AN

HI S T 0 CHE M I C A L A N D
RELATION

BETWEEN

OF A G I N G
LESION

MODPHOLOCICA L INVESTIGATION
THE

GROWTH-PROHOTING

TETRAHYMENA
INDUCTION

GELEII

IN T H E

OF

THE

PROPERTIES

(STRAIN

W)

AND

CHORIOALEANT01S

By
Frederick- O t t o

AN

Submitted
State

to t h e
College

in Par t i a l

School

MarzIce

ABSTRACT

of G r a d u a t e

Studies

of

Michigan

Sciences

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

and

Applied

Fulfillment

the

Requirements

for

DOCTOR

the

OF

of

Degree

of

PHILOSOPHY

Department of Zoology

1953

ABSTRAC T

A

semi-quantitative

distribution
dase,

of

D N A , fat,

(strain

W)

throughout

then

conducted

these

various

horn

embryos.

istics

of

Alkaline

tion,
in

maximum
for
mins

in

any

correlation

and
of

The

th e
T.

those

to t h e

decreased

geleii
of

12-14-day

lesions

embryonic

was

in

a

tryptone

lipase

and

was

media

activity

activity and

between

fat

the

various

obtained.

tended

deposition

the

inoculation
White

Leg­

character­

oil,

W were
strain

suspending

media.

in T_. g e l e i i W
cultures;

glycogen

acid

deposi­

phospholipid deposition,
cyclic

Th e

but

was

at

a

No p o s i t i v e r e a c t i o n
addition

alkaline
to

presence
of

geleii

by c r o t o n

cultures.

inhibited
but

to

in 2 3 3 - h o u r

and

the

pedigreed

cultures;

inter­

properties

activity

activity was

3G0-hour

or u r a t e s

and

geleii

investigation, was

morphological

induced

144-hour

DN A

old

the
peroxi-

72-hour

induced by

maximum

cultures;

144 and

and

grafts

and

at

An

VJ s u b s e q u e n t

lesions

phosphatase

lipase

phosphatase

of

at

growth-promoting

histochemical

cultures.

peroxidase

made

to a s c e r t a i n

activity,

72,

was

cultures.

in 4 3 2 - h o u r

504-hour

media

of

lipase,

in T e t r a h y m e n a

the

in t r y p t o n e

phosphatase

phosphatase,

and urates

chorioallantoic
with

determination

of

lymphomatosis,

reared

life

chorioallantois

compared
12

the

suspensions

the

acid

in v a r i o u s

materials

onto

and

glycogen,

reared

vals

of

alkaline

histochemical

of

vita­

phosphatase

increase

DNA

of g l y c o g e n .

and

and a cid
Diffusion

complicated
acid

th e

phosphatase,

attributable
catalyzed
line
gen

histochemical

to

acted

inhibitory
The

physiology

of

but

alkaline

and

the

role
Td

geleii

geleii

relationship
W and

the

existed

extent

chorioallantoic
these

aged

growth

Td

which

for
the

maximal

while

the

Fat

the

s t i m u l a t i o n of

enzyme

salt

of

cell

geleii

solutions

at

A

and

in t h e
pos­

direct

of

size

a

Td
of

maximum

gelei i
induced
in

epithelial

play

an

important

W cultures

tissues

of

induced

th e

produced a

tissues.

synthesis

solution,

culture

may

membrane

solutions

pronounced

synthesis

mesodermal

metabolism

aegree.

the

counter

phosphatase

induction

Ringer's

epithelial

salt

also

glyco­

determined,

the

proliferation and

T.

of

in th e

DNA

lesion

of

was

its

in c a r b o h y d r a t e

some

and

increased alka­

substances

depo s i t i o n was

fat

or

Magnesium

across

of

age

former

on al k a l i n e

of

to

alkaline

deposition

inorganic

study

proliferation.
in t h e

the

definitely

role

in a r e a s

that

inorganic

be

exception

W and

suggested

the

materials

properties

lesion.

both

be c o n c e r n e d w i t h

epithelial

proliferation

allantois
nounced

of

the

vitamins

play a

between

geleii

in c e l l u l a r

may

for

fat.

W c o u l d not

of

magnesium

ana

of

W suspensions,

growth-promoting

of

investigated

may

inocula utilized

sessed

of

nutrient

chorioallantois, with

role

the

phosphatase

T.

activity

effect
of

of

diffusion

deposition

ph os ph at a. se

transfer

acid

other

th e

reaction

Calcium and

phosphatase

activity.

All

negative

complete

decreased

the

while

a

a

end-products.

and acid
bu t

and

localization

Alkaline

chorio-

more

pro­

and acid

3
phosphatase
nutrient
lesions

and

materials
while

metabolism

of

physiological
No

agent.

could

all

across

alkaline

saline

appeared
the

proteins.

reduced

its

or m o r p h o l o g i c a l

be utilized

for

to a i d

vascular

phosphatase

fibrillar

histochemical

lesion

lipase

also

in t h e

membranes
appeared

Suspension

of

lesion-inducing
characteristic

identification

of

transfer
of

th e

to a i d
T.

of

induced
in

th e

gele i i 'A! -in

properties.
of

the

the

induced

induction

AN

HISTOCHEMICAL AND
R E L A T I O N . BE TW EEN
OF

AGING

LESION

MORPHOLOGICAL
THE

GROWTH-PROMOTING

TETRAHYMENA
INDUCTION

INVESTIGATION

GELEII

IN T H E

OF

THE

PROPERTIES

(STRAIN

W)

AND

CHOPIOALLANTOI3

By
Frederick

Otto

Marzke

A THESIS

Submitted
State

to

th e

College

in Partial

School
of

of G r a d u a t e

Agriculture

Fulfillment
for

DOCTOR

the

of

Studies

and Applied
th e

Degree

of

of Zoology

1953

Michigan

Sciences

Requirements

OF P H I L O S O P H Y

Department

of

ProQuest Number: 10008375

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F r e d erick Otto
candidate
Doctor

Final

examination’

for
of

October

Marzke

th e

degree

of

Philosophy

1 'j

, 1953,

2 i 0 0 p.m. . R o o m 404,

Natural Science
Dissertation:

Outline
Major
Minor

of

Studies:

subject:
subjects:

Biographical
Bo rn ,

An Histochemical and Morphological Investi­
g a t i o n of t h e R e l a t i o n b e t w e e n t h e G r o w t h p r o m o t i n g P r o p e r t i e s of A g i n g T e t r a h y m e n a
g e l e i i ( s t r a i n w) a n d L e s i o n I n d u c t i o n in
the Chorioallantois

July

Zoology
Physiology,

Ite ms :
23,

Undergraduate

1 913 ,

M e m b e r of
Society

Lansing,

Studies,

Gradu a t e Studies,
1951-53
Experience:

Entomology

Michigan

Michigan

Michigan

State

State

College,

College,

1938-42

194^-47,

’

cont.

M e m b e r U n i t e d S t a t e s A rm y, 1 9 4 2 - 4 A , E n t o ­
m o l o g i s t , U n i t e d S t a t e s D e p a r t m e n t of A g r i ­
culture, Savannah, Georgia, 1947-51

Phi K a p p a P h i ,
of A m e r i c a

Society

of

S i g m a Xi,

Entomological

A C K N O W L E D G E NTS

Th e
Dr.

author

R i c h a r d A.

stant

wishes
Fennell

encouragement
The

author

Photographer,

to

his

sincere

thanks

for

hi s

guidance,

interest

during

the

course

this

is a l s o

indebted

Agricultural

photomicrographs.

express

to

of

Mr.

Experiment

P.

G.

Station,

to

and

con­

investigation.
Coleman,
for

the

T AB LE OF CON TE NTS

I N T R O D U C T I O N ............................................... 1
MATERIALS AND

METHODS

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

R E S U L T S ..............................................
1.

Histochemical

Properties

of T.

3
.

8

g e l e ii W

Alkaline Phosphatase
L o c a l i z a t i o n a n d variation with age
of c u l t u r e .................................. 8
E f f e c t of t h e a d d i t i o n of v i t a m i n s
to c u l t u r e m e d i u m
.
9
E f f e c t of th e a d d i t i o n of t h e m a g ­
n e s i u m or c a l c i u m i on to the
................. 1 7
c u l t u r e medium.
S p e c i f i c i t y of a l k a l i n e p h o s p h a t a s e
activity. .
18
D i f f u s i o n ....................................19
A c i d Phosphatase
Localization a n d variation with age
of c u l t u r e ................................. 2 0
E f f e c t of t h e a d d i t i o n of t h e m a g ­
n e s i u m o r c a l c i u m i o n to th e
c u l t u r e m e d i u m ....................... . 2 5
D i f f u s i o n ................................. . 2 9
Lipase
Localization and
vari a t i o n with age
of c u l t u r e .........................
39
E f f e c t of the a d d i t i o n of t h e m a g ­
n e s i u m or c a l c i u m i o n t o th e
c u l t u r e m e d i u m ......................... 30
D i f f u s i o n ....................................30

lycogen
Localization and variation with age
of c u l t u r e ........................
35
Effect of the addition of the mag­
nesium or calcium ion to the
culture m e d i u m ...................... 35
D i f f u s i o n ............................... 35

TABLE OF CONTEN TS

(Cont.)

Fat
L o c a l i z a t i o n a n d v a r i a t i o n w i t h age
of c u l t u r e ..........................
E f f e c t of t h e a d d i t i o n of t h e m a g ­
n e s i u m or c a l c i u m io n to t h e
culture medium
.....................
D i f f u s i o n ...............................

39
40

Phospholipids
Lo c a l i z a t i o n a n d v a riation with age
of c u l t u r e
..........................

40

39

Desoxyribonucleic Acid
Localization and variation with age
of c u l t u r e ..........................
E f f e c t of th e a d d i t i o n of the m a g ­
n e s i u m or c a l c i u m i o n t o t h e
culture medium
.....................
D i f f u s i o n ...............................
R e l a t i o n b e t w e e n i n t e n s i t y of
s t a i n i n g r e a c t i o n a n d s i z e of
nuclei.
............................
Peroxidase

.

43
44

44
47

U r a t e s .................................
V a r i a t i o n in S i z e
and Fixation

2.

40

47

I n t e r r e l a t i o n s h i p of A c t i v i t y T r e n d s
for Enzyme A c t i o n a nd Fat a n d
G l y c o g e n D e p o s i t i o n ..................

50

Induction

in

the

Cell

47

wi t h Age
.............

Lesion

of

.

Chorioallantois

F r e q u e n c y of l e s i o n i n d u c t i o n ..........
S i z e o f l e s i o n i n d u c e d ..................
General morphological characteristics
of i n d u c e d l e s i o n s .......................
M o r p h o l o g i c a l c h a r a c t e r i s t i c s of a n
i n d i v i d u a l l e s i o n i n d u c e d b y T.
g e l e i i W ....................................
M o r p h o l o g i c a l c h a r a c t e r i s t i c s of
embryonic grafts
.......................
E p i t h e l i a l nests
..........................
Inclusion bodies
..........................
H i s t o c h e m i c a l p r o p e r t i e s of i n d u c e d
l e s i o n s ....................................

50
55
58

72
73
74
75
77

TA B L E OF CONTENTS

(Cont.)

Section
IV

V
VI

DISCUSSION
1.

Histochemical

3.

Lesion

induction

S U M M A R Y ............. .
LITERATURE

properties

CITED

in

th e

of

T.

geleii

W

.

80

chorioallantois

.

87

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

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

. . . . . .

95
100

LIST OF TEXT-FIGURES
Text-f igure
I

II

III

IV

V

VI

VII

VIII

Relation between age and alkaline phospha­
tase activity in T_. geleii W. (Organisms
suspended in physiological saline). . . .

10

Relation between age, suspension in
physiological saline and the histochemical
properties of T. geleii W reared in
vitamin-enriched tryptone ................

12

Relation between age and the histochemical
properties of
geleii W reared in
tryptone. (Organisms suspended in
physiological saline) ....................

22

Relation between age and acid phosphatase
activity in TL R-eleii W (Organisms sus­
pended in physiological saline) .........

24

Relation between age, suspension in
physiological saline and acid phospha­
tase activity in T. geleii W .............

27

Relation between age and lipase activity in
Z* Se
W (Organisms suspended in
physiological saline) ....................

31

Relation between age of culture and the
histochemical properties of T. geleii W
reared in t r y p t o n e ...............

33

Relation between a^e and glycogen deposition
in T. geleii W. (Organisms suspended in
physiological saline) . . . . . . . . . .

37

IX

Relation between age and desoxyribonucleic
acid activity in T_, geleii U V ......
41

X

Relation between age and desoxyribonucleic
acid activity in T\ geleii W (Organisms
suspended in physiological saline)
...

45

LIST OF TE XT -FI G U R E S

(Cont.)

Text»figure
XI

XII

XIII

XIV

R e l a t i o n b e t w e e n a g e, h i s t o c h e m i c a l t e c h n i q u e
a n d l e n g t h of
g e l e i i W r e a r e d in
v i t a m i n - e n r i c h e d t r y p t o n e ..................

48

Relation between age
a n d the h i s t o c h e m i c a l
p r o p e r t i e s of T\ g Q l eii W s u b s e q u e n t to
th e a d d i t i o n of C a C l g or M g C l g to t h e
c u l t u r e m e d i u m ( O r g a n i s m s s u s p e n d e d in
p h y s i o l o g i c a l s a l i n e ) .......................

53

Relation b e t w e e n age
a n d the h i s t o c h e m i c a l
p r o p e r t i e s of T\ g e l e ii W r e a r e d in
v i t a m i n a n d magneslum-^cJhloride-enriched
t r yptone (Organisms s u spended in p h y s i ­
.............
ological saline).

5B

R e l a t i o n b e t w e e n age
a n d the h i s t o c h e m i c a l
p r o p e r t i e s of T. g e l eii W r e a r e d in
vitamin and calcium chloride-enriched
t r y p t o n e (Organisms s u s p e n d e d in p h y s i ­
o l o g i c a l s a l i n e ) ...............................-

58

LIST OF TABLES

Table
I

II

A l k a l i n e P h o s p h a t a s e A c t i v i t y in S p e c i m e n s
Z* ^ Q l e i i W R e a r e d i n T r y p t o n e a n d V i t a m i n e n r i c h e d T r y p t o n e .......................

14

C h o r i o a l l a n t o i c Lesions I n d u c e d by S p e c i m e n s
Z* g e l e i i W a n d o t h e r I n o c u l a .............

SI

LIST OF PLATES
Plate
I

II

Alkaline phosphatase activity in specimens of
geleii W suspended in culture medium . . .

103

Fig. 1.

24-hour cells reared in tryptone;

Fig. 2.

144-hour cells reared in tryptone;

Fig.

3.

216-hour cells reared in tryptone;

Fig.

4.

432-hour cells reared in tryptone;

Fig.

5.

288-hour cells reared in tryptone
fortified with GaClg i

Fig.

6.

388-hour cells reared in tryptone
fortified with CaClg (control);

Fig.

7.

288-hour c’ells reared in tryptone
fortified with MgCl 2 ; and

Fig.

8.

288-hour cells reared in tryptone
fortified with MgClg and exposed to
an increased amount of MgClg in the
phosphatase buffer.

Alkaline phosphatase activity in specimens of
i* geleii W suspended in culture medium . . .
Fig. 9.

105

288-hour cells reared in vitaminenriched tryptone;

Fig. 10.

288-hour cells reared in vitamin
and magnesium chloride-enriched
tryptone;

Fig.

288-hour cells reared in magnesium chloride-enriched tryptone;

11.

Fig. 12.

73-hour cells reared in the initial
riboflavin-enriched tryptone;

Fig. 13.

Maximum phosphatase activity after
first transfer to vitamin-enriched
tryptone, 216-hour cells;

LIST OF PLATES

(Cont.)

Plate

III

Fig. 14.

72-hour cells from 2nd transfer to
vitamin-enriched tryptone; and

Fig.

360-hour cells from 2nd transfer to
vitamin-enriched tryptone.

Alkaline phosphatase activity in unwashed specimens
X* gQleii W from 288-hour cultures after a
20-minute exposure to various enzyme
inhibitors......................................... 107
Fig.

IV

15.

16.

0.01M sodium arsenate (specimens
reared in vitamin-enriched
tryp ton e);

Fig. 17.

0.01M sodium arsenate (specimens
reared in tryptone);

Fig. 18.

0.01M sodium arsenate (specimens
reared in magnesium chlorideenriched tryptone);

Fig. 19.

0.01M sodium arsenate (specimens
reared in magnesium chloride and
vitamin-enriched tryptone);

Fig. 20.

0.02M semicarbazide (specimens
reared in magnesium chloride and
vitamin-enriched tryptone;

Fig. 21.

0.025M oxidized glutathione (speci­
mens reared in magnesium chloride
and vitamin-enriched tryptone); and

Fig. 22.

0.025M reduced glutathione (speci­
mens reared in magnesium chloride
and vitamin-enriched tryptone)

Histochemical reactions in specimens of
T. geleii W ......................................... 109
Fig. 23.

Acid phosphatase activity in 73-hour
cells reared in tryptone;

Fig. 24.

acid phosphatase activity in 73-hour
cells reared in vitamin-enriched
tryptone;

LIST OF PLATES (Cont.)
Plate

V

VI

Fig.

25.

acid phosphatase
238-hour cells

a c t i v i t y in
r e a r e d in t r y p t o n e ;

Fig.

28.

a c i d p h o s p h a t a s e a c t i v i t y in 3 8 8 =
h o u r c e l l s r e a r e d in v i t a m i n - e n r i c h e d
tryptone;

F ig .

27.

l i p a s e a c t i v i t y in 3 8 8 - h o u r
r e a r e d in t r y p t o n e ;

Fig.

28.

D N A a c t i v i t y in 2 8 8 - h o u r
in t r y p t o n e ; a n d

Fig.

29.

a l k a l i n e p h o s p h a t a s e a c t i v i t y in 1 4 4 h o u r c e l l s r e a r e d in t r y p t o n e .

cells

cells

reared,

H i s t o c h e m i c a l r e a c t i o n s in s p e c i m e n s of T. g e l e i i
.....................
s u s p e n d e d in culture m e d i u m
Fig.

30.

G l y c o g e n d e p o s i t i o n in 7 2 - h o u r
c e l l s r e a r e d in t r y p t o n e ;

Fig.

31.

g l y c o g e n d e p o s i t i o n in 2 8 8 - h o u r
c e l l s r e a r e d in t r y p t o n e ;

Fig.

32.

g l y c o g e n d e p o s i t i o n in 7 2 - h o u r
c e l l s r e a r e d in v i t a m i n enriched tryptone;

Fig.

33.

g l y c o g e n d e p o s i t i o n in 2 3 8 - h o u r
c e l l s r e a r e d in v i t a m i n enriched tryptone;

Fig.

34.

p h o s p h o l i p i d d e p o s i t i o n in
c e l l s r e a r e d in v i t a m i n enriched tryptone; a n d

73-hour

Fig.

35.

p h o s p h o l i p i d d e p o s i t i o n in
c e l l s r e a r e d in v i t a m i n e nric h e d tryptone.

504-hour

Chorioallantoic lesions
p e n s i o n s of T_. g e l e i i
Fig.

36.

W
Ill

*

induced by saline sus­
W ................................1 1 3

C r o s s - s e c t i o n of c h o r i o a l l a n t o i c
l e s i o n s h o w i n g e p i t h e l i a l n es t
f o r m a t i o n induced by 1 8 -aay old
T . geleii W ;

LIST OF PLATFS

(Cont.)

Plate

VII

Fig.

37.

c r o s s - s e c t i o n of e p i t h e l i a l
nest induced by 1 8-day old
T . gelei i W ;

Fig.

38.

l o c a l i z a t i o n of fat in h y p e r ­
p l a s t i c d o r s a l e p i t h e l i u m of
chorioallantoic lesion induced
b y 1 4 - d a y o l d T_. g e l e i i W ;

Fig.

39.

i n c l u s i o n b o d i e s in e p i t h e l i u m
of c h o r i o a l l a n t o i s i n d u c e d b y
3 - d a y o l d Id g e l e i i W;

Fig.

40.

inclusion
inocula

b o d i e s in u n a b s o r b e d
of 3 - d a y o l d T_. g e l e i i

Fig.

41.

a l k a l i n e p h o s p h a t a s e a c t i v i t y in
peripheral mesodermal fibro­
b l a s t s of c h o r i o a l l a n t o i c
l e s i o n i n d u c e d by 1 2 - day old
Td g e l e i i W; a n d

Fig.

42.

a c i d p h o s p h a t a s e d i f f u s i o n in
chorioallantoic lesion induced
b y 9 “ d a y o l d Id, g e l e i i Vv
e x p o s e d to 5 0 ° C. f o r o n e - h a l f
hour.

Chorioallantoic lesions
p e n s i o n s of Id g e l e i i

W;

i n d u c e d by s a l i n e s u s ­
W .......................

Fig.

43.

C r o s s - s e c t i o n of v e n t r a l s u r f a c e
of c h o r i o a l l a n t o i c l e s i o n i n d u c e d
b y 3 - d a y o l d T. g e l e i i W s h o w i n g
f o r m a t i o n of e p i t h e l i a l n e s t s ;

Fi g.

44.

f a t d e p o s i t i o n in d o r s a l e p i t h e l i u m
of c h o r i o a l l a n t o i c l e s i o n induced
b y 1 4 - d a y o l d T. g e l e i i W; a n d

Fig.

45.

f a t d e p o s i t i o n in e p i t h e l i a l n e s t s
of c h o r i o a l l a n t o i c l e s i o n i n d u c e d
b y 1 4 - d a y o l d T. g e l e i i W

115

INTRODUCTION
Robertson (1923) maintained that protozoa released autocatalytic materials which stimulated growth.

Mast and Pace

(1946) demonstrated that cultures of Chilomonas paramecium
produced a substance which was not a B vitamin but which in
low concentrations accelerated growth but in high concentra­
tions retarded it.

Other living cells,

including those of

higher animals, have also been found to elaborate substances
wh ich facilitate growth.

Duran-Reynals

(1929) demonstrated

that the addition of testicular extracts enhanced the patho­
genicity of a neurovirus.

Margoliash, Tennenbaum and Doljanski

(1948) noted that the reduction in growth-stimulation by the
chicken heart subsequent to dialysis was not accompanied by
the appearance of growth substances in the dialyzate itself.
Webb and Loofbourow (1947) found that the supernatant from
ultra-violet damaged cells exhibited higher concentrations

of

biotin, folic acid, nicotinic acid, pantothenic acid and
riboflavin and possessed greater growth-promoting properties
than supernatants from undamaged cells.
Errera,

Loofbourow and Yeats

Loofbourow, Oppenheim-

(1947) found that enhanced growth-

promoting properties of suspensions of damaged cells could be
correlated with an increase in the amount of nucleotides and
nucleosides.

Linser and Kaindl (1951) believed that growth-

inhibiting or promoting materials were adsorbed by molecules

2
present

in the protoplasm and converted into a part of the

living substance.

These materials must fit available spaces

in order to stimulate growth and must not accumulate to
excess or growth inhibition may occur.
Fennell
materials

(1951) demonstrated that the growth-promoting

in Tetrahymena geleii

tive lesions

(strain W) induced prolifera­

in the chorioallantois of the chicken embryo

and investigated the relationship between age of culture,
lesion induction and concentration of possible growth-promoting
substances.

As very little information is available concerning

the effect of protozoan growth-promoting materials on verte­
brate tissues,
of that study.

this present

investigation is a continuation

It has as its objectives:

(l) a semi-

quantitative histochemical estimation of the distribution
of alkaline and acid phosphatase,

lipase, peroxidase, fat,

glycogen, urates and DNA at various intervals during the life
of cultures of T. geleii W;

(2) a clarification of the role

of these materials in the physiological processes of aging
T. geleii W; and (3) an ascertainment of any correlation
between the presence of these substances and the growthpromoting properties of

geleii W subsequent to inoculation

onto the chorioallantois of the chicken embryo.

M A T E R I A L S AND METHODS

All histochemical studies were made on specimens of
Tetrahymena geleii

(strain W) cultured at 20-30°.

culture media were used:
Bacto-Tryptone
1 gm.

Two basic

(l) tryptone made with 15 gm.

(Difoo Laboratories,

Detroit,

iCHgPO^ in 1,000 cc. distilled water;

Michigan) and

(2) vitamin-

enriched tryptone made in an identical manner as the preced­
ing but enriched with the following vitamins:
flavin,

1 mg. thiamine,

micrograms biotin.

1 mg. ribo­

100 micrograms nicotinic acid and 0.5

Additional media were prepared in which

either 0.448 gm. of CaClg or 1.07 gm.

MgClg was added to each

of the basic solutions.
Cultures of organisms were established by inoculating a
125 cc. Ehrlenmeyer flask containing 75 cc. of the sterile
culture medium with 1 cc.
culture.

of a sterile heavily populated stock

In most experiments tests were made with organisms

72 hours subsequent to inoculation and at 72-hour intervals
thereafter throughout the life of the culture.

In all investi­

gations age of culture refers to hours subsequent to inocula­
tion.

Histochemical studies were made both on X- gelQii W

suspended in the original culture medium and on specimens
suspended in physiological saline for 30 minutes subsequent
to removal from the culture medium.

4
Alkaline and aci d phosphatases were localized by the
methods of Gomori
(1943).

(1941) and lipase by the method of Gomori

Peroxidase was ascertained by methods of both

McJunkin and Armitage

(Glick 1949).

The deposition of

glycogen was studied by mean3 of the Feulgen-Bauer reagent
(Bensley and Bensley 1938).

Desoxyribonucleic acid was

identified by the method of Bensley and Bensley (ibid.)

The

Hollande modification of the Courmont-Andre method was utilized for testing the presence of urates

(Glick ojo. c it. ).

The deposition of phospholipids was determined by the SmithDietrich reaction (Lison 1936) although the reliability of
this test has been questioned (Cain 1950).

Aqueous suspen­

sions of cells were stained with Sudan IV to ascertain the
amount of fat at various ages.
.Estimates of enzymatic activity and deposition of fat
a n d glycogen in T. geleii W were made by measurements of the
length of the cell3 and the approximate area of the positive
reaction subsequent to the histochemical tests.

With the

exception of desoxyribonucleic acid, the following formula
was used for establishing a comparative index of activity:
Activity factor = LgP
LIT
in which L^,
cells,

L2 , P and T represent the average length of the

the approximate area of the positive reaction, the

total number of positive cells and the total number of cells
counted,

respectively.

The term glycogen/ or fat/cytoplasm

5
ratio is used in the discussion on fat and glycogen deposi­
tion, but

it was ascertained in the same manner as the ac tiv­

ity factor for enzymes.

However,

on all graphs the term

activity factor is u s e d synonomously with glycogen and fat
deposition.

Each factor or ratio was determined after a

count had been made of the total number as well as the total
positive cells in 10 microscopic fields

(800X) and after the

average total length and length of positive area of 10-15
cells on each slide had been measured with a micrometer.
fields and cells were selected at random.

All

It should be

emphasized that this activity factor is an approximation,
a similar method was devised and used by de Robertis
in a study of thyroid tissues in vertebrates.

but

(1949)

In the estima­

tion of nuclear desoxyribonucleic acid an activity factor was
established on intensity of the staining reaction.

A nega­

tive reaction was given a value of 0; a light nuclear stain­
ing reaction, a value of 1; a medium reaction, a value of 2;
and a heavy reaction, a value of 3.

Each of these values

was then multiplied by the total number of nuclei placed in
each category, and the sum of these products for each slide
divided by the total number of nuclei counted.

Each activity

factor for DNA was obtained by the classification and count­
ing of all nuclei in 10 randomly selected microscopic fields

(soox).
Pedigreed White Leghorn hatching eggs were used exclus­
ively for the study of lesion induction by T. geleii W.

6
Organisms used for inoculation of the chorioallantois were
wa shed in two changes of physiological saline.

After each

washing the cells were centrifuged and the supernatant removed.
A suspension was then made of a 1-10 dilution of the concen­
trated washed organisms in physiological saline and left for
2 hours to several days.

The chorioallantois of 12-14-day

old embryos was then inoculated with 0.05 c c . portions of
the suspension by the artificial air sac method of Burnet
(1936).

A few eggs were inoculated with organisms suspended

in the original culture solution.

Also in several experi­

ments the organisms were heated for one-half hour at 50° C.
prior to inoculation.

Other inocula were prepared in which

Ringer's solution or 0.02N HOI or 0.005N CaO in physiological
saline was substituted for physiological saline as the wash­
ing and suspending medium.
Some embryos
portions of a 1:4

were also inoculated with

0.05 or0.1 c c .

saline dilution of strain 12 lymphomatosis

mince or 0.05 cc. portions of a 0.05-0.25 per cent croton
oil emulsion in 0.03N NaOH in physiological saline.
Approximately 16 square millimeter sections of embryonic
heart, head ectoderm or liver washed in normal saline were
grafted onto the chorioallantoic membrane through a triangular
window cut into the shell.

The section of shell was then

replaced and sealed with scotch tape.

The embryonic tissue

was

maintained in saline at either room temperature

for

15 minutes at 50° C. prior to grafting.

or heated

7
In each series of experiments,

0.05 cc.

solutions use d as the suspending medium for

portions of all
gelei i W and

other inocula were u s e d as c o n t r o l s .
Lesions were fixed in acetone for the histocheinical
localization of enzyme activity.

Acid and alkaline phospha-

tase, lipase and desoxyribonucleic acid activity and the
deposition of glycogen were determined by the methods utilized
for T_- geleii W.

Leaions were fixed in acetic-bicbromate to

ascertain the site of fat accumulation (Bensley and Bensley,
op. c i t . ).

All morphological investigations were made with

lesions fixed in Zenker-formol and stained with haemotoxylineosin or haemotoxylin-eosin azure.

RESULTS
1.

HISTOCHEMICAL PROPERTIES OF TETRAHYMENA GELEII W,
All histochemical tests were at least duplicated ana

were conducted with specimens of T. geleii W obtained from
various culture media 24 to 576 hours subsequent to inoculation.

In all tests control slides were stained.

Results

in

these experiments represent trends in activity rather than a
quantitative estima.tion of the activity at definite hours,
as variations may exist in the activity at identical hours
the duplicated studies,

in

but the trend in activity usually

remains fairly constant.
ALKALINE PHOSPHATASE
Localizat ion and variat ion w ith age of cult ure .

Alkaline

phosphatase activity in young (24-hour) unwashed cells reared
in tryptone was characterized by small black spherical entitie
wide l y scattered throughout the cell but most heavily concen­
trated near the nucleus or posterior end of the organism
(Fig.

1).

At 72 hours these discrete particles disappeared

a nd the positive area was concentrated in the vicinity of the
nucleus.

A diffuse grayness noted throughout

the cytoplasm

of many cells was not considered as definitely positive.
sequent to 144 hours,

Sub­

there was still phosphatase activity in

the vicinity of the nucleus,

but the most highly concentrated

9

site occurred in the posterior end of the cell
an d 4).

(Figs. 2, 3

The tendency for the phosphatase to be concentrated

in this area remained fairly constant until the age at which
enzymatic activity disappeared except for occasional spherical
senescent

cells in which the phosphatase activity was located

in the center of the organisms.

The initial addition of vita­

mins alone to the tryptone solution did not materially alter
the localization of alkaline phosphatase

(Fig. 9).

There was

a tendency for any nuclear phosphatase to decline in activity
in organisms washed in physiological saline.
A study of Text-figure

I indicates that in specimens

of

2.* gslQii W cultured in tryptone alkaline phosphatase activity
increased slightly between 72 and 288 hours at which time a
maximum activity factor of 0.05 was obtained.

A rapid decline

then occurred and subsequent to 330 hours of age all activity
disappeared.

The initial addition of vitamins to the tryptone

solution tended to induce maximum activity earlier

(72 hours)

and to delay the complete cessation of any visible reaction.
With the exception of 360-hour cultures,

enzynatic activity

was greater in the organisms reared in tryptone than in those
reared in vitamin-enriched media.

It is evident from Text-

figure II that alkaline phosphatase activity was higher in
organisms

in which washing in saline was omitted.

Effect of the addit ion of vitamins to the culture media..
Early in the course of this study it became apparent that
the cultures of T. geleii W were becoming increasingly short­
lived and less productive.

It was to counteract this trend

10
T E X T - F IG U R E

I

elation between age and alkaline phosphatase activity in
* gQiQii W,
All specimens were suspended in physiological
saline for 30 minutes prior to fixation and staining.
Activity
factor determined by formula given under materials and methods.
O ©

i-

gelei i W

“ Z*

geleii W
tryptone;

CD-

1

®

" T.

©

” T.

reared in t ryptone;
reared in calcium chloride-enriched

geleii W reared in magnesium chloride-enriched
tryptone;
geleii W

reared in vitamin-enriched tryptone;

geleii W reared in vitamin and magnesium
chloride-enriched tryptone; and

Z' gQlQli W reared in vitamin and calcium
chloride-enriched tryptone.

CM
1^-

ro

O

O

M010VJ

CM*

o

A1IAI10V
14 4
AGE

216
288
360
OF CULTURE IN HOURS

432

12
TFXT-FIGURE II
Relation between age, suspension in physiological saline and
the histocheraical properties of _T. geleii W reared in vitaminenriched tryptone.
Activity factor determined by formula
given under materials and methods.

o-

Alkaline phosphatase activity in T. geleii W
suspended in physiological saline;

4 ^ - alkaline phosphatase activity in T. geleii W
suspended in original culture medium;
acid phosphatase activity in T. geleii W
suspended in physiological saline;
©

- acid phosphatase activity in _T * geleii W
suspended in original culture medium;
glycogen deposition in T. geleii W suspended
in physiological saline; and
glycogen deposition in T. geleii W suspended
in original culture medium.

a o io V d

AX IA I I O V
AGE

144
OF

288
CULTURE

216

IN HOURS

360

432

504

14
TABLE I
Alkaline Phosphatase Activity in Specimens of
X* geleii W Reared in Tryptone and Vitaminenriched Tryptone
Cays Subsequent
to Inoculation
into Initial
Vitamin-enriched
Culture Medium

Culture
Medium

Age of
Culture
in Days

Vitamin
enr iched

3-15
18-31

1

0

8

18

3-24

8

145

3= 15

9

165

3“ 30

10

188

3-34

11

209

13

212

3

13

223

3
6-31

17

264

19

272

6

30

278

6

21

286

6-27

22

292

6-15

23

299

7

34

306

7-14

25

313

7-14

Tryptone

Enz yme
Reaction

++'

3

*■
++

Vitamin
enr iched

4*^

15
TABLE I (Cont.)

Transf er
No.

Days Subsequent
to Inoculation
into Initial
Vitamin-enriched
Culture Medium

Culture
Medium

Age of
Culture
in Days

Enzyme
React ion

26

320

Vitamin

7

-

27

327

li

7-14

-

28

334

II

7

+

29

341

it

14

-

30

355

it

14

-

31

367

n

7

-

32

369

It

7

-t-

^With the exception of those in which only
listed, tests were conducted in Transfers
intervals and in Transfers 23-32 at 7-day
p
-►-Slightly positive;
Moderate to heavy

one day is
1-22 at 3-day
intervals.
positive reaction.

13
that vitamins were first added, to the tryptone cultures in
the amount stated under materials and me t h o d s .

In the first

series of histochemical studies subsequent to the addition
of vitamins a slight but not significant decline in the level
of alkaline phosphatase activity was apparent
13).

(Figs.

12 and

However, as noted in Table I, upon inoculation of

subsequent series of vitamin cultures and conduction of additional series, all evidence of alkaline phosphatase activity
disappeared (Figs.

14 and 15).

This inhibition was at first

believed to be attributable to factors other than the presence
of the added vitamins, but investigations utilizing triple
distilled water, fresh chemical solutions,
negative for alkaline phosphatase.
approximately seven months'

etc. , remained

After 10 transfers and

exposure to vitamins, the organ-

isms were transferred to tryptone medium.

Three days after

this first transfer phosphatase activity was still inhibited,
but after subsequent

inoculations, activity again became

vis i b l e .
After six transfers and approximately two months in the
tryptone solutions the organisms were again transferred to
media containing vitamins.

Alkaline phosphatase activity was

still visible after eight transfers and a two months'
in the vitamin-enriched media.

exposure

On the ninth transfer inhibi­

tion of phosphatase activity again occurred.

With the excep­

tion of a weakly positive reaction on the 11th transfer,

com­

plete alkaline phosphatase inhibition was maintained until

1?

the 15th transfer in the vitamin-enriched media.

Subsequent

to this transfer the organisms have shown erratic results and
have become alternately positive and negative.

Although the

presence of vitamins appeared to have an inhibitory effect on
phosphatase activity, which could be counteracted by the
addition of

magnesium, future research is required to clarify

existing results.
Effect of the addition of the magnesium or calcium ion
to the culture m e d i u m .

The magnesium ion has been widely

described as an activator of alkaline phosphatase
a n d Furth 1941).

(Kabat

The results obtained in this present investi­

gation demonstrated that the addition of magnesium or calcium
chloride to either of the basic culture media increased
alkaline phosphatase activity, but that the calcium ion was
the more effective

(Text-fig.

A definite shift

I and Figs.

5 and 7).

in the site of alkaline phosphatase

activity from the posterior towards the nuclear region of
the cell occurred in organisms reared in vitamin-enriched
tryptone to which magnesium chloride had been added

(Fig.

10).

Calcium activation of phosphatase activity was more
pronounced in organisms reared in tryptone and reached a
maximum at 144 hours at which time the phosphatase activity
factor increased from 0.03 to 0.32.

Control slides from

cultures

containing magnesium or calcium chloride were

negative

(Fig.

6).

To determine the effect of the concentration of magnesium
in the glycerophosphate buffer on alkaline phosphatase activity,

18
0.0137 gra.

of magnesium chloride was added to each ^50 cc.

of the substrate.

Increasing the concentration of the mag­

nesium did not alter the site of phosphatase activity in
organisms reared in tryptone
enhanced the reaction

(Fig.

8), but occasionally

in cells from 72 to 144 hours of age.

Eliminating magnesium from the buffer also did not alter the
site of phosphatase activity but decreased the intensity of
the reaction in 433-hour cultures.
In addition to its effect on alkaline phosphatase activ­
ity, magnesium chloride extended the life of the culture for
10-15 days.
Specificity of alkaline phosphatase a c t i v i t y .

The shift

in alkaline phosphatase activity from the posterior region
towards the center of the organism (Fig.

10) subsequent to

the addition of magnesium chloride to the vitamin-enriched
media gave ri3e to the possibility that several substrate
specific enzymes hydrolyzing phosphate esters might be present
in T_. geleii W.
which 1 cc.

Enzyme inhibition tests were conducted in

of one of the following inhibitors was applied to

a series of <T. geleii W slides for 20 minutes prior to immer­
sion in the phosphatase buffer:

0.02M semicarbazide, 0.Q25M

oxidized or reduced glutathione and 0.01M sodium arsenate.
Organisms

were obtained from the various culture media 144

hours after seeding and from additional cultures every 144
hours thereafter.

19
All

inhibitors completely inhibited enzyme activity in

organisms reared in tryptone cultures

(Fig.

17).

Occasionally

a diffuse grayness especially pronounced in the nucleus was
visible.

The addition of magnesium chloride to the tryptone

culture medium enhanced this diffuse grayness but did not
counteract the inhibition

(Fig. 18).

A slight positive reac=

tion was visible in the distal end of a few cells from 433hour vitamin-enriched cultures subsequent to exposure to sodium
arsenate,

but cells of all other ages were negative

(Fig.

13).

X* KQisii W from vitamin-enriched cultures containing mag­
nesium chloride exhibited a variation in alkaline phosphatase
activity in accordance with the type of inhibitor used (Figs.
19-22).

Semicarbazide and glutathione inhibited all activity,

but a moderate to heavy reaction in the area between the nucleus
and the posterior end of the cell was visible in 10-15 cells
subsequent to exposure to sodium arsenate.

From the preceding

it was apparent that no definite conclusions could be drawn
as to the existence of two separate enzymes on the basis of
the inhibitor studies alone.
Diffus ion of alkaline pho sphatase.

During the course

of this study it al 30 became evident that factors other than
age,

washing and concentration of the magnesium or calcium

ion were operative in the localization of alkaline phosphatase.
Pronounced activity was frequently visible in areas of highly
concentrated cells but entirely absent in the more widely dis­
persed cells on the same slide.

In addition,

many slides

20

containing organisms which had not been washed in saline
exhibited a heavy black extracellular precipitate subsequent
to the histochemical localization of alkaline phosphatase.
The aforementioned suggested that a diffusion of the enzyme
or enzymatic end-products
occurred.

into the surrounding medium had

Supernatant fluid from centrifuged cultures exhib­

ited this black precipitate,
organisms were negative.
as evenly as possible,
able.

but culture media not exposed to

In all tests cells were distributed

but some congestion was always inevit­

On slides containing highly active organisms this

difference

in activity between concentrated and widely dis­

persed cells was not apparent.

In general, however, the

greater the concentration of the cells,
phatase activity.

the greater the phos­

This apparent difference may play a role

in the high concentration of activity noted in 215-388-hour
organisms,

as it is at that age that the cultures are usually

the most highly concentrated with organisms and the tendency
towards diffusion may decrease.
ACID PHOSPHATASE
Localization and variat ion with age of culture.

A heavy

extracellular precipitate interfered with an accurate locali­
zation of the site of acid phosphatase activity in unwashed
organisms.

However,

in most 72-hour cells reared in tryptone

large discrete positive particles were visible thoughout the
organism but heaviest along the sides and posterior end.

A

31

few cells were visible
fille d the organism.

in which the positive area completely
In all cells the nucleus was usually

the last to show a positive reaction.

No restricted site for

acid phosphatase activity was apparent at any age.
In washed cells from tryptone cultures acid phosphatase
activity was most highly concentrated in the posterior end
of the organism (Fig. 33).

This remained the site of greatest

activity until 388 hours at which time it shifted towards the
nucleus

(Fig.

35).

Highest activity in 73-hour unwashed organisms from
vitamin-enriched cultures occurred in the vicinity of the
nucleus.

This positive area increased as the organisms aged

an d eventually filled the entire cell.

After the cells had

been suspended in physiological saline, acid phosphatase
appeared to be most active in the posterior end of 73-hour
cells

(Fig.

; but with an increase in the age of the

culture, acid phosphatase activity shifted towards the nucleus
(Fig. 26).
It is evident from Text-figures III and IV that the
activity factor for acid phosphatase in washed organisms
cultured in tryptone was at a maximum 144 hours subsequent to
inoculation.

This activity factor of 0.39 decreased to 0.06

during the next 72 hours but then gradually increased again
to 0.18 at 432 hours.
declined to 0.05.

At 504 hours the activity factor

22

TEXT-FIGURE III
Relation between age and the histoehemical properties of
T_. gelei i W reared in tryptone.
All specimens suspended in
physiological saline for 30 minutes prior to fixation and
staining.
Activity factor determined by formula given under
materials and methods.

0

-

Alkaline phosphatase activity;

( J r acid phosphatase activity;
€>0

lipase activity;

“ glycogen deposition; and
fat deposition

COm

o

10

o

o
MOJLOVJ

ro
o

OJ

o

A 1 IA I1 0 V

o

34
TEXT-FIGTJRE

IV

Relation between age and acid phosphatase activity in _T.
geleii W . All specimens were suspended in physiological
saline for 30 minutes prior to fixation and staining.
Act ivit y factor determined by formula given under materials
and methods.
X- g e lQii W reared in tryptone;
@

- T . geleii W reared in calcium chlorideenriched tryptone;
i geleii W reared in magnesium chlorideenriched tryptone;
~ X- geleii W reared in vitamin-enriched
tryptone;

C -

1- g e l 33-1 w reared in vitamin and magnesium
chloride-enriched tryptone; and
X* geleii W reared in vitamin and calcium
chloride-enriched tryptone.'

*>

yOXOVJ

CSI

A1IAI10V

«

o
144
AGE

216
288
360
OF CULTURE IN HOURS

432

504

26

In vitamin-enriched tryptone an alternate rise and fall
in activity appeared throughout the experiment

(Text-fig.

IV).

No positive reaction was visible in cells from 73, 216 or
3 60-hour cultures,

but at 144, 283, 433 and 504 hours acid

phosphatase activity was greater than in cells not exposed to
vitamins.

The maximum difference in phosphatase activity

between organisms cultured in tryptone and those cultured in
vitamin-enriched tryptone occurred at 238 hours at which time
the activity factor for acid phosphatase in cells from tryp­
tone was 0.13 and from vitamin-enriched tryptone 0.72
fig.

(Text-

IV).
Washing organisms

in physiological saline appeared to

influence the activity factor for acid phosphatase to a greater
extent than the factor for any other substance studied.

The

maximum effect occurred at 504 hours at which time the activity
factor for unwashed cells reared in tryptone was 1.0 and for
cells suspended in physiological saline 0.04
In organisms from vitamin-enriched media,

(Text-fig.

V).

the effect of sus­

pension in physiological saline was less pronounced (Textfigs.

II and V).
Effect of the a ddition of the magnesium or calcium ion
culture m e d i u m .

The addition of calcium chloride to

tryptone media increased acid phosphatase activity in all
cells suspended in physiological saline with the exception of
those aged 432 hours

(Text-fig.

I V ).

The maximum increase

was obtained at 288 hours at which time the activity factor

27
TEXT-FIGtJPE V
Relation between age, suspension in physiological saline and.
ac i d phosphatase activity in T. geleii W.
Activity factor
determined by formula given under materials and methods.
O -

®

T . geleii W reared in tryptone and fixed and
stained immediately after removal from
culture medium;
“ T. geleii UV reared in tryptone and suspended
in physiological saline for 30 minutes prior
to fixation;
” X* % e 3-e i i W reared in vitamin-enriched tryptone and stained immediately after removal
from culture medium; and
X- geleii ^ reared in vitamin^-enriched tryp=
tone and suspended in physiological saline
for 30 minutes prior to fixation.

to

a o io v d

AXIAIXOV

o
144
AGE

216
288
OF CULTURE IN

360
HOURS

432

504

29
increased, from 0.13 to 0.80.

The addition of calcium chloride

to media containing vitamins decreased acid phosphatase activity
(Text-fig.

IV).

The activation

phosphatase activity

by magnesium chloride of acid

in organisms reared in tryptone reached

a maximum at 432 hours at which time the activity factor increased from 0.18 to 0.28.

In vitamin-enriched cultures the

magnesium ion stimulated activity at 144 and 380 hoars but
decreased it at 288 hours.

Nuclear acid phosphatase exhibited

the maximum stimulation by the magnesium ion.
Diffusion.

Acid phosphatase or its catalyzed end products

diffused readily from T_. geleii W and resulted in the forma­
tion of a heavy extra-cellular precipitate during the histochemical localization of this enzyme.

There was no evidence for

a complete diffusion of acid phosphatase from any organism.
Occasionally acid phosphatase activity was entirely absent,
but undetermined factors other than diffusion appeared to be
respons i b l e .
LIPASE
Localization and variation with age of cultur e.

Lipase

activity in T_. gelQii W less than 218 hours of age and reared
in tryptone was restricted almost entirely to the nucleus.
However,

a few cells exhibited slight activity in the distal

end of the organism.

In 288-hour cultures the positive sites

of lipase activity became more widely dispersed but remained
the most heavily concentrated in the vicinity of the nucleus
(Fig. 27).

Subsequent to 288 hours,

lipase activity was again

30
restricted almost entirely to the nucleus.
ing the cells

Neither suspend­

in physiological saline nor adding vitamins to

the culture medium shifted the site of lipase activity.
The first positive reaction for lipase was obtained in
cells aged 144 hours

(Text-fig. VI).

The maximum activity

factor for w;-shed organisms reared in tryptone was 0.11 and
for cells suspended in the original culture medium 0.04
(Text-figs.

VI and VII).

The former was obtained 288 hours

an d the latter 380 hours subsequent to seeding.

The addi­

tion of vitamins to the tryptone medium decreased lipase
activity and the activity factor never exceeded 0.03
fig.

VI).

(Text-

Suspension in physiological saline appeared to have

little effect on the amount of visible lipase activity.
Iffeet of the addition of the magnesium or calcium ion
to the culture medium.

The addition of either calcium or

magnesium chloride to vitamin-enriched media shifted lipase
activity from the nucleus towards the distal end of the cell.
Calcium had little effect on the amount of activity in the
vitamin-enriched cells,

but magnesium increased the activity

factor at 288 hours from 0 to 0.1b (Text-fig.
Diff u s i o n .

VI).

All slides containing T. gelpji W exposed to

calcium chloride exhibited a heavy brown extracellular pre­
cipitate when stained for lipase activity.

This may be attri­

buted to either a diffusion of the enzyme or the presence of
the calcium ion.

In these present experiments

it is believed

that diffusion played only a minor role in the localization
of lipase activity.

TEXT-FIGURE VI
Relation between age and lipase activity in _T. geleii W.
All
specimens were suspended in physiological saline for 30 minutes
prior to fixation and staining.
Activity factor determined
by formula given under materials and methods.
geleii W reared
O -

Z*

gQleii W reared

in tryptone;
in vitamin-enriched tryptone

€ > - T. Keleii Vtf reared in magnesium chloride
vitarnin-enriched tryptone; and

and

i- RQloii W reared in calcium chloride and
vitamin-enriched tryptone.

CM
1^

CM

o

a o io v j

A 11A I10V
144
AGE

216
288
OF CULTURE IN

360
HOURS

432

504

33
TEXT-FIGURE VII
Relation between age of culture and the histochemical proper­
ties of _T, geleii W reared in tryptone.
All specimens stained
immediately after removal from culture medium.
Activity
factor determined by formula given under materials and methods.
Alkaline phosphatase activity;
acid phosphatase activity;
^

° lipase activity; and
glycogen deposition.

3

oo
cs

8
H O lO Vd

m

o

o

ro
O

A 1IAIX0V

CVJ
O

-

o

35
GLYCOGEN
Localiza.tion and variation with age of cultu re.

In

unwashed cells from tryptone cultures the highest glycogen
concentration was visible between the nucleus and the pellicle
of the organism (Figs.

30=33).

The nucleus and proximal tip

remained negative in all cells but those in which the entire
organism exhibited a positive Feulgen-Bauer reaction.

In

73-hour organisms suspended in physiological saline glycogen
was located in scattered discrete sites which were most highly
concentrated in the distal end of the cell .

These sites

enlarged and coalesced as the amount of glycogen increased,
but deposition remained at a maximum in the posterior portions
of the cell.

The addition of vitamins to the culture medium

did not alter the localization of glycogen.
Glycogen denosit ion in unwashed cells from tryptone
cultures was at a maximum between 144 and 288 hours during
which time the glycogen/cytoplasm ratio averaged approximately
0.61

(Text-fig.

VII).

The addition of vitamins decreased the

glycogen/cytoplasm ratio

(Text-fig.

II).

In organisms from

tryptone cultures and suspended in physiological saline a
maximum ratio of 0.57 was obtained at 432 hours
III and VIII).

(Text-figs*

In these cells glycogen deposition was gradual

between 72 and 288 hours but increased rapidly as it approached
a maximum.

Maximum glycogen deposition occurred earlier in

organisms from vitamin-enriched cultures, and a glycogen/
cytoplasm ratio of 0.57 and 0.53 was obtained at 216 and 360
hours,

respectively (Text-fig.

VIII).

36
In general,

suspending cells

in physiological saline

decreased the glycogen/cytoplasm ratio.

The maximum decline

n o ted in cells from tryptone cultures occurred at 144 hours
at which time the ratio decreased from 0.63 to 0.13 (Textf i g s . Ill and VII).

The maximum decline in cells from vitamin-

enriched media occurred at 288 hours at which time the ratio
decreased from 0.50 to 0.32

(Text-fig.

II).

Effect of the addition of the magnesium or calcium ion
to the culture m e d i u m .

The addition of calcium chloride to

vitamin-enriched tryptone increased the amount of visible
glycogen

in all cells studied (Text-fig.

VIII).

The addition

of magnesium chloride increased glycogen deposition in all
cells but those aged 316 and 360 hours.

The glycogen/cytoplasm

ratio in organisms from the magnesium-enriched media reached
a maximum of 0.86 at 144 hours and in the calcium-enriched
media it reached a maximum of 0.84 at 216 hours
VIII).

(Text-fig.

The magnesium ion appeared to be more effective in

increasing glycogen deposition in cells 144 hours of age or
less and the calcium ion in increasing deposition in older
cells.
Diffusion.

Many slides containing cells which had been

wa shed in saline exhibited an extracellular positive reaction
for glycogen.

Apparently handling of organisms during the

washing process was an important factor in cytolysis and the
escape of glycogen from the cell.

No extracellular positive

reaction was noted on slides of unwashed T. geleii W, and,

3?
TEXT-FIGURE VIII
Relation between age and glycogen deposition in T. gelei i W.
All specimens were suspended in physiological saline for 30
minutes prior to fixation and staining.
Activity factor
determined by formula given under materials and methods.
• -

T. geleii W reared in tryp ton e;

o-

T. geleii W reared in vi tamin-enriched
tryptone;

© -

T, geleii W reared in magnesium chloride
and vitam in--enriched t ryptone; and
T. geleii W reared in calcium chloride and
vitamin"enriched tryptone.

CM
K

co%

<a

d O lO V d

CM

AJLIAIIOV

o
AGE

144
OF

360
IN HOURS

288
CULTURE

216

432

504

39

therefore,

glycogen did not appear to diffuse naturally

into the culture media.
FAT
Localization and variat ion with
organisms were positive

for fat with

age of cu ltu re.

All

the exception of an

occasional cell in the 73-hour cultures.

The Sudan-positive

area was first noted at the anterior end of the organism and
consisted of distinct granules.
hours,

After the cells had aged 31^

these granules co&lescea to form a compact mass which

eventually expanded distally to fill nearly the entire organism.

Neither the addition of vitamins nor the washing of

the organisms

in saline prior to staining affected the distri­

bution or amount of fat visible in T. geleii W.
An examination of Text-figure III indicates that the
fat/cytoplasm ratio increased from 0.09 in 34-hour cells
reared in tryptone and suspended in physiological saline to
a maximum of 0.49 in organisms from 504-hour cultures.
extension to this curve

would indicate a further rise in

deposition as the cells

aged.

An
fat

Fffect of the addition of the magnesium or calcium ion
to the culture m e d i u m .
media did not affect

The addition of calcium to tryptone

the localization or amount of fat in

organisms from cultures 144 hours or less in age.
hours,

the fat/cytoplasm ratio decreased.

After 144

An inhibition of

fat production by magnesium was less pronounced.

40
Diffus i o n .

No extracellular reaction for fat was visible

on any slides and the suspension of the cells in saline did
not materially alter the amount of fat present.

Diffusion

was not responsible for any variation which might occur in
the fat/cytoplasm ratio.
PHOSPHOLIPIDS
Localization and variation with age of cultu re.

The

Smith-Diet rich test was used in all experiments on the deter­
mination of the presence of phospholipids in T_* gelei i W, and
only discrete black particles as specified by Kaufmann and
Lehmann

(cited in Cain, 1950) were regarded as positive.

The

validity of this test has also been questioned by Cain (ib i d .).
No lipid/cytoplasm ratio was determined.
The first positive entities appeared in cells from
cultures 504 hours of age.
particles,

These spherical,

randomly located

from one to seven in number, were noted in 5.1

per cent of the organisms(Figs. 34 and 35).

At 575 hours of

age 7 per cent of the cells contained positive granules,
which were most heavily concentrated in the vicinity of the
nucleus.

Occasionally a gray or brown extracellular precipi­

tate was visible, but this was not considered as evidence for
diffusion of the phospholipids.
DESOXYRIBONUCLEIC ACID
Localization and variation with age of cultur e.

DNA active

ity was investigated by the Feulgen reaction as cited in
Bensley a n d Bensley (1938) .

41
TEXT-FIGURE

IX

Relation between age and. desoxyribonucleic acid activity in
T. geleii W.
All specimens fixed and stained immediately
after removal from the culture medium.
Activity factor determined by formula given under materials and methods.
X* ^Qlei i W

reared in tryptone; and

T_. geleii W
tryptone.

reared in vitamin-enriched

o

m

o
<0
rO

IN HOURS

CM
fO

00

CM

(£>

AGE

CM

OF CULTURE

00

CM
1^-

rO

CM

HOJLOVd

A 1 IA I1 0 V

43
A positive Feulgen reaction in specimens of T. geleii W
was restricted to the nucleus

(Fig. 28).

Maximum DNA activity

in unwash ed organisms from tryptone cultures was noted 72>
144 and 380 hours subsequent to inoculation (Text-fig.

IX).

No activity was visible at 216 hours and after 360 hours the
Feulgen reaction decreased in intensity until most of the
organisms were negative at 504 hours.
Organisms reared in vitamin-enriched tryptone exhibited
maximum DNA activity 72 hours subsequent to seeding (Text-fig.
I X ).

Between 72 and 144 hours a slight decrease in activity

occurred and then remained constant until the cells had aged
360 hours.

After 360 hours another decline occurred and all

activity disappeared 504 hours subsequent to inoculation.
Suspending organisms in physiological saline prior to
staining produced no significant alteration in the intensity
of the Feulgen reaction in cells reared in tryptone.
activity in organisms

DNA

reared in vitamin-enriched media.was

decreased by the washing process and reached a maximum at 216
hours at which time the activity factor decreased from 1.85
to 0.3 (Text-fig. X).

A cyclic rise and fall in DNA activity

occurred in all cells except unwashed organisms from vitaminenriched tryptone

(Text-figs.

IX and X).

Effect of the addition of the magnesium or calcium ion
to the culture m e d i u m .

The addition of calcium or magnesium

chloride to vitamin-enriched tryptone induced minimal DNA
activity at 144 and 360 hours and a maximum reset, ion at 316

44
hours

(Text-fig.

X).

This is the inverse of that noted in

organisms from solutions

in which calcium or magnesium was

omitted.
Diffus i o n .

No evidence of diffusion occurred on any

slides stained for DNA.
Relat ion between intensity of staining react ion and size
of n u c l e i .

According to Bradfield (i960) fluctuations in

nuclear staining for DNA may be due to changes
volume.

in nuclear

Approximate measurements made of nuclear area in the

course of this study indicated that,

in general,

the smaller

the nuclei, the greater the intensity of staining.

This would

indicate that in the smaller nuclei the DNA was more highly
concentrat ed.
Many nuclei were

irregularly shaped, but all areas were

calculated, as the product of the length and the width.
sequent to the measurement of 30 cell nuclei,

Sub­

the average

area of those placed in categories 1, 2 and 3 (see materials
and methods) was 59, 54 and 31 square microns,

respectively.

According to these results the average area of the lightest
staining nuclei was approximately twice that of the heaviest
staining ones, while no significant difference was apparent
between the light and medium staining nuclei.

The difficulty

in classifying the nuclei into the latter two categories may
account for this small difference.

45
TEXT-FIGURE X

Relation between age and desoxyribonucleic acid activity in
X* ffele i i W.
All specimens were suspended in physiological
saline for 30 minutes prior to fixation ana staining.
Activity
factor determined by formula given under materials and methods.
X* g e leii W reared in tryptone;
O -

i- geleii W reared in vitamin-enriched
tryptone;
X*
leii W reared in vitamin and magnesium
chloride-enriched tryptone; and

( ^ “ X* gelQii W reared in vitamin and calcium
chloride-enriched tryptone.

fO

CJ

aOlDVd

Ail AliOV
144
AGE

216
288
360
OF CULTURE IN HOURS

432

504

47
PEROXIDASE
No positive reaction for peroxidase was visible on any
slides of T. geleii W.
URATES
No positive reaction for urates was obtained.
VARIATION IN SIZE OF CELL WITH AGE AND FIXATION
It is evident from Text-figure XI that specimens of _T.
g e l e i i W reared in vitamin-enriched tryptone decreased in
average length as the culture aged.

Both fixation and the

staining technique tended to decrease the size of the cell.
With the exception of organisms aged 380 and 504 hours,
unfix ed cells,
length.

those stained for fat,

exceeded all cells in

Organisms of the former age stained for lipase and

of the latter age stained either for lipase or acid phospha­
tase were longer than unfixed cells of the same age.

A maxi­

mum average length of 77 microns was exhibited by unfixed
organisms 144 hours of age and a minimum of 33 microns by
organisms 504 hours of age and stained by the Feulgen-Bauer
reaction for glycogen.
In general,

the average length of all fixed cells

increased between 72 and 144 hours subsequent to inoculation.
From 144 to 288 hours of age the length usually remained at a
maximum and then gradually decreased until a minimum length
was reached at 504 hours.

An exception was noted in the cells

stained for alkaline phosphatase, as a decline in length

48
T E X T - F I G TJRE XI

Relation between age, histochemical technique and length of
Z.*
i W reared in vitamin-enriched tryptone and fixed
and stained immediately after removal from culture medium.
Unfixed T. geleii W 3 tained with Sudan IV
for f a t ;
“ !L* £ Q l 0 i i W stained by Gomori technique for
alkaline phosphatase;
Z* g e l ^ ii W stained by Gomori technique for
acid phosphatase;

0

-

O"

1- geleii W stained by Gomori technique for
lipase; and
—•
w stained by Feulgen-Bauer tech­
nique for glycogen.

O
IO

o

iP

00
00
CM

AGE

(£
CM

OF CULTURE

IN

to

HOURS

CM
tO

CM
N-

o
u>
SNOdOm

O
iO

$

Nl 1 1 3 0

8

o

CM

O

dO HJL0N31

O

50
occurred between 144 and 215 hours of age and a pronounced
increase between 350 a n d 432 hours.

The unfixed cells

followed the same general trend as those stained for alkaline
phosphatase.

INTERRELATIONSHIP OF ACTIVITY TRENDS IN BIOLOGICAL SUBSTANCES
INVESTIGATED
In unwashed organisms from tryptone cultures acid phos­
phatase activity and glycogen deposition closely paralleled
one another

(Text-fig.

exception was n o t e d a s

VII).

At 144 hours, however, an

acid phosphatase activity decreased

as glycogen deposition increased.

The deposition of glyco­

gen increased slightly between 350 and 504 hours while acid
phosphatase rapidly approached a maximum activity factor of
1.0.

Fat deposition increased gradually with the increase

in the age of the culture.

Alkaline phosphatase activity

reached a maximum at 215 hours at which time acid phosphatase
was also highly active and glycogen deposition near a maximum.
In cells from cultures aged more than 350 hours acid phospha­
tase activity increased rapidly while any visible reaction
for alkaline phosphatase disappeared entirely.

Lipase activ-

ity was at a maximum at 350 hours at which time a rapid decline
in acid phosphatase activit-y and the deposition of glycogen
was apparent.

Acid phosphatase activity exceeded that of

alkaline phosphatase

in all organisms.

DNA activity was at a

maximum 72, 144 and 360 hours subsequent to seeding and at a
minimum at 215 and 504 hours

(Text-fig.

IX).

In cells more

51
than 72 hours of age DMA activity bore an inverse relationship
to glycogen deposition and acid and alKaline phosphatase
activity

(Text— figs.

VII and IX).

In older cells a slight

parallel between lipase and DNA activity was apparent.
In general,

the activity factor for acid phosphatase in

unwashed cells exceeded that for glycogen denosit ion.
sequent to suspension

in physiological saline this inter­

relationship was reversed (Text-figs.
less,

Ill and VII).

Neverthe-

in washed organisms a parallel between acid phosphatase

activity and glycogen deposition was still apparent
fig.

Sub­

III).

(Text-

At 144 hours acid phosphatase activity increased

as glycogen deposition decreased,

but in all cells 216 hours

of age or older a parallel increase in the amount of both
occurred.

Acid phosphatase activity and glycogen deposition

approached a maximum as fat deposition increased, but dis­
appeared rapidly at the time fat deposition approached its
maximum.

Both lipase and alkaline phosphatase were at a

maximum in cells 288 hours of age and decreased at the time
acid phosphatase activity and glycogen and fat deposition
were approaching a maximum.

Alkaline phosphatase activity

exceeded lipase activity in cells 144 hours or less in age,
but in the older cells lipase became more active.

Glycogen

deposition exceeded fat deposition in cells 72, 360 and 432
hours of age, but in other cells the activity factor for fat
usually exceeded that of all substances studied.

DNA activity

in washed cells appeared to be inversely related to alkaline

53
phosphatase an d lipase activitv,

but the relationship was

not as pronounoed as that noted in the unwashed cells

(Text-

f i g s . Ill and X ) .
Acid phosphatase in unwashed organisms frorn vitaminenriched tryptone exhibited a cyclic rise and fall in activity
(Text-fig.

II).

In each period the activity factor increased

rapidly from approximately 0 to a maximum and then declined
to 0 again.

The activity factor for glycogen deposition

increased gradually to 0.58 between 72 and 216 hours and
then declined to 0.18 at 504 hours.

DNA and alkaline phos­

phatase activity and glycogen deposition followed parallel
trends

(Text-figs.

II and IX).

However, the activity factor

for alkaline phosphatase did not exceed 0.04.
Suspension in physiological saline decreased the maxi­
mum peaks

in the cyclic rise and fall in acid phosphatase

activity in cells 216 to 360 hours in age (Text-fig.

II).

Glycogen deposition.was decreased but maintained the same
general trend as in the unwashed cells.

Alkaline phosphatase

activity practically disappeared in cells 216 hours of age or
older while lipase activity increased slightly between 288
and 504 hours.

Glycogen deposition and alkaline phosphatase

activity appeared to decrease as lipase activity and fat
deposition increased.

The cyclic behavior of acid phospha­

tase did not appear to be directly correlated with the activ­
ity of any of the substances studied.

Alkaline phosphatase

and DNA activity decreased between 72 and 216 hours, but no
additional parallel trend was noted (Text-figs.

II and X).

53
TEXT-FIGURE XII
Relation between age and the histochemical properties of
X* geleii W subsequent to the addition of calcium or magnesium
chloride to the culture medium.
All specimens reared in
tryptone and suspended in physiological saline for 30 minutes
prior to fixation and 3taining.
Activity factor determined
by formula given under materials and methods.
Alkaline phosphatase activity in T_. geleii
W reared in magnesium chloride-enriched
tryptone;
alkaline phosphatase activity in T, geleii
W reared in calcium chloride-enriched
tryptone ;
O -

acid phosphatase activity in T. geleii W
reared in magnesium chloride-enriched
tryptone; and
acid phosphatase activity in T. geleii
reared in calcium chloride-enriched
tryptone.

o
in

00
00
CM

CD

CULTURE

O
CD
rO

IN HOURS

CM
ro

«■

CM

9*0

in

CM

d O lO V J

A 1 IA I10V

o

AGE

OF

CM

55
All organisms reared in media containing magnesium or
calcium chloride were suspended in physiological saline.

The

cells irom tryptone cultures containing magnesium chloride
exhibited maximum acid phosphatase activity at 432 hours and
maximum alkaline phosphatase activity at 238 hours
XII).

(Text-fig.

In organisms from cultures containing calcium chloride

the maximum activity factor for the former occurred at 288
hours and for the latter at 144 hours.

Acid phosphatase was

more active than alkaline phosphatase in these cells.
In washed cultures containing both vitamins and magnesium
chloride a correlation between acid phosphatase activity and
glycogen deposition was again apparent

(Text-fig.

XIII).

Both approached a maximum at 144 hours, declined in activity
between 144 and 216 hours,

increased between 216 and 288

hours and then decreased again between 288 and 360 hours.
Lipase activity was absent in younger cells but between 216
and 360 hours

it followed the same trend as acid phosphatase

and glycogen activity.

The alkaline phosphatase activity

factor decreased gradually from 0.05 at 72 hours to 0 at 288
hours.

A negative correlation between DNA activity and acid

phosphatase activity and glycogen deposition was aoparent
cells 288 hour 3 or less in age (Text-figs. XIII and X).

in
Lipase

activity increased between 216 and 288 hours at which time DNA
activity was decreasing.
Subsequent to the addition of calcium chloride to vitaminenriched media glycogen deposition reached a maximum in

53

TEXT-FIGURE XIII
Relation between age and the histochemical properties of
T. geleii W reared in vitamin and magnesium chloride-enriched
tryptone.
All specimens suspended in physiological saline
for 30 minutes prior to fixation and staining.
Activity
factor determined by formula given under materials and
methods.
C -

Alkaline phosphatase activity;
acid phosphatase activity;
- lipase activity; and

O -

glycogen deposition.

0>

CO

ro

dO lO Vd

AJLIAIIOV

csj

o
144
AGE

216
28 8
360
OF CULTURE IN HOURS

432

58
TEXT-FIGURE XIV
Relation between age and the histochemical properties of
X* gQleii W reared in vitamin and calcium chloride-enriched
tryptone.
All specimens suspended in physiological saline
for 30 minutes prior to fixation and staining.
Activity
factor determined by formula given under materials and
methods.

CD
^

Alkaline phosphatase activity;
- acid phosphatase activity;

C P - lipase activity; and
O -

glycogen deposition.

r-CJ

1^

a O l D V J

A H

A I

1

DV

AGE

144
OF

IN

28 8
CULTURE

216

HOURS

360

432

60
organisms aged 216 hours

(Text-fig. XIV).

additional peaks in the activity curve.

There were no
Both acid and alka­

line phosphatase activity was at a maximum at 144 hours and
lipase activity at 216 hours.

In cells 72 to 144 hours of

age DNA activity bore an inverse relationship to acid and
alkaline phosphatase activity and glycogen deposition
figs.

XIV and X).

(Text-

Between 144 and 216 hours the same relation­

ship between DNA and acid and alkaline phosphatase was apparent,
but glycogen deposition paralleled that of DNA activity.

The

activity factor for glycogen deposition far exceeded that for
any other substance.
2*

LESION INDUCTION IN THE CHORIQALLANTOIS
Pedigreed White Leghorn hatching eggs were used exclu-

sively for the study of lesion induction by protozoan cultures.
X* gelQii W were reared in the various culture media described
under materials and methods and washed by centrifugation in
two changes of physiological saline prior to inoculation onto
chorioallantois of 12-14-day old chicken embryos by the arti­
ficial air sac method of Burnet

(1936).

Frequency of les ion induct i o n .

It is evident from Table

II that at least 66.7 per cent of all embryos survived inocu­
lation by the various suspensions of T. gelei i W and other
substances utilized in this study.

The only exceptions oc­

curred in embryos inoculated with croton oil or a heated sus­
pension of unwashed T_. Re 1 eii W in which a mortality of 100
an d 75 per cent,

respectively, was obtained.

The maximum

the

e i

TABLE

Type of
Inoculum
T.

Chorioallantoic Lesions

Induced by

FTo of
^er cent Per "cent Av Vol of
Embryos
Survival Develops
Lesion
Injected
of
ing
mm^
Embryos
Lesions

A’v*K3ax
Height of
Epithelium
mm

geleii W
3“ day cultures

37

85-2

7-9-day

17

76.4

9-day
+ 50°C

n
"

30

85

4

25

9 -day
"
in 0.005N CaO

10

80

12-14-day

"

49

14-24-day
n
in 0.02N HC1
18-day

56.5
100

30

„
D 0.139“
V 0.130
D 0.051
V 0.042

30

D 0.273
V 0.168

29 7

D 0.073
V 0.030

87.5

46

D 0.161
V 0.073

79.6

^9.2

111

D 0.245
V 0.191

26

84.6

86.4

81

D 0.137
V 0.133

n

47

80.9

68.4

118

D 0.140
V 0.135

18-20-day
"
with MgCla

33

93.9

51.6

25

D 0.122
V 0.155

18-20-day
"
with CaCl2

37

94.6

68.6

29

D 0.142
V 0.070

24-d ay

60

81.7

57.1

120

D 0.161
V 0.154

9 - day
"
+ 50°C
(No saline)

"

88.2

60

100

62

II
Specimens of T. gelei i W and Other Inoculai

“ Per cent
Approx tio Per cent of Lesions Showing Hyperplasia~ of
Lesions
of
Mesoderm
Mesoderm + Epithelium
with
IncluM & E =
M> E
E > M
E>^M
Inclusions
sions—
83.3

100

0-+
A v 12+

19-85
Av 52

o

—

50

0=+
Av Ot

40

0-25
Av 5

50

0-13
Av 3

22.2

44.4

37.5

37.5

25

18.2

36.3

18.2

0-+
A v 15+

22.2

18.2

9.1

100

50

50

11.8

52.9

11.8

28.6

57.1

14.3

7.7

15.4

16.7

6^.7

11.1

53.8
66.7

20

33.3

40

15.4

23.1

15.4

23.5

23.1
16.6
6.7
15.4

30.8

63
TABLE

Type of
Inoculum

~Wo~ of
"Per cent Per cent Av Vol of
A v Wax
Embryos
Survival Develop­
Lesion
Height
of
Injected
ing
Epithelii
Embryos
Les ions
mm
0

1.4

12

D 0.039
V 0.018

78.6

54. 5

13

D 0.024
V 0.063

6

66. 7

50

90

D 0.033
V 0.016

0.02N NaOH

5

80

100

984

D 0.087
V 0.034

0.005N GaO

2

100

50

90

D 0.236
V 0.243

57.1

28

D 0.155
V 0.302

0.05-0.25
croton oil
emulsion

73

0.5-1.0 cc strain
12 lymphomatosis

14

0.02N HC1

Physiological
saline
F?j.nger 1s
solut ion

18

77.8

6

66. 7

0

0

—

i Embryos exposed to 0.5 cc. portions of the various inocula unless
strain 13 lymphomatosis 1:4 with physiological saline.
All
3 Upper figure refers to range in number of inclusion bodies;
3 D-Dorsal epithelium; V-Ventral epithelium.

34
II

(Cont.)

^ er cent
Approx No Far cent of Le~310ns Showing Hyperplasia of
Lesions
of
Mesoderm
Mesoderm t Epithelium
with
Inclu^
M & E M>E
E> M
Inclusions
sions___________ ______________________ __________ _

0

0

0

0

100

33.3

63.7

100
0

0

100

*

0

0

0

0

25

50

25

100
20

40

40

otherwise specified.
All T_. gele i i W w e r e diluted 1:10 and
inorganic salts dissolved in physiological saline.
lower figure to average number of inclusion bodies.

35
survival rate occurred subsequent to inoculation with 0.005N
CaO or 18“ 20“ day old T. geleii W reared in media containing
calcium or magnesium chloride.
All embryos surviving inoculation with either 0.02N
NaOH or 7-9-day old _T. geleii W suspended in either the ori­
ginal culture medium and heated for one-half hour at 50° C.
or in unhea ted physiological saline developed a chorioallantoic
lesion at the site of inoculation.

From 55.5 to 39.2 per cent

of the embryos surviving exposure to unheated physiological
saline suspensions of all other inocula of T. geleii W devel­
oped lesions.

The suspension of 14-24-day old cultures in

0.03N HC1 in physiological saline increased the rate of lesion
induction to 86.4 per cent.

The addition of calcium chloride

to the culture medium did not materially alter the per cent
of lesions developing subsequent to inoculation with 18-20day old organisms,

but the addition of magnesium chloride

decreased the number induced to 51.6 per cent.
All concentrations of croton oil killed the embryos
although one developed a lesion before death.

The strain

12 lymphomatosis agent produced lesions in 54.5 per cent of
the exposed embryos.

With the exception of Ringer's and

0.02N NaOH, all solutions used for suspending T. geleii W
produced lesions in approximately 50 per cent of the inocu­
lated embryos.

No lesions developed in any embryos inoculated

with Ringer's solution.

66

Size of lea Ion induced.

Subsequent to removal from the

ohorioallantois the length, width and depth of each lesion
was measured in millimeters and the total volume used as an
index for growth induction.
Lesions produced in these experiments ranged in average
size from approximately 13 cubic millimeters
allantoic membranes

inoculated with croton oil to approximately

984 cubic millimeters
(Table II).

in the chorio­

in those inoculated with 0.Q2N NaOH

The latter far exceeded in size lesions produced

by any other inocula.
An examination of the effect of age of the T_. geleii W
culture on the size of lesion produced indicated that,

in

general,

An

the older the culture the larger the lesion.

exception was noted in lesions induced by 7-S-day old cultures
suspended in physiological saline, as they averaged only onethird the size of those induced by 3-day old cultures.

The

average size of lesion induced by 12-14-day old organisms was
approximately twice that produced by 3-day cultures and six
times that produced by 7-9-day old organisms.

After 12 days

the lesion-inducing properties of T. geleii W varied but
slightly with an increase in age.
Suspending organisms in physiological saline decreased
the lesion-inducing properties of T. geleii W.

The average

volume of lesion produced by 9-day old organisms suspended
in the original culture medium and subjected to a temperature
of 50° C. for one-half hour was 397 cubic millimeters.

On

67
the other hand,

the inoculation with organisms of the same

age subsequent to suspension in physiological saline and
subjection to heat produced an average lesion size of only
30 cubic millimeters.

Similar aged cultures which remained

unheated but suspended in saline induced lesions averaging
20 cubic millimeters.

The latter would indicate that washing

not heating was the principal factor involved in the difference in size of lesion produced.

This effect of washing

warrants further investigation in the future, as the results
might be attributable to growth-inducing substances diffusing
into the culture medium and being removed in the washing
process,

or it may indicate that the culture medium itself

may have growth-promoting properties.

No study was made of

the effect of Bacto-tryptone on the chorioallantoic membrane.
In cultures 14 days of age or older the addition of mag­
nesium or calcium chloride to the culture medium or HC1 to
the physiological saline utilized as a suspending medium
decreased the lesion-inducing properties of Id geleii 'ft.
The addition of 0.005N CaO to the physiological saline used
for suspending 9-day old unheated cultures of Td geleii W
doubled the average size of lesion produced.

Physiological

saline alone induced an average sized lesion of 28 cubic
millimeters while the addition of either 0.005N CaO or 0.02N
HC1 to the saline increased the size of lesion to approximately
90 cubic millimeters.

Croton oil and the strain 12 lymphoma­

tosis agent produced the smallest lesions in this study.

68
Ringer*s solution wa3 the only suspending medium to induce
no lesions.
General morphological characteristics of induced l e s ions.
Two types of lesions were induced by the inocula used in these
investigations •*

(l) gray thickened areas in the chorioal-

lantois a n d (2) umbilicated.
following inocula:
saline,

The former was induced by the

7-9-day old T. geleii TP in physiological

cultures of 18—20-day old _T. geleii W to which mag­

nesium chloride had been added,

14-34-day old T. geleii W

suspended in 0.02W HC1 in physiological saline, croton oil
and physiological saline.
types of lesions.

All other inocula induced both

Umbilicated lesions usually exhibited the

greater degree of hyperplasia of the various tissue elements
and were frequently characterized by a complete destruction
of the dorsal epithelium near the site of inoculation.

Gray

thickened areas tended to follow the course of the circulatory
system and either varied but slightly from normal tissue or
exhibited varying degrees of tissue proliferation.

Hemorrhages

were visible in many embryos, but no correlation was apparent
between the type of inoculum and the amount of hemorrhaging.
It is also evident from Table II that lesions resulting
from inoculation with croton oil were characterized by necrosis
and slight mesodermal hyperplasia.

Those induced by 0.02N HC1

and 0.005N CaO exhibited both mesodermal and epithelial pro­
liferation in equal ratios.

All other inocula produced lesions

characterized by mesodermal proliferation alone and in

69
conjunction with varying degrees of epithelial hyperplasia.
No lesions were noted in which hyperplasia was restrictei
solely to the epithelium.
In general,

maximum epithelial hyperplasia was induced

by saline suspensions

of T. geleii W and increased with an

increase in the age of the culture.

An exception was noted

in the 18-day old cultures which produced lesions in which
23 per cent exhibited extreme proliferation of the epithelium
but over 50 per cent exhibited the most extensive hyperplasia
in the mesoderm.

Maximum epithelial proliferation occurred

in lesions produced by 24-day old cultures in which 30,3 per
cent of the lesions exhibited a considerably more extensive
hyperplasia of the epithelium than of the mesoderm.

This

epithelial hyperplasia varied from a simple increase in number
of layers

of cells to an extreme foliate type of growth.

The

addition of calcium or magnesium chloride to the culture
medium or HC1 to the saline used for suspension of the organ­
isms decreased the amount of epithelial and stimulated the
degree of mesodermal hyperplasia.

The addition of CaO to

physiological saline stimulated epithelial rather than meso­
dermal hyperplasia.
Unwashed 9-day old cultures subjected to 50° C. stimulated
epithelial growth to a greater extent than mesodermal, but
suspending the organisms

in physiological saline prior to

exposure to heat reversed this growth-promoting relationship.

70
Two-thirds of the lesions produced by strain 12 lympho­
matosis virus showed equal hyperplasia of both mesodermal and
epithelial tissues, while one-third of those examined exhibited
hyperplasia only in the mesodermal elements.

Physiological

3aline and most of the suspending media induced maximum hyper­
plasia in the mesoderm.
Maximum height of dorsal epithelial growth was obtained
by inoculation with 9-day old washed T. geleii W subjected to
heat,

12-14-day old T. geleii W in physiological saline and

0.QQ5N CaO.

Minimal proliferation was induced by the strain

12 lymphomatosis agent,

croton oil, 0.02N HC1 and 7-9-day

geleii W suspended in physiological saline.
nation of Table II indicates that,

An exami­

in general, the average

height of maximum growth was greater in the dorsal

(ecto=

dermal) than in the ventral (endodermal) epithelium.

The

only exceptions noted were, in those lesions produced by TP.
geleii W cultures to which magnesium chloride had been added
and by the strain 12 lymphomatosis agent, 0.005N CaO or
physiological saline.

In many lesions the endodermal epi­

thelium exhibited more extensive proliferation than the
ectodermal epithelium although the height of the latter may
be the greater.

Investigations

indicated that the most

active site for growth stimulation was to be found in the
endodermal or ventral epithelial tissues and that the growthstimulating substances may diffuse through the dorsal epi­
thelium to induce their maximum effect upon the endoderm.

In

71
several

lesions

outpocketings were visible ranging in size

from small knobs to large mushroom-like growths.

These out-

pocketi ng 3 were ch i e f l y mesodermal in origin with no exten­

sive p r o l i f e r a t i o n of the dorsal epithelium.
Maximum mesodermal cellularity was visible in lesions
produced by unwashed 9-day old organisms subjected to 50° C.
for one-half hour.

In most lesions the site of greatest

fibroblast proliferation was to be found in the ventral half
of the lesions adjacent to the endodermal epithelium.

Pro­

nounced fioroblast activity was also visible adjacent to the
dorsal epithelium.

These active sites tended to increase in

concentration and area as the age of the T. geleii W used in
the inoculum increased.
The endodermal epithelium was frequently vacuolated,
the significance of which was unknown but may be an indica­
tion of fatty degeneration.

Also in lesions stained with

haemotoxylin-eosin a blue and a brown precipitate were fre­
quently noted.

The former occurred along capillaries and in

areas in which necrotic tissue was present and was heaviest
in lesions produced by 3-day old T,. geleii W.

The latter was

restricted to the epithelial ne3ts described in a subsequent
section and may represent the breakdown product of some of
the blood constituents.
A positive correlation appeared between the age of the
T. geleii W cultures used for inoculation and the amount of
necrosis visible in the induced lesion.

Congestion was

72
heaviest

in lesions induced by cultures of T. g e 1ei i W aged

from 3-9 days.

Lymphocytes and macrophage-like cells were

visible in most lesions.

The former were at a maximum in

lesions produced by 3-day old T. gelei i W and the latter in
those induced by 14-34— day old cultures.
cells were

Vascular endothelial

in the process of capillary formation in the

larger lesions.

Neutrophils were notea only in lesions induced

by 0.02N NaOH.
No distinct characteristics could be isolated to dif­
ferentiate histologically the lesions produced by the various
inocula use d in this study with the exception of those induced
by croton oil in which extensive necrosis was visible.

Varia­

tion was as extreme among lesions produced by the same inocula
as between those induced by different ones.
Morphological characteristics of an individual lesion
induced by

geleii W.

A microscopic examination of a lesion

induced by 3-day old T. geleii W and stained with haemotoxylineosin revealed pilose projections

in areas of maximum pro­

liferation of the dorsal epithelium.

Adjacent to this area

deep pits were visible which divided the epithelium into
broad-based foliate projections with truncate distal surfaces.
The more distal the epithelium to the site of maximum pro­
liferation the less convoluted and hyperplastic it became.
The nuclei of the cells forming the most extensive epithelial
growth were smaller than those in the less proliferative areas.
In the latter the cells were segmentally arranged but in the

73

former t h e
thin

pattern was

nuclear

surface

of

membranes

the

dorsal

Proliferating
eosinophils
dorsal

more

Whorls
were

or

of

visible

T he
an

or

could

latter

was

onset

lymphocytes

to

little

chromatin.

The

outer

stai ne d an
mesenchymal
t he

cells

identified.
staining

the

themselves
but

surface.

blood

corpuscles,

Aggregates
cells

of

ectoderm

to t h e
re d

few

membrane

proliferating

by nu merous

blue.

and a

arranged

lie p a r a l l e l

b lue

intense

basement

fibroblasts

be

small

part

with a

one
of

had

to t h e

not

of

in f i b r i n o u s

filled

cells

were

of

dispersed

lesion.

central

phages

the

tended

other

the

increase

Proximal

surrounded

but

epithelial

beneath

perpendicular

cells,

throughout

very

All

epithelium

visible

distal

lymphocytes

and

fibroblasts,

epithelium.

obliquely
those

were

irregular.

of

t he

material

pale

the

lesion

note d .

less

first

cells

eosinophils

to a p p e a r

process.

were

macro-

visible.

subsequent

A few

present

cellular a n d

Numerous

brown substance were

the proliferative
and

became

The
to

scattered

in t h i s

area

of

the

mesoderm.
The
ity and

ventral

portion

fibroblast

appeared

to

along

the

ventral

phils

and

lymphocytes

embryonic

liver

the

lesion

a c t i v i t y w a s at

be w a l l i n g

?/iorpho l o g i c a l

of

off

surface.
were

necrotic

maximum.

present

exhibited

in c e l l u l a r -

The

latter

areas whi c h were

Hemorrhage,

characteristics

grafts

a

increased

congestion,

near areas
of

of

visible
eosino­

necrosis.

e m b r y o n ic g r a f t s .

pronounced

necrosis,

All

were

74
adherent
nection

to

the

to t h e

ectodermal

blood, s u p p l y .

tissues

proliferation
hemorrhagic
sequent

cnorioallantois

to

of

and

the

was

adjacent

to

All

which

adherent
areas

several

The

of

small

Feulgen

liferating
The

used

gates
of

t he

of

area
a

heavy
of

heavy

may

were
dub -

blue

the

pre­

graft

exhibited

and was

latter

grafts

pro­

concentretion
the

site

of

be p r e c u r s o r s

in t h e

of

chorio-

grafts.

liver

grafts

phosphatase

intensely

a c t i v i t y was

portions

DNA w a s

were

most

restricted

of n e c r o t i c

intense

in the

t i s sue.
pro­

cells.

to

lesion

that

adjacent

induced

by t he

to all

embryonic

various

inocula

study.

Epithelial

examination

liver

cellular

lymohocytes.

at a m a x i m u m

circumscribed

similar

of

This

and head

induced

suriace

activity,

The

embryonic

for

All

ventral

embryonic

acid

haa

with

fibroblasts

were

chorioallantoic

in t h i s

formation

to

reaction
blood

grafts was

the

formation.

s u d a n o p h i l i c , but
to

along

phosphatase

nests

but

heart

haemotoxylin-eosin a

proliferating

tubule

allantois

adherent

infiltrate;

with

established a con­

embryonic

chorioallantois.

nounced alkaline

epithelial

The

chorioallantoic.

visible
the

of a c t i v e l y

n ot

heavily

staining

cipitate

peculiar

were

ana had

nests.

nests
of

cells

ventral

t he

of

All

inocula a ppear ed

cells

various

(Figs.
lesions

resulted from
(endodermal)

3^ a n d

43).

indicated

stimulation and
epithelium.

to

stimulate
A

t hat

t he

microscopic
these

aggro*

outpccaetings

Apparently

either

75
g rowth-promo ting
the

dorsal

maximum

portion

effect

capacity

for

membrane.
the

upon

maximum

to

number
of

number

for

inoculation

carcinomas

similarity

43)

in t h i s

elements,

(Fig.

geleii

W.

area

of

The

the

of

the
to

exhibiting
were

found

average

by unhea t e d

A slight

t he a g e

or a g r e a t e r

nests

3^).

their

proximity

produced

epithelial
in the

visible

in w h i c h

decline

in

culture u s e d

A

n e gts

surrounding

in w h i c h

cellular
definite

were

a

and

vicinity.

distinct

aggregates

larger

lumen h a d

similar

determination

of

Oc­

to

this

blood
tissue

made.
examination

aforementioned

which

cholanthrene

the

those

apparent.

t he

produce

lesions

28 a n d was

o l d T.

were

A comparative
d uc ed by

mesodermal

to

in c l o s e

occasional

o c c u r r e d as

than

been formed and

be

in

were

d i f f us i ng throu^i

increased.

nests

cells w e r e

of

(Fig.

inherent

nests

was

constituting

casionally

surface

epithelium

7-9-day

vacuolated

c o u l d not

of

nests

produced

Cells

of

but

capable

chorioallantois

is a c t u a l l y

dorsal
of

wore

ventral

majority

the

the

it w a s

the

surface,

suspensions

visible

the

prolifera.tion

adjacent

more

of

growth

The

ventral

extreme

substances

developed

in the

the

former

1952)

epithelial

and

in c h i c k e n s

two.
at

the

inocula

(Duran-Reynals

between

of

the

nests

squamous

treated with

pro­

cell
methyl-

revealed a superficial

However,

the s t a g e

of

no

keratinization

development

was

in w h i c h

studied.

Inclusion
cytoplasmic

bodies.

Fennell

inclusion bodies

in

(1951)
the

noted

intranuclear

epithelial

tissues

of

and

76
lesions
that

induced

the

by c u l t u r e s

combination

of

of T.

preformed

w h i c h a c c u m u l a t e d as

the

soaps

insoluble

w h i c h w o u l d be

characteristics

In t h i s
bodies

were

of

entities

could

plasmic

These

to

(Fig.

could

40)

or

extracellular

duced by

3-day

identical
tained,

to

but

All

suspensions
bodies,

in a

lumen

present

inclusion

bodies

while

9— day

old unwashed

T_. g e l e i i

agent,

NaOH a n d

The

0.02N

maximum

number

of

at

a

could

they

not

numerous

Spherical

W and

and cyto­

periphery
lesion
in

of

of
the

itself.

lesions

pro­

particles

were

be d e f i n i t e l y a s c e r ­
not

be

separated.

C aO a n d h e a t e d
W contained

oil,

in

frequently

these

in l e s i o n s

croton

the b a s a l

geleii

in t h e

maximum

could

by 0.005N

visible
W,

the

lar-e aggregates

on the

Whether

o l d T_. ^ e l e i i

none w e r e

calcium

haemotoxylin

39).

of T.

areas

either

were

most

intranuclear

found

g e l e i i W.

of

fat

assume

in

were also

be

produced

th e

inclusion

with

37 a n d

remnants

o l d T.

lesions

They were

In s o m e

morphologically

with

produce

concentrated

from the

bodies

could

stained

(Figs.

inocular

inclusion b o d i e s .

salts

cytoplasmic

inclusion bodies

distinguished

particles

lesion

epithelium.

in un a b s o r b e d
be

and

heavily

areas

postulated

i n c l u s i o n bodies.

in l e s i o n s

most

hyperplastic

similar

not

these

dorsal

aged

He

in w a t e r a n d m i g h t

visible

visible

W.

calcium

organisms

intranuclear

a n d were

the

moderately

visible

study
also

eosin-azure
cells

of t h e

geleii

inclusion

induced by 9-day

strain

normal

saline

12

Ringer's

or

saline

cellular

i n c l usion bodies

lymphomatosis
solution.
occurred

in

77
lesions
of

T.

age

induced

geleii

induced

sion

W,

possibility

The
of

of

a

inclusion

third

were

of

those

geleii

W

50°

for

in

C.

dorsal

(Figs.
face

45) .

one-half

and

of

nests

the

by

magnesium

had

by

14-24-dey

per

cent

of

those

day

old

obtained.

the

saline a n d
most

or

old

In a l l
Alkaline

T.

of

of

T.

phosphatase

fat

geleii
p er

Epi­

(Fig.

scattered

droplets

cent

most

in l e s i o n s

calcium

ot

and

saline

a positive

was

sur­

W to w h i c h

in 0 . 0 3 N H C 1

to w h i c h

lesions

fat

were

visible

by ph ys iol ogi cal
W

proliferation

for

cells
and

to

a n d vein s .

in 2 5

cultures

o l d T_-

fragments.

positive

a c t i v i t y was

cultures

one-

epithelium and

inocular

in a r t e r i e s

re 1 eii

other

A ll

concentrated

maximum

lesions,

b e e n addea,

induced
of

the

subjected

highly

ventral

epltheloid

most

phosphatase
old

exception

exhibiting

tissue

of

visible

produced

been added.

the

frequently highly

chloride

inclu­

calcium and the

induced l e s i o n s .

Fat w a s

tissues

mesoderm

cultures

of

physiological

hour.

18-20-day

fat,

inoculation with 9-aay

sudanophilic

Mo a l k a l i n e
induced

by

necrotic

occasionally

of

t e n d to support

between

fat w i t h

and along

were

Intensely

throughout
were

44),

in a r e a s

thelial

in

epithelial

38

for

produced

suspended

minimum number

bodies.

properties

positive

the

results

relationship

3— day o l d c u l t u r e s

and

Organisms f r o m 1 2 - 1 9 days of

containing

preceding

Histochemical
lesions

C a O and. 24

respectively.

lesions

bodies.

production

by 0 . 0 0 5 N

those
in 3 3 . 3

or

chloride

reaction

highly

18-20had

wrs

concentrated

78
in the

ventral

half

of

capillaries

and

Mesenchymal

cells

exhibited

slightly

a

in t h e

u s u ally apparent

ing

t he

epithelial

lesions
50

per

suspended
calcium

nests,

of

in 0 . 0 2 N

chloride

HC1

physiological

occurred

in sites

with

exception

chymal
bu t

cells

acid

was

Lipase
produced
normal

but

t he
A

lesions

3-day

saline

was

more

to

old

the

C.

active

positive
studied

or

to

in

tissue,

includ­

a positive

Acid

be d e t e c t e d
of T.

^eleii

14-34-uay

old

55.7

those

per c e n t

of

W,

in

to which

of

alkaline

in a n y

old cultures

cultures

phosphatase

mesodermal

those

pro­

activity

phosohatase

fibroblasts

and mesen­

phosphatase was usually

present

Th e

intense

for a c i d phosphatase.

t h a n that

visible

cultures

for

old

the

of

one-half

less

in a l l
T.

same

W

as

prior

except

alkaline

those

suspended

suspended

hour

sites

r e a c t io n for

lesions

geleii

cultures

to

alkaline

in s a l i n e

in
and

inoculation.
phosphatase,

intense.

Feulgen— Bauer
with

^rayncss

e ntirely absent.

in t h e

r e a c t i o n was

41).

A diffuse

considered

could

18-30-day

alkaline

and 9— day
5 0°

not

and

frenuently

epithelial

by e i t h e r

saline.

activity was

by

.subjected
Lipase

in w h i c h

phosphatase

phosphatase

or

similar
of

(Fig.

lesion

old c u l t u re s

aa.ed

duced by

the

was

activity

induced

was

t he

arteries

phosphatase.

by 7 - 9 - d a y
those

the

reaction.

but

v e ins,

fibroblasts
of

throughout

phosphatase

induced
cent

center

positive

for alk a l i n e

Wo a c i d

lesion a l o n g

in p r o l i f e r a t i n g

was

reaction

th e

reaction was

exception

of

that

obtained

in a l l

p r o d u c e d by c r o t o n

79

oil.

This reaction was restricted to the dorsal epithelium

and epithelial nests.

This reaction was not eliminated by

saliva and may be due to some polysaccharide other than gly­
cogen from which aldehydes could be released.
Desoxyribonucleic acid was present in the nuclei of all
cells but was most heavily concentrated in the actively pro­
liferating blood and enithelial cells and in peripheral fibroblasts of the mesoderm.
With the exception noted in the discussion on glycogen
(Feulgen-Bauer reaction) all control slides were negative.

DISCUSSION
1.

HISTOCHEMICAL PROPERTIES OF TETRAHYMENA GELEII W.
Data presented in the preceding experiments were diffi­

cult to duplicate possibly due to a heterogeneity of the
cultures,

variations

in temperature or to uncontrollable

environmental factors.

No definite conclusions could be

drawn as to the function of the various substances investi/
gated in T^. geleii W for no distinct correlations were demon­
strable.

However, certain possible relationships were noted,

an d it is interesting to speculate on the functional role of
these substances in the physiology of this organism.
Junquiera

(1950) maintained that acid phosphatase might

be involved in carbohydrate synthesis

in vertebrates.

In

tryptone cultures of T . g eleii W 216 hours in age or older
the curves for glycogen deposition and acid phosphatase
activity were similar if the organisms were stained im­
mediately upon removal from the culture medium.

This sug­

gested that acid phosphatase might play a role in glycogen
deposition.

However,

in organisms reared in vitamin-enriched

media acid phosphatase frequently exhibited a cyclic activity
curve;

i.e.,

it increased from approximately zero to a maxi­

mum and then decreased to zero again.

Glycogen deposition,

on the other hand, was maintained at an approximately con­
stant rate.

This suggested that if a c i d phosphatase played

81
a role in glycogen deposition in these organisms,

excess end

products of dephosphorylation occurred with increased enzymatic
activity and inhibited, acid phosohatase activity.

Eventually

the end products of the reaction might be depleted and acid
phosphatase activity again increased to a maximum.
growing cells,
tryptone,

In rapidly

such as those exposed to vitamin-enriched

the end products of enzymatic activity may build

up rapidly.

In other cultures of _T. geleii W no correlation

was apparent between acid phosphatase activity and glycogen
deposit i on.
Wichterman (1953)

believed that fat under anaerobic

conditions could form as an end prouuct of carbohydrate
metabolism in paramecium.

Results of this study indicated

that the curve for fat and glycogen deposition was similar
in organisms reared in tryptone and suspended in physiological
saline prior to fixation.

In young cells the amount of fat

an d glycogen was relatively low,

but as the cultures aged there

was a corresponding increase in both.

Eventually the organisms

became completely filled with fat, ana a rapid decline in
glycogen deposition was observed.

This suggested that the

synthesis of glycogen may be closely related to that of fat,
and that on completion of the latter the supply of the former
becomes depleted either through utilization in the formation
of fat or through a reduced requirement by senescent cells.
However,

the chemical interrelationsnips in the formation of

these compounds in _T. geleii W are not clarified at this time.

82
Wislocki and Dempsey

(1945) maintained that in vertebrate

tissues alkaline phosphatase played an important role in the
synthesis

of glycogen.

If alkaline phosohatase was imrortant

in carbohydrate synthesis

in T. geleii W, it may have been

functional in organisms fro m tryptone cultures but not in
cells which have become adapted to vitamin-enriched media.
Rothstein and Meier

(1949) maintained that yeast surface

phosphatases played no direct role in either metabolism or
in the mechanisms of phosphate uptake, but that they rnsde
available to cells substances in the medium which ordinar ily
could not be utilized.

Many authors

(Gotnori, 1941, and

Maengwyn- Dav ies , Friedenwald and White,

1952, etc.) have

demonstrated that in vertebrate tissues the duodenum, arteries
and capillaries exhibited maximum alkaline phosphatase activity.

This has led to the conclusion that one of the functions

of alkaline phosphatase is the absorption and transfer of
organic substances across cell membranes.

Alkaline phospha­

tase activity in T. geleii W reared in tryptone was confined,
in general,

to the distal end of the cell adjacent to the

pellicle which suggested that one function of alkaline phos­
phatase

in this organism was also concerned with an increase

in the availability and transfer of nutrients into the cell.
All visible alkaline phosphatase activity disappeared in
T. geleii W which had become adjusted to vitamin-enriched
culture media.

Therefore,

this enzyme may also be involved

in the release of materials essential for the synthesis of

83
certain vitamins.

Most of the vitamins used in this study

regulate carbohydrate metabolism, and the disappearance of
alkaline phosphatase may be correlated with a vitamin-activation of other enzymes associated with the synthesis of carbo­
hydrates and other metabolic processes essential for rapidly
growing cells.

The presence of a negative reaction for alka­

line phosphatase does not eliminate the possibility of the
enzyme being present

in either an inactive state or in too

minute amounts to be detected by the Gomori technique.
Pritchard
phatase

is

both

and

and

nuclear

cell

alkaline

sequent

partial

was

and

salivary

to t h e

n o t e d that

increased

bacteria

and

found

that

acid

which

there was

suggested

synthesis

of

a

with

t he

rate

liver

of

phosphatase
evidence

for

relationship

found

of

that

desoxyrat

sub­

Catcheside

alkaline

phosphatase

b a nas

Drosophila

of

Heden

synthesis

polynucleotide

DeRooertis
occurred
the

a c id,

in p r o t e i n

the

Malmgren and

protein

increased

metazoa.

protein.

(1944)

phos­

c o xitclx 1*10ct t ti0 mo x x mixin 00

aesoxynucleohides.

closely associated

et a l

that

that

nucleic

involved

Feulgen-positive

and

of

Danielli and

showed

growth

believe

a^e

and

in t h e

c h r o m o s o nio s w lix o 1*1 glI s o
of

and

which

hepatectomy.

centration

both

metabolism

phosphatase

(1945)

(194-)

Brues

increased

Krugelis

restricted

the

division.

turnover

(1945)

Danielli

nucleoproteins

nucleotide
to

and

concerned with

nucleotides
synthesis

( 1 947)

and

presence

between

acid

were
content

Schmitt

in a r e a s
of

(194?)

in

(1948)

of a x o n s

in

neurofibrils

phosphatase

and

the

84
In u n w a s h e d
enriched
was

m e d i a alkaline

maintained

were

specimens

at a

360 hours

in w h i c h

DNA

between
latter

DNA
may

role

maximum
This

catalyze

in D NA

etc.

suspended

DNA

and acid

cyclic,
at

360 h o u r s

Acid

phos'ohstase w e r e

in w h i c h

DNA

turnover

hi g h l y active.

The

former

may

aid
On

from

in

the

the

tryptone

phosphatase
tase
and

from
t he

solutions

the

in the

cannot

measurements
indicated

of

that,

D NA

bore an

end

activ­

DNA

in w h i c h

into

activ­

the

nucleotides,

tryptcne

to p l a v

and

a.

cultures

the a c t i v i t y c u r v e

for

in v i t a m i n - e n r i c h e d

rapid, a c i d

phosphatase

that a c i d

was

phosphatase

towards
in DNA
of

size

made

may

Although
involved

this

time.

in the

smaller

of

the

cell

implicate
both

in DNA

acid

acid and
metabolism,

Approximate

course
the

acid

of a c i d p h o s p h a ­

nucleus

activity

be

organisms

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

the

D NA.

to

the

in u n w a s h e d

in l oc a ti o n

c l a r i i i e d at

in g e n e r a l ,

cultures

relationship

from

inverse

shift

appeared

nuclear

cells

time

appeared

similar,

activity

breakdown

be

activity

DNA.

decrease

phosphatase

role

of

DNA

saline

may be

DNA

the

also

suggested

The

posterior

subsequent

alkaline

hand,

activity.

phosphatase

their

synthesis

other

of

organisms

in p h y s i o l o g i c a l

at w h i c h

activity

phosphatase
In

but

phosphatase

inverse

phosphatase
conversion

in v i t a m i n -

from tryptone

alkaline

suggested an

the

turnover.

and

media

was

absent

level u n t i l

In s p e ci me ns

and alkaline

nucleosides,

was

fairly constant

activity

ity disappeared.

g e1ei i W reared

phosphatase

of a g e .

ity approached a

of _T.

of this

nucleus

study

85
the greater the staining intensity for DNA, which may alter
the estimation of DNA activity ma.de on the basis of staining:
reaction alone.
Kabat and Furth

(1941) maintained that the mag­

nesium ion activated alkaline phosphatase.
experiment,

In these present

calcium was even more effective than magnesium

in the activation of phosphatase.

Both ions tended to increase

glycogen deposition and the life of the culture but decrease
fat deposition.

The decrease in the latter by addition of

magnesium to the culture solution may have resulted from a
greater utilization of the stored lipids by the rapidly
growing cells.

The shift

in alkaline phosphatase activity

from the distal end towards the nucleus in cells from magnesium
and vitamin-enriched culture media, may be correlated with the
increased division rate and DNA turnover which must occur in
rapidly growing cell3.
Maengwyn-Davies, Frielenwala and White
man, Feigen,

Wolf and Kabat

(1950)

(i960) and New­

showed that it was possible

to differentiate histochemically substrate specific enzymes
hydrolyzing a number of phosphate esters.

This suggested

that in these present experiments the shift in alkaline phos­
phatase activity from the distal end of the cell towards the
nucleus upon addition of vitamins and magnesium chloride to
the culture medium might be attributable to the presence of
several enzymes.

The results obtained in the experiments on

inhibition, however,

suggested that the shift was a mani­

festation of two different functions of the same enzyme and

"that a
i n 1*

non-specific
g eleii

centrations
is

essential

wof

alkaline

Further
inhibitors

before

phosphatase

w as

probably

investigation utilizing
and an

definite

other

increased number

conclusions

may

be

present

of

con­

substrate

drawn.

LeDuc and Dempsey (1951) and Martin and Jacoby (1949)
demonstrated that, the phenomenon of diffusion complicated the
histochemical reaction for alkaline phosphatase in vertebrate
tissues.

Diffusion was also a prominent factor in the local­

ization of both alkaline ana acid phosphatase

in T. geleii W.

A negative alkaline phosphatase reaction was obtained and
attributed entirely to the diffusion of the enzyme or the
inorganic phosphate resulting from the enzymatic catalysis
of the dephosphorylation of hexose phosphates,

etc.

Acid

phosphatase unlike alkaline phosphatase diffusion was never
complete due to a higher degree of activity for the former.
An extracellular equilibrium was readily established and
diffusion curtailed before catalysis was complete.
Lipase appeared to be concerned with the hydrolysis of
fat

in T. gelei i W, as the addition of magnesium to the

culture medium enhanced lipase activity and decreased fat
deposition.

Moreover, a decrease

in lipase activity occurred

after the cells aged 360 hours during which time fat deposi­
tion rapidly approached a maximum.

The shift

in location of

lipase activity from the nuclear region to the distal end of
the cell subsequent to the addition of calcium or magnesium
to the culture medium cannot be explained at this time unless
fat hydrolysis

is excessive in rapidly growing organisms and

87
lipase aids

in the transfer of the end products of this

reaction across the cellular membranes.
Peroxidase may have been present
inactive state,

in T. geleii W in an

but it could not be demonstrated.

gested that no biological oxidations

This sug­

in T. geleii W require

catalysis by peroxidase.
Purine catabolism in _T. geleii W does not appear to
result

in the formation of urates

or uric acid but is probably

accomplished through other chemical pathways.
3.

LESION INDUCTION IN THE CHQRIQALLANTOIS
All salt solutions,

suspensions of TO geleii W and other

inocula utilized in the experiments on lesion induction,
with the exception of Ringer's solution,
promoting properties

to some degree.

possessed growth-

Apparently most foreign

materials will stimulate cellular proliferation in the
chorioallant ois either by irritation or by release of q uan­
tities of normally occurring growth-promoting materials.
Lesion induction by protozoan cultures undoubtedly involved
both of these processes;

i.e., an irritation by the suspend­

ing medium and the release of growth-promoting substances
by the organisms themselves.

These substances accumulated

as the organisms aged and reached a maximum when the cells
became senescent

(34-day old cultures).

Fennell

(1951)

demonstrated that suspensions of these senescent specimens
exhibited abundant amounts of neutral fat, fatty acids,
cium salts and a high degree of phosphatase activity.

cal­
The

88
largest

lesions

NaOH

and

tion

or

tissue

in the

roust b e
the

as

attributable

release

of

to

either

growth-promoting

NaOH would u ndoubtedly

concentration and
ring

investigation were

could

not

be

not

an

occur

factor.

In these present

experiments

by 0.02N

inflammatory

factors

considered a

growth-promoting

the age

produced

by

the

in n a t u r e
normally

reac­
necrosed

in t h i s
occur­

it was noted that during

interval in which the lesion-inducing properties of

X* geleii W were at a maximum the fat and glycogen content of
the organisms were also approaching a maximum.
however,

Glycogen,

frequently disappeared rapidly when the cells haa

become nearly filled with fat.

Acid phosphatase also ordi­

narily increased in activity during this period.
scopic examination of the lesions

The micro­

induced by these senescent

cells revealed that maximum fat deposition occurred in areas
of maximum epithelial proliferation.
that the role of lipids
been underestimated.

It, therefore, appeared

in the proliferative process may have

The corresponding appearance of maxi­

mum fat concentration in areas of greatest epithelial pro­
liferation and in T. geleii w at the height of its lesioninducing power indicated that although lipids may not be the
dominant factor inducing growth their possible role in this
process should not be overlooked.

Acid phosphatase and gly­

cogen also increased in T. geleii W during the period in which
it manifested its maximum growth-inducing properties and may
play an indirect if not dominant role in the proliterative

89
process.

Experiments with organisms suspended in physiological

saline emphasized the possible role of the phosphatases or
other enzymes

in growth induction as many of them diffused

readily from specimens of Th gel e i i W and could be materially
reduced in the washed organisms prior to inoculation onto
the ch ori oallantois.

Unwashed cultures of _T. geleii W pro­

duced lesions larger than any induced by cultures suspended
in physiological saline.

Apparently the omission of washing

conserved the phosphatases or other substances conducive to
lesion induction.

However,

the culture medium itself may

possess some growth-stimulating properties.
Neither the histochemical nor the morphological investi­
gations of the induced lesions revealed any characteristics
by which the induction agent could be accurately identified.
Extensive variations
same inocula.

existed between lesions produced by the

However, a tendency was noted for certain types

of inducing agents to be specific in their stimulation of
certain chorioallantoic tissues.

In general,

entities induc­

ing maximum epithelial growth appeared to be concentrated in
inocula prepared from protoplasm ana appeared to increase in
concentration as the age of the protoplasm used for inocula­
tion increased.

On the other hand, maximum mesodermal pro­

liferation was stimulated by inorganic salt solutions and
other suspending media.

The addition of metallic ions to

protozoan cultures reduceo the protoplasmic effect on the
epithelial tissues.

The effect of metallic ions on mesodermal

90

tissues may be attributable to an alteration in the concen­
tration of the extracellular fluid of the mesodermal connective
t i s sue.
The endoderm&l epithelium and the ventral half of the
mesodermal tissues exhibited the maximum growth stimulation
and enzyme activity.

The endodermal cells may normally be

active sites of enzyme secretion which would aid in the trans­
port of nutrient materials ana would account for the strongly
positive reaction visible in the ventral portions of the indi­
vidual lesions.

On the other hand, an apparent diffusion of

enzymes or catalyzea ena products from the dorsal to the
ventral surface of the lesion was noted and the subsequent
increased ventral growth may be attributable to the increased
concentration of the diffused growth-promoting materials.
appeared,

however,

It

that all tissues of the chorioallantois

were stimulated equally a.nd that the growth differential of
the ventral surface was normally greater than that of the
dorsal.

The increased amount of phosphatase activity present

in the ventral half of the lesion might also have been a mani­
festation of induced stress, as Moog

(1952) found that phos­

phatase in embrvonic tissue was normally increased at times
of stress .
Acid and alkaline phosphatase were visible in all lesions
with the exception of several induced by physiological saline
or protozoan cultures
added.

to which HC1, MgClg or CaClg had been

The heaviest concentrations of both ot the phosphatases

and lipase were visible in the walls of arteries,

veins and

91
capillaries
that

in r a p i d l y

increased
across
of

of t h e

to

the

growing

enhance

vascular

substances
The

induced

fibroblasts

tissues

the

in t h e

suggested

a

proteins

of

proliferation.

alkaline
of

phosphatase

nucleic

Fell

and

acid and

Danielli

healing wounds
alkaline
was

more

and

alkaline

the

acid

which

growth

cated

that

phosphatase

important

positive

thelial

t ha t

promotion
role

in

in g r o w t h

might

of a l l

of

at
act

was
by

a

various

the

mesodermal

off

of a r e a s

the

liberation
complex.

formation
increase

phosphatase

lesion than
geleii

of

s u g g e s t e d that

marked

in

in
activity

in the

W w h e n the

induc­
l a t t e r ’s

a maximum.

This

as

inhibitor

a growth

fibrillar

geleii

metabolism

metabolism

collagen

proteins

materials.

highly active
Th

the

nucleoprotein

growth-indueirg

during

W was

suggested

to w a l l
On t h e

the

period

pronounced and

physiological

other

processes

be a b s e n t

in a l l

r eaction was

lesions.

induced

obtained

may

However,

the

lesions.

in t h e

in

indi­

promotion.

Feulgen— Bauer

tissues

Td

materials

walling

Alkaline

were

the f o r m a t i o n
and

in the

(1950)

the

induced

properties

G l y c o g e n a p p e a r e d to
A

from

notea

in the

in

concerned with

protein

phosphatase

its

role

Bradfield
be

either

st re am.

subsequent

disappeared from

irritants

hand,

be

may

therefore,

were

nutrient

phosphatase

associated with

pronounced

stimulating

off

the

(1943)

wa s

g r o w t h - i n d u c t io n

by

blood

phosphatase activity.

tion agent

that

and

of

enzymes

to catalyze

possible

fibrillar
active

or

of a l k a l i n e

It a p p e a r e d ,

these

transfer

membranes

present

presence

lesions.

epi­

reaction was

93
not

eliminated

by saliva

saccharide

or

other

released.

In

the

as

rapidly as

by

ordinary

glycogen

it

and was

substance

induced
is

formed,

ordinarily

from which

lesions

histochemical

is

attributed

the

so t h a t

highly

mucopoly­

aldehydes

glycogen

it c o u l d

techniques.

most

to a

could

be

m a y be u t i l i z e d

not

be

detected

In e m b r y o n i c

concentrated

tissue

in th e

yolk

sac.
Th e

localization

relationship
turnover

between

in t h e

DNA

activity

alkaline

proliferating

pronounced

in the

and

essential

may be

of

vascular
in

inter-

phosphatase activity and
tissue.

tissue

the

suggested an

Both were

and

especially

peripheral

formation

DNA

fibroblasts

of c y t o p l a s m i c

desoxy-

ribonucleotides.
The

calcium

investigated
older
ties

ion w h i c h

in t h i s

the

appeared

senescent

to

increase

o n fat

production may

effect

on

lesion

Groton
irritant,
by

oi l

and a

cultures.
lesion

The

strain

12

small

virulence

of

of

the

e n z ym es

production

lesion-inducing

In y o u n g

induction.

cultures

The

effect

for

its

in

proper­

calcium
of c a l c i u m

inhibitory

induction.
at

all

concentrations

detailed

ncmber

resistance
th e

the

partially account

lymphomatosis

inherent

most

d e c r e a s e d fat

decreased

comparative

carcinogenetic agents

ma de .

an

study and

T_. g e l e i i W a l s o
of

activated

viral

was
study

and protozoan
and

size

agent
of

the

was

of

of

powerful

lesions

could

induced

undoubtedly
eg g s

an

lesion=induction

cultures

inoculated

culture.

too

due
or a

not

by

be

th e

to e i t h e r
loss

in

93
Ringer
normal

s

s o l u t i o n was

extracellular

induction
R i n g e r 13

processes
solution

Epithelial
appeared
sional

to

be

nests
could

ences

between

nated

any

ficial

nests

similarity

suggested

agents

and

Further

clusions

can

be

from

those

and
were

most

24-day

pothesis
be

cium

salts

and

the

growth

to a l l

medium.
studied

two.

a n d the

Occa­

in w h i c h

blood

differ­

system

elimi­

A super­

squamous

(195?>) w i t h

cell

methylcholan-

carcinogenetic

encompass

are

research

morphological

circulatory

might

scrowth-

lesions

lumens

the

the

enithelium.

s t i m u l a t i o n by

cultures

cytoplasmic

could
in

in

by

required

similar

pro­

before any

lesions.

distinguished

con­

were

Other

present

All

W.

(19 51)

by the

noted

in

morphologically
aggregates
o n th e

of

periphery

aforementioned bodies

produced by calcium

TT. g e l e i i

Fennell

No

inclusion bodies

infections.

lesions
of

be

entities

produced
fat.

not

viral

similar

specimens

with

extreme

by D u r a n - R e y n a l s

of s e v e r a l

soaps

contained

future

suspending

ventral

The

simulate

d ra wn .

suggested

calcium

common
the

investigations

numerous

old

of

the

nests

tha t

visible

in l u m e n s

were

In al l

between these

lesions

morphologically

as

to

i n f l a m m a t o r y or

between

Intranuclear and
the

No

correlation

protozoan

cesses.

of

which

nests

produced

threne

many

be u s e d

detected.
these

inocula

stimulated.

noted which

possible

carcinomas

were

invaginations

be

only

environment.

should

were

cells

the

Tnis
that

supports

the

or
hy­

inclusion bodies

combination

i n v e s t i g a t i o n was

oxide

of

made

preformed
to

may
cal­

determine

24
the

presence

requires

of

living

production

any

virus

cells,

within

it w o u l d

of t h e s e b o d i e s

the
be

inocula,

difficult

b y a CaO s o l u t i o n .

but
to

as

the

explain

virus
the

SUMMARY

1.

Cultures

throughout

the

p h 0 3 phata.se,
A

life

each

were

T_. g e l e i i
of

lipase,

semi-quantitative

for

or

of

the

determination

of

made

in t ryptone

chemical

No

functions
no

of

and

the

the
Th

conclusions

substances

phosphatase

in

tryptone

was

most

of

the

and

reached a

cell

Alkaline
synthesis
brane
thesis
tests

of
of

phosphatase
and the

highly

transfer

Th g e l e i i W.

It

carbohydrates

i n d i c a t e d this

phosphatase

histotests

were

original

be d r a w n as

in T.

to t h e

in T.

g e l e ii

However,

geleii

in t h e

in c u l t u r e s

role

may also

to

noted.

in DNA

nutrients

in y o u n g

o n th e

in t h e

concentrated

of

chloride

demonstrable.

activity

may play a

organisms

tryptone alone

investigated

were

maximum

The

s a l in e.

could

were

of a c t i v i t y

Histochemical

suspended

relationships

Alkaline

W.

and acid

th e a m o u n t

medium

72 h o u r s

glycogen and urates.

calcium

culture

correlations

certain possible

or

%eleii

every

in te r v a l .

physiological

various

fat,

vitamin-enriched

organisms

definite

distinct

3.

of

in

each

magnesium

of

both with

medium

2.

W as

effect

properties

conducted
culture

the

and

at

stained

for alkaline

DNA,

in c o m b i n a t i o n w i t h

ascertain

culture

peroxidase,

substance was

reared

W were

was

distal

end

aged 283 hours.

metabolism, v i t a m i n

across

play a role

T.

W reared

gele_ii W.

the

cell

in t h e

mem­

syn­

Inhibition

non-specific.

4.

A

accurate

heavy

extracellular

localization

of a c i d

suspended

in t h e

reared

tryptone and

in

phosphatase
end

of

original

activity

organisms

cultures

acid

Maximum activity
play an

role

phosphatase

and

reached

geleii

nucleus

to

be

A

proximal

tip

senescent
8.
and
9.

geleii

of

most

Acid

the

nucleus

phosphatase

synthesis.

Cyclic

relationship

between

highly

organisms

hours

concentrated

reared

subsequent

c o n c e r n e d w i t h the

T.

geleii

m a x i m u m at

positive

d i s t a l l y as

W.

of

reached a
7.

in

was

G l y c o g e n d e p o s i t i o n was

pellicle

tone

In o l d e r

shifted towards

hours.

distal

may

curves
acid

to

in th e

in t r y p t o n e

seeding.

hydrolysis

of

and

Lipase

fat

in _T.

W .

6.
and

of

maximum 360

appeared

144

saline a c i d

in the

in age.

in c e l l s

metabolism.

activity

a

activity

possible

Lipase
the

concentrated

an

in o r g a n i s m

However,

in p h y s i o l o g i c a l

in g l y c o g e n

suggested a
DNA

medium.

than 288 hours

in activity

vicinity

highly

o c c u r r e d at

important

5.

culture

phosphatase

interfered with

phosphatase activity

suspended

was

l es s

precipitate

between

in or g an is ms

W and

the

reared

nucleus

in t r y p ­

432 h o u r s .

reaction for

fat

of

3-day

o l d T.

geleii

the

cells

ag e d .

Fat

first

appeared

W and gradually

deposition

in the
expanded

reached a maximum

cel ls.

Phospholipids

were

dispersed

first

appeared

in c e l l s

DNA

activity

was

W and

heaviest

exhibited

aged

throughout

504

r e s t r i c t e d to

cyclic

changes

T.

geleii

hours.
the

nucleus

of

in c o n c e n t r a t i o n .

Id
An

97
inverse

relationship

existed

and

intensity

the

the
10.

No

visible

positive
geleii

The

addition

biotin

to

phosphatase
gen

deposition

the

medium

Suspension

decreased

alkaline

deposition
13.
medium
the

of

to

14.

W,

The

of

T.

and acid

of g l y c o g e n but

alkaline

W

magnesium

inhibitory

phenomenon

dispersed

of

chloride

effect

to

of

saline

a c tivity and the

of

salt

of £.

solutions

and

the

salts

the c u l t u r e

deposition
shifteu

of

the

lipase activity.
complicated

the

histo-

and acid phosphatase.

phosphatase

Croton

geleii

were

chorioallantois

embryos.

to

phosphatase activity and ■

diffusion

alkaline

added

occurred

in

c e l ls .

Suspensions

the

and acid

in p h y s i o l o g i c a l

these

phosphatase

alkaline

Leghorn

of

decreased

media

diffusion

of

riboflavin,

activity.

and acid

Complete

tion

was

inhibited alkaline

phosphatase

of

other

acid,

i n c r e a s e d DN A

th e

geleii

localization

15.

nucleus

lipase a c t i v i t y and g l y c o ­

but

phosphatase

chemical

and

medium

The a u d i t i o n

In v i t a m i n - e n r i c h e d
of

the

or u r a t e s

nicotinic

decrease

increased alkaline

widely

peroxidase

C a l c i u m a n d .m ag ne s i u m c h l o r i d e

location

of

glycogen.

deposition

f at .

culture

counteracted

on alkaline

12.

size

reaction.

thiamine,

i n T_. geJLeii

activity.

vitamins

of

tended

phosphatase
culture

th e

W.

tryptone

and

Feulgen

r e a c t i o n for

i n T.

11.
and

of

between

of

oil,

W

in p h y s i o l o g i c a l

prepared and used
12-14-day
the

strain

old
12

for

saline
inocula­

pedigreed

White

lymphomatosis

98
agent,

embryonic

u s e d as

comparei

13.

All

exception

of

properties
17.

Two

(2)

gray

an

inocula

some
types

culture

of

T.

in

areas

in t h e

these

reaction and

(3)

to

the

existed

between

size

of

the

properties

of

T.

the

deposition approaching

induced a
direct

maximal

W.

cultures

tissues

proliferation and the
22.
orior
size

to
of

23.

Suspension
inoculation
lesion

epithelial

may

play a

role

age

of

T.

onto

of

processes:
normally

geleii
the

geleii

the

le sion.

W were

at

increasing and

maximal
salt

mesodermal

a
fat

prolifera­

solutions

tissues.

extent

g e l e ii W u s e d
W

of

maximum.

b e t w e e n th e

of T.

age

induced

inorganic
of

the

of e p i t h e l i a l
for

inoculation.

in p h y s i o l o g i c a l

chorioallantois

A

saline

r e d u c e d th e

induced.

Maximal

mal

while

existed

a

stimulated

proliferation

relationship

two

presence

and

epithelial

the

(l) u m b i l i c a t e d

involve

acid phosphatase act i v i t y was

of

with

chorioallantois.

maximum when

tion

experiments,

substances.

gelei i W a n d

T_. g e l e i i

cultures.

induced:

induction appeared

relationship

31.

were

possessed growth— promoting

were

Lesion-inducing

glycogen

media

1 esion-indueing

protozoan

lesions

thickened

direct

of

utilizea

growth-promoting
A

20.

those

suspending

and their

solution,

of

inflammatory

19.

various

degree.

Lesion

occurring

th e

inocula

with

Ringer's

to

IS.
(l)

and

chorioallantoic

properties

and

grafts

fat

deposition was

proliferation
in c e l l u l a r

and

visible

suggested

proliferation.

in a r e a s
t ha t

fat

of

maxi­

synthesis

99
24.

The

phosphatase
veins

and

play a

25.
blasts
a

lipase

in t h e

Alkaline
might

23.

the

concentration
activity was
of

induced

transfer

of

of a l k a l i n e
noted

lesions.

nutrient

and acid

in w a l l s
These

of a r t e r i e s ,

enzymes

materials

may

across

membranes.

subsequent

induced

and

capillaries

role

vascular

heaviest

be

phosphatase activity

related

to

fibrillar

inhibition

of

growth.

Intranuclear
lesions

hypothesis

and an
of

inclusion

protein

bodies

investigation

Fennell

(1951)

in m e s o d e r m a l

that

of

metabolism and

were

these

fibro­

visible

bodies

t h e y m a y be

in

supported

calcium

soaps.
27.
the
of

No

induced
the

histochemical
lesions

or m o r p h o l o g i c a l

c o u l d be u t i l i z e d

i n d u c t i o n agent.

fo r

characteristic
the

of

identification

LI TFRAT-JRE

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Co mp. P h y s i o l . 34: 9 7 - 1 1 4 , 1949.
W e b b , A. M. a n d L o o f b o u r o w ,
J. Rf a c t o r s of t h e V i t a m i n B c o m p l e x
B i o c h e m . J. 41:
1 1 4 - 1 1 9 , 194 7.
Wichterman,
New Y o r k ,

R.
The
1953.

Biology

R e l e a s e a n d s y n t h e s i s of
b y d a m a g e d l i v i n g cells.

of P a r a m e c i u m .

W i s l o c k i , G. B. a n d D e m p s e y , E. W.
of the e n d o m e t r i u m in p r e g n a n c y .
403, 1 9 4 5 .

The

Blakiston

Co.,

Histochemical reactions
Am. J. An at . 77:
36 5 -

103
PLATE I
A l k a l i n e p h o s p h a t a s e a c t i v i t y i n s p e c i m e n s of T.
s u s p e n d e d in c u l t u r e m e d i u m .
All figures X60Q7
scale insert:
1 s p a c e = 0 . 0 1 mm.

geleii W
Micrometer

Fig.

1.

24-hour

Fig.

2.

144-hour

cells

reared

in t r y p t o n e >

F ig .

3.

216-hour

cells

reared

in t r y p t o n e >

Fig.

4.

432-hour

cells

reared

in t r y p t o n e >

Fig.

5.

2 88-h o u r cells r e a r e d
f i e d with CaOl^ »

in t r y p t o n e

f o r t i-

Fig.

6.

2 8 8 - h o u r c e l l s r e a r e d in t r y p t o n e
fie d with CaCl2 (control) ■
>

forti-

Fig.

7.

2 8 8 - h o u r cells reared
fied with MgCl2 ; and

in t r y p t o n e

f ort i-

Fig.

8.

2 8 8 - h o u r c e l l s r e a r e d in t rvp t one f ort if i e d w i t h M g C l 2 a n d e x p o s e d to a n i n c r e a s e d
a m o u n t of M g C l 2 in the p h o s p h a t a s e buffer.

cells

reared

in t r y p t o n e ;

105
PLATE II
A l k a l i n e p h o s p h a t a s e a c t i v i t y in s p e c i m e n s of T.
s u s p e n d e d in cult u r e medium.
A l l f i g u r e s X600.
scale insert:
1 s p a c e - 0 . 0 1 mm.
rearea

geleii W
Micrometer

Fig.

9.

2 8 8 - h o u r cells
tryptone;

in v i t a m i n - e n r i c h e d

Fig.

10.

2 8 3 - h o u r ce l l s r e a r e d in v i t a m i n a n d
m a g n e s i u m chloride-enriched tryptone;

Fig.

ll.

2 8 8 - h o u r c e l l s r e a r e d in m a g n e s i u m
c h l o r i d e - e n r i c h e d tryptone;

Fig.

12.

7 2 - h o u r c e l l s r e a r e d in th e i n i t i a l
r i b o f l a v i n - e n r i c h e d tryptone;

Fig.

13.

Maximum phosphatase activity after
f i r s t t r a n s f e r to v i t a m i n - e n r i c h e d
t r y p t o n e , 2 1 6 - h o u r ce lls ;

Fig.

14.

7 2 - h o u r .c e l l s f r o m 2 n d t r a n s f e r
vitamin - e n r i c h e d tryptone; and

Fig.

15.

3 6 0 - h o u r ce l l s f r o m 2 n d t r a n s f e r
v i t a m i n - e n r i c h e d t r y p to ne .

to

to

107
PLATE III

c+pq

A l k a l i n e p h o s p h a t a s e a c t i v i t y in u n w a s h e d s p e c i m e n s of T.
e l s i i W f r o m 3 8 8— h o u r c u l t u r e s a f t e r a 2 0 - m i n u t e ex no s ur e
o various enzyme inhibitors.
A l l f i g u r e s X800.
Micro­
m e t e r s c a l e insert:
1 s p a c e = 0 . 0 1 mm.
Fig.

18.

0.0 1 M sodium arsenate (specimens
r e a r e d in v i t a m i n - e n r i c h e d t r y p t o n e ) ;

Fig.

17.

0.01M sodium arsenate
r e a r e d in t r y p t o n e ) ;

Fig.

18.

0.01M sodium
in m a g n e s i u m
t r y p t o n e );

Fi g.

19.

0.01M sodium arsenate
in m a g n e s i u m c h l o r i d e
enriched tryptone);

Fig.

20

Q.03M semicarbazide (specimens reared
in m a g n e s i u m c h l o r i d e a n d v i t a m i n enriched tryptone);

Fig.

21.

0 . 0 2 5 M oxidized glutathione (specimens
r e a r e d in m a g n e s i u m c h l o r i d e a n d
vitamin-enriched tryptone); and

Fig.
w

22.

0. 0 2 5 M reduced glutathione (specimens
r e a r e d in m a g n e s i u m c h l o r i d e a n d
v i t a m i n - e n r i c h e d tryptone).

(specimens

arsenate (specimens
chloride-enriched

reared

(specimens reared
a n d vit am in -

Iumdk

109
PLATE IV
H i g t o c h e m i c a l r e a c t i o n s in s p e c i m e n s of T. g e l e i i W.
Specim e n s i n F i g s . 2 3 - 2 % s u s p e n d e d in p h y s i o l o g i c a l s a l i n e a n d
in F i^ s. 3 7 = 2 9 i n t h e c u l t u r e m e d i u m .
Al l f i g u r e s X600.
Micrometer scale insert:
1 s p a c e = 0 . 0 1 mm.
Fig.

23.

Acid phosphatase activity
c e l l s r e a r e d in t r y p t o n e ;

Fig.

34.

a c i d p h o s p h a t a s e a c t i v i t y in 7 3 - h o u r
c e l l s r e a r e d in v i t a m i n - e n r i c h e a
tryptone;

Fig.

25.

acid phosphatase activity
c e l l s r e a r e d in t r y p t o n e ;

Fig.

26.

a c i d phosphata.se a c t i v i t y in 2 8 8 - h o u r
c e l l s r e a r e d in v i t a m i n - e n r i c h e d
tryptone;

Fig.

27.

l i p a s e a c t i v i t y in 2 8 8 - h o u r
r e a r e d in t r y p tone;

Fig.

28.

DPA a c t i v i t y
in tryptone;

Fig.

29.

a l k a l i n e p h o s p h a t a s e a c t i v i t y in 144h o u r c e l l s r e a r e d in t r y p t o n e .

in 2 8 8 - h o u r
and

in 7 2 - h o u r

in 2 8 8 - h o u r

cells

cells

reared

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PLATE

V

H i s t o c h e m i c a l r e a c t i o n s in s p e c i m e n s of Th ge 1 eii W susp e n d e d in cult ure medium.
All f i g u r e s X600T
Micrometer
scale i n s e r t :
1 s p a c e - 0 . 0 1 mm.
Fig.

30.

Glycogen deposition
r e a r e d in t r y p t o n e ;

in 7 3 - h o u r

Fi g.

31.

glycogen deposition
r e a r e d in t r y p t o n e ;

in 2 8 8 - h o u r

Fig.

32.

g l y c o g e n d e p o s i t i o n in 7 2 - h o u r c e l l s
r e a r e d in v i t a m i n - e n r i c h e d t r y p t o n e ;

Fig.

33.

g l y c o g e n d e p o s i t i o n in 2 8 8 - h o u r c e l l s
r e a r e d in v i t a m i n - e n r i c h e d t r y p t o n e ;

F ig.

34.

phospholipid
cells reared
tone; and

Fig.

35.

p h o s p h o l i p i d d e p o s i t i o n in 5 0 4 - h o u r
c e l l s r e a r e d in v i t a m i n - e n r i c h e d
tryptone.

cells

cells

d e p o s i t i o n in 7 2 - h o u r
in v i t a m i n - e n r i c h e d t r y p ­

30

31

113
PLATE VI
C h o r i o a l l a n t o i c l e s i o n s i n d u c e d b y s a l i n e s u s p e n s i o n s of
1* £ e l Q i:L
L e s i o n s in Figs. 3 6 , 37, 39 a n d 49 f i x e d in
Z e n k e r s f l u i d a n d s t a i n e d with h a e m o t o x y l i n e o s i n - a z u r e .
L e s i o n s in Fig. 3 s f i x e d in a ce t i c - b i c h r o m & t e a n d in Figs.
4 1 a n d 42, i n a c e t o n e .
Figs. 36 a n d 42 X 1 0 0 . Figs. 3 7 - 4 1
X690.
M i c r o m e t e r scele insert:
1 s p a c e - 0 . 0 1 mm.
Fig.

36.

C r o s s - s e c t i o n of c h o r i o a l l a n t o i c
l e s i o n s h o w i n g epithelial nest f o r m a ­
t i o n i n d u c e d by 1 8 - d a y o l d T_. g e l e i i W;

Fig.

37.

c r o s s - s e c t i o n of e p i t h e l i a l nest
i n d u c e d b y 1 8 - d a y o l d T_. g e l e i i W;

Fig.

33.

l o c a l i z a t i o n of fat in h y p e r p l a s t i c
d o r s a l e p i t h e l i u m of c h o r i o a l l a n t o i c
l e s i o n i n d u c e d by 1 4 - d a y o l d T. g e l e i i

Fig.

39.

Fig.

40.

Fig.

41.

a l k a l i n e p h o s p h a t a s e a c t i v i t y in p e r i ­
p h e r a l m e s o d e r m a l f i b r o b l a s t s of
c h o r i o a l l a n t o i c lesion i n d u c e d by 12d a y o l d T. g e l e i i W; a n d

Fig.

42.

a c i d p h o s p h a t a s e d i f f u s i o n in c h o r i o ­
a l l a n t o i c lesion induced by 9=day old
T. g e l e i i W e x p o s e d to 50° C. for oneh a l f hour.

W;

i n c l u s i o n b o d i e s in e p i t h e l i u m of c h o r i o ­
a l l a n t o i c l e s i o n i n d u c e d by 9 - d a y old
T. g e l e ll W®
i n c l u s i o n b o d i e s in u n a b s o r b e d i n o c u l a
of 3 - d a y old X- g e l e i i

115
PLATE VII

Chorioallantoic lesions induced by saline suspensions of
T. gelei i W.
Lesion in Fig. 43 fixed in Zenker's fluid and
stained with haemot.oxylin eosin-azure; Figs. 44 and 4.5 fixed
in acet i e-bichrornat e . All figures X1700.
Micrometer scale
insert:
1 space - 0.01 mm.
Fig.

43.

Cross-section of ventral surface of
chorioallantoic lesion induced by
3-day old T. ge1eii W showing forma­
tion of epithelial nests;

Fig.

44.

fat deposition in dorsal epithelium
of chorioallantoic lesion induced by
14-day old T. ge1 eii W; and

Fig.

45.

fat deposition in epithelial nests
of chorioallantoic lesion induced by
14-day old T. geleii W.

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