THE RELATSON BETWEEN METHQCEL
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Thesis 1‘0: the Degree :3! M. S.
MlCHMAN STATE COLLEGE
Naifiie Marie Efiaukama

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The relatimn between methocel concentration,

frequency cf fond ingestion, rate of I
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locomotion, and 561/901 ratio in ‘
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ETI‘IOCEL

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THE RELATION BETWFE}
CON ENTRATION FREQUENCY OF FOOD
’
IN EQTION, RATE OF LOCOMOTION, AND

GEL/SOL RATIO IN MOI-373A PTIOTEUS

 

By

NELLIE MAZIE FEUKENA
,-

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Avriculture and Applied Science
in partial fulfillment of the requirements

for the degree of

.TH iii 5*;-

 

ACKNOWLEDGMENTS

Sincere acknowledgment is given to Dr. R. A. Fennell for
his invaluable aid and assistance in the preparation of this
thesis, both during the experimental work and the preparation
of the manuscript, and for his continued advice and encour-
agement.

Acknowledgment is also given to Dr. H. R. Iunt for pro-
viding the Opportunity and the materials necessary, as well
as for numerous tokens of encouragement.

Acknowledgment is made to Dr. Henry Zylstra for reading
the manuscript. Acknowledgment is also given to Miss Berna-

dette McCarthy (Mrs. Dale Henderson) for her many favors.

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The Relation Between Viscosity of the Culture
Solution and Rate of Locomotion in Amoeba

ElfioteL‘I-S...OOOOOOOOOOOOOOOO.OOOOOOOOOOOOOOOO...

The Relation Between Viscosity and Ingestion
of Food by Amoeba proteus.....................

 

The Relation Between Viscosity and Gel/sol
Ratio in Amoeba proteus.......................

 

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summar‘drOOOOOOOOO000......OOOOOOOOOOOOOOOOOOOOO

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TABLES
Page

I. The Relation Between Viscosity and the Rate
of Locomotion (in microns)....................... 11

II. The Relation Between Viscosity and the
Frequency of Ingestion of Food by Amoeba
DIroteus.OOOOOOOOOOOOOOO...OOOOOOOOOOOOOOOOOOOOOOO15

III. The Relation Between Viscosity and the
Frequency of Ingestion of Food by Amoeba

proteus.......................................... 16

IV. The Relation Between Viscosity and the Gel/sol
Ratio in Amoeba proteus.......................... 21

 

v. Showing the Width (in microns) of the Plasmagel,
Plasmasol, and the Gel/sol Ratio................. 22

ILLUSTRATIONS

Figure I ...................................... follows Page
Figure II ............... ’. ..................... follows Page

Figure III..... ............................... follows Page

I. INTRODUCTION

Hopkins (1928) found a close correlation between the
rate of locomotion and salt concentration of the culture
medium. In buffered CaCl2 or SrCl2 solutions, the rate of
locomotion increased from about zero in .0002 N CaCl2
solution to a maximum in .0005 N solution, remained fairly
constant until the concentration was increased to .005 N
solution, and then rapidly decreased to zero in .05 N CaCl

2

solution, and to almost zero in SrCl2 solution. Further, the
nature of the substratum was an important factor in rate of
locomotion. Rate of locomotion was highest in vessels coated
with paraffin, lower in ordinary glass vessels, lower in
quartz vessels, and lowest in Pyrex glass vessels. In mono—
valent (NaCl, K01, LiCl or RbCl) buffered cation solutions,
rate of locomotion reached a maximum in .0009 N solution,
remained fairly constant until the concentration was .01 N,
and then decreased to zero in .1 N solution.

Mast and Prosser (193 ) studied the effects of temper-
ature, salts, and hydrogen ion concentration on rupture of

the plasmagel sheet, rate of locomotion and gel/sol ratio in

Amoeba proteus. They demonstrated that the thickness of the

 

plasmagel sheet was increased by decreasing the temperature
and increasing the concentration of inorganic salts and
hydrogen ions. As a consequence the frequency of rupture of
the plasmalemma was decreased, but rate of locomotion was
not Specifically correlated with either the frequency of

breaks in the plasmalemma, or the gel/sol ratio.

iast and Pitts (1933, 1934a, 1934b) found that in a
balanced salt solution the rate of locomotion increased
from 0 at pH 5.0, to 257 microns per minute at pH 5.6,
increased to a maximum of 281.8 microns at pH 6.2, decreased
slightly to a median minimum at pH 7.0, and then decreased
fairly rapidly to 148.0 microns at pH 8.0. Gel/sol ratio
decreased as the hydrogen ion concentration decreased, but
the rate of decrease was greater in the acid range than in
the alkaline range.

In .001 N NaCl solutions, rate of locomotion increased

2
from 240 microns per minute to a maximum of 265 microns at
pH 6.8, then decreased to 90 microns at pH 8.0. Gel/sol
ratio rapidly decreased to a minimum as the hydrogen ion
concentration decreased. In .001 N CaCl2 solutions, rate
of locomotion increased from 0 at pH 4.7 to 265 microns at
pH 5.0, and then remained fairly constant to pH 8.0.

In solutions in which the concentration of K was .005
N, and the CaCl2 concentration was .001 N, rate of locomo—
tion increased from 1 at pH 4.1 to a maximum of 265 microns
at pH 6.2, decreased to a minimum of 195 microns at pH
7.0, increased to a second maximum of 265 microns at pH
7.5, and then it remained fairly constant to pH 8.0.

Mast and Fennell (1938) found that frequency of inges-

tion by Specimens of Amoeba proteus increased from zero in

 

Chalkley solution to a maximum at 25 degrees, decreased to
a median minimum at 30 degrees, increased to a second maximum
at 35 degrees, and then rapidly decreased to zero at 40

degrees. These results strongly suggest that the factors

which control ingestion are essentially the same as those
which control locomotion.

It is evident from the preceding review of the litera-
ture that the relation between inorganic salt concentration,
hydrogen ion concentration, and frequency of ingestion of

food by Amoeba proteus has been studied in considerable

 

detail. Further, the relation between environmental factors
and rate of locomotion and gel/sol ratio has been investi-
gated extensively. However, there is no information concern-
ing rate of locomotion, ingestion of food, and gel/s01 ratio
in balanced salt solutions with variable viscosities. Thus
it is the objective of the present study to obtain additional
information concerning these various physiological processes

in Amoeba proteus.

 

r-
q...

II. METHODS AND MATERIALS

Dow Nethocel was used to study the effects of viscosity

on physiological processes in Amoeba proteus. Nethocel is a

 

cellulose ether manufactured in the form of white fibers.

It is soluble in cold water, insoluble in hot water, satu-
rated salt solutions, and most organic solvents, and is unaf-
fected by oily or greasy materials of animal, vegetable or
mineral origin. It is stable in alkalies and dilute acids.
Aqueous solutions are stable and ordinarily do not require

a preservative.

Methocel (l5 centipoise) was prepared by thoroughly mix-
ing 40 grams of the material with 500 cc. of Chalkley solu-
tion at a temperature of about 950. It was left to soak for
30 minutes and then a 4 per cent solution was made by adding
500 cc. of Chalkley solution to the mixture. This solution
was cooled to room temperature and stirred until smooth.

0n analysis of the Methocel viscosity concentration
chart, it can be seen that with a solution of 15 centipoise
Methocel, the viscosity of the experimental solutions used
varies considerably, reaching about 5.8 centipoise in a 1.0
per cent Nethocel solution by weight. Since water has a
viscosity of 1 centipoise, the addition of even a .1 per
cent solution of Nethocel will raise the viscosity consider-
ably. As the concentration in percentage by weight increases,
the viscosity increases markedly, so that in a 15 centipoise
Methocel solution at a concentration of 4 per cent, the vis-

cosity increases to 80 centipoise at a temperature of 200.

The viscosity of Methocel solutions in centipoise is
based on the viscosity of a 20 per cent aqueous solution at
200. There are six viscosity types with varying centipoise
values of 15, 25, 100, 400, 1,500 and 4,000. The 15 centi-
poise type was selected for experimentation.

All glassware used in the following experiments was
thoroughly cleaned with cleaning solution (potassium-dichro-
mate—sulfuric acid), rinsed 15 times with tap water and 3
times with distilled water. Distilled water used for prepa-
ration of Chalkley solutions and cultures was redistilled
through Pyrex glass. C.P. chemicals were used exclusively.

Amoeba proteus was used exclusively in the following

 

experiments. All organisms were cultured in Chalkley solu-
tion (100 mg. Na01, 4 mg KCl, 6 mg. CaClQ, and 1,000 cc. glass
distilled water)1. Fifty amoebae were selected from a stock
culture and transferred2 to 200 cc. of Chalkley solution in
a clean finger bowl. Three grains of rice were added to the
culture and then the finger bowl was set aside until the
amoebae were abundant. Essentially the same procedure was
used for the preparation of all stock cultures used in these

experiments.

Tetrahymeni geleii was used exclusively for study of the

 

frequency of food ingestion by Amoeba proteus. A bacteria
free stock culture was obtained from Dr. A. M. Elliot, De—

partment of Zoology, University of Michigan. Subcultures of

 

1 Galtsoff: Culture Method For Invertebrate Animals

2 Transferred by means of a pipette in order to reduce the
amount of handling of the amoebae and thus the possibilities
of mechanical injuries.

this organism were made in the following manner: About 50
cc. of culture solution (15 gms. Bacto-Tryptone, 1 gm.

{H2P04 and 1,000 cc. distilled water) were added to each of

5 sterile 125 cc. Erlenmeyer flask . The flasks were auto-
claved at 15 pounds steam pressure for 15 minutes, left to,
cool; then about 5 cc. of sterile stock culture were added

to each of the flasks. They were then set aside and left at
room temperature until the organisms were abundant. All
subcultures used for experimentation were made by essentially
the same methods.

Organisms were prepared for experimentation by the fol-
lowing method: An Erlenmeyer flask containing 50 cc. of
Tryptone solution, in which specimens of Tetrahymeni geleii
were abundant, was used for obtaining the necessary food for
the study of ingestion by Amoeba proteus. To each of two 15
cc. centrifuge tubes 15 cc. of the culture solution were
added, and these were then.p1ugged with sterile cotton. The
tubes were centrifuged at a moderate speed until the organ-
isms were concentrated in the bottom of the tube. The tubes
were removed from the centrifuge, 14 cc. of the supernatant
fluid were poured off, and the volume of solution in each
tube was again made up to 15 cc. by adding 14 cc. of sterile
Chalkley solution. The tube was gently agitated until the

Specimens of Tetrahymeni geleii were uniformly distributed

 

throughout the Chalkley solution. This procedure was repeated
two additional times. Before transfer of the organism to the
vessel in which ingestion was to be studied, the organisms

were again concentrated by centrifuging and 14 cc. of the

solution were removed with a pipette. Thus the organisms
were suspended in 1 cc. of solution. Essentially the same
procedure was used for preparation of organisms that were to
be used in experimental solutions, but the viscosity of the
experimental solution was kept constant by a washing of the
organisms in Chalkley solution to which the desired quantity
of Methocel had been added.

The amoebae were prepared for experimentation by the
following method: Two hundred amoebae were selected from a
stock culture and transferred to 5 cc. of .l per cent Metho-
cel in Chalkley solution and left for 24 hours. After 4 cc.
of the Methocel solution were removed, 4 cc. of fresh .1 per
cent Methocel in Chalkley solution were added to the vessel
containing the amoebae. This procedure was repeated two
additional times. The amoebae were left for 20 minutes and
then 4 cc. of the Methocel solution were removed with a pipette.

Specimens of Tetrahymeni geleii suSpended in 1 cc. of .1 per

 

cent Methocel in Chalkley solution were added to the medium
in which the amoebae were suspended. The volume of the solu-
tion in the vessel was made up to 5 cc. by the addition of

3 cc. of .l per cent Methocel in Chalkley solution. After

20 minutes, 25 amoebae were removed from the vessel and put
on glass slides. They were covered with a cover slip and
sealed with vaseline, and then the organisms ingested by the
amoebae were counted. The number of organisms ingested in

60 and 90 minute periods was ascertained in essentially the

same manner .

Rate of locomotion and gel/sol ratio in Amoeba proteus

 

was ascertained in various concentrations of Methocel in
Chalkley solution. Organisms3 were prepared by essentially
the same procedure as described for study of ingestion, but

in these experiments the addition of food to the experimental
solution was omitted. Rate of locomotion was measured in
microns per minute with a camera lucida and a stepwatch.
Readings were made at 1 minute intervals for 5 minute periods.
The average rate of locomotion for the 20 individuals will be
presented in the following pages.

Amoeba proteus is characterized by a relatively rigid

 

outer layer of plasmagel and a more fluid layer of plasmasol.
B th layers exhibit granules of various Sizes, but the con—
centration of granules is higher in the plasmagel. In mono—
podal Specimens the plasmagel is tube-like and the plasmasol
flows in the direction of locomotion. The width of the
plasmasol and plasmagel was measured by means of a camera
lucida. The gel/sol ratio was calculated by obtaining the
average width of the plasmasol and plasmagel in 20 or more
individuals.

In order to present all the experimental evidence in a
form which could be easily read and compared, it seemed ex-

pedient to determine the mean control of each experiment, and

 

3 MonOpodal amoebae were used exclusively. An animal with a
single pseudOpod, moving continually, is described as a
monOpodal amoeba. Rate of locomotion in such Specimens is
fairly constant and the variation between measurements on
the same animal and measurements on different animals in
the same medium is negligible.

to place in preportion to this average control the experimental
results obtained in the various percentage concentrations of
Methocel in Chalkley solution. The results placed in this

preportion were then plotted on graphs.

10.

III. TH RELATION BETWEEN VISCOSITY F THE CULTURE SOLUTION

til

AND RATE OF LOCOMOTION IN ANOEEA PROTEUS

 

All organisms used in the experiments were prepared for
experimentation by the methods described in the preceding
pages. Analytical grade chemicals and glass distilled water
were used exclusively for preparation of Chalkley solution.
The various concentrations of Methocel were prepared by dilu—
tion of 4 per cent stock solution. Temperature was maintained
at 220. Rate of locomotion was adjusted by use of the fol-
lowing formula: X : E x (M/C). X, adjusted rate of locomo-
tion; E, rate of locomotion in each experimental solution;

M, arithmetic mean for locomotion in control solution; and
0, rate of locomotion in individual control solution. The
results obtained are presented in Table I, and Figure I.

It is evident in Table I, Figure I, that rate of locomo—
tion increased from 337 microns per minute in a .l per cent
Methocel solution to 365 microns per minute in a .2 per cent
solution, decreased to 274 microns per minute in a .3 per
cent solution, again increased to reach 333 microns per min-
ute in a .5 per cent solution, and then gradually decreased
to 234 microns per minute in a 1.0 per cent Methocel solution.
It is evident from this analysis of data that locomotion
neither consistently decreased or increased in concentrations
below .5 per cent but in concentrations above this figure
rate of locomotion consistently decreased as the concentra—

tion of Methocel was increased to 1.0 per cent.

d

The .L\.

 

 

elation Between Viscosity

I

and tn

(in microns)

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i Range 01 h

 

 

 

 

 

 

 

11.

Rate of Locomotion

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.L___hmm_

 

 

 

 

 

 

 

 

Per Cent Avg. Rate ate of Adjusted
Concentra- Organ- of Loco- Locomotion in Rate of
tion of isms motion in per minute Locomo-
Methocel Tested per min. Range tion
-1% 23 283 123 598 337
_--__.__-_._--_.. I l _._.. ._--_ - ._.___ _ l. ._ , _ -_1
Control 19 32 104 800 92
.H-fi—‘T‘fi— -_,.-.. . l w... ._-_._ _ we. _..._ .— ..._.._- w—h— .._.._....._.._.. ._-... ._ _. ..__E _.. __._. K
.2% 22 312 77 015 305
1."- -__ll. - _ _____ilul __1
Control 18 335 143 800 352
.35 23 2+1 51 500 274
___ _ -_. _ -1 ._ -l.. , l- __-l. -—--—4
Control 20 345 189 800 392
.45 24 322 113 613 325
, ._ .-._ _. _ ._-. _ _. ._ - . - _ _.__ .4
Control 23 x88 113 000 392
________________l _ .__- _ ___. - _ ..-_ t __._..___._- .........
(2’ '3 it ) ’1 “i r" o
.5p 21 32% l4; (80 333
Control 22 381 163 800 392
.65 2 270 123 710 322
Control 23 338 120 800 392
.7fl 23 333 165 633 317
.,.._____--__ .. .- - . ._ . __ _ __ __ _. _._.—__.J.
Control 23 412 148 800 392
.8% 22 321 110 603 262
__ ._ — _._- ..._.,. __ ___1 -_________.________+
Control 24 480 310 800 392
l p11- - . -l-
.9% 26 292 92 563 248
Control 26 A61 280 810 392
~--————— t-- —.-—-—1 e - - . -—t
1.0% 25 276 5 _J 550 23A
t———.—_.— ——-—_. —- —— — —— —— 1 —— - —-—- —— L———-_. --.—— — —-—-————-—-———11
Control 26 A61 310 820 392

 

 

 

FIGURE I

The relation between viscosity and the rate.

of locomotion (in microns) in Amoeba proteus.

 

Ordinates Show the microns per minute; ab—
scissae the per cent concentration of Meth-
ocel. GD , rate of locomotion in various
concentrations of Methocel in microns per
minute; C) , rate of locomotion in control

solutions.

 

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The question immediately arises whether or not the ob—
served fluctuations in rate of locomotion are significant in
low concentrations of Methocel (.1 to .5 per cent). It was
found during the course of experimentation that environmental

T»

factors such as abundance of food, age of culture, and methods
of handling affected rate of locomotion. Thus it is possible
and also probable that the observed fluctuations in rate of
locomotion in low concentrations of Methocel can be attrib-
uted to environmental factors.

The results presented in the preceding paragraphs strongly
suggest that in higher concentrations of Methocel (.6 to 1.0
per cent) an inverse relationship exists between rate of
locomotion and viscosity, 34e3, as the viscosity of the cul-
ture solution increases, rate of locomotion decreases. This
View is consistent with that of Stiles (1947) who found an

inverse relationship between viscosity and rate of locomotion

in Paramecia.

14.

IV. THE RELATION BETWEEN VISCOSITY AND IEGESTION

OF FOOD BY ANOEBA PROTEUS

 

Mast and Fennell (1938) found that ingestion of chilo-

monads by Amoeba proteus is not closely correlated with the

 

number of organisms in the solution. Specimens of amoebae
ingested an average of 80 organisms in 120 minutes in a solu-
tion containin- 300,000 chilomonads per 00., and only 81
organisms in 120 minutes in a solution in which the number of
chilomonads was 600,000 per cc.

Since there can be great variations in the number of
organisms without appreciably altering rate of ingestion in

the amoebae, the number of Specimens of Tetrahymeni geleii

 

added to the various solutions of Methocel was not ascertained.
However, a Special effort was made to have an abundance of
food organisms in both the experimental and the control solu-
tions.

Mast and Fennell (1938) found that as the temperature
increased, frequency of ingestion increased to a maximum at
250, and then decreased to zero at about 400. Care was taken
to maintain the temperature of the experimental solutions at

about 220. All amoebae and Tetrahymeni geleii used in these

 

experiments were prepared for experimentation by the methods
described in the preceding pages.

The results obtained in various concentrations of Metho-
cel solutions are presented in Tables II and III. The raw
results obtained in these experiments are presented in Table

II, and the corrected or adjusted ingestion rates are presented

TABLE

II

The Relation Between Viscosity and the Frequency of

Ingestion of Food by Amoeba proteus

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Per Cent No. of Average No. of Tgtrgf No. Ingested
Concentra— Organ— hymeni geleii per Individual
tion of isms Ingested in 90 minutes

Methocel Tested 20 Min 60 Min 90 iln Range
.1s 86 1.55 2.25 2.75 0 9
Control 77 2.12 4.24-33.53l6 0 l5
-2% 82 1:26-___?:35. 2e56- _9- --- 8-
Control 74 2.29 3.64 4.20 L. 0 10
.35 90 1.65 1.91 1 953"" 0 9
Control 933—333—32—2’35“3 33.95— 34:25"“H_"“D——34 16
.is 81 "“3f33"’ ”3'333‘ “o“og‘ ‘”’_6__"‘"'駓 *
Control as 3" “2'33"“ 1""11‘137'117 339 "Mo
.35: 103"" 32:33.91 5—08 0 14
Control 110 3.85 3 5:41 7.88 0 17
.6% 99 2.67 3.50 4.18 0 E 18
Control 333" 3.34” 465 3.o “o“’;'“;‘"“
.7% 104 1.99 3 50 3.79 0 12
Control L 102 3.65 .5372 6.25 0 l5
3.8% -- _Liéé: 3 2.44 3.74 5.41 0 : 15
| Control 107 5.40 6.36 7 55 0 15
r_______--_--_--- ._~n_~._--_____ n.-- - _l
i «3 :31.-- _ea 39}. -___2_-_:1_-, O 10
i Control I 113 3.27 r 4 34 4.98 0 19
3 1.0% 103.333 _ .91 1.58 2.25 0 11
Control 111 3.29 4.26 5.10“—‘ 0 19

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

16
TABLE III
The Relation Between Viscosity and the Frequency of
Ingestion of Food by Amoeba proteus
Per Cent Adjusted No. of Tetrahymeni geleii ingested
Concentration IIWHTHWIM'MJWN .---_-_-“_.-M-____l___
Time in Minutes
of -_-_l-__-_--W-. _
Methocel 1 20 60 90
.19 i 2 3» 2.52 2.71
.2% 3 2 23 i 3.07 3—323 -_-
. s E 2 04 3 2 29 2.49
4% .1 (3.3“ -2 57m _ in- 2.94 4 01
5% i 2 74 3 4.3 3 37
.63 i 2.61 . 3.43 3.,2
.79 i 1 78 3 2.91 q 3 29
.8s 3 1 48 T 2.79 i 3.89
.95 i 1.23 i 2.19 2.52
1.05 91 1.74 2 40
Control . 3.27 4.75 5.44

 

 

 

 

 

l7.

FIGURE II

The relation between viscosity and the fr —

quency of food ingestion by Amoeba proteus.

 

Ordinates Show the adjusted number of in-
dividuals ingested; abscissae, per cent
concentration of Methocel. C» , adjusted

number of Tetrahymeni geleii ingested in

 

twenty minutes; 0 , adjusted number in-

gested in sixty minutes; (5 , adjusted num-
ber ingested in ninety minutes; C) , aver—
age number ingested in control solution in

ninety minutes.

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in Table III and Figure II. Adjusted rate of ingestion was
obtained by the formula presented in Section III.
It is evident in Table III that the number of organisms

ingested by Amoeba proteus in 90 minutes increased from 2.71

 

in a .1 per cent solution of Methocel to 4.01 in a .4 per

cent solution, remained fairly constant until the concentra—
tion reached .8 per cent, and then decreased to 2.40 in a

1.0 per cent solution. A maximum number of 2.74 and 4.31
organisms was ingested in a .5 per cent solution in 20 and

60 minute periods respectively. As the concentration of
Methocel increased from .5 per cent to 1.0 per cent solutions,
ingestion decreased from 2.74 to .91 in 20 minutes, and from
4.31 to 1.74 in 60 minutes.

It is evident in Table II, that the number of organisms
ingested by the amoebae reached a maximum of 5.08 organisms
in .5 per cent Methocel solution. These results strongly
suggest that this concentration of Methocel increased the

availability of Tetrahymeni geleii for ingestion by decreas-

 

ing ciliary activity, i.e., by retardation of locomotion.
The availability of food particles was found to be an impor—
tant factor in ingestion by Fennell (1944). He demonstrated

that low concentrations of strychnine sulphate in NaCl solu-

tion (.0029 M NaCl plus .000069 N strychnine sulphate) inac—

 

tivated Specimens of ‘hilomonas paramecium and increased
adhesiveness of the plasmalemma. Because of this existential
relationship between inactivation and adhesiveness, large
numbers of chilomonads.adhered to the cell surface. The num—

ber of chilomonads ingested increased from 29 in 10 minutes

to 46 in 20 minutes. These results were considered highly

1

significant since the t value in the former was 10.45 and

in the latter it was 7.45.

 

1 By t value is meant the difference between the means divided
by the standard error of that difference. When the t value
/ O 0 I 0
is over 2.0 the results are conSidered Significant.

20.

V. THE RELATION BETWEEN VISCOSITY AND GEL/SOL

RATIO IN AMOEBA PROTEUS

 

The gel/sol ratio was measured by methods essentially
the same as those used by Mast and Prosser (1932). All
measurements were made on monopodal specimens. The diameter
of the plasmasol and the widths of the plasmagel were meas-
ured after projection on a sheet of black paper of the image
of the organisms under observation. An average for the re-
gions was obtained by marking the projected image at the
posterior, middle and anterior ends. Then the mean diameter
of the plasmasol and the mean widths of the plasmagel were
carefully measured with the projected image of a micrometer
slide which was calibrated in .01 mm. units. The mean diameter
divided by the sum of the mean widths is presented as gel/sol
ratios. Each figure denoting gel/s01 ratio is an average
obtained by measuring the plasmasol and plasmagel in 24 to
28 specimens. The results obtained are presented in Tables
IV and V, and in Figure III, along with the corrected or
adjusted ratios obtained by the formula presented in Section
III.

It is evident in Table IV, Figure III, that the gel/sol
ratio decreased from 1.21 in a .l per cent Methocel to a
minimum of 1.09 in a .3 per cent Methocel, increased slightly
to reach 1.45 in a .5 per cent Methocel, decreased to 1.35
in a .7 per cent Methocel, increased to a maximum of 1.76 in
a .9 per cent Methocel, and then decreased to 1.42 in a 1.0

per cent Methocel solution.

.TAELE IV

21.

The Relation Between Viscosity and the Gel/sol Ratio

in Amoeba proteus

 

>.——_ ..,_.-.—.,.. -.—._._.

 

 

..-._._._‘.___

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Per Cent Gel/Sol Ratios Arith— Adjusted
Concentra— in the Various Experiments metic Gel/Sol
tion of mean Ratio
Methocel No. 1 No. 2 No. 3 No. 4
-l% 1328 1.56 1.28 1.11 1.31 1.21
Control 1.11 1.38 1.36 1.16 1.25 1.15
.23 1.31 1.34 _iifii-t-i;§8- 1.32 1.22
Control 1.11 1.50 1.50 1.16 1.24 1.15
.3% F 1.21 1.21 1.26" 1.16 1.21 1.09
Control 1.51 1.36 1110 1.16 1.28 1.15
.4% 1.49 1.28 1.19 T 1.10 1.27 #1 1.28
Control _[3 1.51 .82 1.05ij. 1.16 1.14 l 1.15
. s i 1.56 1 25 1 22 1.22 1.31 i 1.45
Control 1.08 1.03 .87 1.16 1.04 1.15
.6% ___1.34_}A 1.19 .86 1.29 1.17 1.45
1 Control 1.08 . .81 .76 1.16 .95 I 1.15
3 .7% 1.58 ' 1.00 1 03 1.53 1.29 1 1.35
gControl 1.45 .95 H .83 1.16 1.10 i 1.15
E .83 1.67 1.21 1.06 1.15 1.02 i 1.04
LControl 1.45 1 13 .83 1.16 1.13 i 1.15
I .9% 1.36 1.99 1.91 1.24 1.823 1.76
LControl 1.04 1.29 1.27 1.16 1.19 f 1.15
i 1.0% 1.43 1.43 1.55 1.45 1.47 g 1.42
i Control 1.04 1.29 1.27 1.16 1.19 1.15

 

 

 

 

 

 

 

 

TABLE V

Showing the Width (in microns) of the Plasmagel,

Plasmasol and the Gel-Sol Ratio

22.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Per Cent No of Average Diameter in Microns Gel-
Concentra- Organ- Sol
Plasmagel Plasmasol
tion of isms _ _*“_ Ratio
Methocel LTested Range Average Range *3Average
.13 i 26 60 - 158 92 44 115 81 1.31
Control 3 25 43 - 143 74 53 103 68 1.25
.23 24 55 - 118 84 40 103 64 1.32
Control 20 43 - 118 82 57 90 67 1.28
-3% j 23 25 — 115 76 25 103 67 1.21
Control .33 25 30 - 113 77 28 83 62 1.28
.49 25 58 - 115 91 50 951 72 1.27
Control 24 53 - 113 81 53 92% 72 1.14
.5% 27 60 - 113 84 45 933 65 1.31
Control 28 40 - 150 73 50 1103 76 1.04 I
.65 23 37 — 112 79 35 as} 68 1.17
Control 27 15 - 135 72 35 1283 75 .95
.7s 24 42 — 13 88 45 103i 70 1.29
Control 23 50 — 138 82 50 1073 73 1.10
.89 24 58 - 123 85 50 85 66 1.02
Control 25 51 - 138 84 53 107 72 1.13
.95 24 5o - 125 90 20 85 58 1.82
Control 27 65 - 117 83 60 105 72 1.19
1.05 24 60 — 117 87 38 78 61 1.47
Control 27 . 65 — 117 86 60 105 72 1.19

 

 

 

 

 

 

 

 

 

 

FIGURE III

mu

_nc relation between viscosity and the

.gel/sol ratio in Amocha grotcus. Ordinates

 

1

show the adjusted gel/sol rati 3 aoscissae

O
h

per cent concentration of I-Iethocel. (D ,
adjusted gel/sol ratio in various concen-
trations of Kethocel; C) , gel/sol ratio in

control solutions.

QV

% .0

\u0 o fix «t
\o §§\\§\x ‘uusxob \sxoUKUK

b6 ‘6

‘6

Q6

 

 

 

 

 

 

 

 

 

 

 

 

 

0,493 . /os~ yag

24.

A review of the results given in Table V shows that the
mean diameters of the plasmagel in all concentrations of
Methocel was consistently higher than it was in the control
solution. Further, the mean diameter of the plasmasol neither
consistently decreased or increased as Methocel concentration
increased from .1 to .5 per cent, but in solutions in which
Methocel was varied from .5 to 1.0 per cent, the mean diameter
of the plasmasol was consistently lower than it was in control
solutions. These results demonstrate a direct relationship
between plasmagel width and viscosity, and an inverse rela-

tionship between plasmasol width and viscosity.

25.

VI. DISCUSSION

It was shown in Table II and III, that the number of

Tetrahymeni geleii ingested by Amoeba proteus reached a max-

 

 

imum in .5 per cent Methocel solution. Facilitation of inges—
tion in this concentration of Methocel seems to be closely
correlated with decreased rate of locomotion in Tetrahymeni
geleii. This view is consistent with the views of Fennell
(1944) who studied amoeboid ingestion in strychnine sulphate
solutions. He found that low concentrations of this drug

inhibited flagellar activity and locomotion in Chilomonas

 

paramaecium. As a consequence the number of organisms made

 

available to Amoeba proteus was increased by decreasing the

 

frequency of escape of Chilomonas paramaecium from food vacuoles

 

during the ingestion process. Stiles (1947) likewise found
that Methocel decreased ciliary activity and rate of locomo-
tion in paramaecia.

Mast and Fennell (1938) suggested that there was a close
correlation between amoeboid locomotion and amoeboid ingestion.
It is evident in Table I that rate of locomotion is fairly
uniform in concentrations of Methocel varying from .1 to .5
per cent and that ingestion of food reaches a maximum of 5.08
organisms in .5 per cent Methocel. If this increase in inges-
tion was determined by another factor, i;§,, an interaction
between Methocel and cellular substances, it is possible that
this condition would be manifest by observable changes in

1

gel/sol ratio and morphological alterations in amoebae. The

results presented in Tables II and III and also repeated

26.

observations made on amoebae revealed a constancy in both
gel/sol ratio and morphological features in low concentrations
of Methocel (.l to .5 per cent). For these reasons a corre-
lation between this factor and ingestion is not tenable.

It was demonstrated in Table V that the diameter of the
plasmagel increased and the width of the plasmasol decreased
as the concentration of Methocel increased from .5 to 1.0
per cent. Obviously several explanations could be offered
for these observed cytOplasmic variations: (1) It is pos-
sible that the width of the plasmagel may depend on entrance
and union of inorganic salts with some cytoplasmic constituent,
and that rate of entrance may vary with concentration of
Methocel; (2) increased width of the plasmagel may depend on
entrance of Methocel into the cell and rate of entrance may
vary with concentration of the Methocel; or (3) increased
width may be due to loss of water from the cell and the rate
of loss may be dependent on Methocel concentration. There is
little evidence to support the first two hypotheses, but it
is well established that plasmasol can be transformed into
plasmagel or vice versa by loss or gain of water. East (1926)
demonstrated that during amoeboid locomotion there was a
solation of the plasmagel at the posterior end and a gelation
at the anterior end. These reversible cytoplasmic changes
were correlated with intace of water at the posterior end
and loss of water at the anterior end. These results suggest,
but do not demonstrate, that increased width of the plasmagel
and decreased width of the plasmasol are closely correlated
with loss of water, and that this loss of water is dependent

on Methocel concentration.

2.

27.

VII. SUMMARY

An inverse relationship exists between rate of locomotion
and viscosity of the culture solution.

A direct relationship exists between viscosity and inges-
tion in low concentrations of Methocel, but an inverse
relationship exists between viscosity and ingestion in
higher concentrations.

A direct relationship exists between plasmagel width and
viscosity, while an inverse relationship exists between
plasmasol width and viscosity. Increased width of the
plasmagel and decreased width of the plasmasol seems

closely correlated with loss of water from the cytoplasm.

28.

VIII. LITERATURE CITED

Fennell, R. A., 1944, I. "The Relation Between Attachment to
the Substratum and Ingestion by Amoeba in Strychnine
Sulfate Solution and Conditioned Media.” II. "Bio-
logical Assay of Conditioned Media." Jour. Gen.

thsiol., Vol. 27, 339-354.

 

Galtsoff, P. S., 1937, "Culture Method for Invertebrate Animals."

HOpkins, D. L., 1929, "The Effects of the Substratum, Divalent
and Monovalent Cations on Locomotion in Amoeoa pro—
teus. Jour. Morph. and Physiol. ., Vol. L8, 371-383.

 

Mast, S. 0., 1926, "Structure, Movement, Locomotion, and
Stimulation in Amoeba." Jour. Morph. and Physiol.,

Vol. 41, 347-425.

 

 

, and R. A. Fennell, 1938, "The Relation Between
Temperature, Salts, Hydrogen Ion Concentration, and
Frequency of Ingestion of Food by Amoeba." Physiol.
g22,, Vol. 11, 1—18.

 

, and C. L. Prosser, 1932, "Effect of Temperature,
Salts, and Hydrogen Ion Concentration of Rupture of
the Plasmagel Sheet, Rate of Locomotion, and Gel/sol
Ratio in Amoeba proteus." Jour. 9f_Cell. and Comp.

Physiol., Vol.1, 333-354.

Pitts, R. F., and S. O. Mast, 1933, ”The Relation Between
Inorganic Salt Concentration, Hydrogen Ion Con-
centration and Physiological Processes in Amoeba
proteus." I. "Rate of Locomotion, Gel/sol Ratio,
and Hydrogen Ion Concentration in Balanced Salt
Solutions. Jour. of Cell. and Comp. Physiol.,
Vol. 3, 449-462. '—"

 

 

 

 

 

, 193 4a, "The Relation Between Inorganic Salt

 

Concentration, Hydrogen Ion Concentration and Phy-

 

siological Process es in Amoeba proteus. II. "Rate
of Locomotion, Gel/sol Ratio and Hydrogen Ion Con-
centration in Solutions of Single Salts. Jour. g£_

Cell. and Comp. Physiol., Vol. 4, 237— —256.

, 1934b, "The Relation Between Inorganic Salt

 

Concentrations, Hydrogen Ion Concentration and
Physiological Processes in Amoeba proteus." III.
”The Interaction Between SaltsTAntagonism) in
Relation to Hydrogen Ion Concentration and Salt
Concentration." Jour. 9£_Cell. and Comp. Phys iol.
Vol . 4, 435-455 .

 

 

 

29.

1e Rate of Locomotion in Amoeba
Jour. of Morph. and

Schwitalla, A. M., 1925, ”T1
at Different Temperature.”
Vol. 41, 45—58.

 

Physiol.
C. A., and D. A. Hawkins, 1947, ”Immobilizing Para-
Science,

Stiles,
mecia For Study in the Introductory Course."
Vol. 105, No. 2717, January 24.

(,0
O

CURRICULUM VITAE

Nellie Marie Beukema was born March the 11th, 1927, in
Hollywood, California. She attended Grammar School, Junior
High School, and Culter Academy in that city, and graduated
from the latter in 1944. She entered Calvin College at Grand

Rapids, Michigan, in the fall of 1944 and received the A.B.

(D
D

degre in Education from this institution in 1948. She was a

graduate assistant in Zoology at Michigan State College in
1948-1949, and in the fall of 1949 and the winter of 1950 she
returned to Michigan State College to complete work on her
Master's degree. She majored in Zoology and minored in
Chemistry and Nutrition.

J

During the summer of 1949 sne was employed as a Dietician

5

at Blodgett Memorial Hospital in Grand Rapids, Michigan. At

.‘

k

the present time she is a member of the faculty at Calvin
College in Grand Rapids where she is serving as Assistant in

Biology.