THE

R E A C T I O N OF

SPLENIC

LISTERIA

TISSUE IN C U L T U R E

TO

M O N O C Y TO G EN ES

By
ESTHER

M.

SMITH

A THESIS
S u b m i t t e d to th e School of G r a d u a t e S t u d i e s of M i c h i g a n
Sta te C o l l e g e of A g r i c u l t u r e a n d A p p l i e d S c i e n c e
i n p a r t i a l f u l f i l l m e n t of t h e r e q u i r e m e n t s
f o r the d e g r e e of

DOCTOR

OF PHILOSOPHY

D e p a r t m e n t of A n i m a l P a t h o l o g y

1955

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F rontispiece
A s e v e n - d a y - o l d c u l t u r e of s p l e e n .
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ABSTRACT

The
to genes

was

reaction

s tu d y of t i s s u e

in v i t r o in o cu lated with L i s t e r i a m onocy-

undertaken because

resulting fro m

of a n a p p a r e n t d i f f e r e n c e i n t i s s u e

i n f e c t i o n w ith t h i s

organism .

Tissue

cultures

p r o v i d e d a m e d i u m w h e r e b y th e d e v e l o p m e n t of the l e s i o n c o u l d be
o b s e r v e d u n d e r c o n tro lle d conditions.
E xplants

of s p l e e n w e r e p l a c e d i n p l a s m a c l o t s ,

w i th L . m o n o c y t o g e n e s

and s e a le d in d e p r e s s i o n c u lt u re

c o r d i n g to the m e t h o d of M a x im o w .
m a d e to m a t c h the

0.5 t ub e

m anner,

the fluid u s e d th ro u g h o u t th is

of t h e t i s s u e

was

culture.

Control c u ltu re s

om itting the b a c t e r i a .

Medium

sus­
were
199 w as

study.

of l i v i n g t i s s u e

cultures

r e v e a l e d t h a t the g r o w t h

not i n h i b i t e d by the p r e s e n c e

In a ll i n o c u l a t e d t i s s u e

ac­

su sp e n sio n was

A p p r o x i m a t e l y 0.01 m l of t h i s

p e n s i o n wa s p l a c e d in e a c h t i s s u e

O bservations

slides

of t h e M c F a r l a n d n e p h o l o m e t e r w h en

c o m p a r e d in a p h o t e l o m e t e r .

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

The b a c t e r i a l

inoculated

of L . m o n o c y t o g e n e s .

c u l t u r e s the b a c t e r i a w ere

always m otile

w h e n o b s e r v e d u n d e r the m i c r o s c o p e .
W h en i n o c u l a t e d t i s s u e

cultures

w e r e p l a c e d in a r t i f i c i a l

b a c t e r i o l o g i c a l m e d i u m , g r o w t h of L . m o n o c y t o g e n e s

d id n ot o c c u r

unless

the

tissue

cultures

were

The

s t u d y of s t a i n e d

refrigerated

at

4°C

for

several

days .

tures

serial

The

la t i o n and w as
th e lio id cells

r e a c t i o n b e g a n as e a r l y

surroundin g

a vessel.

over a six-day period.

f i e d as

a granulo m ato us

ula te d control c u ltu re s
F urther

studies

re a c tio n was

a result

as

c h a r a c t e r i z e d by the p r e s e n c e

in size

as

of i n o c u l a t e d t i s s u e

cul­

d i s c l o s e d w hat a p p e a r e d to be a p r o l i f e r a t i o n of th e v a s c u l a r

endoth eliu m .

tous

sections

The

24 h o u r s

after inocu­

of two l a y e r s

affected

area

in creased

T he p a t h o l o g i c a l c ha n g e w as

inflam m atory reaction.
exhibited this

None

specific f o r L . m onocytogenes

v

classi­

of the u n i n o c ­

reaction.

m u s t be done to d e t e r m i n e

of a n o n s p e c i f i c i r r i t a n t .

of e p i ­

if the g r a n u l o m a ­

o r if it w a s p r o d u c e d

ACKNOW LEDGMENTS

The

author is

g r a t e f u l to D r . F r a n k

of A n i m a l P a t h o l o g y , f o r h i s

Thorp, J r . ,

D epartm ent

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

t h e l ong a n d d i f f i c u lt p e r i o d of s tu d y a n d p r e p a r a t i o n of t h i s m a n u ­
script.

He h a s b e e n a v e r y i n s p i r i n g a n d e n t h u s i a s t i c t e a c h e r

w h o m to

s t u d y a nd a s s o c i a t e .

L. G ra y ,

D e p a r t m e n t of A n i m a l P a t h o l o g y , f o r h i s

sym pathetic
pleasure
edge.

for

The aulhor is i n d e b t e d to D r . M i t c h e l

attitude thro ughout th is

stu d y .

to w o r k w i t h one who gave

c o n s t a n t h e lp

this

so f r e e l y of h i s t i m e

c h e c k i n g th e m i c r o s c o p i c

a nd k n o w l ­
of

sections p r e p a r e d

w o r k a nd r e a d i n g th e m a n u s c r i p t .

T he w r i t e r w i s h e s
partm ent

of P h y s i o l o g y

structive

criticism

to t h a n k D r .

of

the D e ­
a nd c o n ­

m anuscript.

and s i n c e r e

t e n d s h e r g r a t i t u d e to D r . M.
of A n a t o m y .

L. F .W olterink

a nd P h a r m a c o l o g y f o r h is e f f o r t s

of the

It i s w i t h d e e p

thinking

and

It h a s i n d e e d b e e n a

M a ny t h a n k s to D r . R. A. R u n n e l l s , H e a d , D e p a r t m e n t

A nim al P ath olo gy, fo r

w ith

Lois

ap p re cia tio n that
Calhoun

the au th o r

ex­

H e a d of the D e p a r t m e n t

She h a s e x e r t e d a c o n s t a n t s t i m u l a t i o n f o r c r e a t i v e

and h i g h e r l e a r n i n g .

tinuous in s p ira tio n , this

entire

W ithout h e r

aid,

confidence,

and c o n ­

e n d e a v o r would h a v e b e e n i m p o s s i b l e .
vi

M a n y t h a n k s to D r .
partm ent

R u hla nd , P a t h o l o g i s t ,

f o r th e i n v a l u a b l e

the

S t a f s e t h a nd D r .

of M i c r o b i o l o g y , f o r t h e i r h e l p f u l

to D r . H a n s

M iss

H. J .

assistance

Ada B e i s t e r ,

interlibrary

reference

loans;

W alter

Mack, D e ­

suggestio ns

and a s s i s t a n c e

M ichigan D ep artm en t

of A g r i c u l t u r e

in the m a n y G e r m a n t r a n s l a t i o n s ;
librarian, for her

to M r s .

J o a n V anLue ;

to

w i l l i n g n e s s to o b t a i n

and o th e r te c h n ic a l a s ­

s i s t a n t s , f o r t h e i r p a r t in t h i s w o r k .
Thanks

are

K u p s k y of P a r k e
tation .

a l s o e x t e n d e d to D r . A r n o l d Hooke a nd C h a r l e s

D a v i s a n d C o., D e t r o i t , M i c h i g a n , f o r t h e i r

consul­

VITA

E s t h e r M. S m i t h
candid ate f o r the d e g r e e
Doctor

F in a l exam ination:
D issertation:

O u tl i n e

of

of P h i l o s o p h y

1:00 p . m . , M a y

13,

The R e a c t i o n of Sple nic
Monocytogene s

1955, R oo m
Tissue

346, G i l t n e r H a l l

i n C u l t u r e to L i s t e r i a

of S t u d i e s
M ajor

subject:

M inor

subjects:

A nim al Pathology
A n a t o m y and P h y s i o l o g y

Biographical Item s
Born:

M ay

1Z,

19Z3, H i g h la n d P a r k ,

Michigan

U n d e r g r a d u a t e S tu d ie s:
M i c h i g a n State C o l l e g e , E a s t L a n s i n g ,
M i c h i g a n , 1 94 1 -1 9 4 6 , B . S . , 1946
G r a d u a t e Studie s:
M ich igan State College,
i g a n , 1 9 4 8 -1 9 5 1 , M .S ., 1951

E a s t Lansing,

M ich­

Experience:
B a c t e r i o l o g i s t I, G a m e D i v i s i o n , M i c h i g a n D e ­
p a r t m e n t of C o n s e r v a t i o n , L a n s i n g , M i c h i g a n , 1946-1950;
B a c t e r i o l o g i s t III, D e p a r t m e n t of B a c t e r i o l o g y a nd P u b ­
lic H e a l t h , M i c h i g a n State C o l l e g e , E a s t L a n s i n g , M i c h ­
i g a n , 195 0-1 95 3; on l e a v e , 1953- 1954; G r a d u a t e A s s i s t a n t ,
D e p a r t m e n t of A n i m a l P a t h o l o g y , M i c h i g a n State C o l l e g e ,
E a s t L a n s i n g , M i c h i g a n , 1954-1955
M e m b e r of S o c i e t y of S i g m a Xi; S i g m a D e l t a E p s i l o n ;
S o c i e t y of M e d i c a l T e c h n o l o g i s t s

A m erican

TABLE

IN T R O D U C T I O N

OF

CONTENTS

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

H IS T O R I C A L BACKGROUND
R E V IE W O F L I T E R A T U R E

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

M A T E R I A L S AND M E T H O D S ...............
G lassw are

and I n s t r u m e n t s

Physiological

Salt S olu tio ns

Phosphate buffered
M edium

. . . .
. . . .

saline

(PBS)

1 9 9 ................................................

P l a s m a .................................................................
Em bryonic
B acterial
Tissue

E x t r a c t ..................................
C ultures

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

C ultures

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

Procurem ent

of t i s s u e s ...............

M e t h o d of c u l t u r e ...............................
Methods

of Study

Cytological

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

studies

B acteriological

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

studies

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

P h o t o g r a p h y .................................................

P age
R E S U L T S AND D I S C U S S I O N ................................................................................
B a c t e r i o l o g i c a l S tu d ie s
Effects

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

of f l u i d s ...................

R efrigeration

studies

33
33
33

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

D e s c r i p t i o n of L i v i n g C u l t u r e s .............................................................
....................................................................

D e s c r i p t i o n of F i x e d C u l t u r e s

33
41
53

Whole m o u n t s

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

53

Serial

,

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

65

sections

SUMMARY AND CONCLUSIONS
L IT E R A T U R E CITED

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

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

x

100
102

LIST OF P L A T E S

P LAT E
I.
II.

p ag e
Dou ble c o v e r s l i p c u l t u r e

.

29

A p i e c e of s p l e e n f r o m a n i n f e c t e d c u l t u r e
s m e a r e d on an a g a r p l a t e and i n c u b a t e d 48
hours
.................................................................

37

T y p i c a l L . m o n o c y t o g e n e s c o l o n i e s on a n a g a r
p l a t e ......................................................................................................................

39

IV.

I n o c u l a t e d l iv i n g

splenic culture

three

43

V.

In o c u la te d living

splenic

s i x d a y s old

III.

VI.

VII.

VIII.

IX.

X.
XI.

XII.

XIII.

cultu re

days

old

L . m o n o c y t o g e n e s s t a i n e d s u p r a v i t a l l y with
J a n u s G r e e n in a f i v e - d a y - o l d l i v i n g c u l t u r e

. .

45

. .

48

M o n o n u c l e a r p h a g o c y t e s i n a l iv in g t w o - d a y .............................................................................., ....................
old c u l t u r e

50

A. s i x - d a y - o l d l i v i n g c u l t u r e s h ow ing good
g r o w t h of t i s s u e e v e n t h o u g h i n f e c t e d w ith
bacteria
................................................................................

55

A s e v e n - d a y - o l d l i v in g c u l t u r e showing h e a v y
g r o w t h of f i b r o b l a s t s
....................................................................
An u n i n o c u l a t e d c o n t r o l c u l t u r e
B iz a rre nuclear fo rm s
cultures
..........................

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

.

57
59

se e n in 48-hour
62

C e l l s w i th v e s i c u l a r n u c l e i s e e n in t h r e e d a y - o l d c u l t u r e s .............................................

64

A. s e c t i o n of e x p l a n t a f t e r

73

xi

24 h o u r s ’ i n c u b a t i o n

PLATE
XIV.
XV.
XVI.

XVII.
XVIII.
XIX.
XX.
XXI.

XXII.

Page
H ig h p o w e r

of a r e a m a r k e d i n P l a t e

S e c t i o n of a t w o - d a y - o l d

77

79

S e c t i o n of t h r e e - d a y - o l d c u l t u r e .......................................

81

Hig h p o w e r

83

of s e c t i o n in P l a t e

S e c t i o n of f o u r - d a y - o l d

culture

H ig h p o w e r of s e c t i o n in P l a t e

X V I I ........................
...........................................
XIX

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

85
87

S e c t i o n of u n i n o c u l a t e d c o n t r o l c u l t u r e f o u r
d a y s old
......................................

89

S e c t i o n of s i x - d a y - o l d c u l t u r e

91

High p o w e r

XXIV.

Infected c u ltu re s

XXVI.

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

75

S e c t i o n of a n u n i n o c u l a t e d c o n t r o l c u l t u r e
two d a y s old ............................................................................................

XXIII.

XXV.

culture

XIII

. . , . .

of s i x - d a y - o l d c u l t u r e

. .

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

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

93
95

S e c t i o n of u n i n o c u l a t e d c o n t r o l c u l t u r e s i x
d a y s old .........................................................

97

H ig h p o w e r of s e c t i o n i n P l a t e

99

xii

X X V ...................

I N T R O D U C T IO N

The b eh av io r
has

alw ays

c o n c e r n e d the

interrelations
ism s
th e

scientist.

between tissue

constitu te a p ro b le m

a id of t i s s u e

carefully

system

i n the p r e s e n c e

Maximow

elem ents

The u s e

(1924)

stated:

''the

s u c c e s s f u l l y a t t a c k e d w i th

of t i s s u e

c u l t u r e to

s tu d y the

u n d e r t a k e n in the hope of

r e a c t i o n of the c e l l s to t h i s

is f r e e f r o m

of b a c t e r i a

and p a t h o g e n i c m i c r o - o r g a n ­

m onocytogenes was

c o n t r o l l i n g the

culture

cells

w h i c h c a n be

c u l t u r e . 1'

b e h a v i o r of L i s t e r i a

tissue

of liv in g t i s s u e

organism .

m any uncontrollable f a c to r s

The
so

o f te n p r e s e n t i n a n i m a l e x p e r i m e n t a t i o n .
Since M u r r a y ,
cy togenes

Webb, a n d Swann (1926) f i r s t d e s c r i b e d L . m o n o -

as the cau sative

fo u n d to h a v e wo r i d - w i d e
This

organism

is

d i s t r i b u t i o n in d o m e s t i c

a gram -positive

other m ic ro o rg a n is m s

in m a n y d i a g n o s t i c

d i f f i c u l t y in i s o l a t i n g L.
characteristics.

of the g e n u s

a contam inant,

laboratories

an d w ild a n i m a l s .

C orynebacte r iu m .

or com pletely overlooked

since m any w o r k e r s have

m onocytogenes

G r a y e t a l . (1948)

m a c e r a t i o n of t h e t i s s u e

has been

r o d and h a s b e e n c o n f u s e d w ith

such as those

It h a s b e e n d i s c a r d e d a s

the

a g e n t of l i s t e r i o s i s , the d i s e a s e

and a r e

reported

qu ite u n f a m i l i a r w it h

d e s c rib e d a m ethod w hereby

w a s f o l l o w e d by r e f r i g e r a t i o n a nd t h i s
1

z
i n c r e a s e d the p ro b a b ility

of i s o l a t i n g t h e

c o n firm e d by m any w o r k e r s .

Perhaps

organism .

because

This has been

of the

confu sion in

i d e n t i f i c a t i o n and d if f ic u l t y i n i s o l a t i o n , t h e o c c u r r e n c e
cytogenes in m an has be en som ew hat overlooked.
in G e r m a n y

reported.

tw enty-seven species

the l is t

of l i s t e r i c

with the in cid ence

of a n i m a l s

outbreaks

of t h i s

a nd t h e r e

in d o m e s t i c

organism

In a d d i t i o n to wide h o s t
ifests

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

of s u s c e p t i b l e h o s t s will i n c r e a s e

currence

Very

re c e n t work

i n d i c a t e s the p o s s i b i l i t y of a m u c h h i g h e r i n c i d e n c e

m a n than prev io u sly
from

of L. m o n o ­

m onocytogenes

(M urray,

has been is o la te d
indications that
1955).

The o c ­

a n i m a l s m a y be a s s o c i a t e d

in wild a n i m a l s

susceptibility, h .

i t s e l f in d i f f e r e n t h o s t s i n a v a r i e t y

the fact that h .

are

in

causes

monocytogenes m a n ­

of w a y s .

For

exam ple,

a s e v e r e m o n o c y to s is in r a b ­

b i t s b u t not in c a t t l e , i s in no wa y c o n n e c t e d w ith a d i f f e r e n c e in
strain.
duces

L.

m o n o c y to g en es in fectio n in g o a ts ,

encephalitis

oblongata.
of c a t t l e

B rains
do not.

a nd th e

organism

c a n be i s o l a t e d f r o m the m e d u l l a

of i n f e c t e d s h e e p h av e p u r u l e n t l e s i o n s w hile t h o s e
Inoculated la b o r a to r y

rabbits

alized infection and exhibit focal n e c r o s is
L. m o n o cytogenes
m ononucleosis.

c a t t l e , a nd s h e e p p r o ­

usually

of the l i v e r

show g e n e r ­
and s p l e e n .

of ten h a s b e e n a s s o c i a t e d with i n f e c t i o u s

The o r g a n i s m h a s b e e n i s o l a t e d f r o m

the b lo o d of

individuals

suffering f r o m

infectious

S c h m i d t and N y f e l d t (1938), P o n s
(1943).

However, this

c y te a g g l u t i n i n s
A bortions
cattle,

erythro­

1951).

due t o L . m o n o c y t o g e n e s h av e b e e n d e s c r i b e d in
sw i n e,

a nd r a b b i t s

o c c a s i o n a l l y a b o r t e d d u r in g

(M urray,

1955).

Rabbits

a b o r t e d a f t e r the f i r s t e x p o s u r e

a succeeding gestatio n p e rio d as

i n i t i a l i n f e c t i o n ( G r a y e t a l ., 1955,

organism

a p p a r e n tly p e r s i s t e d in the

1955a).

in h u m a n s

reported from

The f a c t t h a t

reproductive

the e a s t e r n

b e e n a t t r i b u t e d to L.. m o n o c y t o g e n e s
disease

sector

and P o t e l

tra c t for

some
A bor­

of G e r m a n y h a v e

(195 4) d e s c r i b e d a

of th e n e w b o r n w h i c h he c a l l e d g r a n u l o m a t o s i s

T h i s w o r k i n d i c a t e d the o c c u r r e n c e

a nd

a result

t i m e m a y i n d i c a t e th e p o s s i b i l i t y of a n i n t r a c e l l u l a r e x i s t e n c e .
tions

(1929),

(19 39), a nd Webb

r a b b i t ( G i r a r d and M u r r a y ,

infected during g e sta tio n always

the

and J u l i a n e l l e

by N yfe ldt

o rg a n is m has n e v e r p ro d u ced sheep

in th e

sheep, goats,

of t h i s

m ononucleosis

of a g r a n u l o m a t o u s

inf a n t i s e p t i c a .
inflam m atory

response.
From
genes

the foregoing

d i s c u s s i o n it is e v id e n t t h a t L.

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

for fu rth er
reaction

research.

In v iew of the

resulting fro m

suggested that tis s u e

m onocyto ­

o ff er c o n s i d e r a b l e

challenge

apparent differences

in t i s s u e

a n i n f e c t i o n with L,. m o n o c y t o g e n e s , it wa s

cultu re

would a f f o r d a m e d i u m w h e r e b y the d e ­

v e l o p m e n t of t h e l e s i o n c o u ld be o b s e r v e d u n d e r

c o n t r o l l e d c o n d i t io n s

H IS T OR I C A L BACKGROUND

In a n a t t e m p t to s t u d y c e l l f u n c t i o n s

a nd the e f f e c t s

of e n v i r o n ­

m ent,

Roux i s o l a t e d t h e m e d u l l a r y p l a t e

of a c h i c k e n e m b r y o i n a

w arm

saline

This

solution as e a r ly

as

1885.

1923 w h en he p r o v e d t h a t the c l o s u r e
f u n c t i o n of i t s

constituent cells

w as not r e p o r t e d u n til

of the m e d u l l a r y tube w a s

a

an d not of m e c h a n i c a l p r e s s u r e f r o m

nearby structures.
I n 1897, L j u n g g r e n was

a b le to k e e p t r a n s p l a n t s

of h u m a n s k i n

alive f o r w eeks in a s c itic fluid.
Loeb

(1902)

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

g r o w t h of c e l l s i n p l a s m a
the

epiderm is

to o b s e r v e

of th e e a r

clots

o r on a g a r

of a r a b b i t .

sheets pla ced b en eath

H a r r i s o n (1907) w a s t he f i r s t

c e l l g r o w t h u n d e r th e m i c r o s c o p e .

v e l o p m e n t of n e r v e f i b e r s

He

r e c o r d e d th e d e ­

after implanting fra g m e n ts

of e m b r y o n i c

f r o g n e u r a l t u b e in f r o g l y m p h .
Burrow s
Burrow s

(1910)

(1910)

o b se rv e d cell m ito sis

in v i t r o and C a r r e l

developed the co agulated m e d iu m technique

and

as it is

known today.
It w a s
t a i n a ll t h e

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

substances

n e c e s s a ry for
4

c o n ti n u o u s

did not c o n ­

cell growth.

Carrel

5
(1912) f i r s t
possible.
thirty

used em bryo
As

extracts

and m a d e l o n g - t e r m

a r e s u l t he m a i n t a i n e d a s t r a i n

tissue

culture

of f i b r o b l a s t s f o r

over

years.
Lew is

a nd L e w i s

b y employing
and s e r u m .

various
L ew is

(1911) p i o n e e r e d the f l u i d m e d i u m t e c h n i q u e

com binations

(1916)

used

of s a l t

sea w ater

solutions, buffer
as

system s,

a m edium for tissu es

in v i t r o .
The im portance
when C a r r e l
the

(1913)

of p r o t e i n s

d is c o v e re d that e x tr a c ts

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

beef plasm a,
cose,

The

use

Baker

cystine,

and E b e l i n g

(1939)

acids

had

r e p o r t e d . The n u t r i t i v e

was

i m p r o v e d w it h t h e s e s u p p l e m e n t s .
The f o r e g o i n g

studies

and E r l i c h m a n .

than previo us

m a i n t e n a n c e va lue

w orkers

of the m e d i u m

d e m o n s t r a t e d the i m p o r t a n t r o l e

teins

a nd p r o t e i n d e g r a d a t i o n p r o d u c t s

cells

in tis s u e

culture;

glu­

d e v i s e d a f l u i d c o n ta i n in g m o r e

and nucleic
and

accelerated

insulin, thyroxin e,

r e p o r t e d in 19 33 by V o g e l a a r

acids, h o rm o n e s ,

extract

of t i s s u e s

realized

of a s o l u t i o n c o n ta in in g i r r a d i a t e d

W itte's peptone, hem in,

and sa lts was

amino

in c e l l n u t r i t i o n was

of p r o ­

in the n u t r i t i o n of a n i m a l

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

or em bryo

c o u ld not be e l i m i n a t e d f r o m t h e m e d i u m .
Sim m s

of s e r u m

(1936)

ultrafiltrate

and S im m s
as a b a s i c

a n d S a n d e r s (1942)

r e p o r t e d the use

m edium for tissu e

cultures.

T his

6
f l u i d c o n t a i n e d a s u b s t a n c e w h i c h s t i m u l a t e d the
adult ti s s u e s

in vitro.

F ischer

(1946)

s u m m a r i z e d the w o r k on t i s s u e

p re v io u s twenty y e a r s .

It w a s

this p erio d considerable
w it h s p e c i a l

As ea rly

of t i s s u e
as

1926, B a k e r a n d C a r r e l

w as

substances

a nd t h e n i t r o ­

s t u d i e d the p r o t e i n f r a c ­

e x t r a c t a nd f ou n d t h a t f i b r o b l a s t s

juice

grew

T his p o r t i o n c o n s i s t e d of a

of n u c l e o p r o t e i n a nd g l y c o p r o t e i n .

ated em b ry o tis s u e

review that during

cultures.

in this fra c tio n than in o th e rs.

m ixture

this

c u l t u r e f o r the

a t t e n t i o n h a d b e e n g i v e n to c e l l u l a r n u t r i t i o n

t i o n of e m b r y o n i c t i s s u e

factor

apparent fro m

r e f e r e n c e to g r o w t h - p r o m o t i n g

gen m etab o lism

faster

g r o w t h of d o r m a n t

Fischer

(1941) f r a c t i o n ­

and r e p o r t e d t h a t the g r o w t h - p r o m o t i n g

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

a nd t e r m e d it

emb ryonin.
F i s c h e r e t a l . (1948), e m p l o y i n g a m i x t u r e
im portant

substances,

d e v i s e d a m e d i u m k no w n as

the m o s t im p o r t a n t am ino
since its
and ly s in e

acids

V -6 05 .

One of

in V -605 w as f ou n d to be c y s t i n e ,

absence p r e v e n ted growth.
were

of b i o l o g i c a l l y

Arginine, trypto phane,

found to be n e c e s s a r y in t h i s

glutam ine,

o r d e r of d e c r e a s i n g

im portance .
M organ,

M orton,

and P a r k e r

(1950), u s in g t h e b u i l d i n g - u p

m e th o d , d evelo ped a v e ry c o m p lic a te d m e d iu m known as

199-

This

7
is

c h a r a c t e r i z e d by a c o m p l e t e

m ins

i n a d d i t i o n to n u c l e i c

and p e n t o s e s .

supplem ent

of a m i n o a c i d s

a nd v i t a ­

acid co n stitu en ts, p u r in e s , p y ra m id in e s ,

T w e e n 80 w a s

u s e d in t h i s m i x t u r e t o su p p ly a s o u r c e

of f a t t y a c i d an d a i d e d i n d i s s o l v i n g f a t - s o l u b l e

vitam ins

a nd c h o l e s ­

terol .
An adequate
devised.

m edium ,

com pletely

synthetic, has

The e l a b o r a t i o n of s u c h a m e d i u m i s t h e g oa l of t he t i s ­

s ue c u l t u r e w o r k e r but the a t t a i n m e n t of t h i s
close

g r o w n on c o v e r s l i p s

(1912) p r o v i d e d a s a t i s f a c t o r y m e t h o d f o r
C arrel

(1913a)

with a th in coagulu m

cultivated t is s u e s

e v e r , G e y (1933)
P orter

m a d e th e

rack.

Sometime

i n t u b e s w h ic h w e r e
extract.

roller-tube
a practical

This

lined
early

technique.
system

et a l . (1945) d e v i s e d a f l a s k with a long

to f it a r o l l e r - t u b e
senior

roller-tube

as used by C a r r e l

cultivation.

of p l a s m a a nd e m b r y o

w ork was the b a s is fo r the p r e s e n t - d a y

tion.

g oa l d oe s not s e e m

a t h and.
P l a s m a clot c u ltu re s

later,

not y et b e e n

How­

of c u l t i v a ­

s t r a i g h t n e ck

T o d ay t h i s f l a s k b e a r s t h e n a m e

of the

auth or.
U n til r e c e n t l y t h e p l a s m a c l o t c u l t u r e w a s th e m o s t

t o r y m e th o d a v a ila b le , but t h e r e
d i f f i c u l t to h a n d l e b e c a u s e t h e

are

many lim itations.

satisfac­

The c lo t i s

o p e r a t o r m u s t be a ble to c o n t r o l th e

8
clotting m e c h a n i s m .
of i t s t h i c k n e s s ,
th e n the

The

c lo t i n t e r f e r e s

w ith m i c r o s c o p y b e c a u s e

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

clot m u s t be

repaired.

during

E v a n s a nd E a r l e

t h e p l a s m a c l o t w i th p e r f o r a t e d c e l l o p h a n e .
m a k e s p o s s i b l e the u n if o rm
t he

culture flask

cellophane
cells

interface

outside the

possible

m eans for

c a n be

c o n f i n e m e n t of t h e

as

clo t,

surface

of

s c r a p e d off the g l a s s With the

s i n g l e c e l l c u l t i v a t i o n was

g r o w n on g l a s s

or

cellophane p ro v id e s

q u a n t i t a t i v e w o r k w ith r e p l i c a t e

after

The need for
s tim u la te d Sim m s

of c e l l s

cultures

a

( E va ns

originally planted

su sp en sio n -cell cultures for virus propagation

a nd S t i l l m a n (19 37,

1937a) to d e v i s e
These

a method fo r

authors

s t i m u l a t e d i n i t i a l g r o w t h due to t h e r e m o v a l

itory m a te ria l from

tissues

a nd E n d e r s

Dulbecco

It

incubation or e x p e rim e n ta l manipulation.

t h e t r e a t m e n t of k i d n e y t i s s u e w i th t r y p s i n .

bins, W eller,

replaced

N u c l e a r c o u n t s c a n be m a d e i n a h e m o c y t o m e t e r .

made

that try p sin

over the

a suspension.

i s t h e n p o s s i b l e to c o m p a r e the n u m b e r
with counts

(1947)

1948).

of c e l l s

accurate,

1951).

cells

g r o w t h and

T h i s new s u b s t r a t e

d i s p e r s i o n of c e l l s

a nd t r a n s f e r r e d

( S a n f o r d e t a l .,

T he u s e

et al.,

so t h a t the

cellular

as

a result

(1952) m a d e

(1952), a nd D u l b e c c o

m eth od f o r try p sin iz in g tis s u e

of a p r o t e o l y t i c

a sim ilar

reported

of a n i n h i b ­
action.

Rob­

report.

and Vogt (1954)

to o b t a in a m o n o l a y e r

described a
c u l t u r e to

s tu d y

9
anim al v iru se s.
pare

Y o u n g n e r (1954)

adapted D ulbecco's

tr y p s i n - d i s p e rs e d monkey kidney cell

s t a n d a r d i z e d a n d u s e d f o r the p r e p a r a t i o n
cate

cultures.

Y o u n g n e r (1954a)

su sp en sio n s which w ere
of l a r g e

num bers

of r e p l i ­

u t i l i z e d the t r y p s i n - d i s p e r s e d m o n k e y

k i d n e y c e l l s f o r t h e t i t r a t i o n of p o l i o m y e l i t i s
cultures.

m e t h o d to p r e ­

virus

in r o lle r - tu b e

T h i s i s th e b a s i c m e t h o d u s e d t o d a y f o r the p r o d u c t i o n of

the p o lio m y e litis

virus

vaccine.

R E V U E OF L I T E R A T U R E

Although the b e h a v io r
C a r r e l and o th e rs
ologists
the

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

r e a c t i o n of t i s s u e s

m ade little

to b a c t e r i a

use

in t h i s

of the

w a s not p a t h o g e n i c f o r

p roperties;

organism
B.

g e n i c to t h e

tissue

cul­

a nd it w as f ou n d t h a t t h e o r g a n i s m
Smyth re a so n e d that

several hours

since

of i n c u b a t i o n w ith t i s s u e

g r e w w e l l and h a d a s p e c i a l

coli v eru s

af finity f o r the

a nd B. p r o d i g i o s u s , w h ic h w e r e n o n p a th o of the t i s s u e f r a g ­

s e e m to h a v e any i n f l u e n c e on t he g r o w t h of t h e b a c i l l i .

cultures

grew freely

on t i s s u e

s t r a i n of B. t y p h o s u s

c h i c k e n , c o l o n i z e d a n d th e p r e s e n c e

m e n t did not
In tis s u e

The

aureus

did not r e m a i n a l i v e , the p l a s m a c o n t a i n e d b a c t e r i c i d a l

how ever, after

e x p l a n t s t he
new c e l l s .

chickens

chickens.

in f r e s h c h i c k e n p l a s m a .

organism

o p p o r t u n i t y to s t u d y

s t u d i e d t h e e f f e c t of B . d i p h t h e r i t i c u m , B. t y p h o s u s ,

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

d id not l i v e

s t u d i e d by

m anner.

c o li v e r u s , B. p r o d i g i o s u s , an d M i c r o c o c c u s

tures

the

c e l l s in v itr o was

i n the e a r l y p a r t of the t w e n t i e t h c e n t u r y , p a t h ­

S m y t h (1915)
B.

of t i s s u e

i n f e c t e d w i th M i c r o c o c c u s

in t he p r e s e n c e

growth.

S m y t h (1916)

of th e t i s s u e ,

a u r e u s , the b a c illu s

and s o m ew h at d i s p e r s e d

e l a b o r a t e d on s o m e

10

of h i s

earlier

11
experim ents

a n d d e s c r i b e d th e e f f e c t v a r i o u s t i s s u e s

e x e r t e d upon the

g r o w t h of b a c t e r i a .
Lewis

(1920)

chicken em bryo

i n t r o d u c e d B. t y p h o s u s

intestine.

into t i s s u e

cultures

He w a s u na b l e to k e e p t i s s u e

of

cells

alive

m u c h b e y o n d 24 h o u r s .

Soon a f t e r i n t r o d u c i n g t h e b a c t e r i a into the

cultures, large

vacuoles

a p p e a r e d i n the c e l l s , t o g e t h e r w i th a n i n ­

crease

size

in th e i r

and n u m b e r .

b a c t e r i a w h i c h r e m a i n e d a l iv e
S m i t h e t a l . (1922)
tissue
were

a nd m o t i l e

vacuoles

w i t h i n the v a c u o le .

b a c i l l i a nd c o n c l u d e d t h a t t h e c e l l s

not a t t r a c t e d by the b a c i l l i a nd t h a t the p r e s e n c e
d id n o t i n d u c e

of w o r k w a s

r e a c t i o n of m a m m a l i a n t i s s u e s

tum w ere

Lym phoid tis s u e ,

strains

highly v iru le n t

r e p o r t e d by M a x i m o w (1924)

m esenteric

lymph nodes,

other was

a nd o m e n ­

and i n o c u l a t e d w ith one of

of h u m a n t u b e r c l e b a c i l l i .

a nd t h e

avirulent.

One

s t r a i n was

A f t e r the t i s s u e s

b e e n in c u l t u r e f o r t h r e e to fiv e

days the b a c illi w e re

i m o w f ou n d t h a t the b e s t p i c t u r e

of th e

a nd t h e b a c t e r i a w e r e

or­

in v i t r o to M y c o b a c t e r i u m

g r o w n in p l a s m a c lo t c u l t u r e s

two d i f f e r e n t

of t h e

any c h a n g e i n the c u l t u r e s .

An outstanding piece

tuberculosis.

contained

s t u d i e d the b e h a v i o r of c h i c k e n e m b r y o

containing avian tu b e rc le

ganism

on t h e

Some of the

added.

had
Max­

relationship betw een cells

s e e n a t the p e r i p h e r y

of the e x p l a n t i n the

1Z
%
zone

of n e w l y f o r m e d t i s s u e .

virulent

s tra in were

to e i g h t d a y s
e v e r , the

only a few b a c i l l i

w e e k s in v i t r o .

He

cultures

displayed a se rie s

specific tu b ercu lo u s
th is

c ould b e

i n f e c t i o n a nd a f t e r
s e e n in the

i n o c u l a t e d w ith the

of r e a c t i v e

changes

inflam m atory p ro cess.

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

and w ere

were

seven
How­

cultures
cells

virulent

did not

s t r a i n but

w h ic h d u p l i c a t e d the

It was p o i n t e d out t h a t
of c e l l s

organism s,

u na b le to r e s i s t the i n f l u e n c e

w h ic h b e c a m e
since in this

of t h e s e

intruders

soon killed.

A s a r e s u l t of t h e s e
t h a t the

cultures.

r e p o r t e d t h a t t he t i s s u e

w^.s i n m a r k e d c o n t r a s t to the b e h a v i o r

c a se the c e lls

i n f e c t e d with the

s t r a i n c o m p l e t e l y o v e r g r e w t he t i s s u e

p e r i s h i n any of the
rather

w hic h w e r e

ab le to o v e r c o m e the

avirulent

after th ree

C ultures

reticular

significant

cells

reaction.

and m a c ro p h a g e s
i nto t he m e d i u m
and g ia n t c e lls

experim ents,

M a x i m o w (19Z4)

of the l y m p h o i d t i s s u e

concluded

d i s p l a y e d the m o s t

T h e y m o b i l i z e d a nd f o r m e d w a n d e r i n g

w h ic h w e r e n a m e d p o l y b l a s t s .

The c e l l s m i g r a t e d

and p h a g o c y t i z e d the t u b e r c l e b a c i l l i .
d eveloped and so m e

of t h e s e

cells

cells

Epithelioid

contained a golden

(

y e ll ow p i g m e n t w h i c h wa s
lar

b e l i e v e d to be a r e s i d u e f r o m

intracellu­

d i g e s t i o n of t u b e r c l e b a c i l l i .
Lang

Lu ng t i s s u e

(19Z5)
w as

i n o c u l a t e d lung t i s s u e

used because

in v i t r o with M . t u b e r c u l o s i s .

it p r o v i d e d a t i s s u e

which was

capable

13
of p r o d u c i n g
and septal

an exudative

cells.

reaction,

He c l a i m e d t h a t the e n d o t h e l i u m of lung t i s s u e

not p l a y a r o l e b ut t h a t th e
elem ents.

and c o n ta in ed c a p i l l a r y e n d o th eliu m

The l a t t e r

septal

c e l l s w e r e the p r i n c i p a l a c t i v e

c o r r e s p o n d e d to the m o b i l i z e d h i s t i o c y t e s

were

the p h a g o c y t i c

am eboid cells

w ere

then t r a n s f o r m e d

w ere

a r r a n g e d in t u b e r c l e - l i k e

clusters.

tissue

c u ltu re s influenced the e le m e n ts

means

of t h e

tissues

the endoth elium

L a n g did not m a k e

The e p i t h e l i o i d c e l l s

The t u b e r c l e b a c i l l i i n the
of the

e x p l a n t e d t i s s u e by

of m e t a b o l i s m .

of b l o o d v e s s e l s

In c u ltu re s

any a t t e m p t to e x p l a i n t h i s l a s t finding, b u t i t is

response

of l e u c o c y t e s

S t r e p t o c o c c u s , King et a l . (1928)
The

organism s

lu te d in T y r o d e 's .
per

w ere

These

in t i s s u e

used cultures

of the buffy c o a t f r o m

w orkers

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

cells

g r o w t h c o n t i n u e d f o r f iv e d a y s .

150,000 b a c t e r i a

did not g r o w b e y o nd

H o w e v e r , if only f i v e b a c t e r i a p e r m i l l i l i t e r w e r e

cultures

c u l t u r e to a

g r o w n 18 to 24 h o u r s in b r o t h an d d i ­

m i l l i l i t e r w e r e p r e s e n t the

of the

results

work.
To s tu dy t h e

blood.

of l y m p h o i d

a s s u m e d an em bryonic fo r m ,

m e n t i o n e d h e r e b e c a u s e it s e e m e d p e r t i n e n t i n v iew of t h e
of t h i s

and

w hic h e n g u l f e d th e b a c i l l i a nd

into e p i t h e l i o i d c e l l s .

soluble p ro d u c ts

did

It w a s

also

i n f e c t e d w ith the b a c t e r i a w as

12 h o u r s .

added, cellular

r e p o r t e d th at the p e r i p h e r y
coarse

and g r a n u l a r in

14
c o n t r a s t to t h a t
form

of u n i n o c u l a t e d c u l t u r e s

cell distrib u tio n .
Utilizing p u re

saprophyte,
nies

w h i c h h a d a s m o o t h and u n i ­

cultures

Tannenberg

of f i b r o b l a s t s

(1929)

r e p o r t e d that individual b a c t e r i a l

g r e w i n t h e c e n t e r of a f i b r o b l a s t i c

d e f i n e d , b ut h a d f i n g e r - l i k e
these fibroblastic

and a g r a m - p o s i t i v e

appendages.

"ring"

of d e a t h .

g r o w w e l l a nd e v i d e n c e

were

of t h e s e
really

organism s.

C ultu res

A r o n s o n (1931)
culture.
spleen,

tuberculin from

reported.

sues tak e n f r o m
same

s t u d i e d t h e s p e c i f i c c y to t o x i c a c t i o n s
Using tu berculous
and t e s t e s

nontuberculous

of t u b e r ­

w e r e c u l t u r e d i n the p r e s e n c e

anim als

tu b e rc u lin , th e r e was

(1932)

cells

guinea pig s, explants

t h e h u m a n a n d b ov in e t y p e .

In o r d e r to
Foldvari

As a

of f i b r o b l a s t s .

were

of
of

It was f o u n d t h a t the

t u b e r c u l i n w as t o x ic to the c e l l s , but w hen e x p l a n t s

of t h e

stain, indicating

i n o c u l a t e d w ith b a c t e r i a did not

of f a t t y d e g e r n a t i o n w a s

degenerated fo rm s

bone m a r r o w ,

revealed

The f i b r o ­

s t u d i e s , i t w as c o n c l u d e d t h a t m a c r o p h a g e - l i k e

c u lin 'in tissue

sharply

c e l l s to be f i l l e d with b a c t e r i a a n d he t h e r e f o r e

c o n t a i n i n g b a c t e r i a did not t a k e t h e n u c l e a r

th e p o s s i b i l i t y

result

w h i c h w as not

Stained p r e p a r a tio n s

a s s u m e d t h a t th e f i b r o b l a s t s p h a g o c y t i z e d the
blasts

colo ­

of t h e

same t i s ­

c u l t u r e d i n the p r e s e n c e

no to xic e ff e ct .

s tu d y the b e h a v i o r

an d K a s t and K o l m e r

of T r e p o n e m a p a l l i d u m i n v i t r o ,
(1933) a t t e m p t e d to g r o w t h i s

15
organism

in th e p r e s e n c e

u n s u c c e s s fu l in th is
in it e

of liv in g t i s s u e .

endeavor and w ere

c o n c l u s i o n as to the

relationship

These

investigators

were

u n ab le to c o m e to any d e f ­
of t h i s

o r g a n i s m to t h e h o s t

cell.
I n a n e f f o r t to o b s e r v e the
c le a r cells,

chem otropism

M c C u t c h e o n a n d Dixon (1936)

of p o l y m o r p h o n u ­

r e p o r te d that these

exhibited a positive

c h e m o t r o p i s m to Staph, alb u s , S t r .

M. t u b e r c u l o s i s , E).

t y p h o s u s , a n d T. h i s t o l y t i c a .

It i s w e ll k n o w n t h a t p o l y m o r p h o n u c l e a r
to a p p e a r i n t h e
vital
(1938)
cytic

area

of i n f l a m m a t i o n b u t m o n o c y t e s

role in combating
used tissu e

infections

cultures

and r e m o v i n g

agents.

i n c u b a t i o n , th e m i g r a t i n g
polym orphonuclear cells
and l y m p h o c y t e s f r o m
were

te s te d for the

e x p l a n t and v a r i o u s

also play a
Herzog

study the p h a g o ­

of m o n o c y t e s w ith t h a t

a nd l y m p h o c y t e s .

sm all pieces

the f i r s t

C o m a n (1940) u s e d c u l t u r e s

of m o n o c y t e s to c o m p a r e t he c h e m o t a x i s
p o ly m o rp h o n u c le a r le ucocytes

hem olyticus,

are

debris.

of h u m a n l e u c o c y t e s to

activity to w ard various

c y t e s by e x p l a n t i n g

cells

cells

of r a t o m e n t u m .

of

He o b t a i n e d m o n o ­
A f t e r two d a y s

cells w ere m ononuclear phagocytes.

of

The

w e r e g r o w n f r o m the buffy c o a t of b lo o d

the

rat lym ph nodes.

chem otaxic

The

substances

w h ic h

e f f e c t w e r e p l a c e d 0.5 m m f r o m

m igrations w ere recorded.

It w as

concluded

the

16
that polym orphonuclear

cells

exhibited a positive

a u r e u s a n d M. t u b e r c u l o s i s but w e r e
M onocytes
either
taxis

and ly m p h o c y te s

organism

r e v e a l e d t h a t the

organism

p o s s ib ility that they w ere
reaction.

if t h e t i s s u e

It w a s

c u l t u r e s by P e r r y

s u r v i v e d f o r t e n d a y s and t h a t

a s s o c i a t e d w ith a g r a n u l o m a t o u s

also

inflam m a­

r e p o r t e d t h a t T . p a l l i d u m did not g r o w

died.

on t h e m i g r a t i o n

(1949)

s t u d i e d th e e f f e c t of t u b e r c l e b a c i l l i

of p h a g o c y t e s i n v i t r o .

l e u c o c y t i c m i g r a t i o n was

cells

w ere

b a c i l l i t h i s m i g r a t i o n w a s not i n h i b i t e d .
of A r o n s o n

T h e s e w o r k e r s f ou nd t h a t

i n h i b i t e d if t he c u l t u r e s

v i r u l e n t b a c i l l i b u t if w hite

were

T hi s

somewhat c o rro b o ra te s

(1931).

r e v i e w on L. m o n o c y t o g e n e s p e r
and L i n z e n m e i e r
superfluous.

i n f e c t e d w ith

i n o c u l a t e d w ith a v i r u l e n t

It w a s not t he i n t e n t i o n of th e w r i t e r to p r e p a r e

be

chemo­

T h is g i a n t c e l l p r o d u c t i o n s u g g e s t e d the

A l l g o w e r a nd B l o c k

the w ork

c h e m o t a x i s to

slight negative

of T. p a l l i d u m i n t i s s u e

giant cells w ere p roduced.

tory

showed a v ery

silica.

silica.

L a t e r in vestigations
(1948)

r e p e l l e d by a l u m i n a

did not show a p o s i t i v e

and m onocytes

to a l u m i n a

c h e m o t a x i s to S.

(1953)

an d G r a y

a literature

se; t h a t h a s b e e n done by S e e l i g e r
(1954)

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

Only t h o s e p a p e r s w h i c h w e r e

r e a c t i o n of t h e t i s s u e to t h i s b a c t e r i u m

will be

would

c o n c e r n e d w ith the
reviewed.

17
A fter M urray,

Webb, a n d Swann (1926)

d e s c r i b e d the

severe

m o n o c y t o s i s p r o d u c e d in r a b b i t s b y L. m o n o c y t o s i s , m a n y i n v e s t i g a t o r s
b e g a n to u s e t h i s

organism

to

s tu d y t h e

c o n t r o v e r s i a l q u e s t i o n of the

o r i g i n of th e m o n o c y t e .
B loom

(1928)

c o n c l u d e d t h a t t he m o n o c y t e d e v e l o p e d by i n d i ­

v id u a l t r a n s f o r m a t i o n of th e l y m p h o c y t e s
th e

sinuses

w i t h L.
He

of t h e

m onocytogenes

Splenectom ized rabbits inoculated

did not e x i s t an d d i f f e r e n t i a t e d m o n o c y t e s

histiocytes.

Lang

(1928)

re v ie w e d the l i t e r a t u r e

on th e m o n o c y t e

question,

did not c o m e to a ny d e f i n i te c o n c l u s i o n .
N y f e ld t (1932)

no c y t o g e n i c

injected rab b its

centers for

tran sitio n al p ro c e ss fro m
came from

the

with L.

m onocytes.

w ith L , m o n o c y t o g e n e s

a n d found

He s t a t e d t h a t t h e r e w as

no

m y e l o b l a s t to m o n o c y t e b ut t h a t m o n o c y t e s

s t i m u l a t i o n of l y m p h a t i c

H e z z e s i (1933)

this

e s p e c i a l l y in

d e v e l o p e d m o n o c y t e s f r o m t h e bone m a r r o w .

stated that m onoblasts

from free

but

s p l e e n a nd l i v e r .

i n the blood;

tissue.

a n d W a l l b a c h (1934),

studying

rabbits

infected

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

disease

w a s th e m o n o c y t e .

Co n w a y

(1938,

1939)

p h a t i c t i s s u e to i n j e c t i o n s

i n v e s t i g a t e d the

r e a c t i o n of r a b b i t l y m ­

of L. m o n o c y t o g e n e s .

M i c r o s c o p i c a l l y the

18
splenic
from
The

response

was

c h a r a c t e r i z e d by the m o v e m e n t of l y m p h o c y t e s

lym phatic tissu e
rate

of the p e r i a r t e r i a l

s h e a t h s into the

of f o r m a t i o n of new l y m p h o c y t e s i n the

was g r e a t e r th an the
ly m p h o cy tes

were

ly m p h o cy tes.

rate

of d e s t r u c t i o n .

sheath s

r e d pulp.
initially

It w a s c o n c l u d e d t h a t the

a s s o c i a t e d w ith the t r a n s i t i o n a l f o r m

T he m o n o c y t e s w h ic h w e r e

s e e n i n t he

of m o n o c y t o i d

re d pulp, d e ­

v e l o p e d by i n d i v i d u a l h y p e r t r o p h y and t r a n s f o r m a t i o n of the l y m p h o ­
cyte s .
It is k n o w n t h a t t h e i d e n t i f i c a t i o n an d i s o l a t i o n of L.
t o g e n e s h a s b e e n o v e r l o o k e d on m a n y o c c a s i o n s .
1936)

B u r n (1934,

1935,

r e p o r t e d the i s o l a t i o n of an u n i d e n t i f i e d g r a m - p o s i t i v e b a c i l l u s

a s s o c i a t e d with m e n in g o e n c e p h a litis .
b l o o d of t h r e e
In two of t h e

The

infants the b a c t e r i a w e re

of t h e g e n u s
Potel

dis e a s e

( 19 5Z) in th e e a s t e r n

a n d f i r s t n a m e d th e

paper,

However,

Seeliger

s p re a d throughout all o rgans.
c o n c l u d e d t h a t t h is

organism

L ist e r i a ,

of th e n e w b o r n

septica.

o r g a n i s m w a s found in the

n e w b o r n i n f a n t s who h a d d ie d of m e n i n g o e n c e p h a l i t i s .

A f t e r m u c h s t u d y a nd r e s e a r c h , it was
was

monocy­

sector

of G e r m a n y

w h i c h he c a l l e d g r a n u l o m a t o s i s

causative

organism

as

reported

a

infantiseptica

C orynebacterium

in fanti­

in a d i s c u s s i o n f ollow ing the p r e s e n t a t i o n of t h i s

s t a t e d t h a t he w a s

of the

o pinion t h a t the e t i o l o g i c

19
a g e n t of t h i s
reports

disease

was

c o n c e r n i n g the

L . m onocytogenes.

many

e t i o l o g y a nd e p i d e m i o l o g y of g r a n u l o m a t o s i s

inf ant i s e p t i c u m h a v e b e e n m a d e
E r d m a n n and P o t e l

Since t h a t t i m e

by P o t e l

(1953,

(1953), H a h n e f e l d a nd N i s o l k

1954), R i e s s

(1953),

(1954), and H a h n e -

f e l d (1954).
The h i s t o p a t h o l o g y of l i s t e r i o s i s
The m o s t

c o m m o n l y d e s c r i b e d l e s i o n is n e c r o s i s

s p le e n , l i v e r , and b r a i n .
of t h e

h a s not b e e n w e l l d e f i n e d.

abscess

Bloom

inv o lv in g m o s t l y the

(1928a), i n d e s c r i b i n g the f o r m a t i o n

a s s o c i a t e d w ith L. m o n o c y t o g e n e s i n f e c t i o n ,

t h a t t h e t r a n s f o r m a t i o n of l y m p h o c y t e s to m o n o c y t e s
at the p e r i p h e r y

of the

splenic a b s c e s s .

th e

v a s c u l a r e n d o t h e l i u m gave

the

abscesses.
J e n s e n and M a c k e y

sheep,
tissue

r i s e to the

(1949),

r e p o r t e d that the changes
proper.

stated

c ou ld be

seen

He did not b e l i e v e t h a t
exudative cells

st u d yi n g l i s t e r i o s i s

s e e n in

in cattle

and

c o n s i s t e d of f o c i l o c a t e d i n n e r v e

T hese foci contained p la s m a c ells, m a c ro p h a g e s ,

and n e u tr o p h ils .

They o b se rv e d phagocytosis

of the o r g a n i s m by t h e

m ononuclear m acrophages.
Julianelle
in t h e e ye
scess

as

a nd M o o r e

consisting

which they

(1942) d e s c r i b e d the p a t h o l o g i c a l

of a r e a s

of f o c a l n e c r o s i s .

changes

In e v e r y a b ­

studied th e r e w ere many m ononuclear

cells

at the

zo
periphery
necrosis

a n d t h e y t ho ug ht t h i s to be
was

c a u s e d by a p y o g e n i c

a s t r a n g e fin ding

since the

organism .

H a g e m a n n e t a l . (195 3) d e s c r i b e d t h e h i s t o p a t h o l o g y c a u s e d
by L . m o n o c y t o g e n e s

i n f e c t i o n in the n e w b o r n .

c h a r a c t e r i z e d by l a r g e ,
network

rounded a r e a s

and a few a tr o p h ic

droplets

containing a dense

parenchym atous

e n d o t h e l i u m p r o l i f e r a t e d a nd s o m e

L iv e r le sions

cells.

of th e c e l l s

and fa te s.

One l e s i o n w a s

increase

in m e s e n c h y m a l c e l l s

w ere

seld om

and was

w orkers

c h a r a c t e r i z e d by a n

resultin g in an in c re a s e

not s e e n i n a ll c a s e s

seen or isolated from these

clas­

a c c o r d i n g to two

c e lls , h is tio c y te s , m o n o c y te s, and epithelioid cells.
a "granulom a"

The c a p i l l a r y

These

s i f i e d t h e l e s i o n p r o d u c e d by L . m o n o c y t o g e n e s
reactions

capillary

contained fa t-lik e

and d e sq u a m a te d n u c le a r fra g m e n ts .

possible

were

in r e t i c u l a r

This was called

of i n f e c t i o n .

lesions.

The

B acteria

s e c o n d a nd

m o s t f r e q u e n t l y o b s e r v e d l e s i o n w a s c h a r a c t e r i z e d by n e c r o s i s .
H a g e m a n n e t a l . b e l i e v e d the n e c r o s i s to be due to the a c t i o n of a
t o x i n o r t o the
capillaries.
tripetal

organism

itself

W ith t h e b e g i n n in g

i n f i l t r a t i o n of the

b e g a n to e n g u lf t h e whole

r a t h e r t h a n to th e
of t h e n e c r o s i s

c a p illa ry cells.
area.

e l o n g a t e d a nd t ook th e f o r m

o b l i t e r a t i o n of the

th e re was a c e n ­

These endothelial cells

At t h i s t i m e the n u c l e i b e c a m e

of r o d s , d u m b b e l l s , h o o k s ,

clubs,

and

21
other b iz a r r e
form ed

shapes.

a proliferating

These proliferating
"granulom a"

s c r i b e d in t h e p r e v i o u s t y p e

of r a i s e d

areas

w e r e the

same

as those

on l i s t e r i a e n c e p h a l i t i s ,

o n the m e n i n g e s

called

described

Md i s c - l i k e "

infiltrations

of a g r a n u l o m a c h a r a c t e r w ith p a c k e d h i s t i o c y t e s

an in c re a s e

of L . m o n o c y t o g e n e s

there
ence

was

a low -grade

of " g r a n u l o m a s "

organism s.

v ascu lar proliferation.
i n the

process

which c a u se d

proliferation.
"listerom a"

and

In the a r e a of the

sulci

He r e p o r t e d th e p r e s ­

o u t e r a nd m e d i a l m e n i n g e a l l a y e r s .

Sim on s ta te d th at in the p e r i v e n t r i c u l a r
contained m any L i s t e r i a e ,

de­

of l e s i o n .

S i m o n (1953), i n r e p o r t i n g
the p r e s e n c e

c a p i l l a r y c e l l s w h ic h

a re a there

w as f lu i d w h ic h

a nd t h a t t h i s p r o d u c e d a m e c h a n i c a l toxic

s o m e h i s t o l y s i s f o l l o w e d by m e s e n c h y m a l c e l l

T h i s new c e l l f o r m a t i o n p r o d u c e d the g r a n u l o m a o r
and t h e s e f o r m a t i o n s

were

c o n n e c t e d w ith t h e c a p i l l a r y

t i s s ue .
H a g e m a n n e t a l . (195 3) r e p o r t e d t h a t i n t h e
prim ary

p r o l i f e r a t i o n w a s the

a c t i o n in w h i c h the
togenes

entire

result

s t i l l b o r n , the

of i n c r e a s e d m e s e n c h y m a l

c o n n e c t i v e t i s s u e took p a r t .

re­

L,. m o n o c y -

w a s not o ft e n o b s e r v e d in the g r a n u l o m a s .
T h e g e n e r a l o p i n i o n of t h e s e G e r m a n w o r k e r s

c a p i l l a r y e n d o t h e l i u m o f te n p l a y e d a v i t a l

w a s t h a t the

r o l e i n th e f o r m a t i o n of

t h a t t h e y c a l l the

disease

g r a n u l o m a t o s i s i n f a n ti s e p t i c a.

M A T E R I A L S AND M E T H O D S

G lassw are

Most g lassw are

and I n s t r u m e n t s

and i n s t r u m e n t s

were

r i n s e d o nc e i n t a p w a t e r f o l l o w e d by t h r e e
a nd s t e r i l i z e d i n a h o t - a i r
140° C. f o r

one h o u r .

w a s h e d in H e m o - s o l , *

distilled-w ater

o ve n at a t e m p e r a t u r e

C a p illa ry pipettes

rinses,

not i n e x c e s s

of

w it h r u b b e r bu lbs w e r e

auto

claved.

P h y s i o l o g i c a l Salt S o lu tio n s

Phosphate buffered
for p re lim in a ry washes
a diluent was trip le
before

using.

saline

(PBS).

of t i s s u e s .

B uffered saline

used

The w a t e r w h i c h w a s u s e d a s

d i s t i l l e d in p y r e x a nd a u t o c l a v e d i m m e d i a t e l y

The c o m p o s i t i o n was a s fo ll ow s :
NaCl

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

8 . 0 g.

K C 1 ......................................................................

0 . 2 g.

N a H P O , .....................................................
2
4
KH P O
.....................................................

1 . 15 g.

0. 2 g.

CaCl2

0. 1 g.

M g C l 2 - 6HOH

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

H O H

* M einecke

wa s

&c C o., I n c . ,

0. 1 g.
1000 .0 m l

New Y o r k ,
23

N.

Y.

24
M edium

199.

The

was

u sed as the b a s ic

was

obtained f r o m P a r k e

of t h i s

solution was

synthetic medium

constituent
D a v is

a d j u s t e d to

of M o r g a n et a l . (1950)

of th e n u t r i e n t f l u i d .

T h is

and C o ., D e t r o i t , M i c h i g a n .

medium
The pH

8.0 w ith s t e r i l e N a H C O ^

P lasm a

H om ologous
A. n o r m a l

rabbit p l a s m a was

r a b b i t w a s b l e d by c a r d i a c p u n c t u r e .

m l of b l o o d w a s d r a w n into a 30 - m l
1:500 i s o t o n i c h e p a r i n
c l o t ti n g f o r
a te

used throughout this

solution.

several hours.

speed fo r ten m inutes.

syringe

This

st ud y .

Approxim ately

containing 5 m l

25

of a

a m o u n t of h e p a r i n d e l a y e d

The blood was

centrifuged at a m o d e r ­

The p l a s m a w a s i m m e d i a t e l y

rem oved

a nd s t o r e d i n a n i c e b a t h .

Em bryonic

Em bryonic
em bryos.
moved.

tissue

e x tr a c t was p r e p a r e d f r o m n in e -d a y -o ld chicken

The e g g s
A fter f i r s t

which often m a k e s
grinder

E xtract

w ere

o p e n e d and t h e e m b r y o s

aseptically

re­

r e m o v i n g t h e e y e s , to e l i m i n a t e t h e p i g m e n t
a cl ou d y e x t r a c t , the e m b r y o s w e r e g r o u n d in a

a nd a p p r o x i m a t e l y

for each crushed em bryo.

2 m l of m e d i u m

The m i x t u r e

199 w as

added

was placed in an in cu b ato r

25
at

37° C f o r

one h o u r ,

w ith a p ip e tte .
m edium

centrifuged,

The e x t r a c t w a s

199, a n d t h e m i x t u r e

and the

is o la te d in

culture

was

s t o r e d i n a d ee p f r e e z e

1951 f r o m the b r a i n

g r o w t h on a 2 4 - h o u r t r y p t o s e
saline.

This

of a c al f .

u s e d in t h i s

g a uge

needle

was

agar

standard.

to a 5 - m l

Tissue

All tis s u e

and t h e d e n s i t y

area

of a

of t h is b a c t e r i a l

c u l t u r e s f r o m t h e t ip of a 27

syrin ge.

C ultu res

culture

room.

s u b j e c t e d to u l t r a v i o l e t

to t he b e g i n n i n g

One d r o p

read

c u l t u r e w o r k w a s c a r r i e d on in a n a s b e s t o s - l i n e d

h o o d , l o c a t e d i n t he
and the

The

s l a n t w a s w a s h e d off w ith s t e r i l e

a d d e d to t h e t i s s u e

attached

s t u d y was

c u l t u r e i n the follow ing m a n n e r .

i n a C e n c o S h e a r d S a n f o r d p h o t e l o m e t e r u s i n g t h e 0.5 tube

suspension

-27° C.

The b a c t e r i a l c u l t u r e

s u s p e n s i o n w a s d i l u t e d w i th s a l i n e

M c F a r l a n d n e p h o l o m e t e r as

at

C ultu res

of L i s t e r i a m o n o c y t o g e n e s

was p r e p a r e d f o r use in tissu e

removed

d i l u t e d with an e q u a l a m o u n t of

B acterial

The

su p ern atan t fluid

of e a c h e x p e r i m e n t .

The hood w as t h o r o u g h l y w a s h e d
ra y s for

several hours p rio r

26
Procurem ent
f r o m the
The

culture

stainless

of the

room , was

on a m e t a l t r a y .
surface

cardiac

A. s m a l l a u t o p s y

used for

room ,

obta in in g the

separate

anim al t is s u e s .

s t e e l b e n c h w as w a s h e d down p r i o r to t h e b e g i n n i n g

operation.

entire

of t i s s u e s .

A norm al

rabbit was placed ventral

The h a i r w a s

c l i p p e d as

sponged s e v e ra l tim e s

p u n c t u r e m a d e , a nd the

s h o r t a s p o s s i b l e , the

with 70 p e r c e n t

alcohol,

anim al exsanguinated.

c o l l e c t e d a s e p t i c a l l y to p r o v i d e p l a s m a f o r p r e p a r i n g
b l e e d i n g , the a b d o m i n a l s u r f a c e

side u p p e r m o s t

w as

a

Blood w as
clots.

A fter

s p o n g e d w ith a l c o h o l , the

skin

i n c i s e d a n d r e f l e c t e d , b e i n g c a r e f u l not to i n c i s e th e a b d o m i n a l m u s ­
cles.

The m u s c l e

s u r f a c e w a s w i pe d w i t h a l c o h o l to r e m o v e

hairs

w h ic h m i g h t c o n t a m i n a t e the

area.

were

i n c i s e d w i th a s t e r i l e k n i f e , the

The m u s c l e s

any

an d p e r i t o n i u m

s p le e n r e m o v e d and p la c e d

im m e d ia te ly in s te r ile P e t r i d ish es.

M e t h o d of c u l t u r e .
to

rem ove

m ately

1 mm

this

study.

square
tim es

T issues
The

s p l e e n w a s w a s h e d t w i c e w i th P B S

a ny s u r f a c e b l o o d , a n d w a s m i n c e d into p i e c e s

washed three
199.

The

w ere

w it h s h a r p

scissors.

The m i n c e d t i s s u e s

w i th P B S f o l l o w e d b y two w a s h e s
a l l o w e d to r e m a i n i n m e d i u m

double c o v e r s lip m e t h o d of M a x i m o w
Large

coyer

glasses,

approxi­
were

with m e d i u m

199 until e x p l a n t e d .
(1925) w a s

u s e d in

40 x 50 m m , w e r e p l a c e d on s t e r i l e

27
black p o rc e la in tiles
P etri
the

dish.

One d r o p

cover glass

on the

drop

and

c o v e r e d with t h e b o t t o m o r top of a s t e r i l e

of s t e r i l e

a nd a s t e r i l e ,

round,

n u trie n t m e d iu m was

a d d e d with a p i p e t t e .

of e m b r y o e x t r a c t .

w i t h one d r o p

of t h e

previously.

A. d r o p

each co rn er

of t h e

Some

large

o v e r the c u l t u r e

ra tio n was in verted.

of

cover glass.

and one d r o p of the

The n u t r i e n t m e d i u m

199, one p a r t of p l a s m a ,

of the t i s s u e

standardized bacterial
of s t e r i l e ,

center

c o v e r s l i p was p l a c e d

round c o v e rslip

c o n s i s t e d of one p a r t of m e d i u m

placed

22-m m

on the

of f l u i d a n d a d h e r e d t i g h t l y to the l a r g e

The e x p l a n t w as p l a c e d on t h e

part

w a t e r was p l a c e d

cultures

and one

were

inoculated

su sp e n sio n as d e s c rib e d

m e l t e d p e t r o l a t u m w as p l a c e d a t

cover glass, a large
and a f te r

clotting

The c u l t u r e w as

to p r e v e n t e v a p o r a t i o n of th e f l u i d s .

depression

s li d e w a s

o c c u rre d , the en tire

prepa­

s e a l e d with m e l t e d p a r a f f i n
A f t e r the p a r a f f i n was

s e t , the

s l i d e w a s p l a c e d i n a r a c k in a n i n v e r t e d p o s i t i o n in o r d e r to p r o ­
vide a ly in g

drop in stead

i n c u b a t e d at

37° C ( P l a t e I).

When the
a r a tio n was

c lo t l i q u e f i e d ,

o p e n e d and the

s l i p w i th the a d h e r i n g
10 m i n u t e s

of a h an g in g d r o p .

The c u l t u r e s

u s u a l l y i n f o u r to five d a y s , the p r e p ­

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

cultu re

were

was

a n d p l a c e d on a s t e r i l e

rem oved.

washed in f r e s h m ediu m
cover glass.

The c o v e r ­
199 f o r

F r e s h p l a s m a wa s

28

P 1a t e I .

D oable c o v e r s l i p

culture.

Mag.

2x

T h e e x p l a n t (A) i s on t h e r o u n d c o v e r s l i p (B) w h i c h i s
a t t a c h e d to t h e l a r g e c o v e r g l a s s (C) a n d i n v e r t e d o v e r
t h e d e p r e s s i o n (D) .
T h e c o v e r g l a s s i s s e a l e d to t h e
d e p r e s s i o n s l i d e w i t h p a r a f f i n (E) .

PLATE I

Plate

I.

Double

coverslip

caM re,

Mag.

2x

T h e e x p l a n t (A.) i s o a t h e r o u n d c o v e r s l i p (B) w h i c h i s
a t t a c h e d to t h e l a r g e * c o v e r g l a s s (C) a n d i n v e r t e d o v e r
t h e d e p r e s s i o n (D) .
T h e c o v e r g l a s s i s s e a l e d to t h e
d e p r e s s i o n s l i d e w i t h p a r a f f i n (E) .

PLATE

I

29

30
a d d e d if too much, of t h e

c l o t w as w a s h e d away .

w a s p l a c e d o v e r th e c u l t u r e

A depression

a n d s e a l e d as p r e v i o u s l y d e s c r i b e d .

Methods

of Study

It w a s n e c e s s a r y to e m p l o y

s e v e r a l t e c h n i q u e s i n o r d e r to

obtain the d e s i r e d i n f o r m a tio n f r o m this in vestig atio n.
w e re the m ethods

studies.

All c u l t u r e s w e r e e x a m i n e d d a il y u n d e r

o r d in a r y light m ic r o s c o p e .

The

w a s n o t e d as w e l l a s t h e p r e s e n c e
m ig r a tio n was

The following

u se d :

C ytological
the

sl i d e

rate

of g r o w t h of th e t i s s u e s

o r a b s e n c e of b a c t e r i a .

C e ll

r e c o r d e d a n d the d i s t r i b u t i o n of t h e b a c t e r i a w as

stud­

ied.
Supravital
(1:20,000)

were

the m a r g i n s

s t a i n s , N e u t r a l R e d (1:10,000)

u s e d to i d e n t if y t h e t yp e

of th e

and J a n u s G r e e n

of w a n d e r i n g c e l l s

s e e n at

explants.

C u l t u r e s w e r e f i x e d and s t a i n e d in t o to i n t h e following m a n n e r .
T he p a r a f f i n
pel blade.

s e a l w a s l o o s e n e d f r o m t h e d e p r e s s i o n s li d e w ith a s c a l ­
The l a r g e

cover glass

r o u n d c o v e r s l i p , w ith t h e c u l t u r e
surface
tissue

of B o u i n ' s f i x a t i v e .
was

After

w as

carefully

r i n s e d a nd t h e

sm all,

a d h e r i n g to it w a s f l o a t e d onto the
a p p r o x i m a t e l y o n e - h a l f h o u r , the

gradually hydrated through distilled w ater.

With the

31
tissue

s t i l l on t h e c o v e r s l i p , the e n t i r e

percent

aqueous

cultures

w ere

in xylene,
th e

s e c t i o n w as

s o l u t i o n of H a r r i s ’ h e m a t o x y l i n f o r

g rad u a lly dehydrated,

12 h o u r s .

s t a i n e d l i g h t l y with e o s i n ,

a n d m o u n t e d w it h P e r m o u n t / 1'

s tudy the h i s t o p a t h o l o g y

s o m e of t h e

i n B o u i n ’s f o r

ap p ro x im ately one-half hour.

th e

w ere

coverslip,
absolute

w i th a s h a r p
changes
Serial

cleared

mounting

s l id e .
To

In the

The

C a r e w as t a k e n to d e h y d r a t e

c l o t c o m p l e t e l y and c l e a r g r a d u a l l y w ith x ylene b e f o r e

on th e

the

s t a i n e d in a 20

d e h y d ra te d in asc ending

a l c o h o l , th e

r a z o r blade.

cultures
The

were

cultures

w ere fixed

The c u l t u r e s , w hile on
strengths

of a l c o h ol .

s h a v e d off the

coverslip

cu ltures w ere p a s s e d through s e v e ra l

of oiL of w i n t e r g r e e n , i n f i l t r a t e d with p a r a f f i n , an d e m b e d d e d .

sections

sections

w ere

exam inations.
both G r a m

of e a c h b l o c k w e r e

c ut at f o u r m i c r o n s .

Some of

s t a i n e d w ith h e m a t o x y l i n - e o s in f o r h i s t o p a t h o l o g i c al

In a n a t t e m p t to id e nt if y L . m o n o c y t o g e n e s in s e c t i o n s ,

W e i g e r t and G o o d p a s t u r e ' s

W eigert-V an Gieson
connective t i s s u e s

stain was

which w ere

stain were

e m p l o y e d to

used (Mallory,

study th e

various types

in v o lv e d in the l e s i o n s p r o d u c e d .

* P e r m o u n t , F i s h e r S c ie n t i f i c

1942).

C o ., New Y o r k , New Y o r k .

of

32
B acteriological

studies.

m o n o c y t o g e ne s t h e e x p l a n t s
o r in b ro th .
the e x p l a n t ,
t he

A fter
w as

were

v i a b i l i t y of B.

c u l t u r e d on e i t h e r t r y p t o s e

opening the p r e p a r a t i o n s , the e n t i r e

r e m o v e d w ith a b a c t e r i o l o g i c a l

appropriate m edia.

Some t i s s u e

In o r d e r to t e s t f o r

cultures

These

were

cultures w ere

re frig erated for

agar

c l o t with

loop a nd p l a c e d

on

i n c u b a t e d at 37° C.

several

days p r i o r to

checking f o r the b a c te ria .

Photography.
and Bomb

M o de l

togenes were

B

P hotom icrographs
cam era.

w e r e t a k e n with a B a u s c h

Photographs

of c o l o n i e s

of B. m o n o c y ­

t a k e n with a n E a s t m a n Kodak View c a m e r a u s in g a

m i c r o - s u m a r lens.

R E S U L T S AND DISCUSSION

B a c t e r i o l o g i c a l Studie s

Effects

of f l u i d s .

e x p e r im e n t s , the
m edium

Before

r e a c t i o n of the

it w a s p o s s i b l e
constituents

to do t i s s u e

of t h e t i s s u e

culture

culture

on L . m o n o c y t o g e n e s h a d t o be d e t e r m i n e d .
M edium

199, t h e n u t r i e n t

i n h i b i t o r y to t h e g r o w t h of the
in m e d iu m

s o l u t i o n , p r o v e d to be i n no way

organism .

A culture

of t h e b a c t e r i a

199 r e m a i n e d v ia b le f o r m o r e t h a n two w e e k s .

d a y 0.2 m l of t h e
grow th always

cultu re

w a s p l a t e d onto t r y p t o s e a g a r

was luxurious

after

18 h o u r s

Each

and the

of i n c ub a ti o n .

W h e n L . m o n o c y t o g e n e s w a s i n c u b a t e d in a p l a s m a clot w it h
m edium
As

199, th e b a c t e r i a

a result

the tis s u e

r e m a i n e d v i a b l e f o r m o r e t h a n e ig h t d a y s.

of the f o r e g o i n g e x p e r i m e n t s

it w a s e v i d e n t t h a t none of

c u l t u r e f l u i d s w e r e b a c t e r i c i d a l to t h i s

R e frigeration

studies.

G r a y et a l . (1948)

organism .

re p o rte d that m a c e r ­

a t i o n of t i s s u e s f o l l o w e d by r e f r i g e r a t i o n i n c r e a s e d the p r o b a b i l i t y
of r e c o v e r i n g the
sim ilar

results.

organism from tissues;
O s e b o l d a n d Inouye

of 47 p e r c e n t in r e c o v e r y

other w o rk e rs

(1955,

reported

1955a) found an i n c r e a s e

of L . m o n o c y t o g e n e s f r o m
33

have

rabbit tis s u e

34
a n d 95 p e r c e n t i n c r e a s e

in re c o v e r y f r o m

a c u r i o u s p h e n o m e n o n w h ic h r e m a i n s
Because
were

of t h i s

studied f o r this

h o u r s , the t i s s u e

strange
same

cultures

tissues

of s h e e p .

u n s o l v e d at t h i s t i m e .

behavior, inoculated tissu e

phenomenon.
were

T his i s

cultures

A fte r incubating f o r

24

o b s e r v e d u n d e r the m i c r o s c o p e f o r

c e l l m i g r a t i o n a nd the p r e s e n c e

of b a c t e r i a .

The g r o w t h of t i s s u e s

a p p e a r e d to p a r a l l e l t h a t of u n i n o c u l a t e d c o n t r o l s but m i c r o o r g a n i s m s
w ere

seen moving f r e e ly

cultures.

about the m a r g i n s

In all o b s e r v a tio n s

always m o tile .

T h i s will be

of t h e i n f e c t e d t i s s u e

of l iv in g c u l t u r e s , the b a c t e r i a w e r e
d i s c u s s e d in g r e a t e r

d e t a i l i n the

sec­

t i o n c o n c e r n e d w i th the d e s c r i p t i o n of th e living c u l t u r e s .
W hen a 2 4 - h o u r

cultu re was unsealed,

and th e c lo t and e x ­

p l a n t p l a c e d i n t r y p t o s e b r o t h , L. m o n o c y t o g e n e s
after

18 h o u r s '

repeated,

u sin g

incubation.

H o w e v e r , if t h i s

a 48-hour inoculated tis s u e

g ro w th a p p e a r e d in the b r o t h a f te r
the b a c t e r i a w e re
procedure
results.
was

was

3 days'

same procedure

i n c u b a t i o n e v e n though

s e e n m o v i n g i n the l i v i n g t i s s u e

Beginning

was

c u l t u r e , no b a c t e r i a l

r e p e a t e d w i th a 3 - d a y - o l d t i s s u e

culture.

culture

This

with i d e n t i c a l

of the f o u r t h day of i n c u b a t i o n , one t i s s u e

r e m o v e d f r o m the

a nd p l a c e d in the

grew lu xuriously

i n c u b a t o r e a c h day f o r f o u r

refrigerator

at

4°C.

culture

consecutive

Sealed cu ltu re s

were

d ays

removed

35
from

the

were

u n s e a l e d a n d th e e n t i r e

the

refrigerator

round c o v e rs lip

these

cultures

incubation.

w ere

16,

employing

When explants w ere

of s p l e e n .

scraped from
All

a f t e r two d a y s '

under a bin ocular

oblique l i g h t i n g .

organism

dis­

L. m o n o c y t o g e n e s

d i s c r e t e , b lu e - g r e e n colonies.

t i n g th e b r o t h , the

cultures

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

a g a r a n d o b s e r v i n g th e c o l o n i e s

sm all,

The

c lo t and e x p l a n t w e r e

id entificatio n has b e e n u sed very

piece

23, a n d 31 d a y s .

positive f o r L . m onocytogenes

section m icro sco p e
as

7,

a nd p l a c e d i n t r y p t o s e b r o t h and i n c u b a t e d .

The o r g a n i s m

on t r y p t o s e

appear

after

This m e t h o d of

s u c c e s s f u l l y by G r a y e t a l . (1948).

s m e a r e d d i r e c t l y on t r y p t o s e
o fte n w a s

agar,

om it­

s e e n g r o w i n g a r o u n d the

P l a t e II i l l u s t r a t e s t h i s

sm all

and sh ow s t h e c h a r a c t e r i s t i c

c o l o n i a l m o r p h o l o g y a n d c o l o r of L. m o n o c y t o g e n e s .
It is k n ow n t h a t u n d e r c e r t a i n c o n d i t i o n s t h i s i n f e c t i o n with
L. m o n o c y t o g e n e s i s

somewhat

self-lim iting.

o c u l a r i n s t i l l a t i o n o fte n r e c o v e r e d
ease

was

R a b bi ts

e x p o s e d by

a nd t h e only s y m p t o m

of th e d i s ­

the i n t e n s e c o n j u n c t i v a l r e a c t i o n a nd a s li g h t m o n o c y t o s i s .

Sheep a n d c o w s
occasionally

r e c o v e r e d , e v e n t ho ug h t h e y w e r e l e f t with p e r m a n e n t

b ra in damage.
cate th at this

s u f f e r i n g f r o m b r a i n i n f e c t i o n s w ith L. m o n o c y t o g e n e s

R e c e n t e x p e r i m e n t s by G r a y et a l . (1955,
organism

c a n r e m a i n v i a b le i n the u t e r u s

1955a)

indi­

of r a b b i t s f o r

36

P late

II.

A p i e c e of s p l e e n f r o m a n i n f e c t e d c u l t u r e s m e a r e d on
an a g a r p la te and in c u b a te d 4 8 h o u r s .
The b a c te ria l
c o l o n y c a n b e s e e n g r o w i n g out f r o m u n d e r t h e t i s s u e .
M a g . 2 4x

PLATE

II

*

38

P late

III.

T ypical L. m o n o c y t ogenes c o lo n ie s on an a g a r p la te .
Isolated fro m a tis su e culture.
Mag. 1.25x

39
PLATE

III

40
many weeks.
larly
the

W hen the a n i m a l s

during g estatio n ,

d oe ,

successive

e v e r y i n s t a n c e , L.

m onocytogenes

ness

survived.

ganism

in such c a s e s

was

H ow ever,

of s u r v i v a l , the

Perhaps

isolated fro m

was is o la te d f ro m

property.

All t h i s

ent tim e .

N evertheless,

is

substance

sim ple

cultures behaved after

It m u s t be
teriological

of

In

of i l l ­

relatively
The o r ­

viscera.

the p h e n o m e n o n of i n c r e a s i n g

re­

r e f r i g e r a t i o n w i th the f a c t t h a t

acts

as

a bacteriostatic

4° C

a gen t at

this b a c te rio s ta tic

s p e c u l a t i o n w ithout p r o o f at the p r e s ­

in v iew of t he f a c t t h a t i n o c u l a t e d t i s s u e

r e f r i g e r a t i o n t he

su g g ests th a t some
at

sym pto m s

ill and died.

m o s t of the

37° C, b u t w h e n r e f r i g e r a t e d no l o n g e r r e t a i n s

activated

recovery

c a n r e m a i n i n the a n i m a l without d i s p l a y i n g s y m p t o m s ?

som e inhibitory

sim ilarly

or o c u ­

aborted fetuses

occasionally after

rabbits becam e

c o v e r y of T.. m o n o c y t o g e n e s f ollo wing
organism

after apparent

did n o t m a n i f e s t c l i n i c a l

Is it p o s s i b l e to c o r r e l a t e

the

orally

r e b r e e d i n g always t e r m i n a t e d in abortio n.

The d o e s

long p e r i o d s

exposed either

abortion occurred;

and cotyledons.
a nd u s u a l l y

w ere

same

as o t h e r t i s s u e s t r e a t e d

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

system

w h ic h i s i n ­

a l lo w s th e b a c t e r i a to g r o w on a r t i f i c i a l m e d i a .

r e m e m b e r e d t h a t in e v e r y c a s e , p r i o r to m a k i n g b a c ­
cultures

of t h e e x p l a n t s , l i v i n g , m o t i l e b a c t e r i a w e r e

s e e n in a l l i n o c u l a t e d t i s s u e

cultures.

T h e re fo re , something made

41
it i m p o s s i b l e to c u l t u r e t h e o r g a n i s m
w ere

h e l d at

4 C for

ing q u e s t i o n s

still

s e v e r a l days.

on a r t i f i c i a l m e d i a until t h e y
The a n s w e r s

to t h e s e p e r p l e x -

r e m a i n a m y s t e r y to be unfolded.

D e s c r i p t i o n of L i v in g C u l t u r e s

A p p r o x i m a t e l y 290 c u l t u r e s

were

g r o w n and o b s e r v e d .

Some

w e r e f i x e d a nd s t a i n e d i n t o t o , o t h e r s w e r e b l o c k e d and s e c t i o n e d .
W h a te v er the fate

of t he

cultures,

a ll w e r e

o b s e r v e d in the liv in g

state.
D u r i n g the f i r s t
m orphonuclear
ually b e c a m e
w ere

leucocytes
inactive

a f t e r i n c u b a t io n , the p o l y ­

m i g r a t e d o u t w a r d f r o m the e x p l a n t , g r a d ­

and d e g e n e r a t e d .

The n e u t r o p h i l s e v e n t u a l l y

o b s c u r e d by t h e g r o w t h a n d m i g r a t i o n of l y m p h o c y t e s

m onocytes.
culture.
have

24 to 40 h o u r s

P l a t e IV i l l u s t r a t e s t h r e e

Large

am eboid cells moved

any p a r t i c u l a r

g r o w t h of a s ple nic

slowly and did not a p p e a r to

a t t r a c t i o n f o r the b a c t e r i a

vio usly m en tio n ed , in all o b s e r v a tio n s
te r ia w ere

days'

always m otile.

On s e v e r a l

and

(Plate

V) .

As p r e ­

of i n o c u l a t e d c u l t u r e s th e b a c ­
occasions the

organism s

d i r e c t l y t o w a r d t h e m a c r o p h a g e s , h a m m e r e d the c e l l m e m b r a n e

moved
sev­

e r a l t i m e s , t h e n b a c k e d aw ay a s if to g a i n m o m e n t u m f o r a n o t h e r
bom bardm ent.

This

r e p e a t e d m e t h o d of a t t a c k ofte n c o n t i n u e d f o r

42

P late

IV.

I n o c u la te d living
200x

splenic

culture

three

days

o ld .

Mag.

PLATE

IV

43

I

* V > “a* v ‘'
** * %% * o ;
® * * « « r*".

<*
*;v?v%
:i£iL
W :

r

: '*

•

; -L r J . ^

>A f e * &SB
-V -

tftxrww' »

:. v *

S.» 1.1**I f •*

44

P late

V.

I n o c u l a t e d l i v i n g s p l e n i c c u l t u r e s i x d a y s old.
(A.) B a c ­
t e r i a a l o n g t h e m a r g i n of t h e e x p l a n t .
(B) M o n o n u c l e a r
phagocytes.
M a g . 9 40 x

PLATE

!*■

V

P late

Y.

I n o c u l a t e d ^ v i n g s p l e n i c c u l t u r e s i x d a y s old.
(A) B a c ­
t e r i a a l o n g t h e m a r g i n of t h e e x p l a n t .
(B) M o n o n u c l e a r
phagocytes.
M ag. 940x

PLATE

45

V

*
f i

*

4

46
several hours

a nd f i n a l l y the b a c t e r i a e n t e r e d the

m acrophages.

These m o v e m e n ts

p oseful r a t h e r than random .

of the b a c t e r i a

Plate

was

s t a i n e d w ith J a n u s

f o r t he d y e .
well as f r e e

the d if fu se d i s t r i ­

T his p a r t i c u l a r c u l t u r e

G r e e n a nd t h e o r g a n i s m s h a v e an a ff in ity

The b a c t e r i a c a n be
in the

of the

s e e m e d to be p u r ­

VI i l l u s t r a t e s

b u t i o n of t h e b a c t e r i a t h r o u g h o u t t h e c u l t u r e .

cytoplasm

seen in s e v e ra l m a c ro p h ag e s

surroundin g m edium .

Plate

as

VII is a n oil i m m e r ­

s i o n p h o t o g r a p h of a t w o - d a y - o l d c u l t u r e .

These

for nearly four hours

several b a cteria penetrated

t he

and d u r i n g t h i s t i m e

cells w ere

observed

cell m e m b r a n e .
A g r e a t d e a l of d if f ic u lt y w as e n c o u n t e r e d i n the i d e n t i f i c a t i o n

of t he

cells

i n the l i v i n g ,

unstained c u ltu re s.

It is

s u f f i c i e n t to say

that they w e re m o n o n u clea r phagocytes.
C u n n i n g h a m e t a l . (1923) i d e n t i f i e d two d i s t i n c t t y p e s
phagocytic

cells

in the

of c l a s m a t o c y t e s ,

s p l e e n of th e r a b b i t .

m acrophages,

i n t e n s e l y to N e u t r a l Red.
w ere
was

relatively

15p to

these

cells w ere

One g r o u p , c o n s i s t i n g

a nd h i s t i o c y t e s

were

s a i d to r e a c t

The n u c l e i w e r e l o c a t e d p e r i p h e r a l l y and

s m a l l i n c o m p a r i s o n to c e l l s i z e .

i d e n t i f i e d as t y p i c a l m o n o c y t e s

w ere

of

The o t h e r g r o u p

of t h e p e r i p h e r a l blood.

These

30p in d i a m e t e r w i th a h o r s e s h o e - s h a p e d n u c l e u s .
s t a in e d with N e u t r a l R ed t h e r e

was

When

a central, clear

47

P late

VI.

L . m o n o c y to g e n e s s ta in e d s u p r a v i t a l l y w ith J a n u s G r e e n
in a f i v e - d a y - o l d livin g c u l t u r e .
The b a c t e r i a a p p e a r
i n V, Y, a n d p a l l i s a d e f o r m s .
M a c r o p h a g e s containing
b a c t e r i a a r e m a r k e d w ith a r r o w s .
M a g . 1500x

PLATE

x jp

r -T
^

VI

%
[ %

W S r te ’

47

P late

VI.

I
L . m o n o c y to g e n e s s ta in e d s u p r a v i t a l l y with ^ a n u s G reen
i n a f i v e ~d a y - o ld l i v i n g c u l t u r e .
The b a c t e r i a a p p e a r
in V, Y, a n d p a l l i s a d e f o r m s .
M a c r o p h a g e s containing
b a c te r ij i a r e p a r k e d with a r r o w s .
M a g . 1500x

PLATE

48

VI

%
M*

’ • t * 'i t v V * y ;:
'

v

/

* v < \> *

vi
•%

-» •

4 /<

* -E

#- *

'* • '

/i-X T
’*

>* ♦ J C C .
—
v• ^
v ,r «

T« 4

„«
C
•£**

#

\ <

»^

v

*1,

* '* •

M

*•

J*

:? J

• 5

-

.

' *

•

K

V/

y

K

V I

»

S

_

^K*rl
*■& \ m.v
’& * •
:^ • !£•* *?

i
r*r*

t _ r /f

f//

7, i&yj

-

t

$*

i ; > •
* /

o;

-

■; S

<*. r

n

A
v f

«%
•• W 1

f&\5
T "

W

*

*

##
# •*
w #
V#

1

^

49

IPlate

VII.

M o n o n u c le a r p h ag o cy tes in a living tw o -d a y -o ld cu ltu re.
B a c t e r i a c a n be s e e n in th o se c e lls m a r k e d w ith a r r o w s .
M a g . 3050x

PLATE

YE

49

\,

P late

VII.

M o n o n u clear phag o cy tes in a living tw o -d a y -o ld cu ltu re.
B a c t e r i a ca n be s e e n in th o s e c e l l s m a r k e d w ith a r r o w s .
M a g . 3050x

PLATE

Vn

50

51
spot o p p o s i t e the Hof of th e n u c l e u s .
was

a rosette

ticles

of s m a l l ,

radiating

aggregations

a n d a ny p h a g o c y t i z e d m a t e r i a l

red granules
Forkner

at t h e p e r i p h e r y

(1930)

Surrounding this

of the

clear

of N e u t r a l

Red p a r ­

c o n g r e g a t e d i n t he zone
rosette.

co n firm ed the ex istence

spot

of the

M c J u n k i n (1926)

and

of t h e s e two v a r i e t i e s

of

m o n o n u c le a r phagocytes.
This
cytes
vi t a l

a u t h o r w a s u n a b le to i d e n t i f y t h e s e two t y p e s

after repeated tria ls
stains.

but t h e

It w a s

rosette

as

of p h a g o ­

u s in g N e u t r a l R e d and J a n u s G r e e n

obvious t h a t the c e l l s

were

supra­

m o n o n u c l e a r i n type

d e s c r i b e d by C u n n i n g h a m e t a l . (1923)

was n e v e r

seen.
M a x i m o w (1927)
cocytes

of the

d e v e l o p e d into p o l y b l a s t s

m e t h o d s , he
of t he

was

opinion t h a t the n o n g r a n u l a r l e u ­
and f i b r o b l a s t s .

r e p o r t e d t h a t the l y m p h o c y t e s

Using t i s s u e

and m o n o cy te s

s e c o n d da y b e g a n to s t r e t c h out and develop long,

shaped,

nonmotile p r o c e s s e s

cells were

d e s c r ib e d as

w i th l a r g e

regular,

at the end
spear-

oval n u c l e i .

1' f i b r o b l a s t - l i k e ' 1 and a s

culture

These

s oo n a s t h e y w e r e

b r o u g h t into f o c u s w ith an oil i m m e r s i o n l e n s w ith s t r o n g l i g h t t h e y
contracted,
cells

r o u n d e d off and r e s u m e d th e f o r m e r

w it h m o t i l e

fibroblasts

m e m b r a n e - lik e pseudopodia.

were p re sen t.

Maximow, th e r e f o re ,

ameboid wandering

A fter

s ix d a y s t y p i c a l

r e a s o n e d th a t

52
lym phocytes
theory

seem s

wrong.

The

still

rem ains

d e v e l o p e d into p o l y b l a s t s t h e n into f i b r o b l a s t s .
r a t h e r p r e s u m p t u o u s , b at t h e r e

is no p r o o f t h a t it is

o r i g i n a nd d e v e l o p m e n t of m o n o n u c l e a r m a c r o p h a g e s
a very controversial

of t h i s w r i t e r to t a k e
t h a t th e c e l l s

T his

s u b j e c t and i t is

a s t a n d on t h i s m a t t e r .

i d e n t i f i e d i n the l iv in g c u l t u r e s

not the p u r p o s e

It c a n only be
were

stated

m ononuclear

phagocytes.
W h e n th e t i s s u e

culture

experim ents

were firs t

s t a r t e d , it

w a s d i f f i c u l t t o d e t e r m i n e how m a n y b a c t e r i a to add to the e x p l a n t e d
tissue.
were

Pilot experim ents

in w h i c h t h i r t y o r g a n i s m s p e r m i l l i l i t e r

u s e d p r o v e d t h i s to be

e x p e rim e n ts by G r a y
from

an i n a d e q u a t e

anim al

(1954), t h e c o n c e n t r a t i o n of b a c t e r i a o b t a i n e d

was f o u n d to be an a d e q u a t e d o s e to p r o d u c e

l e s i o n s t y p i c a l of L.. m o n o c y t o g e n e s

T he

In p r e v i o u s

a s u s p e n s i o n m a d e to m a t c h the 0.5 t u b e of a M c F a r l a n d n e p h o-

lom eter

as

dose.

a b a s is , the

sam e

s u s p e n s i o n wa s

s u s p e n s i o n c o n t a i n e d 2.289 x 10

therefore,

infection.

i n 0.01 m l t h e r e

w ere

9

L ew is

(1920)

in view of t h e

cultures.

organism s p e r m illiliter;

2.289 x 10

reports

and

With t h i s i n f o r m a t i o n

u s e d in t h e t i s s u e

t r a t i o n of b a c t e r i a did not s u p p r e s s the
It i s i n t e r e s t i n g

sym ptom s

7

bacteria.

This

g r o w t h of the t i s s u e
by Sm y th (1915,

w h i c h s t a t e d t h a t the t i s s u e

1916)

concen­
(Plate
and

did not r e m a i n a li v e in

V) .

53
the p r e s e n c e

of b a c t e r i a .

Tissue

cultures

cytogenes n e v e r d e m o n s tr a te d growth
VIII a n d IX i l l u s t r a t e
of c u l t u r e ,

profuse

respectively.

i n o c u l a t e d with L . m o n o -

s u p p r e s s i o n o r i n h i bi ti o n .

g r o w t h of c e l l s

Plate

Plates

after

s i x a n d s e v e n days

X is an illu s tra tio n

of a s i x - d a y u n i n ­

oculated cu ltu re.
P e r h a p s the
cultures
There

trem endous

salt

h a d d i ff i c u lt y in m a i n t a i n i n g t i s s u e

i n o c u l a t e d with b a c t e r i a b e c a u s e

is little

pioneers

early w orkers

of i n f e r i o r n u t r i e n t f l u i d s .

doubt but t h a t a r t i f i c i a l m e d i a

influence

upon th e

s i m i l a r to

199 e x e r t

v i a b i l i t y of t i s s u e s i n v i t r o .

The

in t h i s f i e l d w e r e l e s s f o r t u n a t e i n having to r e l y on s i m p l e

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

and e m b r y o e x t r a c t .

D e s c r i p t i o n of F i x e d C u l t u r e s

Whole m o u n t s .

C u l t u r e s w h i c h w e r e f i x e d in toto w e r e

with h e m a t o x y l i n - e o s i n o r W rig h t's
M a ny p o l y m o r p h o n u c l e a r
first

24 to

36 h o u r s

of c u l t u r e .

d i s t r i b u t e d and s o m e
A fter
M a ny of the
hook,

48 h o u r s
cells

a nd s p i n d l e

stained

s t a in .

cells

w e r e p r o m i n e n t d u r i n g the

T hese cells

were

rather

d i f f u se l y

c o n t a i n e d e ng u lf e d o r g a n i s m s .
the m a j o r i t y of t h e

cells were

m acrophages.

exhibited b i z a r r e f o r m s ,

s uc h as

shapes.

m ost numerous

The c e l l s

were

club, d u m b b e l l ,
at the

54

P late

VIII „

A s i x - d a y - o l d l i v i n g c u l t u r e s h o w i n g g o o d g r o w t h of
t i s s u e e v e n though in fe c te d w ith b a c t e r i a .
M a g . 200x

PLATE

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55

VIII

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56

P late

IX .

A s e v e n - d a y - o l d l i v i n g c u l t u r e s h o w i n g h e a v y g r o w t h of
fib roblasts.
T h e p r e s e n c e of b a c t e r i a d i d n o t i n h i b i t
t h e g r o w t h of t h e t i s s u e .
M a g . 7 5x

PLATE

t<y. - - -

IX

sg p w * 3 , - , -~ c£ ? U ' :

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T T s '—

15&
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58

P late

X.

An uninoculated co n tro l c u ltu re .
.Six d a y s old.
n u c l e a r p h a g o c y te s a r e the p r e d o m in a tin g c e lls .
1 60 Ox

Mono­
Mag.

PLATE

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60
m a r g i n of th e e x p l a n t .

A. s m a l l a m o u n t of c y t o p l a s m w a s

e a c h e n d of t h e c e l l s .

Plate

of t h e s e
as four

cells.

XI, F i g u r e s

These b iz a r r e

cells

s e e n at

1 a n d 2, show e x a m p l e s

were

s e e n in c u l t u r e s

d a y s ; h o w e v e r , t h e y w e r e n ot p r e s e n t

as

old

at all i n u n i n o c u l a t e d

c ont r o l s .
H a g e m a n n a nd S i m o n (195 3) a nd H a g e m a n n et a l , (195 3) d e ­
scribed b iz a rre
cells

arose

as

nuclear fo rm s

in g r a n u l o m a s .

In t h e i r

opinion the

a r e s u l t of h i n d e r e d p r o l i f e r a t i o n of c a p i l l a r y wall

tissue

due to t h e

s t i m u l u s of a to x ic

reagent.

form s

o b s e r v e d i n the i n o c u l a t e d t i s s u e

The

cultures

aberrant cellular

were

s i m i l a r to

t h o s e d e s c r i b e d by H a g e m a n n and S im o n (195 3) and H a g e m a n n et a l .
(1953).

M o s t of t h e s e

a re a im m ediately
A fter th re e
i n e n t and a f t e r

a d j a c e n t to the

p i c t u r e d in P l a t e

o r f o u r d a y s the b i z a r r e f o r m s w e r e not as p r o m ­

stage w ere
XII.

out i n t h e

cytes w ere
M ore

n u c l e i w e r e found in the

e x p l a n t.

six days they w ere

o ft e n s e e n a t t h i s

s u e , w hile

c e l l s w ith b i z a r r e

only r a r e l y

seen.

The

cells

round c e ll s with v e s i c u l a r nuclei as

M o s t of t h e s e w e r e

at th e m a r g i n of the t i s ­

surrounding m edia, p la s m a cells

and ly m p h o ­

m ost prom inent.
s tu d y w ith s t a i n e d whole m o u n t s would be d e s i r a b l e .

However, these p re p a ra tio n s

most

present

s o m e d if f ic u l t y i n h a n d l i n g .

61

P late

XI.

B iz a r r e n u c le a r f o r m s se e n in 48 -ho ur c u ltu re s ,
w ith, h e m a t o x y l i n - e o s i n .
M a g . 16 5 0 x

Stained

PLATE

62

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V

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Figure

1
2

63

P late

X II.

C e l l s w ith v e s i c u l a r n u c l e i s e e n i n t h r e e - d a y - o l d
S tained with h e m a t o x y l i n - e o s i n .
M a g . 18 6 0 x

cultures

PLATE

! «

• >

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64

XII

*.

7
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65
The p r o t e i n i n the c l o t t e n d s to a b s o r b t h e
detail is m a sk e d .
v ery thin clots

Serial
w ere

B e t t e r p r e p a r a t i o n m a y be p o s s i b l e b y the u se

sections.

T he m o s t o u t s t a n d i n g p a t h o l o g i c a l

serial

i n o c u l a t e d at t h e t i m e

sections

of t h e e x p l a n t s .

of e x p l a n t a t i o n , w e r e

s i x c o n s e c u t i v e d a y s , f i x e d and s e c t i o n e d .
w e re t r e a t e d in the

same

sinus

Radiating f r o m

cells

vesicular,

(Plates

s in g le

24 h o u r s , two l a y e r s
a s m a l l blo od v e s s e l .
rows

XIII, XVI).

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

a t t h e e n d of th e

of t h e

to t h i s

is

The n u c l e i of t h e s e
As a r e s u l t of
cells

u ntil t h e i n v ol ve d a r e a was

six -day period

a p p e a r e d as

of c o n s i d e r a b l e

( P l a t e s XV to XXVI).

s i n u s e n d o t h e l i u m h a s b e e n c o n s i d e r e d as

reticuloendothelial

system

of c e l l s i n f i l ­

e l o n g a t e d , and p l e o m o r p h i c .

c o n t i n u e d p r o l i f e r a t i o n of t he e n d o t h e l i u m , the

The v a s c u l a r
part

Uninoculated controls

As e a r l y as

th is blood v e s s e l w ere

surroundin g tis s u e

size

r e m o v e d e a c h day f o r

seen circum scribing

t r a t i n g th e

sw irls

cultures,

m anner.

endothelium.

of e n d o t h e l i a l c e l l s w e r e

the

T issue

changes

r e a c t i o n b e g a n by wh at a p p e a r e d to be p r o l i f e r a t i o n of

the v a s c u l a r

were

of

o r p e r h a p s p e r f o r a te d cellophane.

s e e n in the

The

s t a i n a nd m u c h c y t o l o g i c a l

system .

a

One g r o u p of c e l l s belong ing

c o m p o s e d of l a r g e m o n o n u c l e a r w a n d e r i n g c e l l s .

T h e y a r e f o u n d i n a ll t i s s u e s

and c i r c u l a t i n g f l u i d s .

The d e s i g n a t i o n s

66
of th e
are

cells

change

usually

w ith r e s p e c t to t h e i r l o c a t i o n .

r e f e r r e d to

as t i s s u e

In the t i s s u e s

m acro p h ag es, histiocytes,

m a t o c y t e s , w h il e in t h e c i r c u l a t o r y

s y s te m they a r e

they

or c las-

called m onocytes.

Th e o r i g i n of m o n o n u c l e a r p h a g o c y t e s h a s b e e n a s u b j e c t of c o n s i d e r ­
a b le c o n t r o v e r s y .

M allory

(1898)

and F o o t (1925) w e r e

of the

opinion

t h a t t h e m o n o n u c l e a r c e l l w a s d e r i v e d f r o m the e n d o t h e l i a l c e l l .

On

the

did

o t h e r h a n d , M a x i m o w (1924a), L a n g

not b e l i e v e t h a t t h e e n d o t h e l i u m gave
characteristics.

S a bin e t a l . (1925)

s t a t i n g th a t t h e c l a s m a t o c y t e s
and the m o n o c y te s f r o m

(1926), and B l o o m

(1928a)

r i s e to c e l l s p o s s e s s i n g a m e b o i d
r e p o r t e d still anoth er th e o ry ,

w ere derived from

' ' r e t i c u l a r 1* c e l l s .

Lewis

the e n d o t h e l i u m
(1925), while not

in a g r e e m e n t w ith Sabin e t a l . (1925), f a v o r e d M a xi m o w (1924a) to
som e extent,

a nd a l s o b e l i e v e d a p a r t of M a l l o r y ' s t h e o r y

Today,

so m e t h i r t y y e a r s l a t e r , the questio n r e m a in s

Forbus

(1943)

(1898).

unanswered.

s t a t e d t h a t the m o n o n u c l e a r w a n d e r i n g c e l l s w e r e

d eriv ed e ith e r f r o m p r im itiv e m e se n c h y m al cells or fro m
sinus endothelium.

vascular

The l a t t e r view s e e m s to be th e m o s t widely

a c c e p t e d one t o d a y .
E v e n t h o u g h the
clearly phagocytosis.
m arkable
giant cells

o r i g i n i s m o s t c o n t r o v e r s i a l , the f u n c t i o n is
It i s w e ll kn ow n t h a t t h e s e

cells

c a p a c i t y f o r p r o l i f e r a t i o n a nd p l e o m o r p h i s m .
an d e p i t h e l i o i d c e l l s

are

derived fro m

these

show a r e ­
M ultinucleated
cells.

67
S e c t i o n s t r e a t e d w ith W e i g e r t - V a n G i e s o n c o n n e c t i v e t i s s u e
s ta in ex h ib ited a single e l a s t i c
l u m e n of th e

re a ctiv e blood v e s s e ls

epithelioid cells

c i r c u m s c r i b e d the

S i m o n (1953)
apparently
(1953)

c o n n e c t i v e t i s s u e f i b e r a r o u n d th e
(Plate

XXIV, F i g u r e

a r e a form ing

2).

The

a solid m a s s .

s t a t e d t h a t t h e f o r m a t i o n of th e g r a n u l o m a w a s

connected with c a p illa ry tis s u e .

r e p o r t e d th a t the e n tir e

H a g e m a n n and Sim on

connective tissu e

of th e v e s s e l w as

i n v o l v e d i n t h e p r i m a r y p r o l i f e r a t i o n of m e s e n c h y m a l c e l l s .

Hage­

m a n n e t a l . (195 3) c l a i m e d t h a t the c a p i l l a r y e n d o t h e l i u m p h a g o c y t i z e d
t h e b a c t e r i a a n d a l s o t h a t t he v e s s e l

r e a c t i o n i s o l a t e d the f o c u s of

the i n f e c t i o n a n d s e t up a g r a n u l o m a .
p o n e n t of t h e g r a n u l o m a w a s th ou gh t
The c a refu l d ay -b y -d a y
revealed a sim ila r

reaction.

48 h o u r c u l t u r e s w e r e
of c e l l s

characteristic

w h ic h b l o o d v e s s e l s
these

c e lls have

thelium .

to be v a s c u l a r e n d o t h e l i u m .

s t u d i e s of the t i s s u e
It w a s e s p e c i a l l y

studied.

com­

A.t t h i s t i m e

culture

sections

obvious w he n 24 o r

only one o r two l a y e r s

s u r r o u n d e d the b l o o d v e s s e l s , w ith a fe w c e l l s t r a i l i n g off

into t h e n e a r b y t i s s u e .
that this

T h e r e f o r e , the c e l l u l a r

A noth er in te re stin g
r e a c t i o n n e v e r w as

were present.

some intim ate

This

observation revealed
seen except in a r e a s

in

s e e m e d to i n d i c a t e t h a t

a s s o c i a t i o n w ith the v a s c u l a r e n d o ­

68
At t h e m a r g i n of t h e t i s s u e
exceptionally la r g e ,
a p p e a r e d to be the

cultures

round m a c ro p h a g e s
same

cells

at l e a s t f o u r

w ere

seen (Plate

w o r k w i t h t he t u b e r c l e b a c i l l i in t i s s u e

a s i m i l a r finding.
gested b a c te ria .

He w a s
However,

w ere m a d e with s p e c ia l

the

organism s

(1953)
were

of t h e o p in io n t h a t t h i s
in the p r e s e n t

m ore

Maximow

culture,

m aterial

reported

was

di­

cells

o r i n the

reactionary cells

of the

c u ltu res infected

r e q u i r e d r e f r i g e r a t i o n in o r d e r to d e m o n s t r a t e

in a rtific ia l culture

m edium .

H a g e m a n n and Simon

r e p o r t e d d i f f i c u l ty in finding b a c t e r i a in the g r a n u l o m a s , b ut
a ble to c u l t u r e t h e

m atter

o rg a n is m fr o m these

structures.

As a

of f a c t , t h e g r a n u l o m a w a s ofte n c a l l e d a " L i s t e r i o m a " by

th e se in v e s tig a to rs be cau se they w e re
tu re was

s p e c i f i c f o r t he

In t h e
ent a f t e r

center

of the opinion t h a t t h i s

d isease, granulom atosis

of t h e e x p l a n t s

48 to 72 h o u r s

sim ply f r o m

some n e c ro s is

the m e c h a n ic a l a s p e c t.

controls

struc­

infantiseptica.
was u s u a l l y p r e s ­

of c u l t i v a ti o n ; t h i s was to be e x p e c t e d
The c e n t e r of t he t i s s u e

not r e c e i v e t h e b e n e f i t of t h e n u t r i e n t f lu i d .
in the

T he y

s tu dy r e p e a t e d a t t e m p t s

As p r e v i o u s l y m e n t i o n e d , t i s s u e

w ith L . m o n o c y t o g e n e s

XXIII).

s t a i n s , bu t L, m o n o c y t o g e n e s w a s n e v e r d e m ­

o n stra te d in the p e r i p h e r a l
explant p r o p e r .

old,

a s t h o s e in t h e t i s s u e but w e r e

s p h e r i c a l a nd u s u a l l y c o n t a i n e d a g o ld e n y e ll o w p i g m e n t .
(1924), i n h i s

d ay s

The n e c r o s i s

a s w e l l as i n th e i n o c u l a t e d c u l t u r e s .

did

appeared

69
The endothelial
the tis s u e
the

re a c tio n usually

a n d o fte n long

rows

a r e a of n e c r o t i c t i s s u e a s

sim ilar

r e a c tio n was

o c c u r r e d at the p e r i p h e r y

of the p r o l i f e r a t i n g

cells

capillary

cells

ing a p r i m a r y
f ou nd i n t he

infiltrated

if to d i g e s t th e c e l l u l a r d e b r i s .

A

r e p o r t e d by H a g e m a n n and S im on (195 3) a nd

H a g e m a n n e t a l . (195 3) i n d e s c r i b i n g t h e h i s t o p a t h o l o g y
t o s i s inf a n t i s e p t i c a .

of

of g r a n u l o m a ­

Th e y d e s c r i b e d a c e n t r i p e t a l i n f i l t r a t i o n of Mthe

of r e so r p t i o n ' 1 w h ic h e n g u l f e d the e n t i r e f o c u s f o r m ­
1' p r o l i f e r a t i n g

organs

g ra n u lo m a .'1

These granulom as

of s t i l l b o r n i n f a n t s f r o m

were

which L. m o n o c y t o g e n e s

was iso late d .
M o s t of t h e A m e r i c a n w o r k e r s who h av e d e s c r i b e d the h i s t o ­
p a t h o l o g y a s s o c i a t e d w ith L.. m o n o c y t o g e n e s i n f e c t i o n s ,
necrosis
(1942)

a s b e in g t h e p r i m a r y l e s i o n .

s i n g l e d out

H o w e v e r , J u l i a n e l l e and M o o r e

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

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

a r e a and d r e w a t t e n t i o n to t h e f a c t t h a t t h i s w as a p e c u l i a r finding
to be a s s o c i a t e d w i th a n e c r o t i c
p o rte d that f ib r o b la s ts w e re
were
of t h e

really fibroblasts

present;

im m ature

system .

are

Other w o rk e rs
makes

h av e

re­

one w o n d e r i f the y

actually p ro life ra te d cells

The n u c l e i of young f i b r o b l a s t s

m ononuclear cells.

As p re v io u sly m entioned,
that th e r e

t h is

o r if t h e y w e r e

reticuloendothelial

often r e s e m b l e

reaction.

H a g e m a n n a n d S im o n (1953) b e l i e v e d

two d i s t i n c t c e l l u l a r

re a ctio n s possib le

resultin g f r o m

70
in fe ction with L . m o n o c y t o g e n e s .
lom atous

in fla m m a tio n , the

One w a s

o t h e r by n e c r o s i s .

t h a t a c o m b i n a t i o n of t h e s e two r e a c t i o n s
a r e a.

T hese facts

A c c o r d i n g to F o r b u s
an ''e x u d a tiv e "

of t h i s

(1949)

They also

same

of t he c o n f u s i o n e x i s t i n g

disease.

a p ure granulom atous

a nd ' ' p r o d u c t i v e ' '

reported

c a n o c c u r w i th in the

m a y h e lp to e x p l a i n s o m e

re g a r d in g the h is to p a th o g e n e s is

is

c h a r a c t e r i z e d by a g r a n u ­

inflam m ation

r e a c t i o n in the t i s s u e s

character­

i z e d by t h e a c c u m u l a t i o n a n d p r o l i f e r a t i o n of r e t i c u l o e n d o t h e l i a l c e l l s
from

the circu latin g fluids

c e l l i n the

reactive

Forbus

b e a c u te

or

spaces.

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

a r e a is the m a c r o p h a g e i n c o n t r a s t to o t h e r i n ­

flam m atory processes
v o lv e d .

a n d the t i s s u e

in which neutrophils

(1949)

or lymphocytes

stated that this in flam m atory

are in­

response

can

chronic.

S e v e r a l b a c t e r i a h av e b e e n a s s o c i a t e d with a g r a n u l o m a t o u s
reaction,

such as B r u c e lla s u i s , P a s t e u r e l l a t u l a r e n s i s , M ycobacteriu m

tuberculosis

and M y c o b a c te r iu m p a r a t u b e r c u l o s i s .

g e s t e d by s o m e i n v e s t i g a t o r s t h a t t h e s e
cellularly,

and th e r e

intracellular
produce

is

at t i m e s .

It h a s b e e n s u g ­

organism may exist intra-

s o m e e v i d e n c e t h a t L.. m o n o c y t o g e n e s

is

Since the p r e v i o u s l y m e n t i o n e d o r g a n i s m s

a granulom atous

monocytogenes p o s s e s s e s

i n f l a m m a t i o n it m a y be p o s s i b l e t h a t L.
some

of t he

same c h a ra c te ristic s.

Work:

71
done i n G e r m a n y
c e r t a i n l y th e

s u p p o r t s t h i s t h e o r y to a c e r t a i n e x t e n t ,

r e a c t i o n p r o d u c e d in the t i s s u e s

cultures

and m o s t

was g ra n u lo ­

m atous in n a tu re .
T h e p o s s i b i l i t y t h a t th e
m a y be
lo o k e d .

a nonspecific
Further

s e e n in the t i s s u e

cultures

r e a c t i o n due to an i r r i t a n t m u s t not be o v e r ­

studies

p r o d u c e d in v itro is

response

m u s t be m a d e to d e t e r m i n e if t h i s l e s i o n

specific for L . m o n o c y to g e n e s.

72

P late

XIII.

A s e c t i o n of e x p l a n t
r o w s of p r o l i f e r a t e d
M a g . 400 x

a f t e r 24 h o u r s ' i n c u b a t i o n .
Two
v a s c u l a r e n d o th e liu m c a n be seen .

PLATE

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72

CP
P la te

XIII.

A s e c t i o n of e x p la n t a f t e r 24 h o u r s ' in c u b a t io n .
Two
r o w s of p r o l i f e r a t e d v a s c u l a r e n d o t h e liu m c a n b e s e e n .
M ag. 400x

PLATE

XIII

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P l a t e XIV.

High, p o w e r of a r e a m a r k e d in P l a t e

XIII.

M ag.

120Ox

PLATE

;

9

•

XIV

74

P l a t e XIV.

H igh p o w e r of a r e a m a r k e d in P l a t e

XIII.

Mfag. 120Ox

PLATE

75

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XV.

S e c t i o n of a t w o - d a y - o l d c u l t u r e .
in th e a r e a m a rk e d .
M ag. 400x

R eactio n

can be

seen

PLATE

ft

XV

<

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76

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M ag

culture.
40 0 x

Reaction

can

be

seen

PLATE

XV

*+**!< r

' 5*•# & C . * y*» LM •^ T
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78

P late

X V I.

S e c t i o n of a n
M ag. 400x

un i no c ilia te d

co n tro l

cu ltu re

tw o

days

old.

PLATE

XVI

79

I
II f 'jk

80

P late

X V II.

S e c tio n of t h r e e - d a y - o l d c u l t u r e .
in th e a r e a m a r k e d ,
M ag. 400x

R eactio n

can be

seen

PLATE

X V II

,- 'r

t o

W

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in

t h e

e f
a, r e a

t h r e e - clay - nl d
rn a r k

1

c u l t u r e

Vi a g

40 0 x

R e a c t i o n

c a n

b e

s e e n

PLATE

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82

Plate

XVIII.

H i g h p o w e r of s e c t i o n i n P l a t e XVII.
N o te t h e p r o l i f e r ­
a t i o n a n d i n f i l t r a t i o n of t h e e p i t h e l i o i d c e l l s i n to t h e n e ­
crotic tissu e .
M a g . 1200x

PLATE

XVIII

82

Hi gh p o w e r of s e c t h o n / n P
ation and in f iltr a tin n /fjH fe
crotic tissu e .
M arg .
00 x

XVII.
N o t e the p r o l i f e r e p i t h e l i o i d c e l l s in to th e n e ­
/

PLATE

83

XVIII

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r

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84

Plate

XIX.

S e c t i o n of f o u r - d a y - o l d c u l t u r e .
R eaction a r e a has en­
l a r g e d , i n v o l v i n g a r e g i o n a t t h e l e f t s i d e of t h e s e c t i o n .
M a g . 400x

PLATE

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XIX

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84

XIX

Se c t i
1a r g ^

m /j

of f f o a r - d a v - o l d

cal

re
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s i d e

h a s

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86

P la t e

XX.

H igh p o w e r of s e c t io n in P l a t e
M a g. IZOOx

X IX .

N o te in f iltr a tio n .

PLATE

87

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86

P la t e

XX.

H igh p o w e r of s e c t io n in P l a t e
M a g. 1 20 0 x

X IX .

N o te in filtr a t io n .

PLATE

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S e c t i o n of u n i n o c i l i a t e d c o n t r o l
M a g . 4 00 x

culture fo ur

days

old.

PLATE

XXI

89

90

P late

XXII.

S e c t i o n of s i x - d a y - o l d c u l t u r e .
size.
Note m a r k e d in f iltr a tio n .

A r e a i s of c o n s i d e r a b l e
M ag. 400x

PLATE

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92

P late

XXIII. High, p o w e r of s i x - d a y - o l d c u l t u r e .
Two c i r c u m s c r i b e d
blood v e s s e l s a r e shown w ith p r o l i f e r a t e d c e l l s .
Note
t h e l a r g e m o n o n u c l e a r c e l l s o n t h e p e r i p h e r y of t h e t i s ­
sue.
M a g . 1200x

PLATE

93

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94

Plate

XXIV. I n f e c t e d c u l t u r e s .
F igure

1.

S a m e s e c t i o n a s s h o w n i n P l a t e XXIII.
Note
t h e s l i g h t y e l l o w i s h t i n g e i n t h e c e l l s of t h e
upper left-hand co rn e r.
M a g . 10 5 0 x

F igure

2.

S ection s ta in e d w ith W e i g e r t - V a n G ie so n .
A
g r e e n e la s tic t i s s u e f i b e r c a n be s e e n aro u n d
t h e l u m e n of t h e v e s s e l .
M a g . 800x

PLATE

X X IV

Figure

1

F ig u re

2

96

P late

XXV.

S e c t i o n of u n i n o c u l a t e d c o n t r o l
400x

culture

six days

old.

M a g.

P L A T E XXV

97

98

P late

XXVI.

High, p o w e r

of s e c t i o n i n P l a t e

XXV.

Mag.

1200x

PLATE

XXVI

99

\

V
*.
>

■k

s .

SUMMARY AND CONCLUSIONS

1.

The double c o v e r s lip

of M axim ow w as u sed in a tis s u e

c u ltu r e stu d y of th e r e a c tio n of sp le n ic explants in o cu lated with
L . m o n o c y to g e n e s .
2.

h ied iu m

199* h om o lo g o u s p la sm a , and chicken em bryo

e x tr a c t did not d e p r e s s th e grow th of th is o rg a n ism after rem aining
in t h e s e f lu id s fo r s e v e n d a y s.
3.

A fte r 48 h o u r s in t is s u e cu ltu re the b a c te r ia could not

b e d e m o n str a te d in a r tific ia l m ed ia u n le ss the t is s u e s w ere r e fr ig ­
e r a te d at 4°C f o r s e v e r a l d a ys.
4.

O b se r v a tio n s of liv in g tis s u e

m o n o c y to g e n e s w as a lw a y s m o tile .

cu ltu res

r e v e a le d that L.

The o rg a n ism did not su p p ress

th e g row th oi: the t i s s u e .
5.

Stain ed w hole m ounts of the t is s u e cu ltu res r e v e a le d c e lls

w h ich co n ta in ed m any b iz a r r e n u c le a r fo r m s c h a r a c te r is tic of the
g r a n u lo m a s p ro d u ced in a d is e a s e of the new born, g ra n u lo m a to sis
in f anti s e p tic a .
6.

S e r ia l s e c t io n s of in fe c te d t is s u e cu ltu res

g ra n u lo m a to u s in fla m m a to r y r e a c tio n .
r e a c tio n a p p ea red to a r is e fr o m

The c e lls

v a s c u la r

100

r e v e a le d a

r e sp o n sib le fo r th is

sin u s endothelium .

This

10 1
re a c tio n was

s i m i l a r to t h a t d e s c r i b e d f o r g r a n u l o m a t o s i s inf anti-

septic a .
7.
s e e n in the

It i s

not kn ow n at t h i s t i m e

cultured tissue

it w a s p r o d u c e d as

was

s p e cific f o r L.

if the g r a n u l o m a t o u s
m o n o c y t o g e n e s or if

a r e s u l t of a n o n s p e c i f ic i r r i t a n t .

response

LITERA TU RE

CITED

A l l g o w e r , M . , a n d H. B l o c k .
1949.
The e f f e c t of t u b e r c l e b a c i l l i
on t h e m i g r a t i o n s of p h a g o c y t e s in v i t r o .
Am. Rev T u b e r c
59: 5 6 2 - 5 6 6 .
A r o n s o n , J . D.
in t i s s u e

1931.
Tbe s p e c i f i c cyto to xic a c t i o n of t u b e r c u l i n
culture.
J . Exp. Med. 54: 387-397.

B a k e r , L . E . , a n d A. C a r r e l .
1926.
A c t i o n on f i b r o b l a s t s of the
p r o t e i n f r a c t i o n of e m b r y o n i c t i s s u e e x t r a c t .
J. Exp. Med.
44: 3 8 7- 39 5 .
B a k e r , L . E . , a nd A. A. E b li n g .
1939.
A rtificial maintenance m e ­
d i a f o r c e l l and o r g a n c u l t i v a t i o n .
I. The c u l t i v a t i o n of
f i b r o b l a s t s i n a r t i f i c i a l and s e r u m l e s s m e d i a .
J. Exp. Med.
69: 3 6 5 - 3 7 8 .
Bloom,

W illiam .
1928.
H e m a t o l o g i a . 37:

O r i g i n and n a t u r e
1 -62.

of the m o n o c y t e .

Folia

Bloom,

W illiam .
1928a.
The f o r m a t i o n of a b s c e s s e s in a n i n f e c ­
t i o n w i t h B a c t e r i u m m o n o c y t o g e n e s . A r c h . P a t h . 6 : 993 1007.

Burn,

C. G.
1934.
Unidentified g r a m positive bacillus asso c ia te d
w i th m e n i n g o - e n c e p h a l i t i s . P r o c . Soc. Exp. Biol. & Med. 31:
1 09 5 -1 0 9 7 .

Burn,

C. G.
1935.
C h a r a c t e r i s t i c s of a new s p e c i e s of the genus
L i s t e r e l l a obtained f r o m human so urces.
J . B a c t . 30: 573-

__

Burn,

C. G.
1936.
C l i n i c a l and p a t h o l o g i c a l f e a t u r e s of an i n f e c ­
t i o n c a u se d by a new p a t h o g e n of the genus L i s t e r e l l a . Am.
J. Path.

12:

3 4 1- 34 8 .

B u r r o w s , M.
T.
1910.
T h e c u l t i v a t i o n of the t i s s u e s of the chick
e m b r y o o u t s i d e the b od y.
J . A m , Med. A s s o c , 55. 2057-2058

102

103
C arrel,

A.
1912.
ganism .
J.

The p e r m a n e n t life of t i s s u e s
Exp. Med. 15: 5 1 6- 528.

ou tsid e of the o r ­

C a r r e l , A..
1913.
A r t i f i c i a l a c t i v a t i o n of the g r o w t h in v it r o of
connective tis s u e .
J . Exp. Med. 17: 1 4 - 1 9 .
C a r r e l , A.
1913a.
N eu e U n t e r s u c h u n g e n iiber das S e lb sta nd ig e L e b e n
d e r G e w e b e und O r g a n e .
B e r l i n . Klin. W c h n s c h r . 50- 10971011.
C a r r e l , A . f and
and org a n s
1381 .

T. B u r r o w s .
1910,
C u l t i v a ti o n of adult t i s s u e s
o u t s i d e the body.
J . A m . Med. A s s o c . 55: 1379-

C o m a n , D. R.
1940.
C h e m o t a x i s of m o n o c y t e s c o n t r a s t e d with t ha t
of p o l y m o r p h o n u c l e a r l e u k o c y t e s and l y m p h o c y t e s .
Arch.
P a t h . 30: 8 9 6 - 9 0 1 .
Conway, E . A.
1938.
R e a c t i o n of l y m p h a t i c t i s s u e in e a r l y s t a g e s
of B a c t e r i u m m o n o c y t o g e n e s in fe c ti on .
A r c h . P a t h . 25: 200227.
Conway, E . A.
1939.
R e a c t i o n of l y m p h a t i c t i s s u e s of r a b b i t s to
r e p e a t e d i n j e c t i o n s of B a c t e r i u m m o n o c ytog e n e s . J. Inf. Dis.
64: 2 1 7 - 2 4 0 .
~
C u n n i n g h a m , R. S., F . R. Sabin, a nd C. A.. Doan.
1923.
The d i f f e r ­
e n t i a t i o n of two d i s t i n c t t y p e s of p h a g oc y ti c c e l l s i n the s p l e e n
of t h e r a b b i t .
P r o c . Soc. Exp. Biol.
Med. 21: 330-333.
D u l b e c c o , R.
1952.
P r o d u c t i o n of p l a q u e s i n m o n o l a y e r t i s s u e c u l ­
t u r e s of s i n g l e p a r t i c l e s of an a n i m a l v i r u s .
P r o c . Nat.
A c a d . S c i e n c e . 38: 7 4 7 -7 5 1 .
D u l b e c c o , R . , and M. Vogt.
1954.
P l a q u e f o r m a t i o n and i s o l a t i o n
of p u r e l i n e s w i th P o l i o m y e l i t i s v i r u s e s .
J . Exp. Med. 99.
16 7- 1 9 9 .
E rd m a n n , G., and J. P o te l.
1953.
L i s t e r i o s e d e r N e u g e bo ren en :
G r a n u l o m a t o s i s inf a n t i s e p t i c a.
Z e i t s c h r i f t f. Kinde rhe ilkunde
73:

1 1 3- 132.

104
Evans,

V. J . , a n d W. L. E a r l e .
1947.
The use of p e r f o r a t e d c e l ­
l o p h a n e f o r t h e g r o w t h of c e l l s in t i s s u e c u l t u r e .
J . Nat.
C a n c e r I n s t . 8 : 10 3-119.

Evans,

V. J . , W, R. E a r l e , K. K. S a nf o r d, J. E. Shannon, a nd H. K.
W altz.
1951.
The p r e p a r a t i o n and h andling of r e p l i c a t e t i s ­
sue c u ltu re s f o r quantitative studies.
J . Nat. C a n c e r In st .
11: 9 0 7 - 9 2 2 .

F i s c h e r , A..
1941.
The n a t u r e of the g r o w t h - p r o m o t i n g s u b s t a n c e s
i n the e m b r y o n i c t i s s u e j u i c e .
A cta. P h y s i o l . Scand. 3 : 54-70.
F i s c h e r , A.
1946.
B io lo gy of t i s s u e c e l l s , C o p e nh ag e n, Gyldendal.
i n M e t h o d s of T i s s u e C u l t u r e .
R. C. P a r k e r , 2nd Ed., P a u l
B . H o e b e r , New Y o r k , N. Y., p. 121.
F i s c h e r , A.., T. A .strup, G. E h r e n s u a r d , and V. O e h l e n s c h l a g e r .
G r o w t h of a n i m a l t i s s u e c e l l s in a r t i f i c i a l m e d i a .
Proc.
E x p . B i o l . & Med. 67: 40-46.

1948.
Soc.

Foldvari, F ra n c is .
19 32.
The conduc t of S p i r o c h e t a p a l l i d a i n t i s s u e
explantations.
A.m. J . Syph. G o n or . Ven. Dis. 16: 145- 154.
Foot,

N a t h a n C.
1925.
The e n d o t h e li a l p h a g o c yt e.
v ie w .
A n a t . R e c . 30: 15-51.

F o r b u s , W i l e y D.
of D i s e a s e .

A critical

re­

1943. R e a c t i o n to I n j u r y .
P a t h o l o g y f o r Students
W i l l i a m s a nd Wilkins Co., B a l t i m o r e .

F o r b u s , Wiley D.
1949. G r a n u l o m a t o u s In fl a m m at i o n :
Its N a t u r e ,
G e n e r a l P a t h o l o g i c a l S ig n if ic a n c e and C lin ic al C h a r a c t e r .
M o n o g r a p h i n A m e r i c a n L e c t u r e s in P a t h o l o g y .
53 pages.
C h a r l e s C. T h o m a s , S p r i n g f ie l d , I llin o is .
F o r k n e r , C. E .
1930.
The o r i g i n of the m o n o c y t e s in c e r t a i n ly mp h
n o d e s , t h e i r g e n e t i c r e l a t i o n to o t h e r c on ne ctiv e t i s s u e c e l l s .
J.

E x p.

Gey, G. O.
Am.

Med.

1933.
J.

52:

3 8 5- 40 1 .

An i m p r o v e d t e c h n i c f o r m a s s i v e t i s s u e

Cancer

17:

752-756.

culture.

105
G irard,

Kenneth F . ,

a nd E. G. D. M u r r a y .

1951.

L is te ria monocy-

a s ^■^ie c a u s e of d i s e a s e in m a n and a n i m a l s and its
r e l a t i o n s to i n f e c t i o u s m o n o n u c l e o s i s f r o m an e ti o lo g i c a l and
im m unological aspect.
A m . J , Med. S c ie n c e s 221: 243-352.
Gray,

hi. L .
1954.
L i s t e r i a m o n o c y t o g e n e s : A r e v i e w and colonial
dissociation.
Unpublished Ph.D. th esis.
M i ch i g a n State C o l ­
le g e.
120 numb, le a v e s .

G r a y , M. L . , C h i n t a m a n i Singh, and
F . Thorp, J r .
1955. A b o rtio n,
s t i l l b i r t h a n d d e a t h of young
of r a b b i t s in du c ed by L i s t e r i a
m o n o c y t o g e n e s . I. O c u l a r i n s t i l l a t i o n . I n P r e s s .
Gray,

M. L . , C h i n t a m a n i Singh, and F . T h o r p , J r .
1955a.
A b o r tio n,
s t i l l b i r t h and d e a t h of young
of r a b b i t s i n d uc ed by L i s t e r i a
m o n o c y t o g e n e s . II. O r a l e x p o s u r e .
In P r e s s .

G r a y , M. L . , H. J . S t a f s e t h , F . T h o r p , J r . , L. B. Sholl, and W. F .
Riley.
1948.
A ne w t e c h n i q u e f o r i s o l a t i n g L i s t e r e l l a e f r o m
t h e b ov in e b r a i n .
J . B a c t . 55: 471-476.
H a g e m a n n , U ., an d H. S im o n.
1953.
F ruchttod oder F ru h g e b u rt
d u r c h d i a p l a c e n t a r e L i s t e r i e n i n f e k t i o n . G e b u r t s h i l f e und
F r a u e n h e i l k u n d e 12: 1089-1095.
H a g e m a n n , U., H. S im on , a n d A. B i e n e n g r ab e r . 1953.
Die L i s t e r i o s e
der F ruhgeburten.
Z e n t r a l b l a t t f u r A l l e g e m e i n e P a t h o l o g i e 90:
1 7 - 23 .
Hahnefeld, H o rst.
1954.
E r g e b n i s s e von F i i t t e r u n g s u e r s u c h e n an
T i e r e n m i t L i s t e r i a m o n o c y t o g e n e s (Pinie) P r o p h y l a x e : Z e i t s c h r i f t f u r M i k r o b i o l o g i e , E p i d e m i o l o g i e , Hygiene und Sozialhygiene

4:

164- 169.

H a h n e f e l d , H . , a nd E . N i s o l k .
1954.
U b e r das V o r k o m m e n d e r L i s ­
t e r i o s e i n O s t m e c k l e n b u r g B e o b a c h t u n g e n b e i N e u g e b or en e n .
D e u t. G e s u n d h e i t s w .

5:

149-155.

H a r r i s o n , R. G.
1907.
O b s e r v a t i o n on the living developing n e r v e
fiber.
P r o c . Soc. E x p . Biol. & Med. 4: 140-143.

106
H e r z o g , A. J .
1938.
The p h a g o c y t i c a c t i v i ty of h u m a n l e u k o c y t e s
w i t h s p e c i a l r e f e r e n c e to t h e i r type and m a t u r i t y .
Am J
P a t h . 14: 5 9 5 - 6 0 4 .
^Jensen,

R u e , and D. R. M a c k e y .
1949.
L i s t e r e l lo sis in c a t tl e and
sheep.
J . A m . Vet. Med. A s s o c . 114: 4Z0-4Z4.

u l i a n e l l e , L . A.., a nd E l i z a b e t h k l o o re .
194Z.
P a t h o l o g i c a l ch an g e s
i n L i s t e r e l l a i n f e c t i o n p a r t i c u l a r l y of the eye .
Am. J. P a t h .
18: 813-8Z5.
K ast,

C. C ., a n d J . A. K o l m e r .
1933.
On the c u l ti v a ti o n of S p i r o c h e t a p a l l i d a in liv in g t i s s u e m e d i a .
Am. J . Syph. Gonor .
Ven. D i s . 17: 5Z9-53Z.

King, J . T . , B. S. G r e e n , a nd A. F . H e n s c h e l .
1938.
R e s p o n s e of
l e u k o c y t e s to c o l o n i e s of S t r e p t o c o c c i gro wing in t i s s u e c u l ­
tu r e m edia.
P r o c . Soc. Exp. Biol. & Med. 38: 81Z-815.
Lang, F . J.
19Z5.
The r e a c t i o n of lung t i s s u e to t u b e r c u l o u s i n f e c ­
tio n in vitro.
J . Inf. D i s . 37: 430-44Z.
Lang, F. J.
19Z6.
R ole of e n d o t h e l i u m in the p r o d u c t i o n of p o l y ­
b l a s t s ( m o n o n u c l e a r w a n d e r i n g c e lls ) i n i n f l a m m a t i o n .
A rc h.
P a t h . 1 :4 1 -6 3 .
Lang, F .

J.

19Z8.

Zur m onozyten-f r a g e .

F o l i a H a e m a t o l o g i a 36:

38 3 -3 8 9 .
L e w i s , M.

R.

1916.

S ea w a t e r a s a m e d i u m f o r t i s s u e

A n a t . R e c . 10:
Lewis,

cultures.

Z87-Z99.

M. R. 19Z0.
The f o r m a t i o n of v a c u o l e s due to B ac il lu s
t y p h o s u s i n c e l l s of t i s s u e c u l t u r e s of the i n t e s t i n e of the
chick e m b ry o .
J . E x p. M ed . 31: Z93-311.

L e w i s , M. R ., a nd W. H. L e w i s .
1911.
The c u l t i v a t i o n of t i s s u e s
f r o m c h i c k e m b r y o s in s o l u ti o n s of Na Cl, Ca Cl^, KC1 and
N a H C 0 3.

Anat.

Rec.

5:

Z77-Z94.

L e w i s , W. H. 19Z5.
The t r a n s f o r m a t i o n of m o n o n u c l e a r blood c e l l s
i n to m a c r o p h a g e s , e p i t h e l i o i d c e l l s and giant c e l l s .
Harvey
L ectures

Zl:

77-11Z.

107
L j u n g g r e n , C. A.
1897.
Von d e r F a h i g k e i t des Ha u te pith e l s , a a s s e r h a l b des O r g a n i s m u s se in le b e n zubehalten, mit B e ru c k s i c h t i g u n g d e r T r a n s p l a n t a t i o n . D e u t s c h . Z t s c h r f C hi r
47
608-615.
Loeb,

L.
190 Z.
On t h e g r o w t h of e p i t h e l i u m in a g a r and blood
s e r u m in t h e l i v i n g body.
J . Med. R e s . 8 : 109-115.

M a llo r y , F . B.
1898.
A histological
E x p . Med . 3: 6 11 -6 1 6.
M a llo ry , F . B.
1942.
P h i l ade l p h i a .

study of t yphoid f e v e r .

P a t h o l o g i c a l T e c h n iq u e.

M a x i m o w , A.. A.. 1924. T u b e r c u l o s i s
J . Inf. D i s . 34: 54 9-5 8 4.

J.

E. B. S a u n d e r s Go.,

of m a m m a l i a n t i s s u e in v it r o.

M a x i m o w , A.. A.
1924a. R e l a t i o n of blood c e l l s to c on n e c ti v e t i s s u e s
and endothelium .
P h y s . Rev. 4: 533-563.
M a x i m o w , A.
bryos.

A.
1925. T i s s u e c u l t u r e s of young m a m m a l i a n e m ­
C a r n e g i e I n s t . W a s h . , C o n t r i b . E m b r y o l . 16: 47-113.

M a x i m o w , A.. A.
1927. D e v e l o p m e n t of n o n - g r a n u l a r l e u c o c y t e s
( l y m p h o c y t e s a nd m o n o c y t e s ) into p o l y b l a s t s ( m a c r o p h ag e s)
a n d f i b r o b l a s t s in v i t r o .
P r o c . S o c . Exp. Biol. & Med. 24:
570-572.
M c C u t c h e o n , M ., a n d H. M. Dixon.
1936.
Chemotropic reactions
of p o l y m o r p h o n u c l e a r l e u c o c y t e s to v a r i o u s m i c r o - o r g a n i s m s .
Arch.

Path.

21:

7 4 9 -7 5 5.

Me J unkin, F .
A. 1926. R o s e t t e and g r a n u l e - h y a l i n e p h a go cy te s in
t i s s u e c u l t u r e s s t a i n e d s u p r a v i t a l l y with N e u t r a l Red.
Proc.
Soc. E x p. B i o l. & Med. 24: 84-86.
M o r g a n , J . F . , H. J . M o r t o n , a nd R. C. P a r k e r .
1950. N a t r i t i o n
of a n i m a l c e l l s i n t i s s u e c u l t u r e .
I. Initial s tu d i e s on a s y n ­
thetic m e d iu m .
P r o c . Soc. Exp. Biol.
StMed. 73: 1-8.
M u r r a y , E . G. D.
1955.
A characterization
ofl i s t e r i o s i s in m a n
and o t h e r a n i m a l s .
The C a n a d i a n Med. A.ssoc. J o u r . 72. 99103.

108
M u r r a y , E . G. D., R. A. Webb, and M. B. R. Swann.
1926.
A dis­
e a s e of r a b b i t s c h a r a c t e r i z e d by a l a r g e m o n o n u c l e a r l e u c o c y t o s i s c a u s e d by h i t h e r t o u n d e s c r i b e d b a c i l l u s , B a c t e r i u m
m o n o c y t o g e n e s . J . P a t h , and B a c t . 29: 40 7-439.
N y f e l d t , A.
rend.

1929. E t i o l o g i e de l a m o n o n u c l e o s e
Soc. d e . Biol. 101: 590-592.

i n f e c t i e u s e . Compt.

N y f e l d t , A..
1932. K l i m s c h e und E x p e r i m e n t e l l e U n t e r suchunge n u b e r
die M o n o n u c l e o s i s i n f e c t i o s a .
F o l i a H e m a t o l o g i a 47: 1-144.
O s e b o l d , J o h n W., a nd T a k a Inouye.
1955.
P a t h o g e n e s i s of L i s t e r i a
m o n o c y to g e n e s infections in n atu ral ho sts.
I. Rabbit s tu d ie s .
J . Inf. D i s . 95: 5 2 - 6 6 .
O s e b o l d , J o h n W., a n d T a k a Inouye. 1955a.
Pathogenesis
ofL i s t e r i a
m ono cy to g en es infection in n a tu ra l h o s ts .
II. Sheep s t u d i e s .
J . Inf. D i s. 95: 6 7-7 8.
Perry,

Pons,

W. L . M. 1948.
The c u l t i v a t i o n
tis s u e culture.
J . P a t h . B a c t . 60:

of T r e p o n e m a p a l l i d u m
339-342.

in

C. A., a n d L. A. J u l i a n e l l e .
1939.
I s o l a t i o n of L i s t e r e l l a
m o n o c y t o g e n e s f r o m i nf e c ti ou s m o n o n u c l e o s i s .
P r o c . Soc.
E x p. B i o l. & Med. 40: 360-362.

P o rter,

K. R ., A. C l a u d e , and E . F . F u l l a m .
1945.
A study of t i s ­
s ue c u l t u r e c e l l s by e l e c t r o n m i c r o s c o p y .
Methods and p r e ­
lim in ary observations.
J . Exp. Med. 81: 233-246.

P o tel,

J . 1952.
Zur G ranulom atosis
Abt. I. O r i g . 158: 3 2 9 -3 32 .

Potel,

J . 1953.
X tiologie d e r G ra n u lo m a to s is Infantiseptica.
Z e i t . M a r t i n L u t h e r U n i v e r . 2: 341-364.

Potel,

J. 1954.
Zur epidem iologie der L iste rio se der Neugeborenen
(G ran u lo m ato sis infantiseptica).
D e u t s . G e s u n d h e i t s w . 3: 92-

inf a n ti septic a.

Z e n t r . Bakt.

W is se n.

95.
R e z z e s i , F . D.
1933.
L a in f e z i o n e
e il p r o b l e m a d e l m o n o c i t o .

da " B a c te riu m monocytogenes"
F o l i a H e m a t o l o g i a 14. 239 314.

109
K ie s s , H. J.
1953.
Z ur P a th o lo g isc h e n A natom ie d er K indlichen
L is te r io s is .
K inder a r z tlic h e P r a x is .
W isse n sc h a ftlic h e r
V erh a n d lu n g sb er 1cht d er K m d erarztetagu n g d er jDeuichen
D e m o k r a tisc h e n Republik in L e ip z ig 24. b iz 26 A pril pp

92- 100.
R ob b in s, F . G ., T . H. W e lle r , and J. F . E n d e r s.
1952.
II. The
p r o p a g a tio n of th e P o lio m y e litis v ir u s e s in r o lle r -tu b e c u l­
t u r e s of v a r io u s hum an t i s s u e s .
J. Im m unol. 69: 6 7 3 -6 9 4 .
R oux, W.
1923.
M eine E n tw ick lu n g sm ech a n isch e m ethodik.
In:
A b d erh o ld en , E ., Handbuch der b io lo g is c h e n A rb eitsm eth od en .
B e r lin , U rban and S ch w arzen b erg , Abt. 5 T e il 3 A , 539 -6 16.
Sabin, F . R ., C. A . D oan, and R. S. Cunningham.
1925.
D isc r im in a ­
tio n of tw o ty p e s of p h a gocytic c e l l s in the con n ective t is s u e s
b y th e su p r a v ita l te ch n iq u e.
Gontr. to E m bry. C arnegie
In st. 16: 1 2 5 -1 6 2 .
S an ford , K. K ., W. R. E a r le , and G. D. L ik e ly .
1948.
The grow th
in v itr o o f s in g le is o la te d t is s u e c e l l s .
J. Nat. C ancer Inst.
9: 2 2 9 -2 4 6 .
S ch m id t, V ., and A., N y fe ld t.
1938.
U eber M on on u cleosis in fe c tio sa
und M e n in g o e n c e p h a litis.
A cta O to-laryn g. 26: 680-68 8.
S e e lig e r , H .f and G. L in s e n m e ie r .
1953.
D ie lis t e r io s e und ihre
E r r e g e r (L i s t e r i o s e m o n o c y to g e n e s). Z e itsc h r . f . H ygiene,
136:

3 3 5 -3 7 8 .

S im m s, H . S.

1936.

The e f f e c t s of p h y sio lo g ic a l agents on adult

t i s s u e s in v it r o .

S c ie n c e

83: 4 1 8 -4 1 9 .

S im m s, H. S ., and M. S a n d e r s.
1942.
U se of seru m u ltra filtra te
in t i s s u e c u ltu r e s fo r studying d ep o sitio n of fa t and fo r prop
a g a tio n of v ir u s e s .
A r c h . P a th . 33: 6 1 9 -6 3 5 .
S im m s, H. S ., and N. P . S tillm a n .
1937.
Substances affectin g adult
t i s s u e in v itr o .
I. The stim u la tin g a ction of tr y p sin on f r e s h
adult t i s s u e .

J . G en. P h y s . 20: 6 0 3 -6 1 9 .

1 10
Sim m s,

H. S., a nd N. P . S t i l l m a n .
1937a.
S u b s t a n c e s affecting
adult tis s u e m vitro.
II. A g r o w t h i n h i b i t o r in adult t i s s u e .
J . G e n . P h y s . 20: 6 2 1-629 .

S i m o n , H.
1953.
Ube r die L i s t e r i e n e n z e p h a l i t i s .
u. P a t h . A n a t . 90: 35 3- 359.

Zent. A.llg. P a t h ,

S m i t h , D. T . , H. S. W i l l i s , and M. R. L e w i s .
1922.
The b e h a v i o r
of c u l t u r e s of c h i c k e m b r y o t i s s u e c ontaining a via n t u b e r c l e
bacilli.
A m . Rev.
T u b e r c . 6 : 21-38.
S m y th , H. F .
1915. The i n flu e nc e of b a c t e r i a upon the d e v e l o p m e n t
of t i s s u e s i n v i t r o . Z e n t r . B a k t . Abt. I. O ri g.
76: 12-23.
S m y t h , H. F .
1916. The r e a c t i o n s b e t w e e n b a c t e r i a and a n i m a l
t i s s u e s u n d e r c o n d i t i o n s of a r t i f i c i a l c ultiv atio n .
J. Exp.
M e d . 23: 26 5- 2 8 3 .
Taim enberg, J.
1929.
I. U b e r die E n t w i c k l u n g s p o t e n z e n d e r F i b r o b l a s t e n i n d e r G e w e b e k u l t u r . II. Das V e r h a l t e n von F i b r o b l a s t e n r e i n k u i t u r e n b e i b a k t e r i e l l e r Infektion.
A r c h . E x p e r.
Z e l l f o r s h . 9: 5 2 8 -5 6 8 .
V o g e l a a r , J . P . M., a n d E. E r l i c h m a n ,
1933.
A feeding solution
f o r c u l t u r e s of h u m a n f i b r o b l a s t s .
A m. J. C a n c e r 18: 28-38.
W allbach, G u n ter.
1934.
Sur l a m o n o c y t o s e i n f e c t i e u s e du la p in
p a r l e b a c i l l e m o n o c y t o g e n e . A r c h . d ’Anat. M i c r o . 30: 275294.
Webb,

R. A.
1943.
L i s t e r e l l a monocytogenes isolated fro m
of i n f e c t i o u s m o n o n u c l e o s i s .
L a n c e t 2: 5-10.

a case

Y o u n g n e r , J. S.
1954.
M onolayer tis s u e cultures.
I. P r e p a r a t i o n
a n d s t a n d a r d i z a t i o n of s u s p e n s i o n of t r y p s i n - d i s p e r s e d monkey
kidney c e lls.
P r o c . Soc. Exp. Biol. & Med. 85: 202-205.
Y o u n g n e r , J . S.
1954a.
M onolayer tis s u e cu ltu res.
II. P o l i o m y e ­
l i t i s v i r u s a s s a y i n r o l l e r - t u b e c u l t u r e s of t r y p s i n - d i s p e r s e d
m onkey kidney.
P r o c . Soc. E xp. Biol. & Med. 85. 527-530.