THE GROWTH OF
MTOORACTERIUM PARATUBERCULOS IS
IN TISSUE CULTURES

THE GROWTH OF
MYCOBACTERIUM PARATUBERCULOSIS
IN TISSUE CULTURES

THESIS

S u b m itte d t o t h e F a c u l t y o f th e M ic h ig a n S t a t e
in

p a r tia l f u lfillm e n t

C o lle g e

o f t h e r e q u ir e m e n t s f o r

t h e d e g re e o f M a s te r o f S c ie n c e i n
A n im a l P a t h o lo g y

by
C liffo r d

£
B . L in e

J u n e , 1934

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3

C o n te n ts

In tr o d u c tio n

4

R e v ie w o f L i t e r a t u r e

5

T e c h n iq u e

12

D e s c r ip tio n o f L iv in g

C u lt u r e s

18

D e s c r i p t i o n o f F ix e d C u lt u r e s

20

D is c u s s io n

29

Sum m ary

38

F ig u r e s

39

B ib lio g r a p h y

51

A c k n o w le d g m e n ts

53

102391

4

In tr o d u c tio n
S in e © t h e

d is c o v e r y o f t h e

e tio lo g y

o f J o h n e , s d is e a s e

some 4 0 y e a r s a g o b y J o h n e and F r o t h in g h a m , much h a s b e e n
added t o
s p it e

t h e k n o w le d g e o f t h i s

n a tu re o f th e

c o m p a r a t iv e ly l i t t l e

is

in

th is

know n a b o u t t h e

o r g a n is m and th e r e s u l t i n g

s lo w n e s s o f g r o w th o n l a b o r a t o r y m e d ia i s
th e

B u t,

o f r e s e a r c h e s c o n d u c te d b y m any w o r k e r s b o t h i n

c o u n tr y and a b ro a d ,
tru e

p e r p l e x i n g d is e a s e *

d is e a s e *

The

a g r e a t h a n d ic a p t o

s t u d y o f t h e b i o l o g y o f M y c o b a c te r iu m p a r a t u b e r e u l o s i s ;

lik e w is e ,

th e f a i l u r e

o f a l l a tte m p ts

to p r o d u c e i n

th e

common l a b o r a t o r y a n im a ls a n y d is e a s e r e s e m b lin g i n

th e

J o h n e 's d is e a s e o f c a t t l e

and th e s lo w n e s s w i t h w h ic h t h e

d is e a s e d e v e lo p s i n b o v in e s h a v e g r e a t l y l i m i t e d
w o rk s i m i l a r

le a s t

e x p e r im e n t a l

t o t h a t f r u i t f u l o f such r e v e a lin g r e s u l t s

in

o t h e r d is e a s e s o f a n im a ls a n d hu m a ns*
S in c e H a r r i s o n
v itr o

in

s tu d y o f th e r e a c t io n to

v itr o *

I t h a s been to

r o e n t g e n r a y s w e re t o
in to

th e d ia g n o s t ic ia n ,

g iv in g e n tra n c e to

c y to lo g ic p ro c e s s e s *
c u lt u r e

o f th e

is

in

o r g a n is m s o f t i s s u e

a m eans o f p e e r in g

a k e y to

a h ith e r to

th e s e c r e ts o f v i t a l
t h a t tis s u e

th e s t u d y o f t h e b i o l o g y o f

n o t o n ly b e cau se o f d i f f i c u l t i e s

o t h e r m e th o d s b u t b e c a u s e o f t h e s lo w r a t e

o r g a n is m a n d i t s

v iv o *

m e th o d o f p r o ­

p a r tic u la r ly f i t t i n g

m e th o d s be a p p l i e d t o

Johnef s b a c illu s ,
te re d

It

th is

in

th e e x p e r im e n t e r w h a t t h e

th e v i t a l p ro c e s s e s o f th e b o d y ,

u n o p e n e d g a te w a y ,

in

s u c c e e d e d i n g r o w in g t i s s u e

1 9 0 7 , m any w o r k e r s h a v e u t i l i z e d

c e d u re f o r t h e
g ro w n i n

(1 ) f i r s t

lo w t o x i c i t y

fo r c e lls

encoun­
o f g r o w th

in v it r o

and

5

The p r e s e n t w o r k h e r e i n r e p o r t e d w as in a u g u r a t e d w i t h
th e

id e a o f g r o w in g ,

c u ltu r e s
fu lly

p o s s ib le ,

o f a n im a l t i s s u e

c o n tr o lle d

Johnef s b a c i l l i

v itr o

a n d o f s t u d y in g u n d e r th e s e

in

c a re ­

b o d y , th e r e a c t io n o f in d iv id u a l c e l l s

th e p r e s e n c e o f t h e

o r g a n is m *

It

is

r e a liz e d

in c o m p le t e a n d t h a t th e p r o b le m i s

lo n g e d and d e t a i l e d

in v e s tig a tio n ,

n o t p e r m it o f s u c h d e t a i l *

t h a t th e

to

re p o rt

w o r t h y o f m ore p r o ­

b u t t h e t im e

a llo tte d

does

U n d o u b te d ly w o r t h y r e s u l t s c o u ld

h a v e b e e n o b t a in e d b y s t u d y in g
c u ltu r e s

in

c o n d i t i o n s , f r e e f r o m t h e g e n e r a l s y s te m ic

in flu e n c e s o f th e

is

if

c u ltu r e s

o f lo n g e r g r o w t h ,

i n o c u l a t e d a f t e r g r o w th w as w e l l com m enced, a nd p a r ­

t ic u la r ly

c u ltu r e s

r e p o r t o n th e s e

o f b o v in e

s tu d ie s

tis s u e *

It

is

hoped t h a t a

c a n be made a t a l a t e r

d a te *

R e v ie w o f L i t e r a t u r e
It
o u t s id e

is

g e n e r a l l y a c c e p te d t h a t th e

c u ltiv a tio n

o f th e o r g a n is m w a s s u c c e s s f u l l y a c c o m p lis h e d f o r t h e

f i r s t t im e b y H a r r i s o n

(1 )

in

1 9 0 7 , w o r k in g

D e p a rtm e n t o f J o h n s H o p k in s U n i v e r s i t y *
Loeb

(2 )

s ta te d

as in s id e

t h a t he h a d c u l t i v a t e d

in

th e

t h e A n a to m y

h is

o u t s id e

(3 )*

tis s u e

o f th e

fro g

in

In

s k i n o f g u in e a p i g

( i*

c o a g u la b le ly m p h *

e *,

Leo

as w e l l

in s i d e

of
em­

th e m i n t o

H a r r i s o n f s m e th o d , p u b lis h e d

c o n s is t e d o f p l a c i n g e m b r y o n ic t i s s u e

th is

m e th o d *

a g a r a n d c o a g u la b le s e ru m and i n s e r t i n g

a d u l t g u in e a p i g s

tis s u e )

c e lls

a m e th o d o f c u l t i v a t i n g

t h e b o d y b y p l a c i n g f r a g m e n ts o f

in

P r e v io u s t o

o f th e b o d y b u t d i d n o t p u b lis h

1 9 0 2 he p u b lis h e d

b ry o

o f tis s u e

in

1907,

c e n t r a l n e rv e

G ro w th o f n e r v e

6

f ib e r s

was o b s e r v e d *

im p ro v e d t h e

L a t e r B u rro w s

t e c h n iq u e ,

a n d a d a p te d i t

to

m a lia n

tis s u e ,

e x tra c t,
tio n

s u b s t i t u t i n g b lo o d p la s m a f o r

c u ltiv a tio n

B o rro w s and C a r r e l (5 )

( 4 ) , w o r k in g u n d e r H a r r i s o n ,

th e n

ly m p h ,

o f c h i c k e m b r y o n ic t i s s u e *

succeeded i n

and C a r r e l ( 6 ) ,

g r o w in g a d u l t mam­

b y a d d in g

c h ic k e m b r y o n ic

o b t a in e d a p e rm a n e n t and u n i n t e r r u p t e d m u l t i p l i c a ­

o f fib r o b la s t s *

Such s t r a in s

m a in t a in e d f o r m any y e a r s

o f f ib r o b la s ts

have been

(7 )*

The c h a n g e s u n d e rg o n e b y ly m p h o id t i s s u e w h e n c u l t i v a t e d
in

v itr o

in

m edium com posed o f b lo o d p la s m a a n d t i s s u e

a r e d e s c r ib e d b y M axim ow ( 8 )
is

show n t h a t t h e

shaped c e lls
new t i s s u e

b lo o d v e s s e l s *
p a r tly

g r e a t a c c u ra c y *

They p r o l i f e r a t e ,

in to

zone,

s u r r o u n d in g t h e
It

and p h a g o c y t i c ,

e x p la n t *

2 . E n d o th e liu m o f

p a r t l y p r o d u c e s new c a p i l l a r y
in to fib r o b la s ts *

b o d ie s ,

t h r o u g h f u s i o n , m u lt i n u c l e a t e d g i a n t

fa c e
p a le ,

o f th e

e x p la n t*

la r g e

th e

o r m a c ro ­

th e

c e lls ,

liq u id

m e dium a n d th e m d e g e n e r a t e ;

s u r­

by th e ir

am eboid p r o t o p la s m a nd th e

s ta in in g

A p a rt of

a n d some­

o n th e

They a re e a s i l y d i s t i n g u i s h e d

t y p i c a l p ig m e n t g r a n u le s ,

a m e b o id

p re s e n c e o f f o r e ig n

n u m b e rs i n

i r r e g u l a r n u c le u s , t h e i r

4 * L y m p h o c y te s *

3* B e tie u la r

p o ly b la s ts

s to r in g d y e s ,

a c c u m u la t in g i n

in

s p ro u ts ,

p r o life r a tin g ,

s o m e tim e s e p i t h e l i o i d
f o r m in g ,

as s p i n d l e -

fra m e w o rk o f t h e

phages,

t im e s

c e l l ty p e s

g ro w r a d i a l l y

T h e y a r e m o b i l iz e d a s l a r g e ,

and i t

"T h e c e l l t y p e s a r e :

t h e p la s m a a n d f o r m t h e

becom es t r a n s f o r m e d

c e lls *

a s e r ie s o f a r t i c l e s ,

t r a n s f o r m a t i o n o f th e v a r io u s

can be fo llo w e d w ith
1* F ib r o b la s ts *

in

e x tra c t

g re e n w it h

th e m m ig r a t e

in to

e o s in - a z u r e *
th e

s u r r o u n d in g

a p a r t r e m a in i n t h e e x p la n t and

7

in

th e lo o s e

new t i s s u e

and tra n s fo r m

zo n e a n d h e r e c o n t in u e

th e m s e lv e s i n t o

p h a g o c y t ic p o l y b l a s t s
A lth o u g h t i s s u e
and o th e r s f o r

th e

p la s m a c e l l s ,

o r in to
c u ltu r e

u p o n th e

c e lls

in t e r p r e t e d

b a c illi

a c tio n o f

a f t e r th e t h i r d

and e p i t h e l i a l c e l l s .

fin d in g s

s u b s titu te d

th e

o f tu b e r c le
s lig h t,

v itr o ,

of

s m a ll ro u n d
of

th e b a c i l l i .

( 1 1 ) c o n f ir m e d

c h i c k e m b ry o n ic

b a c illi.
in

T h e y c o u ld

some o f

in

b a c illi

in o c u la te d w it h

n o t d e te r m in e a n y g r o w th

of

P h a g o c y to s is w as v e r y
s in g le

f a c t t h a t th e

any d e g re e .
in

in

v itr o

to

tu b e r c le

th e

p la s m a

b a c i l l i was

c e lls

w e re n o t

No ch a n g e in d u c e d b y

th e c u l t u r e s

c o u ld b e d is c e r n e d .

A m o s t e x c e l l e n t w o rk o n th e r e a c t i o n o f t i s s u e
r a b b it

He

i d e n t i c a l f o r th e y

t is s u e

t h e m edium u s e d .

a t t r a c t e d b y th e b a c i l l i
o f th e

th e

G -ia n t c e l l f o r m a t i o n w as o b s e r v e d .

T h e y e m p h a s iz e d t h e

th e p r e s e n c e

among w h ic h w a s t h e

a ro u n d c o l o n i e s

a n d o n l y a c c i d e n t a l e n g u lf in g

o b s e rv e d .

P r e v io u s ly

a r t i f i c i a l L e w is m e dium f o r b lo o d

b a c illi

as

s e v e r a l f o r m s o f o r g a n is m s

T h e i r r e s u l t s w e re n o t

m edium a n d c u l t i v a t e d
a v ia n t u b e r c l e

( 9 ) o b s e rv e d

o r g a n is m s b y c o n n e c t iv e t i s s u e

a n d M . R . L e w is

o f S m y th .

s tu d y a c tio n o f

o f tis s u e c u ltu r e s

d a y o f g r o w th w i t h

r e p o r t e d p h a g o c y t o s is o f th e

S m it h , W i l l i s ,

1920 L e w is

He o b s e rv e d a c l u s t e r i n g
as ly m p h o c y te s

b y C a rre l

p a th o lo g is ts

E b e r th e lla t y p h i.

o f c h i c k e m b ry o s i n

tu b e r c le b a c illu s .
c e lls

In

th e c e l l s

th e r e s u l t o f in o c u la t io n w it h
s tu d ie d th e

a m e b o id

m e th o d s w e re u t i l i z e d

th e o p p o r t u n it y o f fe r e d t o

t h a t v a c u o le s w e re fo r m e d i n

p r o life r a te

g r a n u lo c y te s ."

o r g a n is m s u p o n i s o l a t e d c e l l s .

S m y th ( 1 0 )

in to

s tu d y o f c e l l p h y s io lo g y ,

w e re s lo w t o g r a s p

to

o f th e

b a c i l l i was r e p o r t e d b y M axim ow

8

(1 2 )*

L y m p h o id t i s s u e

tis s u e fro m

fro m

th e m e s e n t e r ic ly m p h n o d e s ,

t h e o m e n tu m , and t h e common c o n n e c t iv e t i s s u e

w e re g ro w n and i n o c u l a t e d w i t h
o f human t y p e w e re u s e d *

tu b e r c le

b a c illi#

One w a s r e c e n t l y

s p u tu m a n d h i g h l y v i r u l e n t ;

Two s t r a i n s

is o la t e d fro m

a n o th e r was o f v e r y le w v i r u l e n c e

a n d g re w w e l l o n a r t i f i c i a l m e d iu m .

He o b s e r v e d t h a t t h e

t u b e r c l e b a c i l l i g re w w e l l i n t h e c u l t u r e s a nd w e re a c t i v e l y
e n g u lf e d b y c e r t a i n c e l l s *
to

o v e rc o m e t h e v i r u l e n t

The c u l t u r e s

s tr a in

a f t e r i n o c u l a t i o n o n ly t r a c e s
th e c u lt u r e s ,
p lie d

fr e e ly

so t h a t

of

w ere g e n e r a l l y a b le
in

them c o u ld

a n d c o m p le t e ly o v e rg r e w t h e
in

v itr o *

He e m p h a s iz e d th e f a c t

o f r e a c tiv e

fo r

in

is

i n th e

c a s e s w h e re v a r i o u s
th e

to g e th e r f o r

o r g a n is m *

c e lls

o n e s w e re

and

T h is

to

s a p r o p h y t ic

o r g a n is m s ,

c e lls

and a r e

b la s ts

in to

c u ltu r e s

n o t be k e p t
L ik e w is e ,

c o n ta m in a te d w i t h

a re a b s o l u t e l y u n a b le t o

c h ie f r o le

was p la y e d b y t h e

u s u a lly m o b iliz e d

i n m a rk e d

soon k i l l e d .

M axim ow c o n c lu d e d t h a t th e
ly m p h o id t i s s u e

t u b e r c u lo u s

l e n g t h o f t im e *

c u l t lo re s become a c c i d e n t a l l y

in tr u d e r s

even in

o t h e r m ic r o o r g a n is m s

th e b a c t e r i a c o u ld

a n y a p p r e c ia b le

th e

is

in o c u la te d

w hen t i s s u e

r e s i s t th e

th a t,

d i d n o t p e r i s h b u t d is p la y e d a s e r i e s

t h e b e h a v io r o f t h e c e l l s

(S m y th , L e w is )
a liv e

fo u n d

T he y m u l t i ­

o f t u b e r c le b a c i l l i ,

c h a n g e s w h ic h d u p lic a t e d c l o s e l y t h e

p r o c e s s as i t
c o n tra s t to

tis s u e

in

c u ltu r e s a f t e r th re e

th o s e c u lt u r e s c o n t a in in g g r e a t q u a n t it ie s
o f th e

e ig h t days

be d is c o v e r e d

b u t n o t so th e a v i r u l e n t b a c i l l i *

w eeks o f l i f e

th e c e l l s

seven to

in th e c u ltu r e s o f

r e tic u la r c e lls *

a n d fo rm e d a c t i v e w a n d e r in g c e l l s ,

(m a c ro p h a g e s ), and m ig r a t e d

in to

th e

They w e re
p o ly ­

s u r r o u n d in g m e dium

9

and r e a d ily

e n g u lf e d

th e t u b e r c l e b a c i l l i w h e r e v e r

e n c o u n te r e d th e m .

Those r e t i c u l a r

c e lls

in to

ly m p h o id t i s s u e

and r e m in e d i n

p o ly b la s ts

in

th e y

w h ic h w e re

c o n c e r te d
th e

ex­

p l a n t a ls o e n g u lf e d t h e b a c i l l i w h ic h p e n e t r a t e d e v e ry w h e r e
b e tw e e n t h e c e l l s .
w h i le
to

M any r e t i c u l a r c e l l s e n g u lf e d b a c i l l i

s t i l l m a in ta in in g

th e r e t ic u lu m *

th e ir fix e d

E p ith e lio id

a n d g i a n t c e l l s w e re fo r m e d

a r e s u l t o f p h a g o c y t o s is o f b a c i l l i
c o n ta c t w it h
to

o r g a n is m s .

be th e r e s u l t

T h is

o f d iffu s ib le

h y p e rtro p h y o f r e t i c u l a r
r e s u lts

c e lls

p o s it io n w ith r e fe r e n c e

and a ls o b y c e l l s

in o c u l a t e d

c u ltu r e s

to x in s .

c o n tr o ls .

and p e r i s h e d ;

M ito s is

in o c u la te d

Some o f th e m m ig r a t e d i n t o

th o s e o f r e t i c u l a r o r i g i n

o f r e tic u la r

to r e ta in

th e

o r ig in

and c o u ld

c a s e s w h e re

e p ith e lio id

i n t r a c e l l u l a r d ig e s tio n

g ia n t c e l l .

d e s tro y e d w i t h i n

m u ltip lie d r a p id ly .

p o ly b la s ts

t h a t th e

and
of

in tr a ­

o f b a c i l l i w as c o n t in u e d a nd p r o b a b ly

in c r e a s e d w i t h i n

b a c i l l i w ere n o t

fr o m t h e

M aximow f u r t h e r s t a t e s

c e l l u l a r d ig e s t io n

m edium

s ta in .

a s a r e s id u e

b a c illi.

th e

r e t a i n e d a t y p i c a l g r e e n is h p ig m e n t

A g o ld e n y e l l o w p ig m e n t w as o b s e rv e d i n

h ig h ly

ly m p h o c y te s

o t h e r s t r a n s f o r m e d th e m s e lv e s i n t o

a f t e r t h e e o s in - a z u r e

c u ltu r e s .

c lu m p t o g e t h e r , n o t

n o t b e d is tin g u is h e d fro m th e l a t t e r e x c e p t i n

tu b e r c le

be d i r e c t

o f ly m p h o id t i s s u e ,

w h ic h b e h a v e d e x a c t l y a s th o s e

g ia n t c e l l s

and a p e c u l i a r

were o b s e r v e d t o

show ed a r e m a r k a b le d i s p o s i t i o n t o
o b s e rv e d i n

n o t in

l a t t e r r e a c t i o n w as c o n c lu d e d

o f t h e c h e m ic a l s t i m u l a t i o n i n

In

as

In

o th e r

th e e p i t h e l i o i d

cases
c e lls ,

but

S uch c e l l s f u l l o f b a c i l l i w e re o b s e rv e d

th e ir v i t a l i t y

f o r a lo n g t i m e , move a b o u t as u s u a l ,

10

and even d iv id e m i t o t i c a l l y .
r a p id ly w ith in
th e

th e

B a c i l l i d id

g ia n t c e lls

fo r

n o t m u ltip ly

th e d ig e s tiv e

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l a t t e r w e re p r o b a b ly f a r g r e a t e r th a n th o s e o f

phage*

The p r i n c i p l e

mode o f f o r m a t io n

f u s i o n o f m o n o n u c le a r c e l l s *
as a f u r t h e r s te p

in

th e

p r e v i o u s l y m e n tio n e d *

so

th e m a c ro ­

o f g ia n t c e lls

w as b y

T h is phenom enon w as lo o k e d u p o n

p ro c e s s o f th e c lu s t e r in g

I n a fe w in s t a n c e s g i a n t

o f c e lls

c e lls w ith

c o n s t r i c t e d a n d fr a g m e n te d n u c l e i w e re o b s e rv e d a n d i n t e r ­
p r e t e d a s b e in g th e

r e s u lts

n o t o f common o c c u r r e n c e *

o f a m ito tic

d iv is io n *

T h e y w e re

I n some c a s e s t h e f o r m a t i o n o f

g i a n t c e l l s w a s b e l i e v e d t o b e d ue t o

th e

h y p e r t r o p h y o f ly m p h o c y te s *

in s t a n c e s t h e p o l y -

b la s tic

In th e s e

d i r e c t f u s io n and

s ta g e was o m it t e d *

M axim ow made no r e f e r e n c e t o
b y g r a n u lo c y t e s

in

th e

a n y im p o r t a n t p a r t p la y e d

t u b e r c u lo u s p r o c e s s i n

v itr o .

t i o n e d t h a t t h e y a re o f common o c c u r r e n c e i n t h e
b u t as th e

t u b e r c u lo u s p i c t u r e

a p p a r e n t l y b e in g

s e n s itiv e t o

T he g r e a t e r n u m b e r o f h is
a fte r fo u r o r fiv e

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in itia l

in v i t r o

a f t e r th e

o f ly m p h o c y te s i n a p a s s iv e w a y .
e p ith e lio id

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c e lls ,

s p e c t a c u la r f e a t u r e s h a ve
w o rk e rs

in

th is

c o u n try

Its

e n te r th e

p r o t o p la s m

L a t e r when th e y w e re t r a n s ­

a c tiv e

o n J o h n e f s d is e a s e

t h a n t h a t on t u b e r c u l o s i s .

g r a n u lo c y t e s

a c tiv ity .

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fo r m e d i n t o

d e g e n e r a te *

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d a y s g r o w th

h a d l o s t much o f t h e i r

c u ltu r e s ,

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th e

He men­

p h a g o c y to s is t o o k p l a c e .
i s much le s s v o lu m in o u s

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c u r b e d th e i n t e r e s t

(H a llm a n a n d W i t t e r ,

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IB ).

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11

w i l l be made t o

r e v ie w c o m p le t e ly th e r e s u l t s

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to m s and l e s i o n s •
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(1 3 ).

th e r e s u l t s

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sym p­

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th e r e a d e r i s

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of

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th ic k e n in g

o f th e

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ly m p h n o d e s *

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i n t e s t i n a l w a l l and in v o lv e m e n t
I n th e i n i t i a l

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t h a t o f t u b e r c u lo s is
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m u co sa d i f f u s e l y
and a v a r i a b l e

and r e s u l t i n g f o c a l n a t u r e

num ber o f g ia n t

c e l l s w h ic h

o f a c id - f a s t ro d s *

a t o t a l absence o f any f i b r o t i c

in h is

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e x p e r ie n c e e p i t h e l i o i d

fo r m a tio n o f

r e s u l t o f p h a g o c y t o s is

is

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changes*

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s ta te s th a t

e p it h e lio id

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lo c a l f o r m a tio n o r d i s i n ­

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e p ith e lio id
tim e * "

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c e l l s a lw a y s c o n t a i n a c i d - f a s t

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i n m any c a s e s c o n ­

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r e s u ltin g

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r o d s a n d c o n c lu d e s t h a t no d i f f u s i b l e
th e

and c a s e a t io n ,

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t a in g re a t q u a n titie s

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to

12

t h a t o f H a llm a n

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14

a n d p la c e d

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in h ib ito r y

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j a r c o n ta in in g a b o u t f iv e

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15

s t r a i n h a d p r e v i o u s l y b e e n u s e d f o r th e p r o d u c t io n o f J o h n in *
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e x p la n t *

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s k in

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(8 ).

o f T y ro d e r s

An e q u a l n u m b e r o f c u l t u r e s w a s l e f t u n in o c u la t e d

f o r c o n tr o ls *

g r o w in g

2 cc*

b la c k t i l e s

and c o v e re d w it h

p la c e d u p o n

one h a l f o f a s t e r i l e

16

p e t r i d is h *
c e n te r o f
th is

A d r o p o f T y r o d e 's

each s l i p

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

d ro p *

The c o v e r s l i p

to o la r g e ,

and t h e t i s s u e

c o v e re d w it h
c lo tte d

in

s o l u t i o n was p la c e d

one o r

a d h e re d t i g h t l y

m s p la c e d on t h i s

tw o d r o p s

a s h o r t tim e ,

in v e r te d

and im m e d ia te ly

g la s s s l i d e

P r e v io u s ly a d ro p o f

to

in c u b a to r *

p r e v e n t e v a p o r a t io n
It

is

s te r ile ,
make i t

exposed t o

of flu id

im p o r ta n t t h a t a l l

on as s p e e d il y a s p o s s i b le

m e lt e d
a d h e re t o

o p e r a t io n s h e

and t h a t th e

tis s u e

i n th e
c a r r ie d

a t n o t im e be

d r y in g *

b y o p e n in g t h e p r e p a r a t i o n , r e m o v in g t h e
c u ltu r e ,

w a s h in g

m in u t e s ,

r e tu r n in g to

in

T y ro d e *s

G u in e a p i g
n o t because i t
ly m p h o id t i s s u e
o b ta in a b le .

day

ro u n d c o v e r s l i p

c u ltu r e ,

and

o f 20

c o v e r s lip , p la c in g

m e dium o n t h e

o v e r a h o ll o w

or f if t h

s o l u t i o n f o r a p e r io d

a c le a n , l a r g e

d ro p o f th e f l u i d

in v e r tin g

th e

m e lte d

a n d p la c e d

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

fre s h

w h ic h h a d a

The p r e p a r a t i o n th u s made was s e a le d w i t h

p a r a ffin

T h is

The c o v e r g la s s w as im m ed­

p e t r o la t u m w a s p la c e d a t e a c h c o r n e r t o
s lid e *

d ro p w as n o t

a f t e r w h ic h a d r o p o f th e f l u i d

o v e r a l a r g e , h o llo w

37 mm* c o n c a v it y *

th e

th e

p la c e d o n

o f t h e m edium m ix t u r e *

p h a s e w as p la c e d o n t h e c u l t u r e *
ia te ly

if

in

a

and a g a in

s lid e *

s p le e n m s

s e le c t e d a s

th e t i s s u e

was c o n s id e r e d more p r e f e r a b l e
b u t b e c a u s e th e

H o w e v e r, i t

la t t e r

h a d th e

a n im a l c o n s id e r e d r e s i s t a n t

to

to

be u s e d ,

th a n b o v in e

was n o t e a s i l y

a d v a n ta g e o f b e in g f r o m an

J o h n e ’ s d is e a s e ,

v h ic h

a llo w e d

a s t u d y o f th e m e c h a n is m o f s u c h r e s i s t a n c e *
A l l c u l t u r e s w e re

in c u b a t e d a t

a te m p e r a tu r e o f

3 9° C*

17

T h e y w e re o b s e r v e d t w ic e
s ta g e *

In a d d itio n ,

o b s e r v a t io n i n

per

one c u lt u r e

a m ic r o s c o p e

C u lt u r e s w e re
in

d a i l y u n d e r a m ic r o s c o p e w i t h

fix e d

cent n e u tra l fo rm a l is

g l a c i a l a c e t i c a c id #

was K e p t u n d e r c o n s t a n t

in c u b a t o r *

a t v a r io u s a g e s f o r

M a xim o w ’ s m o d i f i c a t i o n

s u b s titu te d f o r f i v e

per cent

flu id .

In

te n

T h is w a s a c c o m p lis h e d b y r e m o v in g th e

u p o n th e

s tre n g th s

20 m in u te s

i n w h ic h

b e a r in g t h e c u l t u r e

o f d is tille d

15 t o

o f Z e n k e r’ s s o lu tio n ,

ro u n d c o v e r s lip
fix in g

a warm

an d f l o a t i n g

I t w a s n e x t w ashed i n

it

fa c e

down

fre q u e n t changes

w a t e r f o r 24 h o u r s and d e h y d r a te d

in

a s c e n d in g

o f a l c o h o l f o l lo w e d toy a b s o lu t e a l c o h o l f o r 2 4 h o u r s *

t h e a b s o lu t e a l c o h o l th e c u l t u r e

m s remo-ved f r o m t h e

c o v e r s l i p b y s h a v in g o f f w i t h a s h a rp r a z o r b la d e w h ic h w a s
w e t w it h a lc o h o l*
to

s ix h o u rs ,

It

was t h e n p la c e d

in filtr a te d

in

an o v e n i n

m e l t i n g p o i n t o f 5 2 ° C #, em bedded i n
Some o f th e

c u l t u r e s w e re f i x e d

m a l i n a n d s t a in e d

in

to to

M ost o f th e c u ltu r e s

in c e d a r o i l f o r fo u r

in

p a r a f f i n h a v in g

p a r a ffin ,

te n p e r

and s e c t io n e d *

cent n e u tra l f o r ­

f o r b lo o d p ig m e n ts o r
were s e c t io n e d

fa ts *

s e r ia lly

s t a in e d

c a r b o lfu c h s in

f o r 10 t o

15 m in u t e s ,

one m in u te i n

70 p e r c e n t

d e c o l o r iz e d f o r

30 s e c o n d s t o

a lc o h o l c o n ta in in g

one p e r c e n t HC1*

s u r r o u n d in g

and f i b r i n

tis s u e

c o llo id in

f u c h s i n and e o z in - a z u r e

c l o t to a f a in t

and s t a in e d

a fte r

Z i e h l- N e e ls e n
and

T h is d e c o lo r iz e d t h e

h e m a t o x y lin w a s u s e d as a c o u n t e r s t a in *
w e re em bedded i n

in

on t h e

m ic ro to m e a t a t h ic k n e s s o f 7 u * ,
a t ro o m te m p e r a tu r e

a

p in k *

Some o f
in

H a r r is ’ s

th e c u l t u r e s

Z ie h l- N e e ls e n

ih e m e th o d o f M axim ow*

m e th o d w a s a f t e r w a r d a b a n d o n e d f o r p a r a f f i n

s e c tio n s *

c a r b o l­

T h is

18

D e s c r ip tio n o f L iv in g
In

a ll,

a b o u t 200 c u l t u r e s w e re g ro w n a n d o b s e r v e d ,

a b o u t 70 w e re f i x e d
s u ita b le

C u lt u r e s

and s e c t io n e d #

The m edium u s e d w a s fo u n d

f o r th e g r o w th o f g u in e a p i g

s p le e n #

A lm o s t im m e d ia t e ly a f t e r e x p l a n t a t i o n
m ig r a t e

in to

th e

o f le u c o c y te s ,

s u r r o u n d in g m edium #

c e lls

began to

These c o n s is t e d m a in ly

c h i e f among w h ic h w as th e p o ly m o rp h #

W ith in

tw o h o u r s e a c h e x p la n t was s u r r o u n d e d b y a d e n se z o n e o f
m ig r a t e d

c e lls #

The m i g r a t i o n r a n g e w as u p w a rd t o

T h e y show ed a r e m a r k a b le a f f i n i t y

f o r th e

w h e re c lu m p s o f t h e o r g a n is m s w e re w i t h i n
th e e x p la n t,
c y te s #

t h e y w e re

The c e l l s

a lm o s t s o l i d

w a ll,

2 mm#

Johnef s b a c i l l i ,

a r r a n g e d th e m s e lv e s a b o u t ea ch c lu m p i n

an

c o m p le t e ly o b s c u r in g t h e c lu m p o f o rg a n is m s #
u n in o c u la t e d c u l t u r e s

a lt h o u g h

It

be

a d h e r in g t o

each o th e r#

m any o f th e

le u c o c y t e s s u r r o u n d in g s u c h c lu m p s e x h i b i t e d

w as n o t i c e d t h a t th e g r a n u le s

v e r y p ro n o u n c e d B ro w n ia n m ovem ent n o t o b s e r v a b le
ty p e o f c e l l a t some d is t a n c e
c o n t r o l c u lt u r e s #

T h o se i n

and,

m i g r a t i o n ra n g e o f

a f t e r a g r e a t e r la p s e o f t im e a fe w c e l l s w e re s e e n t o

o f th e s e c e l l s

such

s u r r o u n d e d b y d e n s e m a sse s o f le u c o ­

No s i m i l a r c lu m p in g w as s e e n i n

th e

and

in

in
a

t h e same

f r o m th e c lu m p n o r b y c e l l s

A t th e same t im e

it

in

w a s s e e n t h a t m any

w e re s w o lle n and m ore v e s i c u l a r th a n o t h e r s #

c lo s e s t p r o x im it y to

th e

o r g a n is m s s o o n l o s t t h e i r

a m e b o id m ovem ent a n d w e re a p p a r e n t ly

dead.

d o u b t b u t t h a t t h e s e c h a n g e s c o n s is t e d

T h e re

is

little

o f c lo u d y s w e l l i n g

and

a n a c c o m p a n y in g h y d r o p ic d e g e n e r a t io n , and t h e a c c e le r a t e d
m ovem ent o f

th e g r a n u le s w as due t o

th e f a c t t h a t th e

c y to -

19

p la s m s o f t h e
tio n

c e lls

th e

in h ib i­

o f w a te r*
T h is a c t i v e

th e f i r s t

24 to

m i g r a t i o n o f le u e o c y t e s w as o b s e r v a b le
36 h o u r s ,

s t i l l p re s e n t in
of

w e re o f l e s s e r d e n s i t y d u e t o

th e

th e ir a c t i v i t y ,

c u ltu r e ,

e x p la n t.

l o s t m ost
m ig r a tio n

The l a t t e r a r i s e

b o th

t r a n s f o r m a t i o n o f ly m p h o c y te s a n d f r o m

c u la r t is s u e *

These c e l l s

le u c o c y t e s .

They i n t u r n

th e ir f ie ld

th e

a t f i r s t m ig r a t e d f o r t h

e x p l a n t a t a som ew hat s lo w e r r a t e

w ith in

a lt h o u g h

d e g e n e r a te d r a p i d l y ,

and w e re o b s c u r e d b y a n a c t i v e

o f m a c ro p h a g e s f r o m t h e
th ro u g h th e

a f t e r w h ic h t h e s e c e l l s ,

th a n th e

d u r in g

r e ti­

fro m th e

p o ly m o r p h o n u c le a r

s u r r o u n d e d a n y c lu m p o f o r g a n is m s

o f m i g r a t i o n a nd a p p a r e n t ly

in g e s t e d t h e

d e a d le u c o c y t e s a n d c e l l d e b r is a s w e l l as o r g a n is m s w h ic h
th e y e n c o u n te re d .

T h e y c o u ld be s e e n a c c u m u la te d

n u m b e rs a ro u n d c lu m p s o f b a c t e r i a ,
s e rv e d in th e

liv e

c u ltu r e

f o r m in g a m u l t i n u c l e a t e d

th a t

and o f t t i m e s

o r g ia n t c e l l .

th e c u l t u r e

s o lid

b e e n d ig e s te d .
n u c l e i w e re
liq u e f ie d
in

th e

a r e a s i n w h ic h

th e

O f t t im e s v e r y l a r g e c e l l s

se e n c l o s e l y a d h e r in g t o

a re a s .

in o c u l a t e d

The l a t t e r

was o b ­

T h is phenom enon o f

i n th e

in

it

t h e i r c y to p la s m s f u s e d ,

c e l l f u s i o n w a s s e e n m o re f r e q u e n t l y
or

in la r g e

ty p e

th e

flu id

c o v e r in g

fib r in

c l o t had

c o n t a in in g

30 o r 40

c o v e r g la s s i n

such

o f c e l l w a s s e e n n o t o n ly

c u l t u r e s b u t a ls o

i n th e

c o n tr o ls .

W h e re v e r m a c ro p h a g e s e n c o u n te r e d a c lu m p o f b a c i l l i ,
paused to

p h a g o c y to s e th e m , w o r k in g th e m s e lv e s i n t o

a n d g r a d u a l l y b r e a k in g
p r o v id in g

it

up and i n g e s t i n g

t h e c lu m p w e re n o t t o o

W h e re v e r s u c h c lu m p s w e re

la r g e

e n c o u n te r e d ,

a ll

th e

o f th e

th e y

clu m p

o r g a n is m s ,

( F ig s 11 and 1 6 ) .
th e m a c ro p h a g e s seem ed

20

to

lo s e much o f t h e i r m o t i l i t y ,

and b e co m e , f o r
( F ig s

6, 9,

th e

tim e

b e in g

1 0 , and 1 1 ) •

o r 100 u# i n

r e t r a c t t h e i r m any p s e u d o p o d s ,
a t le a s t ,

ro u n d , d a rk

c e lls

M any o f t h e s e c lu m p s u p w a rd

d ia m e t e r may b e s e e n i n

a h e a v ily

to

50

i n o c u la t e d

c u ltu r e •
A t t h e same t im e th e m a c ro p h a g e s w e re m i g r a t i n g
fib r o b la s ts

se n t o u t th e ir

s u r r o u n d in g m edium #
come i s o l a t e d

fro m

p ro c e s s e s

T h e se d i d n o t m ig r a t e ,

r e ta in

th e

o n th e

th e y b e ­
in a

th e

p a r t o f th e

i n o c u la t e d c u l t u r e s #

g r o w th seemed t o b e som ew hat m ore p r o f u s e
it

n o r d id

th e

t h e i r c o n n e c t io n w i t h

No s i g n i f i c a n t r e a c t i o n

f i b r o b l a s t s w as o b s e rv e d i n

H o w e v e r,

in to

th e m a in e x p l a n t b u t a lw a y s g r e w o u t

d e n se m ass and seemed to
m a in e x p la n t #

s p e a r - lik e

fo rth ,

i n th e

m ay b e t h a t t h e r e w a s a s t i m u l a t i o n

T h e ir

c o n tr o ls #
o f t h e g r o w th

and m i g r a t i o n o f m a c ro p h a g e s w h ic h made t h e f i b r o b l a s t i c
g r o w th seem l e s s b y c o m p a ris o n #

D e s c r i p t i o n o f F ix e d C u lt u r e s
An e x a m in a t io n o f f i x e d ,
d is c l o s e s
c e ll to
g r o w th

t h a t th e p o l y m o r p h o n u c l e a r le u c o c y t e

m e e t th e
it

w h ic h t h e

o rg a n is m s #

m ay b e s e e n t h a t

In

c u ltu r e s

le u c o c y t e s

in

is

th e f i r s t

o f a fe w h o u rs
la r g e

n u m b e rs , among

p o ly m o rp h p r e d o m in a te s , have s u r r o u n d e d th e c lu m p s

o f o rg a n is m s #

It

t h e p o ly m o r p h s f o r
c lu m p s t h a t
tu re s t

s e c t io n e d , a n d s t a in e d c u l t u r e s

is
it

e v i d e n t t h a t su c h c o n t a c t s o o n d e s t r o y s
is

d iffic u lt

to f in d

a re n o t a l r e a d y d i s i n t e g r a t e d

O c c a s io n a lly i n t a c t p o ly m o r p h s a re

a n y a ro u n d t h e
in f iv e

hour c u l­

se e n n e a r t h e

21

p e r i p h e r y o f s u c h g r o u p s , and s o m e tim e s o rg a n is m s a r e
w ith in

th e ir

c y t o p la s m s .

It

s a r y f o r t h e p o ly m o r p h t o
c lu m p s a r e s e e n w h e re i t

w o u ld seem t h a t i t

e n g u lf r o d s to
is

is

n o t neces­

d e s t r o y th e m *

b e lie v e d

th o s e

i n th e

Some

o b s e r v a b le t h a t t h o s e r o d s n e a r

th e p e r i p h e r y o f t h e c lu m p h a v e l o s t t h e i r a c i d - f a s t
q u a l i t y w h ile

seen

c e n te r s t i l l

t h a t such d ig e s tio n

is

r e ta in

th e r e s u l t o f

it*

s ta in in g
It

ly tic

is

fe r m e n ts

r e le a s e d b y th e d i s i n t e g r a t i n g c e l l s *

H o w e v e r, when t h e

p o ly m o r p h o n u c le a r le u c o c y t e

is o la te d

s m a ll c lu m p s , th e r o d s a r e
s id e r a b le
a lly

n u m b e rs w i t h i n

e n c o u n te r s

o rg a n is m s

or

d ig e s t e d and may be s e e n i n c o n ­

an a p p a r e n t ly i n t a c t c e l l *

a c i d - f a s t g r a n u le s a r e a ls o

seen w i t h i n such

O c c a s io n ­

is o la te d

c e lls *
The p o ly m o r p h o n u c le a r le u c o c y t e s i n
soon d is in t e g r a t e *
c lu m p r e t a i n
and w i t h
in

it

life

T h o se a t

c o n ta c t w ith

some d is t a n c e f r o m

a n y la r g e

som ew hat lo n g e r b u t s o o n lo s e t h e i r

p r o b a b ly t h e i r

s m a ll n u m b e rs a r e

p h a g o c y tic a c t i v i t i e s *

s e e n s u r r o u n d in g

o rg a n is m s

m o tility

L y m p h o c y te s

c lu m p s , a n d c h a n g e s

i n d i c a t i n g t h a t t h e y becom e t r a n s f o r m e d i n t o m a c ro p h a g e s a r e
seen*

Some a l s o a r e

th e t is s u e

d e g e n e r a te d *

as such b e fo re

and e x h i b i t

an a f f i n i t y

p o ly m o r p h *

They g e n e r a l l y

M a c ro p h a g e s t h a t a r e

tr a n s p la n ta tio n

fo r

a ls o m ig r a t e

J o h n e #s b a c i l l i s i m i l a r t o

in
fo rth
th e

in g e s t b a c i l l i w h e re v e r t h e y a r e

e n c o u n te r e d *
R e tic u la r c e lls
b e g in t o

a re tra n s fo rm e d i n t o

m ig r a t e f o r t h

a b o u t 36 t o 4 8 h o u r s ;

ly m p h o ­

c y t e s w h ic h h a v e a lr e a d y m ig r a t e d f r o m t h e e x p la n t

a re a ls o

tra n s fo r m e d

in to

a fte r

m a c ro p h a g e s a n d

m a c ro p h a g e s *

T h e y in g e s t t h e

dead le u c o -

22

c y t e s a n d c e l l d e b r i s and o f t e n c o n t a i n g r a n u le s o f p ig m e n t
in

th e ir

c y t o p la s m s *

d e a d p o ly m o r p h s

They q u ic k ly

a ro u n d th e

c y to p la s m s o f t e n f u s e

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

c lu m p s o f b a c i l l i

w it h t h o s e

of

b le n d e d

in to

it

is

ih e

c o lo n y i s
and f u s e

d iffic u lt

to

d is tin ­

g r e a t v o r a c i t y a s do

t h e i r ws^y i n t o

in tr a c e llu la r ,
in

th e i n t e r i o r

th e

clu m p u n t i l t h e e n t i r e

and t h e c y to p la s m s o f

s im ila r

to

m eet

o f n u c le i

t h a t s e e n i n t h e b o v in e d is e a s e *

The o rg a n is m s a p p e a r b o t h i n
a s s i n g l e r o d s o r a s c h a in s u p t o
a n o c c a s io n a l a c i d - f a s t g r a n u le
a p p e a ra n c e i s

seen w i t h i n

d iffic u lt

to

c o m p a r is o n o f c u l t u r e s

th e

c lu m p s a n d i n t r a c e l l u l a r l y

12 u * i n

of

fiv e

is

p o s itiv e

o rg a n is m s w i t h i n

o r g a n is m s

e v id e n c e o f
th e s e c e l l s *

d a y s o l d and t h i r t e e n

in

in

th e l a t t e r

c h a in s a n d d e e p e r s t a i n i n g

b e lie v e d t h a t t h e

A lth o u g h

th e m acro p h a g e s a s w i t h i n p o ly m o r p h s ,

show s n o f u r t h e r e v id e n c e o f d i g e s t i o n
on th e c o n t r a r y ,

le n g th *

o r c h a in w it h a b e a d e d

sa y t h a t th e re

d ig e s t io n o r d e s tr u c tio n

o f lo n g

th e c e l l s

o f t h e m a s s , f o r m in g a t y p i c a l m u l t i ­

n u c le a t e d c e l l w i t h a p e r i p h e r a l d i s t r i b u t i o n

is

On th e

s c a t t e r e d m a c ro p h a g e s as t h e y e n c o u n te r i s o l a t e d b a c i l l i *

The c e l l s f o r c e

it

c lu m p s *

c e l l u l a r c y to p la s m and b a c t e r i a l c lu m p *

T h e s e m a c ro p h a g e s in g e s t b a c i l l i w i t h
a ls o

T h e ir

s p h e r e t h u s fo r m e d t h e i r c y to p la s m s a re

t h e c o lo n y so t h a t

g u is h b e tw e e n th e

1 3 )*

a d ja c e n t m a c ro p h a g e s and

f o r m a c o m p le te e n c i r c l i n g b a r r i e r a r o u n d
in n e r s u r fa c e o f th e

( F ig *

days o ld

o ld e r c u l t u r e s ;

show a l a r g e r nu m b e r

q u a litie s *

o rg a n is m s s u f f e r l i t t l e

m a c ro p h a g e s b u t m u l t i p l y w i t h i n

th e

A

So i t

is

h a rm w i t h i n t h e

th e c e l l i n

m any c a s e s *

m ig h t be t h a t th e h e a v y i n o c u l a t i o n has overcom e th e

It

n a tu ra l

23

r e s is ta n c e

o f tlie

c u l t u r e s w h e re

c e lls

i n m any in s t a n c e s *

o rg a n is m s w e re d i r e c t l y

p la n t ,

r a p id m u lt ip lic a t io n

and i n

19 h o u r c u l t u r e s

w h ic h e x h i b i t

(T 7 0 )

H o w e v e r,

th e e d g e o f t h e e x p la n t

in

a d ja c e n t t o

th is

seen b e tw e e n t h e

r e a c tio n

on th e

o f ly m p h o c y te s a r e

a ls o

e x h ib it a b s o lu te ly

no p h a g o c y tic

lo c a tio n *

seen in

s p le n ic

p a rt o f

They h a ve e v i d e n t l y
A g o o d ly num ber

such l o c a t i o n s ,
p o w e rs *

b u t th e y

M any o f them d e g e n ­

e r a t e , and o t h e r s a r e a p p a r e n t ly t r a n s f o r m e d i n t o
a n d t h e n e x h i b i t t h e same p h a g o c y t i c a c t i v i t i e s

m a c ro p h a g e s
a s th o s e f r o m

s o u rc e s *
A t no tim e w a s a y e llo w

b y M axim ow ( 1 2 ) a s a r e s id u e
b a c illi

seen w ith in

w e re a llo w e d
m ig h t

p ig m e n t s i m i l a r t o
o f th e d ig e s tio n

th e m a c ro p h a g e s *

P e rh a p s

th a t

d e s c r ib e d

o f t u b e r c le
if

t o g ro w f o r a lo n g e r p e r i o d , s i m i l a r

th e

c u ltu r e s

d ig e s tio n

ta k e p la c e *
As p r e v i o u s l y m e n tio n e d , g i a n t

in

th e

in

c o n t r o l c u ltu r e s *

ta in

ex­

p o ly m o r p h s a r e g a t h e r e d a ro u n d

b e e n a t t r a c t e d f r o m t h e s u r r o u n d in g p la s m a *

o th e r

th e

t h e y may b e s e e n g r o w in g i n t o

o n ly a f e e b l e

a d ja c e n t m a c ro p h a g e s *

some o f th e

t o o k p la c e a lm o s t im m e d ia t e ly ,

t h e e x p l a n t so t h a t d e n s e m a sse s a r e
c e lls ,

In

in o c u l a t e d

c u ltu r e s

m a te r ia l*

c e lls

n o t a lw a y s

l a t e d c u l t u r e s m any g i a n t c e l l s
n u c l e i a r e s e e n w h ic h

a r e com m o nly s e e n

and u n d e r c e r t a i n s t a t e d

M any o f th e s e

o r g a n is m s , b u t su ch i s

c e lls

in

th e

c o n t a in in g

c o n d itio n s

th e fo rm e r con­

case.

In

u p w a rd t o

in o c u ­
18 o r 20

c o n t a in no a c i d - f a s t o r g a n is m s o r

N e ith e r a re th e y

i n th e v i c i n i t y

c t a g ro u p

of

fo rth

th e

o r g a n is m s o f a p p r e c ia b le s i z e *
F ib r o b la s t s and e n d o t h e lia l c e l l s

g ro w

fro m

24

o r i g i n a l e x p la n t,

and t h e l a t t e r

s p r o u t s o r becom e t r a n s f o r m e d
w ith

th e

seen in
fiv e

fib r o b la s ts .

in

a tta c h e d t o
and,

c e lls

a p p a r e n tly

Ho s i g n i f i c a n t a c t i o n
In

some c u l t u r e s b a c i l l i u p to

h o w e v e r, n o p a r t i c u l a r

th is

fin d in g f o r

th e re fo re ,

is

it

is

th e

c y to p la s m

s ig n ific a n c e

o b s e rv e d i n

p r o b a b ly a c c i d e n t a l .

t u r e s w h ic h w e re a llo w e d t o
T41.

g ro w f o r v a r i o u s

In o c u la te d

F ix e d i n

m o d ifie d

g ro w th .

T h is c u l t u r e

is

o n ly a f e w

T h e re f o l l o w s a d e s c r i p t i o n o f a fe w t y p i c a l ,

C u lt u r e

id e n tic a l

on t h e ir p a r t is

num ber a re o c c a s io n a lly seen i n

o f fib r o b la s ts ,

c e lls

in t o

in o c u la t e d c u lt u r e s .

o r s ix

o f t e n f o r m new c a p i l l a r y

f ix e d

le n g t h s

c u l­

o f tim e .

a t tim e o f t r a n s p l a n t a t i o n .

Z e n k e r 's f o r m o l a f t e r f i v e

and a h a lf h o u rs

sh o w s m any p o ly m o r p h o n u c le a r le u c o c y t e s

t h a t h a v e m ig r a t e d f r o m t h e e x p la n t e d t i s s u e .

T h e y show

m a rk e d a f f i n i t y

t h e s u r r o u n d in g

m e dium and a r e

fo r

n e a r th e

in v a r io u s

fr a g m e n te d and p y k n o t ic
m ass a b o u t t h e

c lu m p s ,

have p e n e tr a te d to
n e a r th e
ju r e d ,

c lu m p s

o f o rg a n is m s

in

c o l l e c t e d a r o u n d s u c h c lu m p s i n l a r g e

w h e r e v e r t h e y a re
t h e c lu m p s a r e

th e

its

e x p la n t *

The p o ly m o rp h s s u r r o u n d in g

s ta g e s o f d i s i n t e g r a t i o n , w i t h

n u c le i*

They fo rm

a dense e n c ir c lin g

and an o c c a s io n a l c e l l may be s e e n to
v e ry

c e n te r*

An o c c a s io n a l p o ly m o rp h

p e r ip h e r y o f th e e n c ir c lin g

and a fe w o f s u c h c e l l s

and a c i d - f a s t g r a n u le s *

num bers

c e lls

seems t o

be u n i n ­

c o n t a in p h a g o c y to s e d o rg a n is m s

One l a r g e

clu m p o f J o h n e 's b a c i l l i

s u r r o u n d e d b y m any p o ly m o r p h s show s a d i s t i n c t p e r i p h e r a l
r in g

c o n t a i n i n g no o b s e r v a b le o rg a n is m s a n d v e r y l i t t l e

f a s t m a t e r i a l w h i le

th e c e n t e r

a c id - fa s t ro d s *

a p p e a rs t h a t

It

o f t h e c lu m p sh ow s th e
th e

o rg a n is m s

in

H is

a c id usual
p e r i­

25

p h e r a l a o n e lia v e ‘b e e n ly z e d b y t h e f e r m e n t s l i b e r a t e d
tb e

d is in t e g r a t in g

le s s

d is tin c tly *

p o ly m o r p h s •

O th e r c lu m p s i l l u s t r a t e

T he same phenom enon i s

n o t se e n i n

o f o r g a n is m s u n s u rr o u n & e d b y le u c o c y t e s *
o b s e r v a b le i n

fr o m

c lu m p s n e a r t h e e x p l a n t ,

The l y s i s

th is

c lu m p s
is

m ost

a ro u n d ■which t h e m o s t

le u c o c y t e s h a v e c o l l e c t e d *
L y m p h o c y te s a n d m o n o n u c le a r s h a ve a l s o m ig r a t e d f r o m t h e
e x p l a n t b u t a t a som ew hat s lo w e r r a t e
They a re

o c c a s i o n a l l y s e e n among th e

th e g r o u p s o f o r g a n is m s *

th a n t h e p o ly m o r p h s *
c e lls

c o lle c te d

The ly m p h o c y te s do n o t c o n t a i n

o r g a n is m s , b u t t h e m o n o n u c le a r s h a v e a c t i v e l y
J o h n e 's b a c i l l i *

T h e re a r e

o f w h ic h r e s e m b le

th o s e o f s m a ll ly m p h o c y te s *

no o r g a n is m s .
fib r o b la s ts

fix e d

p h a g o c y to s e d

s e v e ra l g ia n t c e l l s ,

th e n u c l e i

They c o n t a i n

The o r i g i n a l e x p la n t show s a h y p e r t r o p h y o f

a n d a n a c c u m u la tio n o f ly m p h o c y te s a t th e

No a c t u a l g r o w th o f o rg a n is m s i s
C u lt u r e

T70*

o b s e rv e d i n

I n o c u la t e d a t tim e

edge*

th e c u ltu r e *

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

i n m o d i f i e d Z e n k e r 's f o r m o l a f t e r 19 h o u r s g r o w th *

T h is c u l t u r e

d is p la y s

a t tr a c tio n o f

J o h n e 's b a c i l l i s i m i l a r t o
m o rp h i s

th a t

c e lls

to

d e s c r ib e d i n

th e

c lu m p s o f

T41*

The p o ly ­

s t i l l t h e p r e d o m in a t in g c e l l a ro u n d s u c h c lu m p s , b u t

ly m p h o c y te s a n d a l s o l a r g e m o n o n u c le a r c e l l s w i t h
dense, e c c e n tr ic a lly
b e rs *

about

C e lls

lo c a t e d n u c l e i a r e seen

o f r e t ic u la r o r ig in

in

s m a ll,

s m a l l e r num ­

a r e s e e n a ro u n d s e v e r a l

g r o u p s o f o rg a n is m s

lo c a te d a t

t h e v e r y edge o f

1he e x p l a n t ,

f r o m w h ic h t h e r e

a n e x t e n s io n b y g r o w th i n t o

th e e x p la n t*

is

The J o h n e 's b a c i l l i o f t h e s e c lu m p s ha ve a p p a r e n t l y e x c i t e d
as y e t v e r y l i t t l e

r e a c tio n

on th e

p a c t o f th e

c e lls

except

26

s l i g h t p o ly m o r p h and m o n o n u c le a r a t t r a c t i o n
th e

c o lo n y f a r t h e s t f r o m t h e

e x p la n t*

a t th e b o r d e r o f

O n ly r a r e l y

g a n is m s be

se e n w i t h i n

t h o s e p o ly m o r p h s t h a t a r e

a ro u n d th e

l a r g e r c lu m p s o f o r g a n is m s *

In

r o d s c a n b e s e e n m o re f r e q u e n t l y w i t h i n
v e s i c u l a r m o n o n u c le a r c e l l s *

H o w e v e r,

su ch

th e

can o r ­

c o lle c te d
lo c a tio n s

r a th e r la r g e ,

th o s e

is o la t e d p o ly ­

m o rp h s t h a t a p p e a r t h r o u g h o u t t h e m e d iu m can f r e q u e n t l y be
s e e n to

c o n t a i n J o h n e 's b a c i l l i .

S lid e

13 show s m any b e a u t i ­

f u l e x a m p le s o f p h a g o c y t o s is b y p o ly m o r p h s , a n d a s m any a s
fiv e

to

s e v e n o r g a n is m s a r e

th e s e c e l l s

show b u t

little

s e e n w i t h i n one c e l l *

e v id e n c e o f d e g e n e r a t io n *

h o w e v e r,

show a f r a g m e n t a t io n

d is tin c t

d a r k - s ta in in g

o f th e

n u c le u s i n t o

c h r o m a t in g r a n u le s *

th o s e c lu m p s a t some d is t a n c e f r o m

in to

th e

e x p la n t,

at

th e e x p la n t,

th e v e r y e d g e o f ,

a ro u n d w h ic h a r e

num b e r o f f i b r o b l a s t s
show o n l y s c a t t e r e d
and b e a d e d c h a in s ,
a re le s s e xp o se d to

s u r ro u n d e d b y

s ta in .

a c i d - f a s t r o d s o c c u r r in g
and th e s e m a in ly i n

fro m th e

In

s h o rt,

th e

fe rm e n ts *

tio n s

in t o

fa v o r a b le

th o s e
fo r

and e x t e n d in g

in

s h o r t le n g t h s

c e n t e r w h e re th e y
The r o d s o f

th e

dense m a sses

it

t h e e x p l a n t , t h e y become m ore

a p p e a rs t h a t

e x p l a n t a re b e in g r a p i d l y

le u c o c y t e s w h il e

in

o f th e e x p la n t and ta k e a d e e p a c i d - f a s t

As t h e y e x te n d

s c a tte re d .

th o s e

The f o r m e r c lu m p s

l a t t e r d e s c r ib e d c o l o n i e s , h o w e v e r, o c c u r i n
n e a r th e p e r ip h e r y

o r s ix

a fe w m a c ro p h a g e s a nd a

and ly m p h o c y te s *

le u c o c y tic

fiv e

O th e rs ,

The o rg a n is m s i n

le u c o c y t e s , m a in ly p o ly m o r p h s , d i f f e r g r e a t l y f r o m
s e v e r a l c o lo n ie s o c c u r r in g

M any o f

o c c u r r in g

g r o w th

th o s e a t some d is t a n c e

ly z e d b y t h e

n e a r th e

s u r r o u n d in g

e x p la n t f i n d

and a r e m u l t i p l y i n g

c o n d i­

unchecked by

27

c e llu la r

r e a c tio n s •

te r ia

seen*

in

is

th is

fig u r e

The m axim um m i g r a t i o n r a n g e f o r

c u ltu r e
is

An o c c a s io n a l m a c ro p h a g e c o n t a i n i n g b a c ­

is

about 1 l/ 4

and f i x e d

g r o w th *

A n o c c a s io n a l m i t o t i c

seen*

C u l t u r e s T73 a n d T 7 6 .
ta tio n

mm*

tis s u e c e lls

I n o c u la t e d a t t im e

o f t r a n s p la n ­

i n m o d ifie d Z e n k e r’ s fo r m o l a f t e r

B o th c u l t u r e s

96 h o u r s

d i s p l a y m any s c a t t e r e d m a c ro p h a g e s

w h ic h a re p a c k e d w i t h a c i d - f a s t

o r g a n is m s o c c u r r in g

up to

ih e s e m a c ro p h a g e s a r e w e l l

10 u *

in

ro u n d e d o f f ,

and t h e

c ro w d e d t h e r e
c y to p la s m *

le n g th *

b y th e

M ost o f

n u c le u s
la r g e

The p e r i p h e r y

Is

lo c a t e d a t

i n c h a in s

one s i d e , a p p a r e n t ly

q u a n t i t y o f o rg a n is m s w i t h i n
o f th e

th e

e x p la n t w h e re new c e l l s a r e

j u s t g r o w in g o u t seems t o b e t h e

s e le c t e d p la c e

o f g r o w th f o r

J o h n e ’ s b a c i l l i and o f f e r s t h e b e s t p i c t u r e

of

o f J o h n e ’ s d is e a s e

e x a m in a tio n o f

th e
is

in tr a c e llu la r
p o s itiv e

in

v itr o *

A f t e r a c lo s e

o ig a n is m s ,

it

is

d i f f i c u l t to

th e p a t h o lo g y

say t h a t

e v id e n c e o f d e s t r u c t i o n o f o r g a n is m s w i t h i n

m a c ro p h a g e a n d g i a n t c e l l *

On t h e c o n t r a r y ,

th e re
th e

one g s t s th e

im p r e s s io n t h a t t h e y a r e m u l t i p l y i n g *

S c a t t e r e d p o ly m o r p h o ­

n u c le a r le u c o c y t e s c o n t a i n i n g r o d s a r e

s t i l l to

o f such c e lls
m any o f th e m

show d e g e n e r a t iv e
ta k e a v e r y

g r a n u le s c a n b e s e e n *

fa in t

changes*

The o rg a n is m s

fig u r e s .

M o n o n u c le a r s w i t h s m a l l ,

h y m a c ro p h a g e s i s
C u ltu r e
fix e d

T56*

i n m o d ifie d

P h a g o c y to s is

in

de nse n u c l e i
T h e re

a r e a fe w

o f p o ly m o r p h o n u c le a r le u c o c y t e s

w e ll illu s t r a t e d
I n o c u la t e d

M o st

s t a i n , a n d some a c i d - f a s t

a ls o c o n t a i n o r g a n is e s o f s i m i l a r a p p e a ra n c e *
m ito tic

be s e e n *

in

a t tim e

s lid e

T 7 3 —8 ( F i g * 2 4 ) *

o f e x p l a n t a t i o n and

Z e n k e r ’ s f o r m o l a f t e r 132 h o u rs

g r o w th *

28

The m a c ro p h a g e s i n

th is

h a v e m ig r a t e d w i d e l y
c a l l y a l l o f th e

c u ltu r e

in to

a re seen ( F ig . 1 5 }•
o ccu r e ith e r

th e m e dium and p h a g o c y to s e d p r a c t i ­

c o n ta in in g

la r g e

in

th e

liq u id

c o n t a in i n g

or in

a re p r a c t i c a l l y

th e l i q u i d

a l l n e c r o tic .

is

v e r y s c a n t.

p o r tio n s

C u ltu r e T 7 9 *
fix e d

i n m o d ifie d

h o u rs ).

Jo h n e ’ s b a c i l l i up to
q u ite

th ic k

o f c e lls

tim e

mm. i n

a b o u t 12 u .

a n o th e r c u lt u r e

A t th e b o rd e r s o f

p h a g e s and m any s c a t t e r e d
w h ic h l i k e w i s e

c a tin g

fo rm

g ia n t

c e lls

o f tr a n s p la n ta tio n
12 d a y s g r o w th

and

(2 8 8

th r o u g h o u t t h e m edium c o l o n i e s o f
d ia m e te r a r e s e e n .

in

le n g th .

o f th e

T h is

same age

be s e e n , b u t o n l y a fe w r o d s s u r v i v e

s ta in .

in to

illu s tr a te s

The g r o w t h o f f i b r o b l a s t s

t h e o r i g i n a l eXumps t h a t w e re i n o c u l a t e d

m ass.

The

c e n t e r o f th e e x p la n t e d

and t a k i n g a d e n s e a c i d - f a s t s t a i n ,

a s c h a in s up t o
c o n tra s t to

l/2

th e p e r i ­

a ls o p r e s e n t .

Z e n k e r’ s fo r m o l a f t e r

I n m any p la c e s

at

T h is c u l t u r e

o f th e m e d ia .

I n o c u la t e d a t

fib r in

has b e e n l i q u e f i e d .

i n th e

w e l l th e g r e a t e r te n d e n c y f o r f u s i o n
in

th e

no o r g a n is m s a re

s c a t t e r e d ly m p h o c y te s r e m a in in g
tis s u e

n u m b e rs o f b a c t e r i a

T he g r e a t e s t nu m b e r o f m u l t i n u c l e a t e d

p h e r y o f a n a r e a i n w h ic h t h e f i b r i n
G ia n t c e l l s

They

J o h n e ’ s b a c i l l i w i t h i n m ig r a tio n r a n g e .

M any m u l t i n u c l e a r c e l l s

c e lls

have been v e ry a c t iv e .

is

out o f

may b e s e e n
i n m a rk e d

(T 7 7 ),

in to

H ods,

i n w h ic h

th e c u lt u r e

th e

can

r a th e r la r g e

th e e x p la n t s e v e r a l g ro u p s o f m a c ro ­
o n e s may be fo u n d c o n t a i n i n g r o d s ,

c h a in s u p t o

T h e re a r e m any m i t o t i c
a r a p id m u lt ip lic a t io n

10 o r 12 u . and ta k e

fig u r e s i n
o f c e lls .

th is

a den se

c u ltu r e ,

in d i­

29

D is c u s s io n
The r e m a r k a b le a f f i n i t y
c y te f o r J o h n e *s b a c i l l i

in

o f th e
tis s u e

p o ly m o r p h o n u c le a r le u c o ­
c u ltu r e s

is

to

o u r kn ow ­

le d g e

a phenom enon p r e v io u s ly u n re c o rd e d b y an y o b s e rv e r#

S m ith

(1 4 )

s e rv e d i n
a t th is

s t a t e s t h a t p o ly n u c le a r c e l l in v a s io n
a n y s ta g e o f

in s titu tio n

th e

(1 5 )

it

f r e q u e n t l y a s s o c ia t e d w i t h
p a r tic u la r ly

in

d is e a s e *

In

is

n o t ob­

w o rk p r e v i o u s l y done

w as o b s e rv e d

"that p o ly m o rp h s w e re

m a c ro p h a g e s c o n t a i n i n g b a c i l l i ,

th e g l a n d u l a r t u b u le s

o f th e

in t e s t i n a l m ucosa,

a lt h o u g h p h a g o c y t o s is

o f b a c i l l i b y p o ly m o rp h s was n e v e r o b ­

s e rv e d .

in tis s u e

P h a g o c y t o s is

s e v e ra l w ays*
th e

It

g u in e a p i g ,

m u s t b e re m e m b e re d t h a t

1he

and, th e re fo re ,

o r c u ltu r e s

tis s u e

c u ltu r e s

o f b o v in e

tis s u e .

th e p o ly m o r p h o n u c le a r

s u r r o u n d e d t h e c lu m p s ,

It

is

c e lls

it

tru e

on th e s c e n e ,

th e m a c ro p h a g e s f a i l e d

th a t

in
th e

b u t, even in

a d ja c e n t t o

to r e a c t t o

s e n c e o f t h e o r g a n is m s u n t i l m any h o u rs a f t e r
h a d done s o *

th e

m ig r a t e d i n t o

th o s e c u l t u r e s w h e re o rg a n is m s w e re d i r e c t l y

n u c le a r c e l l s

may be

and in g e s t e d o r g a n is m s some

tim e b e f o r e t h e m a c ro p h a g e a r r i v e d

e x p la n t e d t i s s u e ,

was f r o m

d is e a s e h a s

same c o n d i t i o n m ig h t n o t p r e v a i l i n

b o v in e

p la s m a ,

th e t is s u e

a r e s i s t a n t a n im a l i n w h ic h t h e

n o t b e e n s t u d ie d e x t e n s i v e l y ,
a rg u e d t h a t

c u l t u r e s may b e e x p la in e d i n

th e

th e p r e ­

t h e p o ly m o r p h o ­

The q u e s t io n may be r a is e d

as to

w h e th e r o r n o t th e p o ly m o r p h o n u c le a r le u c o c y t e

is

d e s tr o y in g

c o n tr a r y , th e

J o h n e 's b a c i l l u s

o r w h e th e r,

o r g a n is m s do n o t s o o n d e s t r o y t h e
t o b e e n g u lf e d b y m a c ro p h a g e s .

c e lls

o n th e

c a p a b le

of

a n d a re a g a in l i b e r a t e d ,

O b s e r v a t io n o f

c e lls

con-

30

ta in in g
th o s e

" b a c i l l i d i s c l o s e d t h a t m any p o ly m o r p h s ,

is o la te d

p a r tic u la r ly

o n e s w h ic h h a d e n c o u n te r e d and p h a g o e y to s e d

s c a t t e r e d o r g a n is m s ,
s ta n c e s a t l e a s t ,
th e s e le u c o c y t e s

a p p a r e n t ly d i d

fo r

o fttim e s

d i g e s t them; i n

o rg a n is m s w e re

th a t s ta in e d v e r y f a i n t l y •

some i n ­

se e n w i t h i n
O th e r s

showed a

b e a d e d a p p e a ra n c e and a c i d - f a s t g r a n u le s w e re f a i r l y
On t h e

common*

o t h e r h a n d , w h e re p o ly m o r p h s h a d s u r r o u n d e d l a r g e r

c lu m p s o f o r g a n is m s , i t
c e lls *

T h is ,

tr a tio n

o f to x in s

was d i f f i c u l t t o

I b e lie v e ,

was d u e t o

fin d

o rg a n is m s w i t h i n

t h e f a c t t h a t th e

concen­

a b o u t s u c h c lu m p s o f o r g a n is m s w as so g r e a t

t h a t th e c e l l c o u ld n o t lo n g r e s i s t th e m a n d so o n d ie d and
d is in t e g r a t e d b e fo re
ro d s *
c e lls

T h is

is

it

had a n o p p o r tu n ity

s u b s ta n tia te d b y th e

a c c o m p a n ie d b y t h e h y d r o p ic

d e s c r ib e d l i v i n g

c u ltu r e s *

s e e n g a t h e r e d a b o u t c lu m p s

in ju r y

changes

and d e a th
se e n i n

H o w e v e r, w h e re
i n la r g e

t o p h a g o c y to s e

fe rm e n ts

n u m b e rs , i t

was o b s e rv e d
c lu m p and

t h e le u c o c y t e s h a d a p p a r e n t ly b e e n ly z e d b y th e

lib e r a te d

was a r a t h e r

fro m th e d is in t e g r a t in g

s h a r p ly l i m i t e d

A lt h o u g h

g r a n u le s *

b y th e c a m e ra l u c i d a d r a w in g
b y th e

c a re

p h o to m ic r o g r a p h

( F ig *

( F ig *

T h is i s
23)

and

2 2 ).

s h o u ld be u s e d i n d r a w in g p a r a l l e l i s m s

b e tw e e n t h e r e a c t i o n
and t h e

no

as e v id e n c e d b y b e a d e d

a p p e a ra n c e and p r e s e n c e o f some a c i d - f a s t

le s s d i s t i n c t l y

The r e s u l t

zone w h e re o rg a n is m s had n o t

y e t b e e n ( d e s tr o y e d b u t showed i n j u r y

w e ll il l u s t r a t e d

c e lls *

p e r i p h e r a l zone c o n t a i n i n g

s t a in e d o r g a n is m s and a c e n t r a l

p ig tis s u e

p r e v io u s ly

le u c o c y t e s w e re

t h a t t h o s e o r g a n is m s n e a r th e p e r i p h e r y o f t h e
n e a re s t to

o f th e

o f c e lls

as seen in

d is e a s e a s i t

e x is ts

c u ltu r e s
in th e

of

g u in e a

b o v in e ,

it

31

d o e s n o t seem u n r e a s o n a b le
c e lls

of

th e b o v in e ,

in

to

assum e t h a t t h e

th e e a r ly

le a s t,

p o sse ss id e n t ic a l pow er to

be t h e

c a s e , w hy t h e n do n o t

tio n to

th is

s ta g e s o f th e
ly z e

t h e o r g a n is m s *

th e

in

q u e s t io n we

f r o m th e

r e tic u la r

w it h tu b e r c le
D iffu s ib le

b a c illi,

t u b e r c u lo s is

t o x i n s a ls o

t r a n s fo r m a tio n

in to

w as n o t p o s s ib le
w ith in

H o w e v e r,
to

in

No r e s i d u a l y e l l o w
d ig e s t io n o f

to

c a n n o t be p ro d u c e d
assume t h a t i t

t h e o r g a n is m *
is

is

id e n tic a l

in o c u l a t e d w i ih

c u l t u r e s u p to

two weeks o ld i t

i n M a xim o w *s c u l t u r e s , c o n t a i n i n g

fin d

e ig h t days*-

th a t r e s u ltin g

fr o m

As s t a t e d b e f o r e ,

t h e c y to p la s m o f t h e m a c ro p h a g e s

c o n t in u e d g r o w th and i n
fr e e ly *

some c u l t u r e s

S in c e , h o w e v e r, th e

i n th e g u in e a p i g ,

it

is

d is e a s e

r e a s o n a b le

to

t h e p o ly m o r p h o n u c le a r le u c o c y t e an d i t s

p la y an im p o r t a n t p a r t i n
I n tis s u e

b e li e v e d

o r g a n is m s *

c a u s in g a

P r a c tic a lly

c u ltu r e s

p ig m e n t s i m i l a r to

a p p a r e n tly m u l t i p l i e d

fe rm e n ts t h a t

p r o g e n ito r s ,

tu b e r c le b a c i l l i w as se e n *

th e ir

c o n ta c t

in g e s t e d th e

d ig e s tio n w as w e ll advanced in

t h e o r g a n is m s seemed t o
fa v o r a b le

came i n

s a y t h a t th e o r g a n is m s w e re d ig e s t e d

th e m a c ro p h a g e s w h ile

t u b e r c le b a c i l l i ,

c y te s

im m e d ia te ly

tis s u e

a ro s e fro m

o f in f la m m a t io n and

When th e y

a ffe c te d t h e ir

in

th is

M axim ow and

th e s e c e l l s

e p it h e lio id © e lls *

c o n d itio n s p r e v a ile d
J o h n e 's b a c i l l i *

c e lls #
th e y

If

s h o u ld t u r n o u r a t t e n ­

ly m p h o c y te s a n d m o n o c y te s o n t h e f i e l d
a ls o

at

in fe c tio n ?

th e m a c ro p h a g e o r e p i t h e l i o i d e e l l *

o t h e r s h a v e show n t h a t

d is e a s e

th e p o ly m o rp h s d e s t r o y th e

b a c i l l i a n d p r e v e n t th e a d v a n c e o f
F o r an a n sw e r t o

p o ly m o r p h o n u c le a r

c u lt u r e s th e

to be l i m i t e d

to

th e d e s t r u c t i o n

num ber o f

th o s e

of

s u c h le u c o ­

th a t a re in

th e

32

tis s u e

at

t h e t im e

s h o rt liv e d ,
and t h e i r

it

o f tr a n s p la n ta tio n ,

c a n h e se e n

e f f e c t upon th e

T h e o b a ld S m ith
lo s is
to

(1 4 )

t h a t th e

o rg a n is m s
in

s in c e t h e y a r e

s u p p ly i s
is

s o o n e x h a u s te d ,

tr a n s ito r y *

a c o m p a r is o n o f b o v in e t u b e r c u ­

w it h p a r a tu b e r c u lo s is

th e

and,

s ta te s

absence o f a n y d i f f u s i b l e

t h a t th e

to x in ,

e v id e n c e p o i n t s

an d t h e

d is e a s e p r o ­

g r e s s e s b y c o n t a c t o f b a c i l l i , w h ic h h a v e e s c a p e d fr o m i n f e c t e d
c e lls ,

w ith

fib r o s is

fre s h

s im ila r to

b e e n s u g g e s te d
tu r b a n c e i s
th e

th a t o f

N e it h e r

o f in t e r f e r e n c e

b y th e h y p e r p l a s t i c

A p p a r e n tly th e r e

c e l l n e c r o s is n o r

tu b e r c u lo s is

( 1 7 ) t h a t th e r e s u l t i n g

th e r e s u l t

in te s tin e

(1 5 )*

tis s u e c e l l s *

is

w it h

a b s o r p t io n f r o m

o r to

c a u s e s u n kn o w n

c a p a b le o f k i l l i n g

m a c ro p h a g e s p r o d u c e d e i t h e r b y t h e e x t r a c e l l u l a r
c e l l u l a r g r o w th o f t h e

o r g a n is m s *

e v i d e n t t h a t some d i f f u s i b l e

H o w e v e r,

s u b s ta n c e

o f th e

c o lo n ie s d if f u s e d

bunches o f b a c i l l i *
s tro n g p o s it iv e
d e a th o f th e s e
o th e r b a s is *
th a t,

It

is

d iffic u lt

to

in

th e v i c i n i t y

I t h a s a ls o

v itr o

it

is

o f a ttr a c tin g
in

d ie

account

th e

la r g e r
f o r th e

p o ly m o r p h s a n d th e

o f t h e c o lo n ie s o n a n y

been o b s e rv e d

a n e x t e n s iv e

th e

o r in tr a ­

c e lls

fr o m

c h e m o ta x is e x e r t e d on t h e
c e lls

tfT h e re i s

fo rth

in

c a p a b le

l e u c o c y t e s a n d o f g r e a t l y d a m a g in g c e r t a i n
v ic in ity

has

s e v e re c l i n i c a l d i s ­

tis s u e

is no to x in

s e e n , and i t

(1 4 )

i n th e b o v in e

p h a g o c y to s is o f r e d c e l l s

th e

s p le e n and a g r a d u a l c o n v e r s io n o f

d ie i n c l u s i o n s

m e n t*

damaged w h ile c i r c u l a t i n g

E v id e n t ly th e r e d

in

th e c a p illa r ie s

to

th e p r o d u c tio n o f a t o x i c
S m ith

o f th e

c e lls

(1 4 ) s ta te s

a re

in te s tin a l v i l l i * w

th a t

in to

in

T h is a ls o

p ig ­

p o in ts

s u b s ta n c e *
in h is

e x p e r ie n c e a l l e p i t h e l i o i d

33

c e lls

c o n t a in e d b a c i l l i *

fo u n d some e a r l y
e p ith e lio id

H a llm a n and W i t t e r

c a s e s i n w h ic h ,

and g i a n t c e l l s

a lt h o u g h

w as f o u n d ,

c o n t a in e d n o a c i d - f a s t o r g a n is m s *
c o u r s e , m a c ro p h a g e s w e re fo u n d

in

( 1 5 ) , h o w e v e r,

an a b u n d a n c e o f

th e m a j o r i t y

o f them

M any g i a n t c e l l s

and, o f

in o c u la t e d c u l t u r e s w h ic h

c o n t a in e d n o o r g a n is m s .
I t was o b s e rv e d t h a t g ia n t c e l l f o m a t io n
m any c a s e s w h e re m a c ro p h a g e s s u r r o u n d e d and
as th e r e s u l t o f
phages

( F ig s 11 and 1 8 ) *

t a k e s p la c e w i t h
c e lls

th e f u s i o n

in g e s t e d o r g a n is m s

As p r e v i o u s l y s t a t e d t h i s
in

phenom enon

l i q u i d m e d ia w h e re th e

a r e d e p r iv e d o f a n y s u p p o r t in g fr a m e w o r k o f f i b r i n *
th e

fa c t th a t

becom es a r o u n d e d c e l l ,
a s s u m in g a n o n - m o t i le
lo s in g

its

e ffe c tiv e

o b s t a c le

to

advanced th e

i n s u c h m edium th e m a cro p h a g e

r e tr a c tin g

s ta te ,

its

( 1 9 ) , Tidiieh i s

c e l l fu s io n .
s u rfa c e

te n s io n .

m u tu a l p r o t e c t i o n *
c e l l f o r m a tio n

is

in

e s s e n tia lly

is

a f u s io n

O th e r s b e l i e v e

p ro c e s s

o f c y to p la s m f o r

th a t th e p ro c e s s o f g ia n t

one o f in c o m p le te

th e

liv in g

c e lls

m any a s e i g h t n u c l e i w e re

d iv is io n .

fo r th is
H o w e v e r,

as 24 h o u r s

o f a p e c u l i a r t y p e c o n t a in in g

seen.

In

In

m o st in s t a n c e s

c u ltu r e s .

c u lt u r e s , even a fte r as l i t t l e

g ro w th , m u lt in u c le a t e d

be a n

th e m a n ife s ta tio n o f

th e r e s u l t o f c e l l f u s i o n

c o u ld be o b s e rv e d i n

some f i x e d

p r o b a b ly an

Some o b s e r v e r s h a v e

c u l t u r e s h e r e i n d e s c r ib e d a p p a r e n t ly i n

m u l t i n u c l e a t e d c e l l s w e re

same tim e

T h e re may a l s o

th e o ry t h a t c e l l fu s io n

a m ild c e l l i n j u r y r e s u lt in g

m any p s e u d o p o d s ,

a n d p o s s i b l y a t th e

u n d u l a t i n g m em brane

a c c o m p a n y in g c h a n g e i n

in

in

o f c y t o p la s m o f a d ja c e n t m a c ro ­

m o re f r e q u e n c y

T h is may be d u e t o

th e

t o o k p la c e

as

s e v e r a l in s t a n c e s a d i s —

34

t i n e t L i n k o r f i l a m e n t c o n n e c t in g tw o n u c l e i w a s
th e s e c e l l s *
o f a m ito tic
w ith

T h i s m ig h t b e i n t e r p r e t e d a s n u c l e i i n
d iv is io n *

T h ese c e l l s a r e

some o b s e r v e d b y M axim ow i n

v itr o *

In

c e lls w ith

liq u e fie d

p o r tio n s

th e

b lo o d p ig m e n t , w e re

s lip *

S uch c e l l s

id e n tic a l

t u b e r c u lo u s p r o c e s s i n
n u ltin u c le a te d

see n c lo s e ly a d h e re n t t o

a re n o t s p e c if ic ,

and o t h e r s

th e a c t

c o n t a i n i n g b a c i l l i and e n ­

o b s e rv e d u n d e r s i m i l a r c o n d i t i o n s
L a m b e rt ( 1 8 )

p r a c tic a lly

o f c u lt u r e s la r g e

a s m any a s 40 n u c l e i ,

g u lfe d

tis s u e

seen i n

th e c o v e r-

h o w e v e r, h a v in g b e e n

i n u n in o c u la t e d

o b s e rv e d th e

c u ltu r e s *

same ty p e o f c e l l i n

c u l t u r e s o f s p le e n *

D e ta ile d

e x a m in a t io n c o n v in c e s u s t h a t m u l t i p l i c a t i o n

J o h n e 's b a c i l l i t o o k p la c e w i t h i n
tw o w eeks g r o w th

show i n

th e

c u ltu r e s *

of

C u lt u r e s o f

a lm o s t e v e r y c a s e m any d e e p - s t a in in g

r o d s b o t h i n t r a - and e x t r a c e l l u l a r *

M o st o f th e s e

occur a t

th e b o r d e r o f t h e

e x p la n t e d t i s s u e

g r o w th o f c e l l s *

The m a nn e r i n w h ic h t h e o r g a n is m s p e n e t r a t e d

in to

t h e e x p la n t

D e n se m a sse s o f
m any s c a t t e r e d
s u c h m a sse s*
in

le n g th

12 u *

19 h o u r c u l t u r e s a ls o

new

d e m o n s tr a te s g r o w th *

t h e o r g a n is m s a r e seen b e tw e e n
o rg a n is m s

th e c e l l s ,

a r e o b s e rv e d a ro u n d th e

an d

p e r ip h e r y o f

The o r g a n is m s a p p e a r a s r o d s as s h o r t a s 0 * 5 u *

o r a s a p p a r e n t ly u n b ro k e n c h a in s a s lo n g a s 10 t o

L o n g e r,

son o f t h r i f t y
of

in

among a n d w i t h i n t h e

u n b ro k e n c h a in s a re n o t o b s e rv e d *
in o c u la te d

th e e x p la n t e d t i s s u e

c u ltu r e s

w it h t h o s e i n

was s c a n t y ,

A c o m p a r i­
w h ic h g r o w th

b e ca u se o f u n d u e e x p o s u re

d u r i n g m a n i p u la t i o n o r l i q u e f a c t i o n o f th e p la s m a m edium due
to

a c c u m u la t io n o f m e t a b o l i t e s ,

Johnef s b a c i l l i

in

th e

la tte r

shows t h a t

d id

th e c o lo n ie s

n o t in c r e a s e

in

s iz e

of
or

in

35

num ber o f b a c t e r ia *

In

such

o n ly a fe w r o d s r e t a in e d
o u t o f r e la tiv e ly
T h i s w o u ld

c u ltu r e s

a fte r

t h a t g r o w th

im m e d ia te n e ig h b o r h o o d o f t h e
n o t p r o m in e n t i n

c u ltu r e s

in t o

th e c u l t u r e #

o f Jo h n e f s b a c i l l i depends

upon th e a c t iv e g ro w th o f c e l l s w i t h i n

is

days i n v i t r o ,

t h e ir p r o p e rty o f a c id - fa s t s ta in in g

l a r g e c lu m p s i n o c u l a t e d

in d ic a te

te n

th e

o r g a n is m s *

c u ltu r e

e x p la n t,

to g r o w th ,
t h e y w e re

i* e * ,

o ld e r th a n e i g i t

t h e zo n e

to

th a t e p ith e lio id

s e p a ra te and d i s t i n c t
a d a p t a t io n s

c e lls

a nd t h e

c e lls *

fo rm o r

s u b s ta n c e s i n t h e

s ta in in g

o n ly

due t o

th e

c h a r a c te r is tic s

in d i­

th e

in c e r ta in
in g e s t i o n o f
p re s e n c e o f

in o c u l a t e d c u l t u r e s #

M any m a c ro ­

cannot be d if f e r e n t ia t e d
fro m

ta in in g

no J o h n e f s b a c i l l i o r f r o m th o s e

tu re s #

T h o se w i t h

lik e
in

c e lls

con­

u n in o c u la t e d

c u l­

a n d w it h o u t b a c i l l i a p p a r e n t l y a r e s t a in e d

b y n e u t r a l r e d w it h e q u a l

n u c le a t e d c e l l s

a ro u n d

o r d i n a r y m a c ro p h a g e s a r e

and a h y p e r t r o p h y b r o u g h t a b o u t b y

s u p ra v i t a l l y

lo c a t i o n s

o f new c e l l s

They d i f f e r

p h a g e s c o n t a i n i n g e n g u lf e d b a c i l l i
in

days

a n y in o c u la t e d c u l t u r e s t o

o r g a n is m s o r a c h e m ic a l s t i m u l a t i o n
d iffu s ib le

te n

s o o n s u r r o u n d e d a n d in g e s t e d b y m a c ro p h a g e s .

N o t h in g was o b s e rv e d i n
c a te

th e

E x t r a c e llu la r g ro w th

b e c a u s e , w hen c o l o n i e s o f o r g a n is m s o c c u r r e d i n
fa v o r a b le

in

a re

s t a in e d

fa c ility *

a ls o s u p r a v i t a l l y

to

The m u l t i a b o u t th e

same d e g r e e *
The
d is e a s e

s u g g e s t io n t h a t th e
is

due to

e x tre m e e m a c ia t io n o f Johne *s

a s im p le m e c h a n ic a l i n t e r f e r e n c e w i t h

s o r p t i o n h a s n o t r e c e i v e d w id e
a b s e n c e o f p o s i t i v e e v id e n c e
le d m any t o

d is b e lie v e

in

th e

s u p p o rt*

H o w e v e r, t h e

o f a n y d e g e n e r a t iv e
f o r m a t io n o f

ab­

a p p a re n t

changes has

a t o x i n b y th e

36

o r g a n is m *

A m o s t im p r e s s iv e

m a c ro p h a g e s a n d g i a n t c e l l s

o b s e r v a t io n
in v i t r o ,

is

th e f a c t

c o n ta in in g g r e a t

titie s

o f Johner s b a c i l l i ,

in ju r y

e x c e p t p o s s i b l y a te m p o r a r y l o s s o f m o t i l i t y *

is

th e re

a n y e v id e n c e

t a in e d r o d s *

e x a m p le o f s y m b io s is ,
w ith in
to

th e c e l l ,

d iffu s ib le

to x in s

s u s c e p tib le
c y te s *
in

th e

a ttr a c te d to
to x in s

in te g r a te

is

th e s i t e

e v e r,

a re

g ro w n i n

th e

m ost

v itr o

a re

th e b o v in e

o r g a n is m s *

fe rm e n ts h ig h ly

o r g a n is m s .

th ro u g h

It

b y th e m ,

S in c e a b s o r p t i o n

th e
in

ly tic

T h e re fo re ,

p o s s ib le

th e c a p i l l a r i e s

im p a ir e d a n d th e y a r e
of

is

They d is ­

th e s e l y t i c

of

fo r

th e

f e r m e n ts

i t w o u ld
th e ir h o s t,

th a t e ry th ro ­

o f th e

ly m p h g la n d s e n c o u n te r

in ju r e d

a re

a n d a r e s o o n d e s tr o y e d b y

p r o t e c t e d b y t h e c y to p la s m

p h a g o c y to s in g c e l l s

b a b ility

p e r fe c tly

o th e r c e l l s ,

c u ltu r e s

H o w e v e r, a p p a r e n t ly

m ucosa and m e s e n t e r ic

is

th e

lib e r a te

re a c h th e

c y te s c i r c u l a t i n g

fu ln e s s

to

in tr a c e llu la r

t h e m a cro p h a g e o r g i a n t c e l l *

and a r e g r a v e l y

and m u l t i p l y i n g

b lo o d le u c o c y t e s a n d e r y t h r o ­

o f in fe c tio n

c o m in g f r o m th e

seem t h a t t h e y

be a n e x c e l l e n t

th e p a t h o lo g y as s e e n i n

and. e v i d e n t l y

if

th e c o n ­

b e l i e v e d t h a t p o ly m o r p h o n u c le a r le u c o c y t e s

i n v a d in g o r g a n is m s *
s e ld o m ,

N e it h e r

and p o s s i b l y s e n d in g f o r t h

a re c e r t a in
in

of

h a v e a d a p te d i t s e l f

a re i n j u r i o u s

p e r f e c t h a rm o n y w i t h
It

w h a t a p p e a rs t o

in v a d e r ,

w h ic h

o f w h ic h

o r d ig e s tio n

t h e o r g a n is m l i v i n g

T h e s e o b s e r v a t io n s

d is e a s e *

th e

is

to ,

w h ic h seem s t o

th e p re s e n c e o f

quan­

show: a b s o l u t e l y n o e v id e n c e o f

o f in ju r y

H e re , th e n ,

th a t

in te s tin a l

th e s e

so much so t h a t

same t o x i n s

th e i r

use­

c o n s e q u e n tly e n g u lf e d b y t h e

s p le e n when th e y r e a c h t h a t o r g a n *

th e

in te s tin a l t r a c t i s

a v i t a l p ro c e s s c a r r ie d

on by

c e lls

in

o f th e

a ll p ro ­
in te s ­

37

tin a l

e p ith e liu m ,

in te r fe r e d
is

may b e t h a t

w i t h b y th e

c o rre c t,

c y te s

it

th is ,

th is

same t o x i n *

t o g e t h e r w it h

and e r y t h r o c y t e s , w o u ld

assum es t h e a b i l i t y

o f th e

m a c ro p h a g e a n d t h e

in a b ility

in to

th e

same c e l l ,

v ie w o f t h e f a c t
to

c e lls

t o b lo o d

cases.

to x in to
o f th e

T h is h y p o t h e s is

d iffu s e
ly tic

th e

c e lls

f r o m th e

w it h in th e
fe r m e n ts

b o d y a re

r e s is t a n c e o f th e

g ro w n i n v i t r o

e ffic ie n t
le u c o c y t e *

a b le

l i b e r a t e d fr o m

a b le

t is s u e

to m u lt ip ly

in v it r o ,

o r,

as s ta te d b e fo r e , t o

p h a g o c y t o s is on th e

we may

g u in e a p i g m u st d e p e n d

u p o n some s y s t e m ic , u n d e te r m in e d f a c t o r n o t o p e r a b le
c u ltu r e s

pene-

o r g a n is m .

m a c ro p h a g e s o f g u in e a p i g

c o n c lu d e t h a t th e

fo rth

fe rm e n ts to

S in c e J o h n e * s b a c i l l i w e re a p p a r e n t ly
w ith in

le u c o ­

a n o t u n r e a s o n a b le c o n c lu s io n i n

th a t u n in ju r e d

o f th e

lik e w is e

to e x p l a i n t h e e x tr e m e

p r o t e c t th e m s e lv e s fr o m h e t e r o l y t i c

o th e r

is

th e a b o ve h y p o t h e s is

in ju r y

s e rv e

i n m any b o v in e

tra te

If

th e

e m a c ia t io n o b s e r v e d

fu n c tio n

p a r t o f th e

i n th e

a more

p o ly m o r p h o n u c le a r

38

Summary
1 * M y c o b a c te r iu m p a r a t u b e r c u lo s is
tu re s

in

v itr o

v iv e d

a n d a p p a r e n t ly g r e w b o th

c e llu la r ly

in

2 * E v id e n c e i s

o f g u in e a p i g

a nd e x t r a -

th e p o ly m o r ­

c a p a b le o f p h a g o c y to s in g a n d d i ­

b y th e d is in t e g r a t in g

3 . A d iffu s ib le

c u l­

o r g a n is m s s u r ­

in tr a c e llu la r ly

o r g a n is m i n t r a c e l l u l a r l y

c y t e s a p p a r e n t ly l y z e

to

H ie

p r e s e n t e d w h ic h i n d i c a t e s t h a t

th e

lib e r a te d

s p le e n *

in to

such c u ltu r e s *

p h o n u c le a r l e u c o c y t e i s
g e s tin g

w as i n o c u la t e d

and t h a t fe r m e n t s

p o ly m o r p h o n u c le a r le u c o ­

n e a rb y J o h n e f s b a c i l l i *

s u b s ta n c e ,

s t r o n g l y c h e m o ta x ic f o r a n d t o x i c

p o ly m o r p h o n u c le a r l e u c o c y t e s , was l i b e r a t e d b y t h e

J o h n e 's b a c i l l u s *
4 * M a c ro p h a g e s and g i a n t c e l l s

t h e y e n c o u n te r e d th e m b u t a p p a r e n t ly h a d l i t t l e

pow er o f

ly z in g th e

J o h n e 's

b a c illi

I n th e

e n g u lf e d r o d s *

seemed t o

f a v o r a b le
5*

e n g u lf e d t h e o r g a n is m s w h e r e v e r

fin d

m e dium o f

th e

in

c y to p la s m o f

d e s c r ib e d g i a n t

p la c e b y c e l l f u s i o n a n d ,

g r e a te r fre q u e n c y

c o n tra ry ,

th e

such c e l l s a

g r o w th *

c u lt u r e s h e r e in

b y n u c le a r d i v i s i o n *

On th e

in

to o k

some fe w in s t a n c e s , a p p a r e n t l y

Such c e l l f u s i o n
liq u id

c e l l fo r m a tio n

m edium *

t o o k p la c e w it h

39

F ig *

1*

(a b o v e )
w ith

F ig *

2.

T is s u e

c u ltu r e

o f 5 days

J o h n e 's b a c i l l i a t tim e

( b e lo w )

g r o w th ,

o f t r a n s p la n ta tio n *

A 5 d a y u n in o c u la t e d t i s s u e

p r o f u s e g r o w th

i n o c u la t e d

o f f ib r o b la s ts *

c u ltu r e

( x l6 0 )

( x l6 )

s h o w in g

40

%r
X

m

F ig .

•

(a b o v e )
b iliz e d

Illu s tr a tin g

a g r o u p o f m a c ro p h a g e s

a r o u n d a c lu m p o f J o h n e f s b a c i l l i ,

s c a t t e r e d m a c ro p h a g e s .
F ig .

• ( b e lo w )
tis s u e

( x l6 0 )

a ls o

(b )

( x l6 0 )

G ia n t c e l l f o r m a t io n
c u ltu r e .

( a ) mo­

(a )

in

an in o c u la t e d

41

F ig *

5.

(a b o v e )
tis s u e

F ig *

6*

G ia n t c e l l f o r m a t io n
c u ltu r e *

(a )

in

an i n o c u la t e d

(x 2 3 0 )

( b e lo w )

C lu m p in g o f m a c ro p h a g e s a ro u n d o rg a n is m s

(a),

fir s t

th e

Com pare th e s e

s te p

in

t h e f o r m a t io n

c lu m p e d c e l l s

m a c ro p h a g e s ( b ) .

( x l6 0 )

w ith

o f g ia n t

th e s c a t t e r e d

c e lls *

42

F ig *

7*

(a b o v e )

M a c ro p h a g e s

pseudopods in
F ig . 8 .

( e p ith e lio id

a n in o c u la t e d c u l t u r e *

( b e lo w )

E p ith e lio id

a tta c h e d

th e m s e lv e s t o

c u ltu r e *

c e lls )

(x 2 3 0 )

w ith

lo n g

(x 2 3 0 )

and g i a n t c e l l s w h ic h h a v e
s u r fa c e

o f c o v e r s lip

o f th e

43

F ig *

9*

(a b o v e )

M o b i l i z a t i o n o f m a c ro p h a g e s a ro u n d g r o u p s

o f o r g a n is m s a t p e r i p h e r y o f e x p la n t i n
c u ltu r e
F ig *

10*

( b e lo w )

(a)*
At

( x l6 0 )
( a ) m a c ro p h a g e s a r e m o b i l i z i n g

c lu m p s o f o r g a n is m s a t p e r i p h e r y
tr a tin g
(x 2 3 0 )

th e

in o c u la t e d

a ttr a c tio n

a ro u n d

o f e x p la n t, i l l u s ­

o f m a c ro p h a g e s t o

th e

o rg a n is m s #

44

F ig . 1 1 .

(a b o v e )

H ig h p o w e r v ie w o f

th e m o b i l i z a t i o n o f

m a c ro p h a g e s a ro u n d a c o lo n y o f o r g a n is m s .
c e lls

m ay r e s u l t f r o m t h e f u s i o n o f su ch m o b iliz e d

c e lls .
F ig .

12.

(x 4 3 Q )

( b e lo w )
phages
is

in

G ia n t

P a r a ffin
(a )

s e c tio n o f tis s u e

c o n ta in in g

o r g a n is m s .

a c t o f m ito s is .

A c id - fa s t

c o u n te r s ta in .

(x 9 3 0 )

c u ltu r e .

M a c ro ­

One o f th e m ( a * )
s t a i n , h e m a t o x y lin

45

F ig . 1 3 .

(a b o v e )

P a r a ffin

d a y s g r o w th

s e c t io n

in v i t r o .

F ig .

14.

( b e lo w )

tis s u e

b y a m a c ro p h a g e .

h e m a t o x y lin c o u n t e r s t a i n .
P a r a ffin

s e c tio n o f

g ia n t c e l l c o n ta in in g
J o h n e #s b a c i l l i .
c o u n te r s ta in .

a fte r 6

A c id - fa s t

(x 9 3 0 )

t is s u e

c u ltu r e .

A

l a r g e m asses o f p r o l i f e r a t i n g

A c id - fa s t

(x 9 3 0 )

c u ltu r e

P h a g o c y to s is o f d e g e n e r a te d

p o ly m o r p h o n u c le a r c e l l s
s ta in ,

of

s ta in ,

h e m a t o x y lin

46

*

F ig . 15*

(a b o v e )
P a r a f f in s e c tio n o f tis s u e c u lt u r e *
A
g i a n t c e l l a t t h e edge o f a l i q u e f i e d a r e a o f th e
fib r in c lo t*
I t c o n t a in s a fe w a c i d - f a s t r o d s w h ic h
do n o t show i n t h e p h o to m ic r o g r a p h *
A c id - fa s t s ta in ,
h e m a t o x y lin c o u n t e r s t a i n .
(x 9 3 0 )

F ig *

( b e lo w )
P a r a f f in s e c t io n o f tis s u e c u lt u r e .
M o b i­
l i z a t i o n o f m a c ro p h a g e s ( a ) a ro u n d a c lu m p o f
b a c i l l i a t p e r ip h e r y o f e x p la n t .
T h e c y to p la s m s o f
th e c e l l s b le n d w i t h t h e m ass o f o r g a n is m s , and c e l l
f u s i o n o f t e n t a k e s p la c e t o fo r m g i a n t c e l l s .
A c id f a s t s t a i n , h e m a t o x y lin c o u n t e r s t a i n .
(x 9 3 0 )

16.

47

/
%

F ig *

17#

(a b o v e )
P a r a f f in s e c tio n o f tis s u e c u lt u r e *
M a c ro ­
p h a g e s i n a c t o f p h a g o c y to s in g J o h n e 's b a c i l l i .
N o te b a c i l l i w i t h i n c y to p la s m o f m a c ro p h a g e s ( m ) ;
fib r o b la s ts ( f ) *
A c i d - f a s t s t a i n , h e m a t o x y lin
c o u n te r s ta in .
(x 9 3 0 )

F ig *

18.

( b e lo w )
P a r a f f i n s e c t io n o f t i s s u e e u l t u r e *
Phago­
c y t o s i s o f b a c i l l i b y m a c ro p h a g e s ( a ) .
C y to p la s m s
o f some o f th e m a c ro p h a g e s h a v e f u s e d , f o r m in g a
g ia n t c e l l ( b ) .
F ib r o b la s ts ( f ) *
A c id - f a s t s ta in ,
h e m a t o x y lin e o u n t e r s t a i n *
(x 9 3 0 )

48

\
?

jpff *f* 4w
W
Mr
jfS
A
F
41
*f
w
r
p
f
*
'• 6
1

to

F ig *

19.

(a b o v e )
P a r a f f i n s e c t io n o f t i s s u e c u l t u r e .
C e ll
g r o w th i n new t i s s u e zon e a ro u n d e x p l a n t .
F ib r o ­
b la s t s ( f ) ;
m ito tic fig u r e ( a ) ;
and m a c ro p h a g e s
(m ) c o n t a i n i n g b a c i l l i .
A c i d - f a s t s t a i n , hema­
t o x y l i n c o u n te r s t a i n •
(x930)

F ig .

20.

( b e lo w )
P a r a f f i n s e c t io n o f t i s s u e c u l t u r e .
F i b r o b l a s t s ( f ) i n z o n e o f new t i s s u e a t p e r i p h e r y
o f e x p la n t.
F ib r o b L a s t s ( f f ) w i t h J o h n e f s b a c i l l i
w i t h i n c y t o p la s m .
H e m a to x y lin s t a i n , h e m a t o x y lin
c o u n te r s ta in .
(x 9 3 0 )

49

F ig . E l.

(a b o v e )
P a r a f f i n s e c t io n o f a 5 l / 2 h o u r t i s s u e
c u l t u r e , i l l u s t r a t i n g a c lu m p o f J o h n e 's b a c i l l i
s u r r o u n d e d b y p o ly m o r p h o n u c le a r l e u c o c y t e s . A c i d f a s t s t a i n , h e m a t o x y lin c o u n t e r s t a i n .
(x 9 3 0 )

F ig .

( b e lo w )
P a r a f f i n s e c t io n o f a 19 h o u r t i s s u e c u l ­
t u r e , i l l u s t r a t i n g a clu m p o f J o h n e 's b a c i l l i s u r ­
ro u n d e d b y p o ly m o r p h o n u c le a r l e u c o c y t e s .
B a c illi
a t e d g e o f c lu m p h a ve b e e n ly z e d b y f e r m e n t s
l i b e r a t e d b y d i s i n t e g r a t i n g p o ly m o r p h s .
A c id - fa s t
s t a i n , h e m a t o x y lin c o u n t e r s t a i n .
(x 6 2 0 )

22.

50

A
<
v
1 V;r-r^/K^:?‘.-~= •* U
jSr
, •r ;^

^ C £ #- * * ' - v ;-

v..

4fr

F ig #

23#

(a b o v e )
C am era l u c i d a d r a w in g o f F ig # 2 2 , s h o w in g
p e r i p h e r a l a r e a i n w h ic h o rg a n is m s h a ve been ly z e d #
(a p p # xlO O O )

F ig #

24#

( b e lo w )
C am era l u c i d a d r a w in g o f a p a r a f f i n s e c ­
t i o n , i l l u s t r a t i n g p h a g o c y t o s is o f J o h n e *s b a c i l l i
b y p o ly m o r p h o n u c le a r le u c o c y t e s .
( a ) P o ly m o r p h o n u c le a r le u c o c y t e c o n t a in in g J o h n e 's
b a c illi•
(b ) E x t r a c e llu la r Jo h n e r s b a c i l l i .
( c ) I n t r a c e l l u l a r o r g a n is m s .
( d ) D i s i n t e g r a t i n g p o ly m o r p h o n u c le a r s s u r r o u n d e d b y
a fe w J o h n e fs b a c i l l i .

51

B ib lio g r a p h y

1 * H a r r is o n ,

R . G-.

The o u tg r o w th

of

mode o f p r o t o p la s m ic m ovem ent*

th e n e rv e f i b e r a s a

J o u r * E x p * Z o o l,

ix ,

787,

1910 .
2 . Loeb, Leo*
tis s u e

in

Q u o te d b y C a r r e l l and B u r r o w s *
v itr o

and i t s

t e c h n iq u e *

C u ltiv a tio n o f

J o u r * E x p . M e d *, 1 3 ,

387, 1911.
3* Loeb,

Leo*

U b e r d ie E n ts th u n g v o n B in d e g e w e b e L e u c o c y te n ,

u n d u b e r e in e M e th o d s i s o l i e r t e

G e w e b s te ile zu Z u c h te r n *

C h ic a g o ,

E n tw i eke lu n g smech a n ile d *

1897, p . 41*

O rg a n , x i i i ,

A rc h . f .

487, 1902.

E x p . M e d ., 1 3 ,

C a r r e l & B u rro w s ,

J o u r*

3 8 7 , 1 9 1 1 .)

4 . B u r r o w s , M* T .
e m b ry o o u t s id e

( fr o m

The c u l t i v a t i o n
th e b o d y .

o f t is s u e

of

th e c h i c k -

J o u r . Am. M ed. A s s o c . ,

lv ,

2057,

1910.
5 . C a r r e l , A . and B u r r o w s , M . T .
and o r g a n s
1379,
6*

o u t s id e t h e b o d y .

J o u r * Am* M ed. A s s o c . ,

lv ,

A r t i f i c i a l a c t i v a t i o n o f th e g r o w th i n v i t r o

o f c o n n e c t iv e

tis s u e *

7 * E b e lin g , A . H .

8 , M a xim o w , A *
1916;

J o u r.

35(6),

755,

s tr a in

17, 1 4 , 1913*

o f fib r o b la s ts .

1922.

A r c h , r u s s e s d *A n a t * d ’ H i s t o l *

A rc h , f *

m ik r o s p . A n a t * ,

tr ib u tio n s

to

Z ts c h r. f *

w is s M ik r o s p ,

34,

E xp* M e d *,

A t e n y e a r o ld

J o u r . Exp* Med*,

D is .,

o f a d u lt t is s u e

1910.

C a r r e l, A .

1,

C u ltiv a tio n

e m b r y o lo g y C a rn e g ie

549, 1924.

26,

e t d 'E m b r y o l* ,

96, 494, 1922;
In s t,

177, 1909*

in

Con­

p re s s ;

(In

J o u r. I n f .

52

9 * L e w is , R . L .
ty p h o s u s i n
of

th e

1 0 . S m y th ,

The f o m a t i o n
th e

c e lls

c h i c k e m b ry o .
H. F.

o f tis s u e

1 1 . S m it h ,
1922.

1 3 . H a s tin g s ,

and a n im a l

o f a r t i f i c i a l c u ltiv a tio n .

and L e w is , M. R .

J o u r. I n f .

J o u r.

Am. R e v . o f T u b e r c . ,

6, 21,

D i s . , 3 4 , 5 4 9 , 1 9 2 4 .)

T u b e r c u lo s is

D is .,

o f m am m alian t i s s u e

in v it r o *

34, 149, 1924.

B e a c h , a n d M a n s f ie ld .

tr a n s m is s ib le

1920.

265 & 2 8 3 , 1 9 1 6 .

1 2 . M a xim o w , A . A .
J o u r. I n f .

o f th e in t e s t in e s

The r e a c t i o n s b e tw e e n b a c t e r i a

W illis ,
( in

c u ltu r e s

to B a c illu s

J o u r . E x p . M e d ., 3 1 , 2 9 3 ,

tis s u e s u n d e r c o n d itio n s
E x p . M e d ., 2 3 ,

o f v a c u o le s due

J o h n e 's d is e a s e - a

d is e a s e o f c a t t l e .

W is e . A g r * l E x p . S t a .

R e s. B u i. 8 1 , 1 9 2 7 .
1 4 . S m it h , T .
d is e a s e s .

F o c a l c e l l r e a c tio n

In

t u b e r c u lo s is and a l l i e d

B u i . o f J o h n s H o p k in s H o s p .,

1 5 . H a llm a n and W i t t e r .
J o h n e * s d is e a s e .

Some o b s e r v a t io n s

J o u r . A . V . M. A . ,

53,

197, 1933.

on th e

85, n .s .

p a t h o lo g y o f
36(2),

159,

1933.
1 6 . Z a r k r z e w i s k i and K r a s z e w s k i.
T u m o re n i n v i t r o .
1933.

(abst.

1 7 . M 'F a d y e a n , J .
d is e a s e .

v itr o .

Z ts c h r . f • K re b s fo rs c h ,

The h i s t o l o g y o f

1he le s io n s
x x x i,

653,

1 9 3 4 .)

o f J o h n e 's
73, 1918.

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

J o u r . E x p . M e d .,

1 9 . C a r r e l and E b e lin g .

15,

510, 1912.

The fu n d a m e n ta l p r o p e r t i e s

f i b r o b l a s t a n d th e m a c ro p h a g e .
285, 1926.

3 9 : 4 7 1 -4 9 1 ,

Am. J o u r , o f C a n c e r, x x ( 3 ) ,

J o u r . Com p. P a t h . & T h e r a . ,

1 8 . L a m b e r t, R . A .
in

in

Z u c h tu n g v o n m e n c h lic h e n

o f th e

J o u r . E s p . M e d ., x l i v ( 3 ) ,

53

A c k n o w le d g m e n t

It

is

w ith

g r e a t p le a s u r e t h a t I

deep a p p r e c ia tio n o f

1iLe r e a d y a s s is t a n c e and k i n d

e n c o u ra g e m e n t o f D r * E * T * H a llm a n i n
C r e d it i s

a ls o

e x p r e s s my

th is

due t o D r * E * G-* H a s t in g s ,

w o rk *
o f The

U n i v e r s i t y o f W is c o n s in , D r * W* A * H a g e n , o f C o r n e l l
U n iv e r s ity ,

a n d D r* J * R e ic h e l ,

g ic a l L a b o ra to ry ,
b a c illu s ;

a ls o

f o r s u p p ly in g

o f M u lf o r d B i o l o ­
s tr a in s

o f J o h n e #s

t o M r* F * N* B a r r e t , D e p a rtm e n t o f

P o u l t r y H u s b a n d ry o f t h i s
in c u b a tio n o f eggs*

in s titu tio n ,

f o r a id

in