i a l

h e a t i n g and g r a d u a t e d

into

preparation

The f i n a l

o f e x o t o x i n was

After

B a c t e r i o p h a g e was

The s e w a g e was f i l t e r e d
through f i l t e r

paper

by u l t r a f i l t r a t i o n

su fficien t

rapid

to

transfers

o f t h e b a c t e r i o p h a g e was d e t e r m i n e d by t h e

methods

the

r e c o mme nd e d by d ' H e r e l l e
Krueger

(9)

for

(8)

as p r e l i m i n a r y t e s t s

the f i n a l t e s t s .

After

t h e b a c t e r i o p h a g e had

reached a "fixed"

stage the succeeding f i l t r a t e s

for

inoculations.

fu tu re animal
Another

Mal l mann ( 5 ) .

s o u r c e o f b a c t e r i o p h a g e was t h a t
The b a c t e r i o p h a g e r e a d i l y

18 and a f t e r

10 t r a n s f e r s ,

the t i t r e

lysing

checked a gain

after

titre

Subsequent
increase

in

checks

for

The l a r g e

abundant supply of b a c te r io p h a g e
as to

the r e la t iv e

toxicity

Bacteriophage with
from u n a l t e r e d

sewage.

were r e s e r v e d

obtained

lysed S h ig e lla

o b t a i n e d was

strain

10“ ©.

and 150 t r a n s f e r s

number

This

so th at

g a v e no

a check could

lysed

bacterial

a final

titre

of 10-8.

be made

products.
was

isolated

B a c t e r i o p h a g e t h a t was s i x mo n t h s
larger

doses

10-9

o f t r a n s f e r s g a v e an

of the

produced death w ith s l i g h t l y

f rom

50 t r a n s f e r s w e r e made was

100 t r a n s f e r s

the t i t r e .

and by

than w i t h t h e

old

freshly

prepared b a c te r io p h a g e .
PREPARATION OF BACTERIOPHAGE-EXOTQXIN
B a c t e r i o p h a g e - e x o t o x i n was p r e p a r e d
p h a g e and e x o t o x i n
The M . u . D .

in proportion

to

their

by a d d i n g b a c t e r i o ­

minimum l e t h a l

dose.

o f t h e b a c t e r i o p h a g e - e x o t o x i n was t h e n d e t e r m i n e d .
PREPARATION OF Ba CTEKIQPHAGE-EAOTOXOID

B a c t e r i o p h a g e - e x o t o x o i d was p r e p a r e d a s f o l l o w s :
0.4

per c e n t

form alin (U.tf.P.)

bacteriophage-exotoxin.

CM. a . B . - 4 c c )

was added t o

the f l a s k s

o

The m i x t u r e was i n c u b a t e d
I t was t h e n

checxed for

for

f o u r weeks a t 4 0 ° 0 .

toxicity.

The same q u a n t i t y was u s e d f o r
used i n

bacteriophage-toxin

i m m u n i z a t i o n a s was

immunizations.

PREPARATION OF PURIFIED BACTERIOPHAGE
(a)

Tne P r o c e d u r e f o r t h e P u r i f i c a t i o n

The p r e p a r a t i o n o f r e l a t i v e l y
been presen ted
purification
order

oy K r u e g e r

process,

t o use agar

h u n d r e d grams o f a g a r
liters

boiling
in

eight

was c o n t i n u a l l y

precipitated

form was d i s s o l v e d

at a temperature j u s t

at

white sedim ent.

The m e l t e d

or more l i t e r s

w h i c h had b e e n s l i g h t l y

fluid

Two h u n d r e d and f i f t y

in a double b o i l e r .

a th in stream i n t o

hol,

out t h e

a g a r was u s e d a s a s u b s t r a t e .

i n a powder

of hot water kept

point

To c a r r y

In

t h e au tho r has m o d i f i e d t h e p r o c e d u r e recom­

mended by D o m i n i k i e w i c z ( 1 2 ) .

four

p u r e b a c t e r i o p h a g e has

and Tamada ( 1 1 ) .

purified

o f ^g ar

acidified

stirred

After

the f l u i d

the sediment

had s e t t l e d

and t h e a g a r

s e d i m e n t was p o u r e d

w h i c h was s t r e t c h e d

to

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

a g a r was w a s h e d

traces

of a c e t i c

acid.

with a c e t ic

60°C.

The a g a r was s t o r e d

acid while
rod.

The a g a r

s h a k e n and

t h e a l c o h o l was d e c a n t e d ,
of fine-meshed

ten

liters

i n 95% a l c o h o l

in g l a s s

the

i n t h e f o r m o f a spongy^

jar of

I t was d r i e d

to

o f 95 per c e n t a l c o ­

on a p i e c e

over a b a t t e r y

three

below the

had b e e n v i g o r o u s l y

th e bottom,

in

a g a r was p o ure d

by means o f a g l a s s

o n c e f rom t h e s o l u t i o n

to three

capacity.

t o r e mo v e a l l

i n an o v e n f o r
containers.

linen,

two h o u r s a t

The r e m o v a l o f

traces

of a c e t i c

acid

so t h a t

was n e u t r a l was a p r o b l e m t h a t had t o he s o l v e d .
that

the

particles

o f agar r e t a i n e d

(12).

and f i r m l y p r e s s e d
in

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

ten 250cc q u a n t i t i e s

gave a n e u tr a l

The p r e p a r a t i o n

slight

recomme nded by K r u e g e r and Tamaf i a

is

a well-known

fact

commonl y a s s o c i a t e d

c u l t u r e medium u s e d f o r i t s
emphasized

by B r o n f e n b r e n n e r

that th ese

non-specific

terious.
(14)

i mme r s e d

four times, each,
This p r o c e s s

Andrews,

of R e l a t i v e l y

of relatively

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

phage,

cloth

o f 95 per c e n t a l c o h o l .

Preparation

is

as recommended by Dom-

agar.

(b)

It

even a f t e r

a g a r was a l t e r n a t e l y

out t h r o u g h a l i n e n

agar

I t was f o u n d

co n sid era b le acid

e x c e s s i v e washings w ith n e u tr a l a lc o h o l
inikiew icz

the

Bulloch,

pure b a c t e r i o p h a g e s u s ­

m o d i f i c a t i o n of the procedure
(11).

that

the

lytic

with c o l l o i d a l

preparation.
( 1 3 ) , and i t

protein

agent,

bacterio­

substances

This

point

Feldes,

of the

h as b e e n

appears quite

substances

Dreyer,

Pure B a c t e r i o p h a g e

obvious

may p r o v e q u i t e
Le d i n g h a m,

dele­

and Wo l f

state;
"Since the horse i s a h ig h ly s u s c e p t i b l e animal, a
s t a r t must be made w i t h v e r y s m a l l d o s e s .
The a c t u a l
q u a n t i t y o f t h e t o x i n t o be i n j e c t e d w i l l d e pe nd on
i t s strength.
The s t r o n g e r t h e t o x i n t h e b e t t e r , s i n c e
t h e v o l u me i n j e c t e d w i l l be t h e s m a l l e r .
Aftej. e v e r y
i n j e c t i o n , a c a r e f u l w a t c h must be k e p t on t h e a n i m a l .
The i n t e n s i t y o f t h e l o c a l r e a c t i o n e v i d e n c e d by i n f i l ­
t r a t i o n at the s i t e of i n j e c t i o n , the tem p erature, appe­
t i t e , and g e n e r a l c o n d i t i o n o f t h e a n i m a l a r e t o be n o t e d
and r e c o r d e d .
Among t h e i l l - e f f e c t s t h a t may f o l l o w
t o x i n i n j e c t i o n s , t h e 'most s e v e r e a r e p a r a l y s i s and d e a t h
o f t h e h o r s e w i t h f a t t y d e g e n e r a t i o n o f t h e h e a r t and
d e g e n e r a t i v e changes in the k id n e y .
A c h r o n i c ma l a d y
s o m e t i m e s s u p e r v e n e s in the a n i m a l s w hich have under gon e
f o r some y e a r s c o n t i n u o u s c o u r s e s o f a n t i t o x i n - p r o d u c t i o n .

The f i r s t s i g n i s an e n f e e b l e m e n t o f t h e a n t i ­
t o x i n - p r o d u c i n g power.
The a n i m a l s o o n b e c o me s i l l
and e v e n t u a l l y d i e s w i t h a m y l o i d d e g e n e r a t i o n o f
t h e s p l e e n and l i v e r , and w i t h h e p a t i c and p e r i t o n ­
e a l hemorrhages.'1
Muir
to

made a s i m i l a r

the r e s u l t s

been
is

(15)

but i t

of absorption of toxins

elaborated

as

statement

follow ing

bacterial

in

applies

t h e body,

infection.

more t o

which have

The s t a t e m e n t

follow s;

"Toxins a r e absorbed from t h e s i t e o f b a c t e r i a l
g r o w t h and. a r e c a r r i e d by t h e b l o o d s t r e a m t h r o u g h o u t
t h e body#
Hence t h e r e i s o f t e n seen in i n f e c t i o n s a
g e n e r a l t o x i c a c t i o n , t h e i n j u r y b e i n g more d i f f u s e
but l e s s s e v e r e .
Various r e t r o g r e s s i v e changes are
th us produced in v a r i o u s organs — cloudy s w e l l i n g ,
f a t t y d e g e n e r a t i o n , sometimes f o c i o f a c t u a l n e c r o s i s .
T h e s e a r e mo s t marked i n h i g h l y s p e c i a l i z e d c e l l s , and
in organs concerned in the e x c r e t i o n of t o x i n s , es p e c ­
i a l l y t h e k i d n e y s and l i v e r . " '
The a p p a r a t u s

and m a t e r i a l s

phage are e x p la in e d
dimensions for the

t u b e a r e 15 cm.

stoppers.

The c y l i n d e r

s in c e the pyrex g la ss

of

bacterio­

Convenient

by 5 cm. w i t h a t o t a l v o l u m e

A pyrex g l a s s

w i t h 70% a l c o h o l w h i c h was l a t e r
solution,

purification

in th e accompanying diagram.

o f a p p r o x i m a t e l y 295 c c .
2 one-hole

for

c y l i n d e r was u s e d w i t h

and s t o p p e r s

were s t e r i l i z e d

removed w i t h s t e r i l e

saline

o c c a s i o n a l l y would break w it h

autoclaving.
The b r i d g e s w e r e i n v e r t e d and f i l l e d
3 per c e n t
distal

with s t e r i l e

purified

a g a r c o n t a i n i n g 2 per c e n t NaCl# by a t t a c h i n g t o t h e

end o f e a c h t u b e a p i e c e

of s o f t

rubber

c l o s e d w i t h a Mohr's

tu bin g clamp.

the

lower

s t o p p e r were i n s e r t e d

der

and s u c c e s s i v e

bridge

and

layers

of 3,

2,

t u b i n g w h i c h was

When t h e a g a r wa s h a r d e n e d

1,

into

the

pyrex c y l i n ­

and O.b p e r c e n t

t a i n i n g 0 . 2 p e r cent, NaCl we r e p i p e t t e d

into

the g l a s s

agar c o n ­
cylinder,

but e a c h
tor

l a y e r was f i r s t

b e f o r e adding th e

divided

so th a t

approximately

the

allowed

succeeding

total

one-half

a g a r was c o n g e a l e d

s u s p e n s i o n was
between
ter

its

le n g th of

over i t

position.

A current

100 t o

of

minus th e

inserts.

quantity cf

bacteriophage

allow a space of

125 v o l t s

After

1 t o 2 cm.

s t o p p e r when t h e

and f rom 5 t o

lat­

10 m i l l i a m -

series

four-45

volts

t o w h i c h was a t t a c h e d

direct

current

voltmeter

of

sufficient

a maximum c a p a c i t y

ohms r e s i s t a n c e .

calibrations

o f 0 . 1 0 a mp s ,

radio

and a r h e o s t a t

and a p p r o x i m a t e l y 2 7 , 0 0 0

The c u r r e n t was p a s s e d

t o 24 h o u r s

solutions

to

stopper

to

by c o n n e c t i n g i n

and m i l l i a m m e t e r

20

the c y lin d er

by t h e r u b b e r

was p l a c e d i n

"B" b a t t e r i e s

for

The l a y e r s w e r e e q u a l l y

u p p e r s u r f a c e and t h e r u b b e r

p e r e s was s u p p l i e d

with

layer.

a sufficient

layered

congeal in the r e f r i g e r a ­

s p a c e o c c u p i e d was e q u i v a l e n t

the

s p a c e w h i c h was o c c u p i e d
the

to

through

the apparatus

and t h e c o p p e r c h l o r i d e and s o d i u m c h l o r i d e

i n w h i c h t h e e l e c t r o d e s w e r e i mmers ed w e r e c h a n g e d

once during that
respectively,

period.

The e l e c t r o d e s

were washed t h o r o u g h l y at

u t i o n s were changed.

of s i l v e r

and c o p p e r

t h e same t i m e t h e s o l ­

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

once during

the procedure.
The 0 . 5
sterile
in

spatula

a sterile

per c e n t
as

Petri

it

layers
to g i v e

of

was f o r c e d

dish

physiological saline

a g a r was s l i c e d

into

f rom t h e c y l i n d e r

and was m a c e r a t e d

for future

agar were t r e a t e d

sections

for

purification.

12 t o

with a

and r e c e i v e d
15 h o u r s i n

The r e m a i n i n g

i n t h e same manner and w e r e f o u n d

a n e g a tiv e r e a c t i o n with Hopkins-Cole (16)

reagent.

The p h a g e c o n c e n t r a t i o n

gave a range between

w h i c h was i n marked c o n t r a s t
report
10

—16

.

greater

com pletely
The

lysed

"layer"

susceptible

met hod h a s

o f t h e uDpe r l a y e r o f p a r t i a l l y
for

to

10“ ^ ,

t o K r u e g e r and Tamada ( 1 1 ) ,

a mount o f t h e c o l l o i d a l

may be u s e d

10““®

bacteria

at a d i l u t i o n

the ad van tages
aggregates,
purified

future p u r ific a tio n .

who
of

of removing a

and t h e r e t e n t i o n

b a cterio p h a g e which

F i g . 1.
A ^ p r s t u p f o r n r e p f r p t i o n of r e l a t i v e l y oure
be c t e r i c u b a g e .
b e c t e r i o o h age s u s o e n n i on.
B, g e l s o f 3 p e r c e n t
2 o e r c e n t , 1 o e r c e n t end 0 . 5 p e r c e n t n u r l f i e d a ^ r end
0 . 2 ; oer c e n t c . p . Ne C l i n d i s t i l l e d w a t e r .
end D, g l a s s
b r i d g e s c o n t a i n i n g 3 . 0 p e r c e n t o u r e e g e r g e l w i t h 0. 2.
n e r c e n t c . o . NeCl.
E, e o r c e l e i n c u n w i t h c r y s t a l s o f
CuCl2 e t b o t t o m .
'V eter to l e v e l of d o t t e d l i n e .
Negative
D o l e ( c n p o e r ) o f D. C. c i r c u i t d i n s i n t o c r y s t a l s .
F, n o r c e l e i n c u p w i t h c r y s t r a l s o f NeCl e t b o t t o m .
‘V e t e r t o
le v e l of dotted lin e .
S ilv e r f o i l attended to o o s it l v e
o o l e o f D. C. c i r c u i t d i n e i n t o c r y s t a l s .

k,

C

Adanted

from K r u e g e r,

J.

G -en.

P h y s i o l . , lro l .

1 3.

RREPal Ui l ON OF PURIFIED EXQTOAlN

The p u r i f i c a t i o n
phoresis

of S h ig e lla

made p o s s i b l e

exotoxin.

the r e te n tio n

toxin.

The e x o t o x i n u s e d f o r

of 0 . 0 5

cc

perceptible

reaction

in

5 cc q u a n t i t i e s ,
death.

Since

positive

the re­

a rabbit

(0.5

o f t h e same e x o ­
intravenously,

bacteriophage

per c e n t ) ,

of b acteriophage,

per c e n t

paralysis
layer

of

of exo­

had an k . L . D .

no

filtr­

w h i c h was r e ­
was i n j e c t e d

was f o l l o w e d

in

by

a g a r g a v e a weak

r e a c t io n w ith the Hopkins-Gole t e s t ,

this

by p a s . s i n g t h r o u g h a n o t h e r 0 . 5

and o n l y a s l i g h t
It

retain

lose

and

fraction

per c e n t

layer.

p r o d u c e d an e x o t o x i n w i t h a n e g a t i v e H o p k i n s - G o l e r e ­

action

w hile

purification

When t h e

characteristic

the 0 .5

was r e p u r i f i e d
This

into

agar

the p u r i f i c a t i o n

to the

When 10 cc

occurred.

the top layer of

jected

of the endo-

the p u r i f i c a t i o n

for a 2.0 K g .r a b b it .

p u r i f i e d was i n j e c t e d

of

part

from t o x i n .

The p r o c e d u r e was a p p l i e d

ate

in

by e l e c t r o ­

T h i s p r o c e d u r e o f f e r e d an o p p o r t u n i t y f o r

mo v a l o f e x t r a n e o u s m a t e r i a l s

toxin

bacteriophage

is

difficult

some o f

its

toxicity.

in t o x i c i t y .

t o u n d e r s t a n d why b a c t e r i o p h a g e

toxicity

e x o t o x i n under
its

loss

when p u r i f i e d

i d e n t i c a l methods
T h i s may

be d u e ,

in t h e agar s u b s t r a t e but checks
onstrate

this

cultures

by e l e c t r o p h o r e s i s
purification

however,

cn t h i s

should

to v a r i a b i l i t y

point

failed

tc

dem­

as the c a u s e .

The ma j o r p o r t i o n
ed w i t h

of

should

(Shigella
received

"0"),

of

t h e e x p e r i m e n t a l worx was c o n d u c t ­

s t r a i n No.

from t h e

18,

which i s

one o f

the f i v e

Imperial U n iv e r s it y ,

Japan,

in

January,

1937.

T h i s c u l t u r e was d e m o n s t r a t e d

t o be t h e mos t

v i r u l e n t ..
Of t h e

19 s t r a i n s

13 ( S h i g a M o o r e ) ,
nounced r e a c t i o n

15,

tested,

16,

1 7,

Strains

o b t a i n e d f rom t h e

w e r e a s a g r o u p t h e mo s t t o x i c .
18 was much more t o x i c

tests

Of t h e s e

than the

other

t h e same r e l a t i o n s h i p

8 ( No .

202-Lederle,

to r a b b i t s

15, 16 ,

1 7,

pro­

18 and 19,

Imperial U n iv e r s ity ,
five

strains,

strain

f o u r . By i n t r a v e n o u s i n ­

as i n d i c a t e d by i n t r a d e r m a l

wa s o b t a i n e d .
S t r a i n No.

20 g a v e c u l t u r a l ,

agglutinating,

characteristics

like Strain

18.

Shigella

c u l t u r e No .

8 was u s e d

for

No s .

18 and 19 g a v e

intraderm ally.

which were t h e c u l t u r e s

oculation

strains

antigenic

the s p e c i f i c

study

of b a c t e r io p h a g e .

and t o x i n

in

the p relim inary

At

first

injections

tests
with

b a c t e r i o p h a g e were made d a i l y .

The i n j e c t i o n s w e r e made b o t h s u b c u t a n e o u s l y and i n t r a ­
venously

on a l b i n o

rabbits.

Shigella

Bacteriophage I n je c tio n s;

The a g g l u t i n a t i n g t i t r e
used for

i n j e c t i o n s was

less

of n o r ma l s e r a

than 1 to 5.

The f o l l o w i n g a n i m a l s r e c e i v e d
1Q^9
later,
ative

as

indicated,

prepared

follow ed with S h ig e lla
antigenic

value:

of a l l r a b b its

l Oc c

from S h i g .
No.

of b a c t e r io p h a g e ,

d v s e n t e r i a e No. 8 and,

18 b a c t e r i o p h a g e f o r c o m p a r ­

O

>
e

43 ,

OF RABBITS

WITH

SHIGELLA

BACTERIOPHAGE

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■23

II

a,

continued

00

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O
O

Tables

III

a and b show t h e

data o b t a i n e d

i n g t h e minimum l e t h a l d o s e o f e x o t o x i n .
dose

f o r r a b b i t s w e i g h i n g 2 . 0 Kg.
Tables

Nos.

3 0 and 3 r e s p e c t i v e l y

obtained

for

the

minimum l e t h a l

exotoxin

dose

Tables

IV and V.

t o b e t h e same a s

for

the

for

exotoxin

p r e p a r a t io n of

Rabbits 3g,

the data for

10,

1 0,

mals were i n j e c t e d

the

inject­

be t h e same

determining
of ra b b its

11 and 1 2 .

normal s e r a

‘i t i s

of

in

also,

the ra b b its
f rom any

of the ex p e r im e n t.

into

two g r o u p s ,

one g r o u p

s u b c u t a n e o u s l y and t h e o t h e r
and S q we r e i n j e c t e d

11 and 12 i n t r a v e n o u s l y .
daily

pro­

rabbits

immune s e r a w e r e f r e e

at t h e s t a r t

5 a>

of

alone.

we r e d i v i d e d

injections

and r a b b i t s 9 ,

to

The minimum l e t h a l d o s e s w e r e f o u n d ,

The r a b b i t s

ously.

prior

o u t i n t h e s ame manner a s f o r

in ter ferin g antibodies

receiving

two h o u r s

of normal p o o l e d s e r a

These d em on strate th at
used

of rab­

alone.

®2» 5 a» ^7» anc* s 8 an(^ r a b b i t s 9 ,
c e d u r e was c a r r i e d

determining

when n o r m a l s e r a

d o s e s w e r e f ound t o

T a b l e s VI and VI I p r e s e n t
the

cc.

w e r e added t o d i l u t i o n s

a t 37° C f o r

The minimum l e t h a l

ast h a t

was f ound t o b e 0 . 5

dose of e x o t o x i n ,

e x o t o x i n and i n c u b a t e d
ion.

The minimum l e t h a l

IV and V show t h e d a t a o b t a i n e d i n

t h e minimum l e t h a l
bits

in d e t e r m i n ­

intraven­

subcutaneously

All

of

f o r 7 d a y s and t h e n w e e k l y

the a n i ­
for

10

weeks *
One w e e k a f t e r
bled

from t h e h e a r t

to g e t

a large

the f i n a l

injections,

into clean s t e r i l e

proportion of clear

test

sera,

e a c h r a b b i t was
tubes.

the t e s t

In order
t u b e s "’e r e

examined f o r i n t e r n a l roughness
did not
placed
that
ton

show an e v e n ,
in each tu b e,

smooth s u r f a c e .

After

from t h e t e s t
frigerator
sterile
until

over

the blood c o a g u l a t e d ,

night,

and k e p t

so

of the c o t­

s u r f a c e was f r e e d

then p laced

in r e ­

and p l a c e d

in

in r e f r i g e r a t o r

was p r e v i o u s l y

(Tabl<@ I I

a

ana b . )

antibody t i t r e

for

subcutaneously

against Shigella

T a b l e IX p r e s e n t s
11 and

determined

to have a

o f 0 . 5 c c f o r r a b b i t s w i t n an a v e r a g e w e i g n t

Kilograms

r a b b i t s Nos.

the antibody

.

Table X p r e s e n t s

e r i o p h a g e No.
r a b b its Nos.

Sg,

Shigella

b a c t e r i o p h a g e No.
titre

10,

11 and

T a b le XI I shows t h e
18.

One we e k a f t e r
agglutination

against

the antibody

9,

10,

bacteriophage

f o r r a b b i t s Nos.

the antibody

final
titre

titre

intravenously

bact­

for

against

18.
data fo r

intravenous

E a c h a n i m a l was i n j e c t e d w e e k l y

1 to 5.

.

No s .

Shigella

titre

12 i mmuni zed

injection

e a c h a n i m a l was

injections
for

of

e ig h t weeks.

b l e d and t h e

determined.

The n o r ma l a g g l u t i n a t i o n
than

for ra b b its

8

i mmu n i z e d s u b c u t a n e o u s l y a g a i n s t S h i g e l l a

b a c t e r i o p h a g e No.

s t r a i n No.

the

5a , 8 7 and S g i mm u n i z e d

18 and T a b l e XI p r e s e n t s
9,

of 2 . 0

T a o i c VIII p r e s e n t s

12 i mmuni z ed i n t r a v e n o u s l y

£ 2 > 5 a> S 7 and S 3

less

needles,

slanted

used.

M. L- D.

8

its

when s e r a was drawn o f f

tubes which were s ea le d

The e x o t o x i n

No.

and t u b e s

one or t wo c e n t i m e t e r s

tube w ith t r a n s f e r

that

A p p r o x i m a t e l y 5 c c was

p l u g s we r e i n s e r t e d

t h e b l o o d came w i t h i n
plugs.

and t u b e s we r e d i s c a r d e d

titre

for

all

a n i m a l s u s e d was

The immunizing dose used for each a n t i g e n was o n e - h a l f
the minimum l e t h a l d o s e .

Rabbits LR 1, 2 and 3 were t r e a t e d

wi t h e x o t o x i n ; LR 4, 5 and 6 wi t h e n d o t o x i n , LR 7, 8 and 9
wi t h b a c t e r i n , LR 11, 12 and 13 with e x o t o x i n - b a c t e r i o p h a g e ;
LR 14, 15 and 16 with p u r i f i e d b a ct eri opha g e; LR 17, 18 and 19
w i t h b a c t e r i o p h a g e, LR 20, 21 and 22 w i t h ba c t e r i oph a g e e x o t o x ­
oid.

Nos. LR 10 and 23 were c o n t r o l s .
The a g g l u t i n a t i o n

t i t r e s f or the r e s p e c t i v e a n t i ­

gens were as f o l l o w s ;
Exot oxi n l e s s than 1 to 5
Endotoxin ranged from 1-3000 to 1-5000
B a c t e r i n ranged from 1-1600 to 1-2000
E x o t o x i n - b a c t e r i o p h a g e from 1-20 to 1-25
P u r i f i e d bact eri ophage from 1-50 to 1-60
Bac t e ri ophage 1 to 50
B a c t e r i c p h a g e - e x o t o x i d 1 to 60
In order t o have s u f f i c i e n t e x o- en d o t o x i n to carry
out the t e s t s for e v a l u a t i o n of antibody production o f Shig.
d y s e n t e r i a e , v a r i o u s batches o f the exo - en d ot o x i n were pooled so
t h a t a c o n s t a n t M.L.D. could be o b t a i n e d .

Since the range of

t o x i c i t y of the s e v e r a l batches was 0 . 0 5 to 0 . 2 c c , the d i f f e r ­
ent b a t c h e s were mixed in such pr o p o r t i o ns t hat the M.L.D. of the
f i n a l mixture was 0 . 1 c c .
The method for de t e r mi n at i on of proport ion of the bat­
ches i s i l l u s t r a t e d as f o l l o w s :

( 1)

1

20;

(2)

.05

1

10;

0.1

1
0.2

5

Solution;
0.05

20

1 part
10

0.2

5

10

2 parts

The r a t i o of 0 . 0 5 M.L.D. tc .2 M.L.D. i s 1 part to 2 p a r t s ,
or f or every cubi c c e nt i me t e r of 0 . 0 5 M.L.D., two cubi c cen­
t i m e t e r s of 0 . 2 M.L.D. should be used.
For car e of o p e r a t i o n and c l e a r comparative r e s u l t s ,

animals

of approxi mat el y t he same age and weight were used.

This

made i t p o s s i b l e tc g i v e each animal e x a c t l y the same dosage.

DETERMINATION OF M . L . D .

OF EXOTOXIN FROM STRAIN 1 8 .

TABLE I I I a
Rabbit

Weight

Saline

1

2.08

2
3

Exotoxi n

Result

0.4cc

0.6cc

d . 29 h r .

2.06

0.5cc

0 . 5cc

d. 56 hr.

2.08

0. 6cc

0.4cc

S l i g h t Par­
alysis

DETERMINATION OF M.L.D. OF EXOTOXIN FROM STRAIN 18.

TABLE I I I b
Rabbit
4

RECKECK OF I I I a
Weight

Saline

2.00

Result

0.4cc

0. 6cc

d.39 hr.

0 . 5cc

d . 72 hr.

Q.4cc

Paralysi s

i

Exctoxin

CM

o

0.5cc

6

2.01

Q.tocc

CM

5

DETERMINATION OF M.L.D. OF EXOTOXIN MIXED WITH NORMAL SERUM
OF1 RABBIT NO. SO.
TABLE IV
Rabbit

Weight

Serum

Saline

1

2*02

lcc

0.4cc

0*6cc

d . 38 hr

2

2*00

lcc

0* 5cc

0. 5cc

d . 60 hr

3

2.01

lcc

0* 6cc

0 . 4cc

Paralysii

Exotoxin

Results

DETERMINATION OF M.L.D. OF EXOTQXIN MIXED WITH NORMAL
SERUM OF RABBIT NO. S.
TABLE V
Rabbit

Weight

Serum

Saline

Exotoxin

Results

1

2*00

lcc

0.4cc

Q.6cc

d*40 hrs

2

1*99

lcc

0.5cc

0*5cc

d*59 hrs

3

1.98

lcc

0.6cc

Q.4cc

Lived

DETERMINATION OF M . L . D . OF EXOTOXIN MIXED WITH POOLED
NORMAL SERUM OF RABBITS S 2 , 5 a , S 7 , a n d S 8

TABLE VI
Rabbi t

Weight

1

2.03

loc

0.4cc

0 . 6cc

d.39 hr.

2

2.02

lcc

0 . 5cc

0 . 5cc

d *79 hr.

3

2 . 04

lcc

0 . 6cc

0.4cc

Slight
Paralysis

Pooled Sera

Saline

Exot oxi n

Results

DETERMINATION OF M.L.D. OF EXOTOXIN MIXED WITH POOLED
NORMAL SERUM OF RABBITS 9, 10, 11 and 12
TABLE VII
Pooled Sera

Saline

Exotoxi n

Rabbit

Weight

1

2.01

lcc

0 . 4 cc

0. 6cc

d.52 h r .

2

2.01

lcc

0 . 5cc

0.5cc

d . 92 hr.

3

2.00

lcc

0.6cc

0.4cc

Lived

Results

DETERMINATION OF M.L.D. OF EXOTOXIN MIXED WITH SERA FROM
RABBITS S 2 , 5a, 37 and 38 IMMUNIZED SUBCUTANEOUSLY WITH
SHI GA- BACTER10PHAGE NO. 8
TABLE VIII

Rabbit

Weight
2*08
1*96

1

2
3
4
5

1*93
2*06

6

2* 00

2 * 1 0

Pooled Sera
lcc
lcc
lcc
lcc
lcc
lcc

Saline
0 . 4cc
0.4cc
0.5cc
0*5cc
0 . 6 cc
G. 6 cc

Exotcxi n
. 6 cc
. 6 cc
0#5cc
0 *5cc
0*4cc
0*4cc
0
0

Results
d . 4 8 hr
d *4 6 hr
d. 69 hr
d*70 hr
Survived
Survived

DETERMINATION OF M.L.D. OF EXOTOXIN MIXED WITH SERA FROM
RABBITS 9 , 10, 11 and 12 IMMUNIZED INTRAVENOUSLY WITH
SHIGA-BACTERIOPHAGE NO. 8
TABLE IX

Rabbit
1
2

3
4
5
5

Weight

Pooled Sera

Saline

Exctoxin

Resu I t s

2.04
2.08

lcc
lcc

0.4cc
0.4cc

0

. 6 cc
. 6 cc

d ♦56 hr
d *50 hr

2 .0 1

lcc
lcc

0.5cc
0*5cc

0*5cc
0.5cc

d .84 hr
d • 74 hr,

2 .0 0

0

2*03
lcc
Q. 6 cc
0.4cc
Survived
____ 1 »9 9_________ l c c __________ 0. 6 cc________ Q.4cc____ S u r v i v e d

DETERMINATION OF M.L.D. OF EXOTOXIN MIXED WITH SERA
FROM RABBITS S2, 5a, S7 and S8 SUBCUTANEOUSLY
WITH SHIGA-BACTERIOPHAGE NO.18
TABLE X
Rabbit

Weight

1
2
3
4

1 . 99
2*02
2.04
2*01
2*00
1.98

5
6

Pooled Sera
lcc
lcc
lcc
lcc
lcc
lcc

Saline
0.4cc
Q.4cc
0.5cc
0.5cc
0 . 6cc
0 . 6cc

Exotoxin
0.6cc
0*6cc
0.5cc
0.5cc
0 . 4cc
0.4cc

Results
d .74 hr
d •79 hr
Survived
Survived
Survived
Survived

DETERMINATION OF M.L.D. OF EXOTOXIN MIXED WITH SERA
FROM RABBITS 9, 10, 11 and 12 IMMUNIZED INTRAVENOUSLY
WITH SHIGA-BACTERIOPHAGE NO.18
TABLE XI
Rabbit

Weight

1
2
3
4
5
6

2.03
1.9b
2.02
2.00
2.03
1.99

Pooled Sera
lcc
lcc
lcc
lcc
lcc
lcc

Saline
0.4cc
0.4cc
0.5cc
0*5cc
0.6cc
0 . 6cc

Exotoxin
0 • 6cc
0 . 6cc
0.5cc
0.5cc
0.4cc
0.4cc

Results
d • 83 hr
d .83 hr,
Survived
Survived
Survived
Survived

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DISCUSSIOW

This study was i n s t i g a t e d p ri ma r i l y by the f a c t t ha t
Larkum had demonstrated t h a t a s t a p h y l o c o c c u s b a c t er i op h a g e ,
a l t h o u g h i t was not t o x i c , would s t i m u l a t e in the r a b b i t
t he producti on of a n t i t o x i n .

Inasmuch as the i n j e c t i o n of

S h i g e l l a b a c t e r i n i n t o an animal c a u s e s marked t o x i c e f f e c t ,
i t was hoped t ha t S h i g e l l a b act eri ophage would be n o n - t o x i c
and s t i l l capabl e of s t i m u l a t i n g the production of a n t i t o x i n .
Such an immunizing agent would be of c o n s i d e r a o l e v a l u e in
the c o n t r o l of d y s e n t e r y .
A bact eri o pha ge was prepared for S h i g . d. ysenteriae
s t r a i n 8*

This m a t e r i a l was i n j e c t e d i n t o r a b b i t s d a i l y for

7 days and weekly f o r 12 weeks (Table* I I ) .

The bact eri ophage

was i n j e c t e d in lOcc doses through the e n t i r e p e r i o d .
animal s showed l i t t l e

The

or no t o x i c e f f e c t from the i n j e c t i o n s .

The b a c t er i op h a g e a n t i s e r a were t e s t e d a g a i n s t a known e x o t o x i
(Table VIII and IX),

and were found to co n t a i n no a n t i t o x i n .

A second b a c t er i op h a g e was prepared from S h i g . d.ysenteriae
s t r a i n 18.

This ba c t er i oph a g e in c o n t r a s t to the f i r s t prepar

a t i o n showed marked t o x i c e f f e c t .
of 4cc for a 2 Kg. r a b b i t .

The m a t e r i a l had a M.L.D.

determined.

Accordingl y b a c t e r i n s , e n d o t o x i n s and e x o t o x i n s

were prepared.

These a n t i g e n s were t e s t e d on r a b b i t s in the

same manner (Table X I I ) .
The
u n i t s are

approximate v a l u e s of each a n t i g e n in p r o t e c t i v e
pres ent e d in TablesXIV, XV,XVIII and

XIX.

The

r e l a t i v e v a l u e s were as f o l l o w s :
B a c t e r i n ..........................................

2

Exot oxin .........................................

1

Endotoxin

1

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

B a c t e r i o p h a g e .................................. 2
To obtain the t o x i c o g e n i c e f f e c t o f the endotoxi n con­
t a i n e d in the b a c t er i op h a g e and t he e xo t ox i n prepared from
culture f i l t r a t e s ,

mixtures of the two were prepared.

The

i n j e c t i o n dose o f t h i s mixture was l i m i t ed due to the t o x i c
nature of both s u b s t a n c e s .

The preparat i on of the a n t i ­

s e r a i s shown in Table XII.
To reduce t he t o x i c i t y of the above mixture, a b a c t e r i o p h a g e - e x o t o x o i d mixture was prepared as p r e v i o u s l y o u t l i n e d .
A n t i s e r a f or t h i s m a t e r i a l was prepared as shown in Table XII.
These a n t i s e r a were then t e s t e d a g a i n s t exo - en d o t o x i n to
determine t h e i r r e l a t i v e p r o t e c t i v e v a l u e s .
p res en t e d in Tables XVI and XX.

The r e s u l t s are

The r e l a t i v e v a l u e s of t he s e

mixtures were as f o l l o w s ;
Bacteriophage-exotoxin . . .
Bacteriophage-exotoxoid

.2

. . .5

In the p r epa ra t i o n of t h i s b a c t er i o p h a g e,

the S h i g e l l a

c u l t u r e was allowed t o develop 8 hours bef ore the a d d i t i o n
of t h e b a c t e r i o p h a g e .

Later t e s t s showed t ha t when the

c u l t u r e was allowed to develop f or 24 hours pr i o r to the
i n t r o d u c t i o n of t he b a c t e r i oph ag e , a very t o x i c f i l t r a t e
was o b t a i n e d .

In a l l i n s t a n c e s , the f i l t r a t e s from s t r a i n

18 showed marked t o x i c e f f e c t s .

To e l i m i n a t e the t o x i c

e f f e c t of f r e s h "green" b ac t e r i oph a ge , the f i l t r a t e s were
aged in t he r e f r i g e r a t o r for peri ods as long as 18 months
w i t h o u t any m a t e r i a l diminution of t o x i c i t y .

Exotoxin pre­

pared from t h i s same s t r a i n of S h i g . d y s e n t e r i a e r e t a i n e d
i t s t o x i c i t y for 18 months in a s i m i l a r manner.
The data i n d i c a t e that the p roduc t i on of b act eri ophage
w i t h t o x i c p r o p e r t i e s i s dependent upon the t o x i c p r o p e r t i e s
of the S h i g . d y s e n t e r i a e s t r a i n used.

Shig. dysenteriae

s t r a i n 8, which f a i l e d to show t o x i c e f f e c t s ,
toxicogenic properties.

f a i l e d t o show

On the other hand, a t o x i c s t r a i n

( s t r a i n 13), when l y s e d , produced a t o x i c bact eri ophage which
had marked t o x i c o g e n i c p r o p e r t i e s .

I t would appear from

t h e s e data that t he t o x i c o g e n i c p r o p e r t i e s of S h i g e l l a bact ­
e r i o p h a g e were dependent upon the t o x i n cont ent of t he f i l ­
trate .
To a r r i v e at an e v a l u a t i o n of the t o x i c o g e n i c val ue
of b a c t e r i op h a g e prepared from s t r a i n 18, t o x i c o g e n i c v a l ues
of b a c t e r i n s , e n d o t o x i n s and e x o t o x i n s of t h i s s t r a i n were

I t w i l l be observed from t he s e data t hat t he b a c t e r i o p h a g e - e x o t o x o i d , a l t hough i n j e c t e d in the same amounts as
t he b a c t e r i o p h a g e - e x o t o x i n , produced c o ns i d e r a b l y more pro­
t e c tiv e units.

Not only was the b a c t e r i o p h a g e - e x o t o x o i d

more a n t i g e n i c in t he s t i m u l a t i o n of a n t i t o x i n ,

but i t s

i n j e c t i o n was a t t e nd e d with c o n s i d e r a b l y l e s s t o x i c e f f e c t
d ur i ng the c o u r s e of immunization.

The i n j e c t i o n of the

e q u i v a l e n t of 2 M.L.D, doses of b a c t e r i o p h a g e - e x o t o x i n in
t he form o f the b a c t e r i o p h a g e - e x o t o x o i d f a i l e d to show any
toxic e f f e c t s .
Due to the f a c t t hat b act eri ophage c o n t a i n s c o n s i d e r a b l e
ext ranenous m a t e r i a l from the broth and the lysed b a c t e r i a l
cells,

i t was thought a d v i s a b l e to p u r i f y the b act eri ophage

to be sure t hat t h e s e products and compounds might not i n ­
t e r f e r e wi t h the a n t i g e n i c p r o p e r t i e s of the b a c t er i op h a g e .
A c c o r d i n g l y , p u r i f i e d bact eri ophage was prepared.

This mat­

e r i a l was i n j e c t e d i n t o r a b b i t s as i n d i c a t e d in Table XII.
The antiserum was t e s t e d a g a i n s t e x o - en d o to x i n .
are presented in Table XVII.

The r e s u l t s

The r e l a t i v e p r o t e c t i v e value

was t he same as t h a t obtained wi t h the unp ur i f i ed materi al
i n d i c a t i n g t h a t the p u r i f i c a t i o n did not change t he a n t i g e n i c
value»
The a g g l u t i n o g e n i c and toxicogenic p r o p e r t i e s of the var­
i ous immunizing a ge n t s did not p a r a l l e l each other.

For

example, in Table XII the endot oxi n showed an a g g l u t i n a t i o n
t i t r e o f 1 to 5000 and a

a n t i t o x i n t i t r e ( Table XVIII) of 1,

whereas the b a c t e r i o p h a g e - e x o t o x o i d (Table XII) showed an
a g g l u t i n a t i o n t i t r e of 1 to 60 and an
5 (Table XX).

a n t i t o x i n t i t r e of

S U M M A R Y

The t o x i c o g e n i c va l u e o f b a ct eri oph a ge appeared to be
dependent upon the t o x i c o g e n i c val ue of the S h i g . d y s e n t e r i a e s t r a i n used in t he preparat ion of t he ba c t er i oph a g e .
There appeared t c be no evi de nc e t ha t b act eri ophage
in i t s e l f had t o x i c o g e n i c p r o p e r t i e s .
Ba c t e ri op h ag e appeared to be of v a l u e as an agent for
t he l i b e r a t i o n of e ndo t o xi n and p o s s i b l y e xo t o xi n through
t he a b i l i t y to l y s e the b a c t e r i a l c e l l s .
The aging of bact eri ophage f i l t r a t e s did not d e c r e a s e
i t s toxic properties.
The a g g l u t i n o g e n i c p r o p e r t i e s o f the b a c t er i op h a g e ,
ex o to x in s , endotoxins,

and b a c t e r i n s do not p a r a l l e l the

toxicogenic properties.
A t oxoi d p r e p a r a t i o n of b a c t e r i o p h a g e - e x o t o x i n was
found to be the b e s t means of producing a n t i t o x i n .
The t o x i c o g e n i c v a l u e of p u r i f i e d bact eri ophage was
the same as the u n p u r i f i e d b a c t e r i op h a g e .
The procedure o u t l i n e d in t h i s r e s e a r c h for the pre­
p a r a t i o n of b a c t e r i op h a ge - e xo t o xo i d appears to o f f e r a
p r a c t i c a l means f or immunization a g a i n s t Sh_ig. d y s e n t e r i ae
toxin.

L i t e r a t u r e Cited
1.

S h i g a , K. , Ueber den Erreger der D y s e n t e r i e in Japan
Ce n t r a l Ba k t . , Abt.
I , 23; 599, 1898.

2.

Krueger, A . P . , and Baldwin, D.M., Producti on of Phage
in the Absence of B a c t e r i a l C e l l s . , Soc. Exoer.
B i o l , and Med. 37: 393, 1937.

3.

Larkum, N.W., Producti on of A n t i t o x i n s by Means of
Ba c t e r i o p h a g e , Am. J . Pub. He al t h, 23; 1155, 1933.

4.

Holm, A. , Anderson, J . F . , and Leonard, G. F., Staphyl o­
coc cus Bacteriophage Toxoid, Am. J. Pub. Health 26;
1001, 1936.

5.

Mallmann, W.L., S t u d i e s on Bacteriophage in Re l a t i o n to
Sal monel l a and Pullorum D i s e a s e .
Tech. B u l l . No.
109. Agri. Exp. S t a . , Michigan S t a t e C o l l e g e , 1931.

6.

S t i t t , E. R. , Clough, R.W., and Clough, M.C., P r a c t i c a l
B a c t e r i o l o g y , Haematology, and Animal P a r a s i t o l o g y ,
842; Ninth Ed., 1938.

7.

O l i t s k y . P . K . , and K l i g e r , I . J . , Toxins and A n t i t o x i n s
of B a c i l l u s D y se n t er i a e S h i g a . , J . , Exper. Med., 31;
19, 1920.

8.

B o i v i n , M., Andre, and Mesrobeanu, Lydia, B a c t e r i a l
Toxins, Comptes Rendus, 204; 302, 1937.

9.

Krueger, A.P. , ja method for the Q u a n t i t a t i v e determina­
t i o n o f Ba c t e r i o p h a g e , J. Gen. P h y s i o l . S e p t . 1929.

10.

Sollmann, Torald, A Manual of Pharmacology, F i f t h Ed.,
1936.

11.

Krueger, A . P . , and Taraada, H. T., The Pre par at i on of
R e l a t i v e l y Pure B a c t e r i o p h a g e , J. Gen. P h y s i o l . , 13:
Orig. Abt— I I , 1929.

12.

Domi ni ki e wi cz, M. , Lodz., Zur Frage itber d i e Ei nh ei t
der Zusammensitzung und K e r s t e l l u n g s w e i s e n von
n a h r s u b s t ra t en fur B a k t e r i e n .

13.

Bronfenbrenner, J . , S t u di es on the Bacteriophage o f
d ' H e r e l i e , J . Exp. Med., 14; 873, 1927.

14.

Andrews, F.W., B u l l o c h , W., Dougl as, S . R . , Dreyer, G . ,
F el d e s , P . , Ledingham, J . C . , and Wolf, C . G . ,
D i p h t h e r i a — I t s B a c t e r i o l o g y , Pathology and Immun­
o l o g y . His Majesty*s S t a t i o n e r y O f f i c e , (London)
131: 1923.

15.

Muir, R. , Textbook of P a t h o l o g y . , J . B . L i p p i n c o t t Com­
pany, P h i l a d e l p h i a , 140: 1934.

16.

Hawk, P h i l i p , B. and Bergeim, Olaf , P r a c t i c a l P h y s i o l ­
o g i c a l Chemistry, Tenth Ed. 1931.