THE ANTAGONISTIC E F F E C T OF STRONTIUM I O N S
F O R ANESTHETIZATION O F PARAMECIUM
CAUDATUM WITH NICKEL I O N S
A Thesis
Presented t o
The Graduate Division
Drake University
I n P a r t i a l Fulfillment
of t h e Requirements f o r the Degree
Master of Arts i n Biology
by
C l a i r G . Rausch
August 1964
THE ANTAGONISTIC EFFECT OF STRONTIUM IONS
FOR ANESTHETIZATION OF PARAMECIUM
CAUDATUM WITH NICKEL IONS
Clair G. Rausch
Approved by Committee:
TABLE OF CONTENTS
CHAPTER
.
.................
I1. REVIEW OF THE LITERATURE
...........
111. MATERIALS AND METHODS . . . . . . . . . . . . .
I V . RESULTS AND INTERPRETATION OF DATA
......
V. S W Y . . . . . . . . . . . . . . . . . . . .
V I . BIBLIOGRAPHY
.................
1 1 APPENDIX . . . . . . . . . . . . . . . . . . . .
I
INTRODUCTION
PAGE
1
4
12
20
35
37
42
LIST OF TABLES
TABLE
PAGE
I. Analysis of Charles City Public Water
Supply
11.
...................
The Time Required for Anesthesia of
Paramecium caudatum with 4.2 x
Molal Solutions of Nickel and Strontium
Chloride
21
The Twenty-four Hour Fission Rate of
paramecium caudatum Treated for Two
Minutes with Nickel and Strontium
Chloride Solutions
26
..................
111.
.............
IV.
V.
43
The Twenty-four Hour Population Growth
of Paramecium caudatum Treated with
Nickel and Strontium Chloride Solutions
...
Reproduction and Fission Rate of Paramecium
caudatum reated for Two Minutes in
4.2 x lo-' Molal Solutions of Nickel
and Nickel Antagonized by Strontium Ions,
as Compared with the Control Organism
....
27
28
CHAPTER I
INTRODUCTION
The e f f e c t s of s a l t s of m e t a l s upon protozoans h a s
been a n o b j e c t of i n v e s t i g a t i o n f o r many y e a r s .
Recent
s t u d i e s have been i n t h e a r e a of i o n antagonism, and have
been concerned w i t h t h e d i s p a r i t y between t h e obsenred
e x p e r i m e n t a l r e s u l t s and t h e t h e o r e t i c a l l y p r e d i c t e d
results.
Experimental evidence t o show i o n antagonism
between t h e s t r o n t i u m i o n and t h e n i c k e l i o n has n o t been
presented.
Specimens of paramecia may b e e a s i l y c o l l e c t e d i n
n a t u r e , and c u l t i v a t e d i n t h e l a b o r a t o r y .
Beginning w i t h
a s i n g l e specimen, one may o b t a i n a r i c h c l o n e of t h e
a n i m a l s w i t h i n a r e l a t i v e l y s h o r t p e r i o d of time.
The
paramecia a r e m i c r o s c o p i c , r e q u i r e r e l a t i v e l y l i t t l e
maintenance, and t h e t o t a l space r e q u i r e d f o r l a r g e numbers
i s small.
I n d i v i d u a l animals may be removed from a drop of
c u l t u r e medium, washed, placed i n a t e s t medium, and observed
w i t h l i t t l e e x p e n d i t u r e of time.
The a n i m a l reproduces a s e x u a l l y by b i n a r y f i s s i o n .
'The organisms o f any one c l o n e a r e h e r e d i t a r i l y a l i k e , i n s u r i n g the u n i f o r m i t y o f t h e test animals.
Long tern
e x p e r i m e n t s may be performed upon t h e same c l o n e of animals.
I n a d d i t i o n t o b e i n g a s i n g l e c e l l , t h e paramecium may be
c o n s i d e r e d a s an e n t i r e organism, and a s such c a r r i e s on a l l
t h e v i t a l a c t i v i t i e s o f an organism.
The paramecia may be maintained on a s t a n d a r d i z e d
d i e t , e n a b l i n g one t o l i m i t t h e v a r i a b l e s i n experimental
studies.
Paramecia a r e h i g h l y s e n s i t i v e t o t h e a c t i o n of
t o x i c substances.
It i s d i f f i c u l t t o d e v i s e chemical t e s t s
o f s e n s i t i v i t y t h a t approach t h e s e n s i t i v e r e a c t i o n of
l i v i n g organisms f o r t o x i c a g e n t s .
Paramecia have been i n t e n s i v e l y i n v e s t i g a t e d by many
experimenters.
The a n t a g o n i s t i c e f f e c t of t h e s t r o n t i u m
i o n i s t h e o n l y one of t h e a l k a l i n e e a r t h elements f o r which
p r e v i o u s s t u d i e s have n o t been p u b l i s h e d .
I n t h i s s t u d y , an
a t t e m p t has been made t o supply a d d i t i o n a l i n f o r m a t i o n on
i o n antagonism, w i t h p a r t i c u l a r r e g a r d t o t h e s t r o n t i u m i o n .
I t was t h e purpose of t h i s i n v e s t i g a t i o n (1) t o
compare t h e computed e f f e c t s of s t r o n t i u m i o n antagonism
3
and t h e observed b i o l o g i c a l e f f e c t of t h e s t r o n t i u m i o n f o r
a n e s t h e s i a of Paramecium caudatum (Ehrenberg) w i t h t h e n i c k e l
i o n ; (2) t o compare t h e prolonged e f f e c t of t h e s t r o n t i u m
i o n antagonism upon t h e v i t a l i t y of P. caudatum w i t h c o n t r o l
groups.
CHAPTER I1
REVIEW OF THE LITERATURE
P a r a m e c i a h a v e b e e n t h e o b j e c t of s t u d i e s s i n c e man
h a s h a d t h e o p t i c a l equipment n e c e s s a r y f o r t h e i r o b s e w a tion.
E h r e n b e r g , on J u n e 11, 1932, a s c i t e d by Wichterman, 1
d e s c r i b e d Paramecium caudatum f o r t h e f i r s t time.
The
a c t i o n o f v a r i o u s s a l t s and s a l t antagonisms upon t h e
c i l i a r y action of P. caudatum (Ehrenberg) h a s been s t u d i e d
by many; t h e p e r i o d from 1900 t o 1920 was one o f g r e a t
activity.
Wichterman
2
g i v e s a c o m p l e t e r e v i e w of t h e
l i t e r a t u r e prior t o 1952.
Mast and N a d l e r
3
r e p o r t e d t h a t monovalent c a t i o n
s a l t s and h y d r a t e s t e s t e d ( t h i r t y - o n e )
,
with t h e exception
1
Ralph Wichterman, The Biology of Paramecium.
(New York : B l a k i s t o n Company, I n c . , 19=), p . 7 .
2
Ibid.
-
11
3 ~ 0
. . Mast and J. E. Nadler, R e v e r s a l of C i l i a r y
Ac t ion i n Paramecium caudatum," J o u r n a l Morphology and
P h y s i o l o g y , X L I I I ( J u n e , 1926), 1 1 7 .
of ( N H 4 ) 2 (So41 and NH4C2H302, induced r e v e r s a l of c i l i a r y
action.
None of t h e b i v a l e n t o r t r i v a l e n t s a l t s , w i t h t h e
e x c e p t i o n of CaHP04, MgHP04, and Ba(OH)2 r e v e r s e d c i l i a r y
action.
These i n v e s t i g a t o r s concluded t h a t t h e c i l i a r y
r e v e r s a l i n paramecia i s a s s o c i a t e d w i t h d i f f e r e n t i a l
a b s o r p t i o n of c a t i o n s a s w e l l a s subsequent changes i n
electrical potential.
I n a d d i t i o n , o t h e r f a c t o r s were
b e l i e v e d t o be involved.
They f u r t h e r concluded t h a t t h e
a c t i o n of t h e c i l i a a r e c o n t r o l l e d by t h e neuromotor
apparatus.
.
~ i s e n b e r ~ - ~ a m b ui nr d
~i'c a t e d t h a t P caudatum
r e c o i l from s t r o n t i u m i o n s , w i t h motion a r r e s t e d f o r t e n
t o twenty seconds.
This r e a c t i o n was independent of t h e
a n i o n and c h a r a c t e r i s t i c of strontium.
Von G e l e i , a s c i t e d by P. de Puytorac, C . Andrivon,
1
E . Eisenberg-Hamburg, l ' ~ l i n f 1 u e n c edes S e l s de
S t r o n t i u m s u r l e s Mouvements du Paramecium caudatum. Le
Role d e s S e l s de Calcium e t de l a Concentration en i o n
Hydrogene", Ac t a Biologiae Experimentalis, I V , (December,
1930), 262.
F. serrel, w a s t h e f i r s t t o s t a t e t h a t t h e n i c k e l i o n
p a r a l y z e d t h e c i l i a r y a p p a r a t u s of paramecia, w i t h o u t
immediately k i l l i n g t h e animal.
H e used v a r y i n g concentra-
t i o n s o f n i c k e l c h l o r i d e , n i c k e l s u l f a t e , and n i c k e l n i t r a t e ;
o b s e r v i n g t h e e f f e c t upon t h e b e a t i n g of t h e c i l i a and t h e
rhythm o f t h e c o n t r a c t i l e vacuole.
0 1 i p h a n t 2 s t a t e d t h a t r e v e r s a l of c i l i a r y a c t i o n w a s
due t o , o r a s s o c i a t e d w i t h , an i n c r e a s e i n protoplasmic
viscosity.
0 1 i p h a n t 3 a l s o s t u d i e d t h e e f f e c t s of monovalent and
b i v a l e n t c a t i o n s a l t s upon t h e c i l i a r y a c t i o n of P. caudatum.
H i s r e s u l t s a g r e e d w i t h Mast and Nadler.
4
H e f u r t h e r noted
t h a t t h e e f f e c t s were due t o t h e c a t i o n , n o t t h e anion.
-
1
11
P . d e P u y t o r a c , C . Andrivon, F. S e r r e , Sur action
C y t o n a r c o t i q u e d e s S e l s de Nickel Chez Paramecium caudatum
(Ehrb.), Journal of Protozoology X ( I ) , (February, 1963), 10.
L
3. F. O l i p h a n t , "The E f f e c t of Chemicals and Tempera1I
t u r e on R e v e r s a l i n C i l i a r y Action i n Paramecium,
P h y s i o l o g i c a l Zoology, X I ( ~ a n u a r ~1938),
,
30.
11
Reversal of C i l i a r y Action i n
Paramecium Induced by Chemicals," P h y s i o l o g i c a l Zoology, XV
(October 1942), 452.
3 ~ F.
. Oliphant,
4
Ibid.
Thomas
1
7
measured t h e e f f e c t s o f c o n c e n t r a t i o n o f n i c k e l
s u l f a t e upon t h e t i m e r e q u i r e d f o r a n e s t h e t i c a c t i o n and
recovery.
To o b t a i n i n f o r m a t i o n on t h e r e d u c t i o n of c i l i a r y
movement due t o t h e e f f e c t of n i c k e l s u l f a t e , h e compared t h e
d i s t a n c e t r a v e r s e d by a normal and an a n e s t h e t i z e d paramecium.
P . caudatum as
He r e p o r t e d t h e r a t e of movement f o r normal -
950 m i c r o n s p e r s e c o n d , whereas S e a r s and ~ l v e b a c ks~t a t e d
t h a t t h e mean speed o f P. caudatum, a t t w e n t y - f i v e d e g r e e s
c e n t i g r a d e , w a s 400 t o 500 microns p e r second.
G r e b e c k i and Leszek
3
stated that the cation toxicity
of c h l o r i d e s i s of t h e order:
Mn
> Ba > NH4)Sr ) K )
Hg >Cu > N i >Cd )Pb)
Zn> Co)
Ca >Mg)Na> L i , and t h a t t h e t o x i c a c t i o n
o f a n i o n s i s due c h i e f l y t o pH changes.
'~ayrnond Thomas, 11 L' a c t i o n Anaes t h e s i q u e du S u l f a t e
d e N i c k e l s u r Paramecium caudatum," B u l l e t i n de M i c r o s c o p i e
A p p l i q u e , 111 (September, 1953) , 73.
7
-D. F . S e a r s and L . Elveback, "A Q u a n t i t a t i v e Study
of t h e Movement of Paramecium caudatum and Paramecium m u l t i micronucleaturn," Tulane S t u d i e s i n Zoology V I I I ( 5 ) , ( 1 9 6 1 ) .
-
3 ~ n d r z e jGrebecki and Kuznicki Leszek, he R e l a t i o n
oL L'aramecium caudatum t o t h e Chemical P r o p e r t i e s of i t s
Medium and t h e p r o t 1e1 c t i v e E f f e c t of Aggregation a g a i n s t Ino r g a n i c S u b s t a n c e s , F o l i a B i o l o g i a e , 111 ( Z ) , ( O c t o b e r ,
More r e c e n t l y , Bovee
1
d e s c r i b e d t h e u s e of n i c k e l
s u l f a t e as t h e i d e a l a n e s t h e t i c a g e n t f o r p r o t o z o a .
Lee and McCall
2
found t h a t t h e pH of medium a f f e c t e d
t h e s i z e o f f o o d v a c u o l e s , as w e l l as t h e l e n g t h and w i d t h
of t h e t o t a l a n i m a l ,
Dryl
3
induced c i l i a r y r e v e r s a l i n P.
c a u d a t u m by s i m u l t a n e o u s a c t i o n of barium and c a l c i u m i o n s .
4
Yarbrough and o 1 ~ e l l e y found normal swimming o f P.
c a u d a t u m i n 0.0002 M. c a l c i u m changed t o avoidance upon
b e i n g i n t r o d u c e d t o equimolar s t r o n t i u m .
Equimolar barium
h a d a less pronounced e f f e c t .
1
Eugene
C . Bovee, "Nickel S u l f a t e a s an A n e s t h e t i c
f o r P r o t o z o a n s , I 1 T u r t o x -3News XXXVI ( 2 ) , ( F e b r u a r y , 1 9 5 8 ) ,
78.
2
Warren J . Lee and W i l l i a m McCall, " E f f e c t s of pH
a n d Viscositv
on S
u r f a c e Membranes i n Paramecium m u l t i . -- - - - - m i c r o n u c leaturn, I I J o u r n a l o f P r o t o z o o l o g y , V I (2), (May, 1 9 5 9 ) ,
- - -
3
II
-
S t a n i s l a w D r y l , The C i l i a r y R e v e r s a l i n P a r a mecium caudatum Induced by Simultaneous A c t i o n of Barium
II
o f P r o t o z o o l o g y , V I I I (Suppl. )
and Calcium I o n s , J o u r n a l (December, 1961) , 16.
4
James D. Yarbrough and Joseph C . O 1 ~ e l l e y ,
A l k a l i n e E a r t h Elements and t h e Avoidance R e a c t i o n i n
Paramecium m u l t i m i c r o n u c l e a t u m , J o u r n a l of P r o t o z o o l o g y ,
IX ( 2 ) (May, 1 9 6 2 ) , 135.
11
9
Jahn
1
,
i n h i s s t u d i e s of i o n antagonism and c i l i a r y
r e v e r s a l , a n a l y z e d c i l i a r y r e v e r s a l i n terms o f t h e Gibbs
Donnan r a t i o .
H e proposed t h a t a g i v e n bond a n g l e produces
a specific ionic effect.
T h i s i n t r o d u c e d a new c o n c e p t of
stereochemistry concerning t h e b i o l o g i c a l e f f e c t s of ions.
Leeuwenhoek, i n 1676, a s c i t e d by Dobell 2 , w a s t h e
f i r s t t o o b s e r v e c i l i a , and r e c o g n i z e t h e i r u s e i n locomotion.
Many i n v e s t i g a t o r s h a v e s t u d i e d t h e d e t a i l e d s t r u c t u r e of t h e
P e t e r , a s c i t e d by Wichterman 3 , demonstrated t h e f a c t
cilia.
t h a t t h e c i l i a on t h e fragments of a c i l i a t e w i l l c o n t i n u e
t o b e a t i f they a r e i n connection with a piece of cytoplasm.
G e l e i and K l e i n , a s c i t e d by Wichtennan
4 , studied the c i l i a
by f i x a t i o n and s t a i n e d o r impregnated p r e p a r a t i o n s .
Their
a c c o u n t s d i f f e r , p r o b a b l y due t o t h e i r i n a b i l i t y t o c l e a r l y
''Theodore Louis J a h n , he Mechanism of C i l i a r y
Movement 11 I o n Antagonism and C i l i a r y R e v e r s a l , " J o u r n a l
Cellular and Comparative P h y s i o l o g y , LX ( 3 ) ( O c t o b e r , 1 9 6 2 ) ,
228.
-
C l i f f o r d D o b e l l , Antony Van Leeuwenhoek -and His
L i t t l e Animals (New York: R u s s e l l and R u s s e l l I n c . , 1 9 5 8 ) ,
L
' ' ~ a l ~W
hi c h t e m a n , The Biology of Paramecium
(New York: B l a k i s t o n Company I n c . , 1 9 5 3 , p . 6 4 .
'1bid.
,
p . 50.
10
see t h e minute s t r u c t u r e s w i t h t h e a v a i l a b l e o p t i c a l equipment.
Kudo
1
d e s c r i b e s t h e a c t i o n of t h e c i l i a a s t h e
p r e p a r a t o r y and e f f e c t i v e s t r o k e .
J a k u s and H a l l
2
made e l e c t r o n microscope o b s e r v a t i o n s
o f c i l i a u s i n g a shadow-casting technique w i t h chromium.
T h i s showed a c i l l i u m of paramecium t o c o n s i s t of a bundle
of eleven f i b e r s .
The bundle of d r i e d f i b e r s was between 300
and 500 A . i n d i a m e t e r .
Most of t h e r e c e n t work i n t h e s t u d y
o f c i l i a r y morphology has been w i t h t h e u s e of e l e c t r o n
microscopy.
According t o Wichterman
3
, Maupas
was t h e f i r s t t o
u s e t h e r a t e of f i s s i o n a s an index of t h e v i t a l i t y of t h e
organism.
Calkins
4
s t a t e s t h a t any environmental c o n d i t i o n
which a f f e c t s any one l i n k of t h e v i t a l a c t i v i t i e s has an
'R. R. Kudo, Protozoology ( t h i r d e d i t i o n ; S p r i n g f i e l d ,
I l l i n o i s : C . C . Thomas, 1947), p. 111.
II
Jakus and C . E . H a l l , E l e c t r o n Microscope
O b s e r v a t i o n s of t h e T r i c h o c y s t s and C i l i a of Paramecium,"
Biology B u l l e t i n , X C I (October, 1946), 144.
*M.
A.
-
4 ~ a r yNathan C a l k i n s , The Biology of t h e Protozoa
( P h i l a d e l p h i a : Lea and F e b i g e r , 1933), p . 1.
11
e f f e c t upon t h e g e n e r a l a c t i v i t y , and t h a t v i a b i l i t y i s t h e
sum t o t a l o f a l l protoplasmic a c t i v i t i e s s e t up i n response
t o stimuli.
S e v e r a l i n v e s t i g a t o r s have s t u d i e d t h e e f f e c t of
P . caudatum.
n i c k e l s u l f a t e upon t h e r a t e of r e p r o d u c t i o n of A r e c e n t s t u d y i s t h a t of P . de Puytorac, C . Andrion, and
F. S e r r e
1
,
who found t h a t t h e f i s s i o n r a t e was reduced f o r
a p p r o x i m a t e l y twenty g e n e r a t i o n s a f t e r t h e paramecia were
immobilized by t h r e e grams p e r l i t e r of n i c k e l s u l f a t e .
CHAPTER I11
MATERIALS AND METHODS
The p a r a m e c i a u s e d i n t h e experiment were c o l l e c t e d
from a s m a l l pond w i t h i n t h e c i t y l i m i t s of C h a r l e s C i t y ,
Iowa.
The l o c a t i o n of t h e pond was R-15W, T-96N, Floyd
C o u n t y , N i l e s Township, t h e n o r t h - w e s t one f o u r t h o f s e c t i o n
six.
With t h e a i d o f s e v e r a l t e x t s , 1 , 2 9 3 , t h e organism was
i d e n t i f i e d a s Paramecium caudatum Ehrb.
From t h i s sample, paramecia were removed and
c u l t i v a t e d upon a t i m o t h y hay t e a and Aerobacter a e r o g e n e s
media.
From t h e d e n s e p o p u l a t i o n of paramecia e s t a b l i s h e d
by t h i s p r o c e d u r e , two paramecia were i s o l a t e d .
The
i n d i v i d u a l paramecium was washed w i t h i n d e p r e s s i o n s l i d e s
i n seven s u c c e s s i v e baths of s t e r i l e d i s t i l l e d water.
T r a n s f e r o f t h e paramecium was accomplished w i t h a micro
' ~ h e o d o r e Louis J a h n , How t o Know t h e P r o t o z o a
(Dubuque: Wm. C . Brown c o . , lm):
'R. R . Kudo, P r o t o z o o l o g y ( t h i r d e d i t i o n ;
S p r i n g f i e l d : C . C . Thomas, 1957).
-
3 ~ a l p hWichterman, The Biology of Paramecium
(New York: B l a k i s ton Company I n c . , 1 9 5 3 .
13
pipet.
The paramecium was then placed i n a d e p r e s s i o n s l i d e ,
a few d r o p s o f n u t r i e n t medium was added, and t h e c o n c a v i t y
s e a l e d w i t h a c o v e r s l i p and petroleum j e l l y .
The specimen
and medium were t h e n observed a t twenty-four hour i n t e r v a l s
t o n o t e t h e v i a b i l i t y of t h e paramecium, and t o i n s p e c t f o r
0t h e r
c o n t a m i n a t i n g micro-organisms
.
Species p u r e c l o n e s
one and two were i s o l a t e d and used throughout t h e experiment.
To r a i s e l a r g e p o p u l a t i o n s of t h e two c l o n e s , specimens were
t r a n s f e r r e d t o g l a s s c o n t a i n e r s c o n t a i n i n g one l i t e r of t h e
hay i n f u s i o n medium.
The c u l t u r e s were maintained i n rooms
where t h e y w e r e n o t s u b j e c t e d t o d i r e c t s u n l i g h t .
Tempera-
t u r e s ranged from twenty-five t o t h i r t y degrees c e n t i g r a d e .
P r e p a r a t i o n of t h e medium was a s f o l l o w s : l e a v e s ,
s t e m s , and heads of timothy hay (Phleum p r a t e n s e ) were
chopped i n t o one t o two c e n t i m e t e r s e c t i o n s .
( A l l of t h e hay
was from one s t a n d t o keep t h e mineral c o n t e n t uniform.)
Three grams of t h e chopped hay were added t o one l i t e r of t a p
w a t e r from t h e C h a r l e s C i t y water system.
appendix. )
(See Table I i n
The medium was then autoclaved ( i n a P r e s t o
cooker-canner
,
N a t i o n a l P r e s t o I n d u s t r i e s Inc. ) f o r t e n
m i n u t e s a t f i f t e e n pounds of p r e s s u r e .
Before u s e , t h e
14
medium w a s i n o c u l a t e d w i t h a twenty-four hour c u l t u r e of
Aerobac t e r a e r o g e n e s ( f u r n i s h e d by D r . Rodney A. Rogers,
Drake u n i v e r s i t y ) .
P.
-
caudatum w a s t h e n i n t r o d u c e d i n t o t h e
medium a b o u t t w e n t y - f o u r h o u r s l a t e r .
Once a week, some of
t h e p a r a m e c i a were t r a n s f e r r e d t o a new c u l t u r e medium.
This
was done t o k e e p t h e waste p r o d u c t s , which a r e t o x i c , from
becoming t o o c o n c e n t r a t e d .
Paramecia u s e d i n t h i s experiment
w e r e from c u l t u r e s t h a t w e r e from one t o t h r e e weeks o l d .
W i t h t h e e x c e p t i o n o f some e x p e r i m e n t a l t r i a l s w i t h s t a r v e d
p a r a m e c i a , a l l t h e p a r a m e c i a u s e d i n t h e experiment were from
f l o u r i s h i n g , well-nourished clones.
D i s t i l l e d water ( t h e d i s t i l l e d water used during t h i s
e x p e r i m e n t w a s doubly d i s t i l l e d i n pyrex g l a s s ) , h y d r a t e d
n i c k e l c h l o r i d e , N i C 1 2 . 6H20 and t h e h y d r a t e d s t r o n t i u m
c h l o r i d e , SrC12.6H20,
( c h e m i c a l s o b t a i n e d from Wm. Welch
S c i e n t i f i c Company) were u s e d t o p r e p a r e s t o c k s o l u t i o n s of
8.4 x
l o m 3m o l a l i t y .
A l l w e i g h t measurements were made on a
Voland Company 200 B c h a i n a n a l y t i c a l b a l a n c e (Wm. Welch
S c i e n t i f i c Company).
F u r t h e r d i l u t i o n s were made by
v o l u m e t r i c measurements from t h e o r i g i n a l s t o c k s o l u t i o n .
The c o n c e n t r a t i o n o f n i c k e l and s t r o n t i u m c h l o r i d e i n t h e
15
s o l u t i o n u s e d i n t h e e x p e r i m e n t a l work was 8.4 x
molal
u n l e s s otherwise indicated.
The e x p e r i m e n t a l t e c h n i q u e f o r o b s e n r a t i o n o f
a n e s t h e s i a was as f o l l o w s : Paramecia were t r a n s f e r r e d by
p i p e t t o a depression s l i d e .
The i n d i v i d u a l paramecium was
t h e n washed i n t h r e e s u c c e s s i v e b a t h s of g l a s s d i s t i l l e d
w a t e r , and p l a c e d w i t h i n t h e d e p r e s s i o n of a ground g l a s s
s l i d e w i t h a micro p i p e t .
The p i p e t s were c a l i b r a t e d , a s
n e a r l y as p o s s i b l e , t o g i v e t h i r t y drops t o t h e m i l l i l i t e r .
To t h e d r o p c o n t a i n i n g t h e paramecium was added a drop of
s o l u t i o n c o n t a i n i n g t h e ions i n t h e c o n c e n t r a t i o n t o be
tested.
For low power o b s e r v a t i o n and f o r h a n d l i n g t h e
o r g a n i s m s , a Bausch and Lomb "zoom" s t e r o s c o p i c microscope
w i t h a m a g n i f i c a t i o n o f l o x t o 20x was used.
For d e t a i l e d
o b s e r v a t i o n o f c i l i a r y movement, f u n c t i o n i n g o f t h e c o n t r a c t i l e v a c u o l e , and b l e b f o r m a t i o n , h i g h dry and o i l immersion
l e n s on v a r i o u s compound microscopes were u s e d .
To o b s e r v e w i t h h i g h e r m a g n i f i c a t i o n , a drop of t h e
i o n s o l u t i o n c o n t a i n i n g a paramecium was t r a n s f e r r e d t o a
g l a s s d e p r e s s i o n s l i d e and a c o v e r s l i p p l a c e d t h e r e o n .
T h i s was t h e n viewed under a h i g h d r y o r o i l immersion l e n s .
To r e t a i n a sample f o r prolonged o b s e r v a t i o n , a r i n g o f
p e t r o l e u m j e l l y was p l a c e d upon t h e s l i d e , a drop of t h e i o n
s o l u t i o n c o n t a i n i n g a paramecium w a s p l a c e d upon t h e c o v e r
s l i p , t h e s l i d e i n v e r t e d and p r e s s e d l i g h t l y a g a i n s t t h e
cover s l i p .
T h i s p r o c e d u r e s e a l e d t h e chamber and e n a b l e d
o n e t o o b s e r v e a p a r t i c u l a r specimen f o r t e n t o t w e l v e h o u r s
w i t h o u t t h e o r g a n i s m b e i n g d e s t r o y e d by e v a p o r a t i o n .
For t h i s e x p e r i m e n t , d e a t h of t h e organism was
d e t e r m i n e d by t h r e e c r i t e r i a :
c e s s a t i o n of a l l movement of
t h e c i l i a , c o m p l e t e s t o p p a g e of c o n t r a c t i l e v a c u o l e a c t i v i t y ,
and d i s i n t e g r a t i o n o f t h e c e l l body.
I n j u r i e s and e f f e c t s
o f a l e s s s e r i o u s n a t u r e were determined by o b s e r v i n g :
c h a n g e s i n c i l i a r y a c t i v i t y o r locomotion, changes i n r a t e of
c y c l o s i s , c h a n g e s i n volume of t h e c e l l body, changes i n r a t e
of a n t e r i o r and p o s t e r i o r c o n t r a c t i l e v a c u o l e a c t i v i t y , t r i c h o c y s t e x t r u s i o n , f o r m a t i o n of "blebs" o r b l i s t e r s by
e x t r u s i o n o r r u p t u r e of t h e p e l l i c l e , and disarrangement of
i n t e r n a l organization.
Wichterman.
The above c r i t e r i a was adapted from
1
l ~ a l p hWichtermav, The Biology of Paramecium (New York:
B l a k i s t o n Company, I n c . , 1 9 5 3 ) , p . 430.
17
A n e s t h e s i a w a s c o n s i d e r e d t o have o c c u r r e d when a l l
f o r w a r d l o c o m o t i o n o f t h e organism had c e a s e d , and t h e c i l i a
w e r e b e a t i n g w i t h an abnormal s t r o k e and r e c o v e r y i n an
u n s y n c h r o n i z e d manner.
To s t u d y t h e p r o l o n g e d e f f e c t s upon t h e paramecium's
v i t a l i t y , o b s e r v a t i o n was made of t h e l i f e c y c l e , u s i n g
i s o l a t i o n c u l t u r e s and t h e f i s s i o n r a t e a s an i n d e x of t h e
v i t a l i t y as r e p o r t e d by C a l k i n s .
1
Two t y p e s of v i t a l i t y s t u d i e s were made.
The f i r s t
c o n s i s t e d of o b s e r v i n g t h e f i s s i o n r a t e f o r a twenty-four
h o u r p e r i o d , t h e second was a prolonged s t u d y t o n o t e t h e
f i s s i o n r a t e over a five-day period.
The method of r e c o r d i n g
t h e f i s s i o n r a t e was modified from t h a t of E c k e r t and F e i l e r
a s c i t e d by Wichtennan.
2
The method used c o n s i s t e d of
r e c o r d i n g a number f o r t h e number of d i v i s i o n s i n a twentyf o u r hour p e r i o d .
For i n s t a n c e , i f f o u r paramecia were
found i n a d e p r e s s i o n where one had been i s o l a t e d , t h e r e s u l t
l ~ a Nr. ~C a l k i n s and F r a n c i s M. Summers, P r o t o z o a in
B i o l o g i c a l Research (New York: Columbia U n i v e r s i t y P r e s s ,
194L), p. 527.
2
I b i d . , p . 362.
18
would i n d i c a t e two d i v i s i o n s , and be so recorded.
Twelve
p a r a m e c i a would i n d i c a t e t h r e e and one- f o u r t h d i v i s i o n s , e t c
.
The procedure a s used c o n s i s t e d of i s o l a t i n g a
~ a r a m e c i u mand washing i t t h r e e times i n d i s t i l l e d w a t e r .
The paramecium was t h e n p l a c e d w i t h i n one-tenth m i l l i l i t e r
of d i s t i l l e d water w i t h i n a g l a s s spot p l a t e depression.
The t e s t s o l u t i o n was t h e n introduced i n a o n e - t e n t h
m i l l i l i t e r aliquot.
A t t h e end of two minutes, t h e paramecium
was removed, a g a i n washed t h r e e times i n d i s t i l l e d w a t e r ,
examined f o r i n j u r y , and then placed w i t h i n t h e d e p r e s s i o n i n
a g l a s s spot p l a t e .
One-tenth of a m i l l i l i t e r of hay
i n f u s i o n medium was added t o t h e f l u i d t r a n s f e r r e d w i t h t h e
paramecium.
When t h e n i n e d e p r e s s i o n s i n t h e g l a s s s p o t
p l a t e were s i m i l a r i l y f i l l e d , t h e p l a t e was covered t o p r e v e n t
evaporation.
The paramecia were then observed twenty-four
h o u r s l a t e r , when a count was made t o determine t h e f i s s i o n
rate.
The prolonged s t u d y c o n s i s t e d of d u p l i c a t i n g t h e
above p r o c e d u r e , but a t t h e end of each twenty-four hour
p e r i o d , one i n d i v i d u a l from each depression p l a t e was
i s o l a t e d and p l a c e d w i t h i n one-tenth m i l l i l i t e r of n u t r i e n t
19
medium.
The c y c l e was r e p e a t e d e v e r y twenty-four hours f o r
five-day periods.
The g l a s s w a r e u s e d i n t h e experiment was
i n i t i a l l y a u t o c l a v e d f o r t e n minutes a t f i f t e e n pounds
pressure.
T h e r e a f t e r , i t was r i n s e d t h r e e t i m e s i n h o t w a t e r
and d r i e d w i t h paper toweling.
The pH of t h e medium ranged
from 6 . 7 t o 7 . 2 f o r a l l experiments.
Measurements o f t h e time r e q u i r e d f o r a n e s t h e s i a
w e r e made w i t h a s t o p watch, o r a watch w i t h a "sweep1'
s e c o n d hand.
The mean time f o r a n e s t h e s i a and s t a n d a r d
d e v i a t i o n w e r e c a l c u l a t e d f o r e a c h c l o n e and t h e v a r i o u s i o n
solutions.
The mean r a t e of f i s s i o n and s t a n d a r d d e v i a t i o n
w e r e c a l c u l a t e d for each c l o n e .
Comparison of t h e mean r a t e
o f f i s s i o n of t h e c o n t r o l organisms and t h o s e t r e a t e d w i t h
t h e t e s t s o l u t i o n s were made t o f i n d t h e e f f e c t of t h e t e s t
s o l u t i o n upon t h e o r g a n i s m ' s v i t a l i t y .
The a v e r a g e dimensions of t h e P . caudatum of c l o n e
one w e r e : l e n g t h ; 252.5 m i c r o n s , w i d t h ; 58.4 microns.
c l o n e two: l e n g t h ; 217.8 microns, w i d t h , 4 9 . 5 microns.
was f o r f i f t y i n d i v i d u a l s .
For
This
CHAPTER IV
RESULTS AND INTERPRETATION OF DATA
The paramecia of clones one and two were experimentally
tested for the time necessary for anesthetization with NiC12.
6 ~ 2 0and for NiC12.6H20 antagonized with an equimolal
concentration of SrC12.6H20. For anesthetization, those
paramecia anesthetized with NiC12.6H20 were considered the
control organism.
-4
Introduction of a drop of stock 8.4 x 10
molal solution into a drop of distilled water containing a
paramecium lowered the actual concentration of nickel ions to
-4
4.2 x 10 molal.
The typical reaction sequence of paramecia
introduced into a 4.2 x
rnolal solution of nickel ions
was : avoidance, loss of synchronization of ciliary beat,
ineffective stroke and recovery of the cilia, reduction in
rate of locomotion, swelling (probably due to the reduced
rate of operation of the contractile vacuole), anesthesia,
discharge of trichocyst, bleb formation or blistering, and
death.
The mean time for anesthesia was but slightly
different for clones one and two.
(See Table 11.)
he
TABLE I1
THE TIME REQUIRED FOR ANESTHESIA OF Paramecium
c a u d a t u m WITH 4 . 2 x
MOLAL SOLUTIONS
OF NICKEL AND STRONTIUM CHLORIDE
Solution
Calculated
mean t i m e
of
anesthesia
Observed mean
t i m e of
anesthesia
( i n seconds)
Standard
deviation
( i n seconds)
- -
N i C 1 2 . 6H20
Clone 1 39.7
16.4
Clone 2
29.1
40.5
N i C 1 2 . 6H20
+
SrC 1 2 . 6 ~ 2 0
50.5
seconds
Clone 1 84.20
51.3
seconds
Clone 2
For f i f t y i n d i v i d u a l s
.'.
. ,'F..
4 If)
74.80
33.7
22
c o n c e n t r a t i o n o f n i c k e l i o n s employed was h i g h l y t o x i c t o t h e
paramecia.
Death u s u a l l y r e s u l t e d w i t h i n a few minutes.
P a r a m e c i a i n t r o d u c e d i n t o a 4 . 2 x 10-4 molal s o l u t i o n
o f SrC12.6H20 r e a c t e d i n a manner t h a t was t y p i c a l f o r
strontium ions.
Described a s
11
r e c o i l , I 1 by Eisenberg-Hamburg 1,
t h i s r e a c t i o n w a s m a n i f e s t e d by t h e t e s t organism swimming
backwards f o r a few s e c o n d s , probably i n d i c a t i n g c i l i a r y
reversal.
The c o n c e n t r a t i o n w a s n o t t o x i c t o t h e organism,
n o r d i d i t have any d i s c e r n i b l e a n e s t h e t i c e f f e c t .
P a r a m e c i a i n a drop of d i s t i l l e d w a t e r , t o which a
d r o p o f 8.4 x
m o l a l NiCl2.6H20 and SrC12.6H20 was added,
e x h i b i t e d a r e s p o n s e unique f o r t h a t s o l u t i o n .
The sequence
w a s a s f o l l o w s : a v o i d a n c e , a t o and f r o movement ( a p p a r e n t l y
a backward sweep w i t h t h e c i l i a moving t h e organism forward,
f o l l o w e d by a forward sweep o f t h e c i l i a moving t h e organism
backward.
The n e t e f f e c t was a j e r k y motion of t h e organism
w i t h l i t t l e forward movement.), s l i g h t s w e l l i n g (probably
'E . Eisenberg-Hamburg , I 1 L 1i n f l u e n c e des S e l s d e
S t r o n t i u m s u r l e s Mouvements due Paramecium caudatum. Le
r o l e d e s S e l s d e Calcium e t de l a C o n c e n t r a t i o n en i o n
Ilydrogene, I I Acta B i o l o g i a e E x p e r i m e n t a l i s , I V (November,
1 9 3 0 ) , 261.
due t o r e d u c t i o n i n t h e r a t e of t h e c o n t r a c t i l e vacuole
a c t i v i t y ) , and a n e s t h e s i a .
A t t h a t c o n c e n t r a t i o n of n i c k e l
and s t r o n t i u m i o n s , b l e b s were r a r e l y formed.
The
c o n c e n t r a t i o n was n o t l e t h a l f o r most of t h e organisms,
during t h e observation period.
To c a l c u l a t e t h e t h e o r e t i c a l time f o r a n e s t h e s i a with
4.2 x
molal s o l u t i o n of n i c k e l ions antagonized by
e q u i m o l a l s t r o n t i u m i o n s , use was made of t h e Denye-Huckel
1
e x p r e s s i o n , a s c i t e d by Daniels and Alberty.
-lnYi=
where
ti
=
zi
e
=
=
D =
N =
k =
=
T =
L
312
a c t i v i t y c o e f f i c i e n t of ion s p e c i e s i.
c h a r g e on ions s p e c i e s i.
- 10
charge of an e l e c t r o n = 4.803 x 10
electrostatic unit.
d i e l e c t r i c constant of the solution =
78.56 f o r water a t 298 K .
23
~ v o g a d r o ' snumber = 6.023 x 10
gas c o n s t a n t per molecule = R / N =
1.3805 x 10-16erg d e g r e e - l molecule-'.
i o n i c s t r e n g t h = ) i ( c l z l2+c2zZ2+
c323*+ .
. , t h e summation being taken
over a l l the ions i n t h e s o l u t i o n , where
c i i s t h e conc.entration of ion s p e c i e s
i i n moles per l i t e r .
a b s o l u t e temperature.
.
.
' f a r r i n g t o n , Daniels and Robert A. Alberty, P h y s i c a l
Chemistry (New York: John Wiley and Sons, I n c . , 1955), p . 484.
24
I n t r o d u c i n g t h e mean a c t i v i t y c o e f f i c i e n t
i , and
p u t t i n g i n n u m e r i c a l v a l u e s f o r water a t twenty-five degrees
c e n t i g r a d e , f o r an e l e c t r o l y t e c o n t a i n i n g t h r e e k i n d s of
i o n s , t h e e q u a t i o n becomes -log?
=
0.509 zlz2z3
-.
S u b s t i t u t i n g i n t h i s e q u a t i o n t h e m o l a l i t y of
-4
NiC12.6H20 = 4 . 2 x 10
nickel ion
=
c h l o r i d e ion
,
-4
SrC12.6H20 = 4.2 x 10 , charge of
2, charge of strontium ion
=
=
2, charge o f
1. The e q u a t i o n becomes:
Thus, 79% of t h e n i c k e l i o n s a r e a c t i v e i n t h e
solution.
Using t h i s f i g u r e , one may c a l c u l a t e t h e
t h e o r e t i c a l time f o r a n e s t h e s i a , e . g . , l e t t i n g 40.50 seconds
r e p r e s e n t t h e time necessary f o r anesthesia i n a solution
c o n t a i n i n g n i c k e l i o n s a t 100% a c t i v i t y , a s o l u t i o n
c o n t a i n i n g 792 of t h e n i c k e l i o n s a c t i v e should t h e o r e t i c a l l y
r e q u i r e 5 1 . 3 seconds t o a n e s t h e t i z e t h e organism.
T a b l e 11, p . 2 1 . )
(See
w e r e made.
The f i s s i o n r a t e of t h e c o n t r o l organism,
m a i n t a i n e d on hay i n f u s i o n medium, was used a s an index of
vitality.
The mean r a t e o f f i s s i o n and t h e mean r a t e of
r e p r o d u c t i o n of t h e c o n t r o l organism was used a s t h e
s t a n d a r d i n comparison w i t h t h e t e s t organisms.
111 and IV.)
(See Tables
Clones one and two t r e a t e d w i t h a two-minute
immersion i n 4 . 2 x
molal s o l u t i o n of t h e t e s t i o n s , then
washed and t r a n s f e r r e d t o hay i n f u s i o n medium, gave t h e
following r e s u l t s .
(See Table V . )
D a t a o b t a i n e d from a n e s t h e t i z a t i o n of P . caudatum
w i t h n i c k e l i o n s a n t a g o n i z e d by equimolal s t r o n t i u m i o n s ,
showed t h a t t h e r e was a b i o l o g i c a l l y i n h i b i t o r y e f f e c t
e x h i b i t e d by t h e s t r o n t i u m i o n s f o r n i c k e l i o n s .
Although extreme c a r e was taken t o be a c c u r a t e i n a l l
m e a s u r e m e n t s , i t must b e n o t e d t h a t even t h e s l i g h t e s t
v a r i a t i o n i n the drop s i z e would g r e a t l y a l t e r t h e
c o n c e n t r a t i o n of ions i n solution.
V a r i a t i o n i n drop s i z e
c o u l d r e s u l t from c h i p p i n g of t h e a p e r t u r e of t h e g l a s s
p i p e t , s h a k i n g of t h e o p e r a t o r ' s hand, temperature
d i f F c r e n t i a l s , differences i n solution concentration,
TABLE 111
THE TWENTY-FOUR HOUR FISSION RATE OF Paramecium
caudatum TREATED FOR TWO MINUTES W I T H NICKEL
AND STRONTIUM CHLORIDE SOLUTIONS
Concentration
of s o l u t i o n
Time
immersed
i n solution
Clone
Mean
X
Clone 1
Clone 2
1.86
1.69
2 min.
2 min.
Clone 1
Clone 2
0.79
0.72
2 min.
2 min.
Clone 1
Clone 2
1.35
1.20
Control
4.2 x
molal
N i C 1 2 . 6H20
molal
For f i f t y i n d i v i d u a l s
S. D.
TABLE I V
THE TWENTY-FOUR HOUR POPULATION GROWTH OF
Paramecium caudaturn TREATED WITH NICKEL
AND STRONTIUM CHLORIDE SOLUTIONS
Concentration
of solution
Time
imrner s ed
i n solution
Range
Mean
X
S. D.
Clone 1
Clone 2
1-10
1-9
3.55
3.58
2.18
2.53
2 min.
2 min.
Clone 1
Clone 2
1-4
1-6
1.40
1.44
0.67
0.91
2 min.
2 min.
Clone 1
Clone 2
1-8
1-8
2.62
2.41
1.69
1.55
Control
4.2
Molal
- .6H20
NiCl,,
4.2
Molal
N i C 1 2 . 6H20
+
S r C 1 2 . 6H20
For f i f t y i n d i v i d u a l s
TABLE V
REPRODUCTION AND F I S S I O N RATE OF P a r a m e c i u m c a u d a t u m ,
TREATED FOR TWO MINUTES I N 4 . 2 ~ 1 0 - 4 MOLAL SOLUTIONS
O F NICKEL AND N I C K E L ANTAGONIZED BY STRONTIUM
I O N S , AS COMPARED WITH THE CONTROL ORGANISM
R e p r o d u c t i o n rate
*/
ii-
Control
Ni
Ni
Control
Ni
+-t
~ r
Clone 1
Clone 2
Fission rate
-I+
Clone 1
Clone 2
For f i f t y i n d i v i d u a l s
Ni
*/ S r
+$
29
d i f f e r e n c e s i n v a p o r and b a r o m e t r i c p r e s s u r e of t h e a i r , a n g l e
a t w h i c h t h e p i p e t w a s i n c l i n e d , e t c . , making t h e v o l u m e t r i c
m e a s u r e m e n t s o f d r o p s t h e l e a s t a c c u r a t e measurement of t h i s
experiment .
The p h y s i c a l d i f f e r e n c e s of t h e t e s t organisms v a r y
t o some d e g r e e .
Clone two showed a r a t h e r l a r g e S. D, f o r
a n e s t h e s i a with n i c k e l ions.
(See T a b l e 11, p . 21.)
Two
i n d i v i d u a l s from Clone two d i g r e s s e d markedly from t h e mean,
anesthetization time.
One w a s i n t h e 198-202 seconds r a n g e ,
a n d one w a s i n t h e 103-107 seconds r a n g e .
I n Clone one, f o r
a n e s t h e s i a w i t h n i c k e l i o n s a n t a g o n i z e d by s t r o n t i u m i o n s ,
f o u r i n d i v i d u a l s a r e w i t h i n t h e 208-292 seconds range.
These d i g r e s s i o n s may be due t o e x p e r i m e n t a l e r r o r o r
t o individual variation.
A l i m i t e d number of t e s t s were r u n
on p a r a m e c i a immediately a f t e r they had undergone f i s s i o n .
To t h e d r o p o f medium c o n t a i n i n g t h e organisms, a drop of t h e
t e s t i o n s o l u t i o n was added.
Often t h e r e would be a
v a r i a t i o n i n the t i m e f o r a n e s t h e s i a t o o c c u r , t h e most
e x t r e m e v a r i a t i o n b e i n g n i n e minutes f o r a p a i r of organisms
-4
w i t h i n a 4 . 2 x 10
m o l a l n i c k e l and s t r o n t i u m s o l u t i o n .
30
With t h o s e v a r i a t i o n s i n mind, i t i s i n t e r e s t i n g t o
n o t e t h a t t h e a n e s t h e t i c e f f e c t s o f t h e i o n s o l u t i o n s upon
t h e c l o n e s were very similar.
On t h r e e o c c a s i o n s d u r i n g t h e v i t a l i t y s t u d i e s ,
I1
d o u b l e - m o n s t e r s " w e r e n o t e d i n Clone one c o n t r o l medium.
A t t e m p t s w e r e made t o e s t a b l i s h a p u r e c l o n e of t h e s e
i n d i v i d u a l s , b u t none reproduced.
o c c u r r e d i s n o t known.
Wichterman
Why t h e s e i n d i v i d u a l s
1
c i t e d Balbiani, Calkins,
P e e b l e s , and A l v e r d e s as having observed monsters t h a t
o c c u r r e d a f t e r c u t t i n g and r e g e n e r a t i o n of paramecium.
Wichterman
2
c i t e d H e r z f e l d a s h a v i n g been a b l e t o produce
P . caudatum t o h a r d , c h l o r i n a t e d
double-mons t e r s by exposing tap water.
Wichtennan
3
c i t e d Lloyd a s f i n d i n g monsters
o c c u r r i n g i n c u l t u r e s exposed t o one p a r t p e r t e n m i l l i o n
o f b e n z e n e h e x a c h l o r i d e "gammexane,
11
a compound widely used
'IXalph Wichterman, The Biology of Paramecium
(New York: D l a k i s t o n Company I n c . , 1 9 5 v , p . 355.
2
3
Ibid.
Ibid.
, p . 358.
31
as an i n s e c t i c i d e .
It i s b e l i e v e d t h a t t h e paramecia of Clone
o n e w e r e e x p o s e d t o " m a l a t h i o n , " an i n s e c t i c i d e used i n t h e
l a b o r a t o r y where t h e i n v e s t i g a t i o n w a s c a r r i e d o u t .
It would
b e i n t e r e s t i n g t o f u r t h e r s t u d y t h e e f f e c t o f t h i s compound
t o s e e i f i t c o u l d be r e s p o n s i b l e f o r t h e a b n o r m a l i t i e s n o t e d .
With t h e l a r g e r volumes of medium and t e s t s o l u t i o n s
u t i l i z e d i n t h e v i t a l i t y s t u d i e s , a more q u a n t i t a t i v e
measurement w a s o b t a i n e d .
The r e s u l t s of t h e v i t a l i t y s t u d i e s
show a more normal s t a n d a r d d e v i a t i o n .
P . d e P u y t o r a c , C . Andrivon, and F. S e r r e
1
state that
t h e m e t a l l i c s a l t t o x i c i t y i s a r e l a t i o n between t h e c a t i o n
a n d t h e l i p i d s and p r o t e i n s , w i t h t h e n i c k e l i o n being one of
t h e more t o x i c c a t i o n s .
With weak doses of the n i c k e l i o n ,
the lesions are reversible.
g e n e r a t i o n s t o complete.
T h i s t a k e s about twenty
I n t h e v i t a l i t y s e r i e s of t h i s
e x p e r i m e n t , t h e paramecia were not observed f o r a long enough
period t o confirm t h e i r observation.
Paramecia t r e a t e d w i t h
IF'. d e P u v t o r a c , C . Andrivon, and F. S e r r e , "Sur
1,' a c t i o n C y t o n a r c o t i q u e d e s S e l s d e Nickel Chez Paramecium
c a u d a t u m Ehrb , I 1 J o u r n a l o f Protozoology, X (February, 1 9 6 3 ) ,
-
n i c k e l i o n s a n t a g o n i z e d b y s t r o n t i u m i o n s had r e c o v e r e d
n o r m a l r a t e o f f i s s i o n a t t h e end o f f i v e d a y s .
However
s h o u l d b e p o i n t e d o u t t h a t t h e f i s s i o n r a t e o f t h e two c
employed i n t h e e x p e r i m e n t w a s s l i g h t l y lower t h a n t h a t
p a r a m e c i a o b s e r v e d by o t h e r i n v e s t i g a t o r s a s c i t e d by Ca
and Summers.
1
It s h o u l d a l s o b e n o t e d t h a t i n experiments of
comparison o f t h e a c t i o n of s a l t s of d i f f e r e n t d i l u t i o n s
o r o f o n e s a l t o f d i f f e r e n t d i l u t i o n s , t o t h e a c t i o n of
d i f f e r e n t s a l t s , t h e o p e r a t i o n s h o u l d always c o n t a i n t h e
same number o f o r g a n i s m s i n t h e same p h y s i c a l s t a t e .
A c c o r d i n g t o G r e b e c k i and K u z n i c k i ,
2
there i s a protecti
e f f e c t o f aggregation against inorganic substances.
Thi
p r o t e c t i o n i s due t o t h e c a t i o n s i n s o l u t i o n b e i n g bounc
a n i o n s w i t h i n t h e c y t o p l a s m of t h e organisms.
Death of
l ~ a Nr. ~C a l k i n s and F r a n c i s N. Summers ( e d i t o r s :
P r o t o z o a i n B i o l o g i c a l Research (New York: Columbia
~ l n i v e r s i t ~ l ' r e s s1,9 4 1 ) , p . 528.
L
Andrzej- C r e b e c k i and Leszek K u z n i c k i , "The
l i e l a t i o n o f Paramecium caudatum t o t h e Chemical P r o p e r t :
o f i t s Medium and t h e P r o t e c t i v e E f f e c t o f Aggregation
11
Against Inorganic Substances, t r a n s . A. Pigon, F o l i a
R i o l o g i c a , 111 ( O c t o b e r , 1 9 5 5 ) , 1 2 7 .
individual organism does not immediately release the catid
to the environment.
Oliphant
1
demonstrated that ciliary reversal occur
immediately with all monovalent alkaline earth ions, with
-kt
Ba
t-t
(Sr
tt
,
but not with other non-toxic divalent cati
tt
)
E. Eisenberg-Hamburg stated that in
and Mn
and Mg
2
solutions non-fatal to P. caudatum, there is a reaction o
recoil specific for SrC12.
It was observed that the normal movements of the
paramecia were interrupted for ten to twenty seconds,
immediately after contact with strontium ions.
In this
.
experiment, it was further noted that P caudatum usually
exhibited an avoidance reaction which often consisted of
swimming backwards, indicating ciliary reversal.
1
11
Joseph Oliphant, Reversal of Ciliary Action in
Paramecium Induced by Chemicals,'' Physiological Zoology,
XV (October, 1942), 452.
2
E. Eisenberg-Hamburg, "L'influence des Sels de
Strontium sur les Mouvements du Paramecium caudatum. Le
role des Sels de Calcium et de la Concentration en ion
[Iydrogene,1 1 Ac ta Biologiae Experimentalis, IV (December,
1 9 3 0 ) , 261.
Jahn
1 r a i s e d t h e q u e s t i o n of t h e r o l e of t h e divall
i o n w i t h i n t h e c e l l membrane, and g i v e s two p o s s i b l e
e x p l a n a t i o n s , one morphological and one p h y s i o l o g i c a l :
(
t o m a i n t a i n t h e mechanical s t r u c t u r e of t h e c e l l membrane
and ( 2 ) t o p r o v i d e an e l e c t r o n conductor f o r r e a c t i o n s i n
c e l l membrane.
One might c o n j e c t u r e t h a t t h e more a c t i v e
s t r o n t i u m i o n r e p l a c e s t h e calcium i o n w i t h i n t h e c e l l
membrane, b u t i s n o t as r e a d i l y r e p l a c e d by the n i c k e l i o
t h e r e b y d e c r e a s i n g t h e t o x i c e f f e c t of t h e n i c k e l i o n .
C o n f i r m a t i o n o f t h i s h y p o t h e s i s must await f u t u r e s t u d i e s
'~11eodore Louis J a h n , h he Elechanism of C i l i a r y
Movement . I1 Ion Antagonism and C i l i a r y Reversal ,"
J o u r n a 1 o f C e l l u l a r and Comparative Physiology, LY
( 0 c t o b e c 1 9 6 2 ) , 223.
CHAPTER V
SUMMARY
The p r e s e n t s t u d y c o n s i d e r e d t h e e f f e c t s of s t r o n t i u m
i o n a n t a g o n i s m f o r t h e n i c k e l i o n upon a n e s t h e s i a of
Paramecium caudatum and upon t h e i r r e c o v e r y from a n e s t h e s i a
u s i n g t h e f i s s i o n r a t e a s an index of v i t a l i t y .
The t i m e r e q u i r e d f o r a n e s t h e s i a when t h e organism
-4 molal s o l u t i o n of t h e t e s t
was p l a c e d w i t h i n a 4 . 2 x 10
i o n s was o b s e r v e d and r e c o r d e d .
Using t h e Debye and Huckel
e x p r e s s i o n , t h e t h e o r e t i c a l time f o r a n e s t h e s i a w i t h n i c k e l
i o n s a n t a g o n i z e d by e q u i m o l a l s t r o n t i u m i o n s was computed.
Comparison o f t h e o b s e m e d t i m e f o r a n e s t h e s i a w i t h n i c k e l
i o n s a n t a g o n i z e d by s t r o n t i u m i o n s and t h e t h e o r e t i c a l time
showed t h a t t h e r e was a 67% i n c r e a s e i n t h e time r e q u i r e d
f o r a n e s t h e s i a o f Clone o n e , and a 46% i n c r e a s e i n t h e time
r e q u i r e d f o r a n e s t h e s i a of Clone two.
This demonstrated an
a n t a g o n i s t i c e f f e c t of t h e s t r o n t i u m i o n s f o r n i c k e l i o n s .
Comparison of t h e mean r a t e of f i s s i o n i n v i t a l i t y
s t u d i e s of 1'.
-4
x 10
caudaturn t r e a t e d , f o r two m i n u t e s , w i t h 4 . 2
m o l a l s o l u t i o n s of n i c k e l i o n s a n t a g o n i z e d w i t h
e q u i m o l a l s t r o n t i u m i o n s showed t h a t Clone one had a meal
f i s s i o n r a t e 71% g r e a t e r , and Clone two 67% g r e a t e r than
t h o s e c l o n e s t r e a t e d w i t h n i c k e l i o n s alone.
It w a s e v i d e n t from t h e above r e s u l t s , t h a t the
s t r o n t i u m i o n was b i o l o g i c a l l y a n t a g o n i s t i c t o n i c k e l io!
However, t h e mode o f a c t i o n and s i t e of t h i s b i o l o g i c a l
a c t i v i t y must a w a i t f u r t h e r s t u d y .
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I1
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11
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11
I'rcse~~L
in N o n - g l a s s D i s t i l l e d W a t e r , P r o c e e d i n g s
Soc i e t y o f E x p e r i m e n t a l B i o l o g y and N e d G i n e , C I
( A u g u s t , 1 9 5 9 ) , 682-686.
l l n r b r o u g l ~ , .lames D. , and J o s e p h C . O 1 ~ e l l e Y ." A l k a l i n e
llnr t h 1: 1 cmcn t s a n d 'l'he i r Avoidance React i o n i n Paramecium
I I
o f P r o t o z o o l o g v , I S (Yay,
mu 1 L i m i c r o l ~ t l cl c a t u m , J o u r n a l 1962), 132-135.
41
C.
LEARNED SOCIETY
Ehert, C. F., and E. L. Powers. "The Cell Surface of
Paramecium,1 1 International Review of Cytology, VIII
pp. 97-133. New York: New York ~cademicPress, 1959.
D
.
MISCELLANEOUS
Sears, D. F., and L. Elveback. "A Quantitative Study of
the Movement of Paramecium caudatum and Paramecium
multimicronucleatum," Tulane Studies of Zoology, VIII
(1961), 127-139.
E . UNPUBLISHED MATERIALS
Houser, P. J., "Analysis of Charles City Public Water
Supply ," 1960 (Personal communication).
TABLE I
ANALYSIS OF CHARLES CITY
PUBLIC WATER SUPPLY
STATE HYGIENIC LABORATORY~
June 14, 1960
Well number
1
3
4
5
6
Depth (feet)
1530
1350
1320
185
1550
Aver.
(All results as ~ g s / L )
Minerals
70.4
65.6
62.0
60.4
57.2
63.00
5.8
3.7
6.3
1.5
2.8
4.02
Na
13.7
8.9
11.2
3.7
6.8
8.86
C1
7.0
1.0
1.0
0.5
1.0
2.10
288.0 283.0
249.0
Calcium as
Ca
Potassium as
K
Sodium as
Chlorine as
Bicarbonate as HC03 283.0
Silica as
Si02
Iron as
Magnesium
as
Total
hardness as
9.8
10.4
Fe
22.0
38.0
Mg
19.4
19.4
13.2
11.6
10.68
101.0 150.0
106.0
83.40
21.6
19.56
22.6
14.8
CaC4256.0 244.0 248.0 212.0 232.0 238.40
P11
(Well i t 2 not in u s e )
.
8.4
278.0 276.20
7.45
7.45
7.5
7.45
7.5
7.47
Ilouser , Ilirec tor, Ilublic Health Engineering, Iowa
StaLe I>ep:irtnient of Iical th, I'ersonal communication.
.'1'
T.