STRUCTURAL AKD CYTOCHBCCAL STUDIES CF THE CYTOPLASM
IK THE FAMILY AMOEBIDAE
DISSERTATION
Presented In Partial Fulfillment of the Requirements
for the Degree Doctor of Philosophy in the
Graduate School of The Ohio State
University
By
QEORQE DEKETRIOS PAPPAS, B.A., M.SC.
The Ohio State University
1952
Approved byt
STRUCTURAL AND CYTOCHEMICAL STUDIES OF THE CYTOPLASM
IN THE FAMILY AMOEBITUUE
page
INTRODUCTION
— -----------------------
HISTORICAL REVIEW
-----------------------------------
MATERIALS AND M E T H O D S
----------------------------- 10
The Species Studied — —
---
10
Amoeba proteua (Pal 1 as) Leldy
— ---
Cheoa chaoe (Linnaeus)
Theeamoeba striata (Penard) Schaeffer
Mayprella blgeeana Schaeffer
Amoeba gut tula Dujardln
—
—
— — — —
—
10
—
19
--- 19
— —
21
----- — ------------- 21
-------- — ----
— ---
The Parloaion Trap Technique
Cytochemjcal Methods
23
—
2b
----------------
Protein Reactions
-— —
Carbohydrate Reactions
Lipid Reactions
—
----- -----— — — -— -------- 23
Prepara don of Amebaa
General
3
—
—
3
— — — ------—
33
— ------------
39
-------------------
1*1
Other Methods. Fixing Reagents. Stains, and
Chemicals
---------- — ----------------- 14*
Microscopes and Accessories
RESULTS
—
-— —
—
— — —
1*7
------------------------------------------ 1*8
The Cell Membrane
--- ----— —
i
809656
—
—
—
— —
1*8
Cytoplasmic Inclusions
— --------
52
— --
52
The Alpha Granul es
The Beta Granules
..........
The Spherical Refractive B o d i e s -- --The Contractile Vacuoles
Crystal and Crystal Vacuole
62
The Food Vacuoles
63
Other Inclusions
-— ---- ---............—
—
-- — — ------------—
Cytoplasmic Ground Substance
DISCUSSION
—
—
— — -—
--
------------------------------------------
-----------------------------------------
LITERATURE CITED
PLATES
55
...-- ...----- ..... 58
The Fat Globules--- — -------------- -— —
SUMMARY
$2
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67
70
72
82
87
98
STRUCTURAL AND CYTOCHEMICAL STUDIES OF THE CYTOPLASM
IN THE FAMILY AMCEBXDAE
INTRODUCTION
The classification and nomenclature of the cytoplasmic inclu­
sions of free-living amebas was systematized by S* 0. Mast and his
students In a series of papers from 1926 to 19bl»
However, almost
all of their work was done on two rather similar species, Aaroeba
proteus and Chaos chaos. In recent years cytochemical and quanti­
tative physiological Investigations of these two species, especially
C. chaos, have been conducted by Hoiter and Andresen of the Carlsberg Laboratories In Denmark,
Such information has been almost en­
tirely lacking about other species of amebas, many of which differ
strikingly in their general morphology from the two mentioned.
In
the present investigation, a study of the cytology and cytochemistry
of three very diverse species, Theeamoeba striata. Msyorella blgemaa,
and Amoeba guttula, was undertaken and Amoeba proteus and Chaos chaos
were studied at the same time for purposes of comparison.
Along with the classical cytological techniques, including vital
staining, a cytochemical approach has been employed for a better un­
derstanding of the nature of the cell particulates through a know­
ledge of their chemical composition.
1
Since the majority of present
hlstoshemlcal methods are designed for use with metazoan tissues,
special techniques had to be devised for applying these tests to
protoaoa.
mis Investigation was supervised by Professor W. J. Kostlr
of the Department of Zoology and Entomology, The Ohio State Uni­
versity, whom I warmly thank for his suggestions and criticisms.
To Dr, E. R. Hayes of the Department of Anatomy, The Ohio State
University, and Dr, H, Hoffman, now at the Department of Bacteri­
ology, University of Nebraska, who introduced me to cytochemistry
and its potentialities, I wish to express my gratitude for their
continued interest and invaluable advice throughout the investigatlo
Q.
2
HISTORICAL REVIEW
Many studies have been made on the physiological and physi­
cal properties of the cytoplasm of Amoeba proteus.
The most sig­
nificant feature of the cytoplasm of amebas is its ability to
change from a relatively fluid state (sol) to a more solid jellylike condition (gel).
The thlxotroplc (sol-gel) properties of
some colloids, especially cytoplasm, have been studied extensively
by physical chemical methods (Seifris, 1936).
Sol+gel reversal,
as shown experimentally by Angerer (1936), may be brought about by
mechanical agitation.
He showed that mechanical agitation causes
a decrease In viscosity in the plasmagol and an initial decrease
followed by an increase in the plasmasol.
However, the dynamic
aspects of sol-gel reversibility in amebas, resulting in ameboid
movement, are not yet clearly understood.
Mast (lj?26, 1932),
Vantin (1923, 192U, 1926, 1930)t and more recently £>eBruyn (19U7)
delve into this problem rather extensively.
There has been much discussion concerning the characteristics
of the outermost layer of amebas.
The pellicle of Thee amoeba
verrucosa was removed by Howland (l92lic), leaving the plasma mem­
brane, the physiologically active vital membrane, intact.
She
found the pellicle to be quite elastic and easily stretched.
Likewise Chambers (I92li) was ablo to lift the plasnalemma of A.
proteus by injecting water beneath it.
This caused a blister to
form which burst when punctured, leaving the plaamalenna collapsed.
Mast (1926) also caused blisters to appear which u^on bursting
shoved the frayed ends of the plaamalerama.
He then was able to
measure the thickness of the plaanalemma, and found It to be 0.25
micron*
Seifrls (1936) found that the plasmalenna of 1. proteus
was resistant, elastic, and highly viscous except at the advancing
tip of a pseudopodium.
Lehmann (1950) with the aid of the electron microscope show­
ed that the plasmalemna of A. proteus is composed of small glob­
ular particles.
There was no clue, however, to the nature of
these particles*
Numerous observations have been made on the structure, ori­
gin, and functions of various cytoplasmic inclusions In the amebas,
but there Is considerable disagreement among the Investigators.
However, Mast's Investigations on cytoplasmic Inclusions in Amoeba
protous have been, on the whole, corroborated by other workers.
Mast (1926) maintains that A* proteus consists of a central, gran­
ular, fluid portion or "plasma sol" j a jelly-like granular layer or
"plamnagal" surrounding the fluid portionj and a vory thin, well
differentiated elastic surface layer or membrane, the "plasmalemma" *
According to Mast, the plasmasol consists of a fluid in which the
following structures are suspended! numerous minute spherical
bodies (alpha granules), about an equal number of larger, irregular
bodies (beta granules), crystals, varying greatly In number and
sise, food vacuoles, the nucleus and the contractile vacuole.
tain of these bodies are also found in the plaaraagel.
a few alpha granules are found In the hyaline layer*
adhere to Mast's nomenclature of these inclusions*
U
Cer­
Occasionally
We shall
In centrifuged specimens of A* proteus (Mast and Doyle, 1935,
Singh, 1938, and others) the cytoplasmic constituents are fairly
definitely arranged In strata as follows, beginning with the cen­
tripetal end:
1) fat globules, 2) crystal vacuoles, with an all
crystals or no crystals, 3) the contractile vacuole with beta
granules adhering to It, Ii) hyaline substance, 5) the nucleus,
6) some snail food vacuoles, 7) crystal vacuoles with medium-sized
crystals, 8) alpha pranules, 9) be "-a granules, 10) crystal vacu­
oles with large crystals, 11) some additional beta E^anules, 12)
some additional alpha granules, 13) medium and large food vacu­
oles, and lit) spherical refractive bodies*
The crystals are usu­
ally found at the centrifugal ends of the vacuoles.
In the nucleus
also, the chromatic granules are found at the centrifugal end*
Alpha granules*
Mast and Doyle (1935) described the alpha
granules as being the smallest granules visible under the compound
microscope*
They measure approximately 0.25 micron in diameter*
Mast and Doyle were able to observe very few of these granules in
the centripetal half of amebas which had been centrifuged*
But
because of their small size and the presence of other constitu­
ents in the centrifugal portion of the amebas, it was not possible
to ascertain the exact region In which the granules were localized*
Andresen (19U2) stained these granules In Chaos chaos with Heldenhaln's Iron hematoxylin*
MacLennan (19I4I ) classifies them as the
"unknown granules" of amebas*
Beta granules*
Mast (1926) designated certain granules,
5
measuring about one micron, as beta granules.
They are scattered
uniformly throughout the cytoplasm, except at the s urface of the
contractile vacuole, Where they tend to form a layer.
The out­
line of the beta granules can be seen very distinctly in living
specimens.
By observing them from various points of view, Mast
and Doyle (1935) found that those which are suspended in the cy­
toplasm are nearly spherical or ellipsoidal, while those which are
on the surface of the contractile vacuole are flattened at the
points of contact with the vacuole and with each other.
Metcalf (1910, 1926) observed the "microsomes" (beta granules)
around the contractile vacuole.
He asserted that these granules
are specific, and persist in the same general location after the
contraction of the vacuole.
He believed that when the vacuole
reappears, it is found always in the midst of this same group of
granules.
Prom this, Metcalf then concluded that these granules
are responsible for the functioning of the contractile vacuole
and therefore may be considered "excretory granules."
Mast and Doyle (1935) observed that the beta granules change
their form in living amebas.
They observed local protuberances
resembling small pseudopodia, appearing at intervals as shcrt as
five minutes, but no locomotion was observed.
The spherical
granules sometimes became ellipsoidal or rod-like in form and
vice-versa.
When they were rod-like they appeared to these authors
somewhat similar in outline to bacteria.
In unfavorable culture
media, in intense light, and under ultra-violet rays they were
6
seen to break up into smaller spheres*
The fact that beta granules
became angular when In close contact indicated to these authors
that they are plastic*
Mast and Doyle put the amebas into a 1:100,000 solution of
Janus green*
In two and one—half hours, all the beta granules but
no other structures appeared distinctly green*
It was then clearly
seen in optical section that only the surface of these granules
was stained*
This indicated to Mast and Doyle that these granules
have a differentiated surface layer S i m H r to that in mitochondria*
They then further treated amebas with several other mitochondrial
staining methods*
The beta granules in every case were found to
have all the staining characteristics of mitochondria.
Host of
these extremely Interesting facts revealed by the investigations
of Mast and Doyle have been verified in Chaos chaos by Andre sen
(19^2) and Wilber (191:5 ).
Andresen (l9h2) stainod the beta granules with aniline fuchsln after Champy-Kull fixation and treatment with chromic acid.
The relative weight of beta granules was measured by Mast
and Doyle (1935b), Hoiter and Doyle (1938), Wilber (19U5&) and
others by centrifugation.
The granules were found to bo slightly
lighter than the nucleus*
Mast nnd Doyle (1935b) suggested that the beta granules
govern the transport of enzymes to the food vacuoles and the
transfer of digested substances from the food vacuoles to various
places In the cytoplaan*
They reported that death occurred when
7
nost of the beta granules were removed.
According to Horning (1933), working with an unnamed species
of Amoeba, food Ingested comes into intimate contact with some
nltochondrla*
Later, a vacuole forms which encloses the particle
of food and the adjacent mitochondria*
If the process is followed,
It is seen that with the progress of digestion of the food, the
nltochondrla diminish progressively in size and at the end of three
lours disappear completely*
No other investigators have verified
these observations*
Hast and Doyle (193?b) found in A. proteus that mitochondria
accumulate around a food vacuole, six to eight hours after its ori­
gin and again at sixteen and thirty hours*
They never found the
mitochondria actually entering the vacuole, however*
Hopkins
(1936b) found no beta granules associated with the food vacuoles
in the marine anieba Flab-11ula mira*
Upon centrifugation and nlcrodlssection of Amoeba proteus,
Hoiter and Kopac (1937) found no definite differences in amylase
activity of the various regions of the ameba; however, parts con­
taining dense concentrations of mitochondria also showed a more in­
tense enzymatic activity*
Wilber (l9U5a) also by means of microdissection and cen­
trifugation, was able to remove the beta granules almost com­
pletely from giant amebas*
Under these conditions, he found the
amebas were apparently healthy in their mode of behavior*
Wilber
than concluded that the beta granules do not play an essential
8
part in call functions and that their observed changes in position
are merely a visible sign of submicroscopic ch snges in the hyalo­
plasm*
His results are in harmony with the contention of Bolter
(1936) and Hoitor and Kopac (1937) that specific enzymes in amebas
and in marine eggs are not localised in any formed bodies, but are
present chiefly in the hyaloplasm.
Crystals. Crystals as inclusions in the cytoplrcm have been
found in several species of amebas.
Mast and
(1935) main­
tained that in Amooba proteus there are two types of crystals,
plate-like and bipyramidal. Both types are inj vacuole3.
Hie vacu­
oles contain hyaline substance in which the cr^st^ls are suspended,
usually one in each vacuole.
The vacuoles ant^ the crystals, they
maintained, both vary greatly in size, and the size of the vacuoles
in relation to the size of the crystals in them also varied greatly.
The hyaline substance In which the crystals are suspended in the
vacuoles becomes "reddish yellow" in neutral ided from which they
concluded that it is alkaline.
Concerning the plate-llke crystals. Mast and Doyle staged that
"they resemble crystals of leucine in respect to crystal habit and
extinction angles and they are insoluble in a saturated solution
of leucine".
Mast and Doyle found that the bipyramidal crys tals are optii
cally Inactive.
This, they asserted, ollmin'itjos the possibility
that the crystals consist of such substances
s calcium carbonate,
phosphate, cholesterol, etc., as was postulated earlier by other
investigators*
From several solubility tests, it was concluded that
these crystals are composed of glycine, since glycine is the only
amino acid which is optically inactive*
However, when a saturated
solution of glycine was added to the crystals, they dissolved*
Melting point determinations were then made, the results of itiich
led Mast and Doyle to bellove that these crystals are a combination
of an organic and an inorganic substance*
that they contain a salt of glycine*
It was then postulated
Spectrographic analysis then
demonstrated that magnesium is present in these crystals*
These
investigators then concluded that the bipyramidal crystals consist
of a magnesium salt of a substituted glycine*
Luce and Pohl (1935) reported that the bipyramidal crystals
are doubly refractive*
However, Bernheimer (1938a) found, like
Mast and Doyle, that these crystals are net optically active.
The
melting point obtained by Bernheimer was similar to that of Mast
and Doyle, but differed from that given by Luce and Pohl.
Bernheimer (1938a and 1938b) identified many other crystals in
several different species of amebas and other protozoa*
However,
the exhaustive investigations of Mast and Doyle on the crystals of
Amoeba proteus have not to date, been carried out with other species*
Andre sen (19U2, 19U5) reported the same two types of crystals pres­
ent in Chaos chaos as had been described in Amoeba proteus*
Mast and Doyle suggested that probably the crystals are used
in the formation, in part, of cell inclusions*
Bernheimer, how­
ever, maintained that the crystals remain unchanged and inert in
10
-the cytoplasm,
He was able to see. In a few cases, the egestion of
crystals by the aieba,
Andre sen (19U5) found that in Amoeba proteus during starvation
the number of crystals increased.
On the other hand, Andresen and
Hoi ter (191*5?) found In Chaos chaos that the number of crystals dur­
ing starvation remained unchanged*
Spherical refractive bodies.
Various investigators have ob­
served these relatively large, spherical, highly refractive bodies
in the cytoplasm of amebas, which appear homogeneous in the living
cell.
Mast (1926) called them "spherical refractive bodies"*
Brown (1930) stated that these bodies represent the Golgi apparatus
in Amoeba protons.
On centrifugation of Amoeba proteus (Mast and
Doyle, 1935b) and Chaos chaos (Singh, 1938) these bodies were found
to be the heaviest of all cell inclusions.
Mast and Doyle (1935a) demonstrated that in Amoeba proteus the
spherical refractive bodies stain vitally with neutral red.
The
non-staining inner portion was found to be eccentric in some of
the bodies.
The outer layer became black with osmium tetroxlde
and did not bleach in H2O2 or turpentine.
This layer does not
stain with Sudan stains unless it is first treated with phenol
(Ciacclo*s method).
The fact that it does not stain with Sudan
stains shows that it is not fat.
However, since the cortex does
stain with the Sudan dyes after treatment with phenol, Mast and
Doyle concluded that this cortex is fat like.
for protein gave a faint positive re -ctlon.
11
Millon's reagent
Hence it was concluded
that the outer layer of the refractive bodies consists of a pac­
ts in stroma which is impregnated with a fatty substance.
Within
this layer a thin shell made of carbohydrate ami containing no
starch was found.
Mast and Doyle could ascertain nothing concern­
ing the composition of the fluid within the shell.
They concluded
that the refractive spherical bodies are probably Qolgi substance
or material.
In Chaos chaos Andreson (19b2) has described similar spherical
refractive bodies, but was unable to stain them vitally with neu­
tral red.
They did stain with neutral red, however, after the
death of the ameba.
He calls these bodies "heavy spherical bodies".
Wilber (19U5a) observed that in the giant ameba, during star­
vation conditions the
number
of refractive bodies diminished.
How­
ever, Andresen and Hoiter (19U5) found that the number of refrac­
tive bodies remained constant during starvation.
They found that
as the volume of the ameba decreased, d ense packing and coalescence
occurred, which may acccunt for what appeared to be a diminution
in the number of refractive bodies.
Wilber (19U5a) completely removed the refractive bodies from
Chttoy chaos by centrifugation and subsequent mlcrodlssoction, and
asserted that the amebas were not impaired in their activities.
Most investigators (see MacLennan, 19hl) agree that these
refractive or heavy spherical bodies represent the Qolgi complex
in Pro to so a.
Wilber (19U2, 19l*5b) however, disagrees.
He found
that ttoen the giant ameba Is fixed in Champy's fluid and impreg-
12
nated with osmium tetroxide the outer layer of the refractive
bodies blackened.
However, contrary to the observations of Mast
and Doyle, he found that this blackening was easily removed with
turpentine or hydrogen peroxide.
Wilber further stated that since
these bodies stain on the outer layer with neutral red they cannot
be homologous to metazoan Oolgl substance.
Wilber prefers to call
these bodies volutin or metachromatlc bodies because they stain
with the Schiff's reagent without prior hydrolysis, indicating
that they contain "free aldehydes" and also protein.
Contractile vacuole.
The contractile vacuole has probably
been studied more than any other cytoplasmic inclusion in the
protozoa.
Tet after several decades of Investigation, virtually
nothing has been proven beyond question concerning its structure
or the mode of functioning.
According to Mast (1938) the contractile vacuole in Amoeba
proteus contains at the surface a well differentiated membrane
about 0.5 micron thick.
granules.
Adjoining this membrane is a layer of beta
The fluid content of the contractile vacuole is always
colorless and homogeneous.
It is generally agreed that the sub­
stance discharged by the contractile v acuole is chiefly water, and
that this water may contain small quantities of waste substances.
Metcalf (1910, 1926), as was cited earlier, thought that the
granules surrounding the contractile vacuole are associated with
the origin of the vacuole.
Howland (192Ua) found no concentration
of granules on the surface of the vacuole in the- Thee amoeba
13
verrucosa* however she thought it likely that the vacuole arises
from the coalescence of small hyaline grobules.
The actual mode
of origin of the contractile vacuole is still an unsolved question.
Many investigators (see Wentherby, 19III) have postulated that
the contractile vacuole is part of the Golgi apparatus and is thus
intimately associated with it.
However, in amebas the contractile
vacuole is not surrounded by neutral-red stainable, osmiophllic
bodies such as the spherical refractive bodies.
On the contrary,
the vacuole is either surrounded by b> ta granules or by no granules
at all.
Thus, at least in the amebas, postulating an association
of the contractile vacuole with the Golgi complex does not seem
justified.
Of the numerous theories concerned with the functioning of the
contractile vacuole, two have persisted throughout several decades
of Investigation,
It is generally believed that the contractile
vacuole functions as a hydrostatic organ, that is, helps to keep
the water content of the organism approximately constant.
The
other and older theory is that the vacuole functions in the excre­
tion of metabolic waste products,
Howland (192lia, 192l*b) working with Paramecium caudatum and
Amoeba (Theeamoeba) verrucosa demonstrated that uric acid is present
in old cultures.
However, she could not determine whether the con­
tractile vacuole is at all active in the secretion of uric acid,
Irfeatherby's (1927 and 1929) work on Paramecium was probably
the most accurate in testing for nitrogenous products in the conH*
tractile vacuole*
Some of the techniques employed are of Interest*
He first tested cultures of previously washed organisms for awnonia
(by Nesslerisatlon) and found that in cultures 3h or more hours old,
ammonia was always present, from which he concluded that either
ammonia was secreted as such, or occurred as the hydrolysis product
of some other excrete*
He then injected Nessler's reagent by means
of a micro-pipette, and succeeded in twelve different instances, in
injecting the reagent into contractile vacuoles.
In no case, how­
ever, did Weatherby succeed in getting a positive reactism.
Thus
It was concluded that either the concentration of urea in the con­
tractile vacuole is too low, or that urea was not present at all*
By a calculation based on MaupasJ^ method (that the quantity of water
evacuated by the contractile vacuole in lib minutes at 27 d grees C*,
Is equal to the total volume of the organism), Weatherby found that
the quantity of urea which should occur in the fluid of the vacuole
should be about one part in 2000-3000*
Inasmuch as the reagent used
is sensitive to one part in 12,000, it was concluded that the vacuoles
play no part in the excretion of urea*
The theory of a hydrostatic function has a popular appeal among
investigatcrs*
Hyman (1936) believes that the vacuole in Amoeba
(Mayorella) vespertilio serves in discharging the excess of water
which has entered the cell from a hypotonic medium*
However, Hop­
kins (19U6) found that Amoeba lacerata. a fresh water ameba, is
able to adjust to and live in any concentration of salts of sea
water up to 12$%m
Normal contractile vacuoles were formed in all
1$
concentrations.
Hopkins also stated that the contents of the con­
tractile vacuole Hust necessarily contain other substances besides
water.
He contended that the formation of vacuoles more dilute
than protoplasm seems to be an impossibility and contrary to known
physicochemical lavs.
Tor in the formation and growth of a con­
tractile vacuole, vat or must diffuse into a region of greater hydro­
static pressure and of lesser concentration of solutes.
Hopkins
than postulated two ways by which water can be separated from
protoplasm, i.e. by which forces could be set up to attract or re­
pel water more strongly than protoplasm does:
(1) By chemical
changes occurring in regions of the protoplasm, for instance oxida­
tions which results in localized Increases in chemical and osmotic
forces (vacuoles)j (2) By changes occurring in protoplasm In gen­
eral.
The water would collect in localized regions, but during
this collection, waste products become dissolved and consequently
the vacuoles so formed would contain waste and salts, not "distilled"
water.
Hopkins was able to demonstrate further that when digestion
is completed the food residues are eliminated with little or no
fluid.
Hence he claims that the only way that liquid excretion
products can be eliminated is by means of the contractile vacuole.
The food vacuoles.
The study of the food vacuole -i and diges­
tion is based mainly on vital staining.
The changes in pH as shown
by neutral red coloration in the cycle of digestion of a given food
vacuole was studied in detail by Mast (19h2).
The process of inges­
tion has been studied by Edwards (1925) and Hast (1926) for Amoeba
16
protsus. by Kspner and Edwards (1917) for Chaos chaos (Palomyxa
carolln^Wls). and by Lotsa (1931) for Mayorella blgatnma. and by
Pappus and Koatir (1952) for Thecauaoeba striata.
17
MATERIALS AND METHODS
The Speclea Studied
Amoeba proteus (Pallas, 1766) Leidy. 1878,
Cultures of
Amoeba proteus have been maintained continuously In the Proto­
zoology Laboratory at The Ohio State University for many yoars.
The culture method, using Saprolegnia sp, growing on rice grains,
with Chilomonaa paraaeclum as the chief food organism has been de­
scribed in detail by Handy (19li7)*
Many synonyms exist for Amoeba proteus.
1931.)
(See Mast and Johnson,
However, the only other name used today is Chaos diffluens,
proposed by Schaeffer In 1926.
Amoeba proteus can be distinguished
from two similar species. Amoeba dubia and Amoeba discoides, by the
presence of ridges on one or two of its larger psuedopodla.
Also
Amoeba proteus measured 600 or more microns when elongated, where­
as Amoeba disco ides and Amoeba dubia rarely measure more than UOC
microns,
(See Kudo, 19ii6, and Jahn and Jahn, 19h9»)
The cytoplasm of
proteus has undoubtedly been studied
more often than that of any other species of ameba.
The nomencl­
ature of the various inclusions and structures of the cytoplasm
here aa^loyed is that proposed by Mast (1926) in his first paper
on the structure of this species.
Amoeba proteus consists of a ventral granular fluid portion,
the plasma sol, surrounded by a Jelly-like layer called the plasmagel.
The piaamaleana, the outermost limiting layer or membrane in Amoeba
16
proteus is rather thin but well-different!ated.
The cell inclusions,
found almost exclusively in the plasnasol, arei
l) alpha granules,
numerous minute and spherical; 2) beta granules, larger and very
slightly irregular; 3) crystals in crystal vacuoles, varying greatly
in sise and nisnber, and of two fundamental types, bipyramidal and
plate-like; U) spherical refractive bodies, which also vary in num­
ber and sise; 5) food vacuoles; 6) a single nucleus, usually discoidal; 7) usually a single contractile vacuole*
Chaos chaos (Linneaeus, 1758)»
This so-called giant ameba has,
along with Amoeba proteus* been maintained continuously in the Protosoology Laboratory of The Ohio State University for many years*
The
culture method employed is similar to that for Amoeba proteus* Ex­
cept for its tremendous sise (1-3 ran*) and multinucleate condition,
Chaos chaos is rather similar to Amoeba proteus.
The giant ameba has been designated by several scientific names,
the most common being Chaos chaos Linnaeus and Peloayxa carolinensls
Wilson*
Chaos chaos contains alpha granules, beta granules, crystals
and crystal vacuoles, spherical refractive bodies, and food vacuoles,
■n of these being similar to those of Amoeba proteus * However, it
contains a few to several hundred nuclei which are somewhat smaller
than that of Amoeba proteus* and numerous contractile vacuoles are
present.
Theeamoeba striata (Penard* 1890) Schaeffer. 1926* Theeamoeba
19
striata is characterised by the presence of a pellicle which has
prominent longitudinal ridges or folds on the upper surface dur­
ing locomotion.
In one respect It differs rather strikingly from
an other amebas, both pelllculate and non-pelliculate.
It con­
tains a contractile Tacuole of Irregular and changing shape.
Most
Individuals have a second contractile vacuole which Is always
spherical.
Theeamoeba striata measures up to 120 microns during loco­
motion.
It is limax shaped, seldom forming any pseudopodlaj rather
the whole organism advances with an anterior hyaline cap, making
up as much as one-third of the entire animal.
The anterior hyaline
cap is always indicative of the direction of locomotion,
k smaller
form of Thee amoeba striata was described by Penard (1902) and Pappas
and Kostlr (1952).
Most of the observations recorded in the present
paper were made on the larger of the two varieties.
Clone cultures of Theeamoeba striata have been maintained In
Reynolds* (192U) strained hay infusion.
Ten grams of timothy hay
(Fhleum pratense) are boiled in 250 ml. of distilled water for 15
minutes.
The broth is then strained through cheese cloth and di­
luted with 2500 ml. of distilled water.
Cultures of Chiamydomonas
apiculata in its passive st^ge are used as the food organism for
the ameba.
The cultures are prepared in Syracuse watch glasses.
In the cytoplasm there are alpha granules, beta granules,
spherical refractive bodies, food vacuoles, usually two contractile
vacuoles, and a single nucleus.
20
Mayorella btgamma. (Schaeffer, 1916). Schaeffer. 1926*
Kayo rail a blgeeaaa Is characterised by the presence of small con­
ical pseudopodia which form continuously along the anterior edge
and on the upper surface while the animal Is In locomotion.
These
pseudopodia do not determine the direction of locomotion, but are
carried along by the ameba as a whole.
They are not static, but
rather in continuous extension and retraction during locomotion.
Both the anterior margin of the ameba and the conical pseudopodia
are composed of clear hyaline cytoplasm.
Individuals of Mayorella
blgemma vary from 60-200 microns In length during locomotion.
Clone cultur s of Mayorella blgemua were established under
the same conditions as those for Thee amoeba striata, with Chlamydorivmas apiculata as the food organism in strained hay infusion.
Also Ochromonaa sp. and bacteria have been used successfully as food
organisms in strained hay infusion.
short stender dishes.
The cultures were maintained In
Detailed accounts of culture methods and be­
havior of Mayorella bigemna have been given by Lotse (193U and 1937).
The cell Inclusions present in the cytoplasm of Mayorella
blgemma are alpha and beta granules, dumb-bell shaped crystals
(visible under the polarising microscope), four to nine contractile
vacuoles, numerous permanent vacuoles, food vacuoles, and a single
nucleus.
Amoeba guttula Dujardin, 18U1.
Amoeba gut tula was found
thriving in the top scum formation of hay Infusion cultures a week
or two after inoculation.
The pH of the ten scum was 5.0-5.3.
21
Vlhen
the pH became alkaline, the scum tended to sink to the bottom of
the culture.
At this time Amoeba guttula disappeared completely.
Bacteria are the chief food source of this ox'ganian.
Amoeba guttula is the smallest of the five amebas studied,
measuring only
20-25
microns during locomotion.
This
ameba
has
lobose pseudopodia which are extremely short and composed entirely
of hyaline cytoplasm.
Because they are both lobose and short,
they often appear almost circular,
Each pseudopod appears and
develops with characteristic abruptness.
At the anterior end of
the ameba about one-third to one-half of the cytoplasm is hyaline.
In the cytoplasm, the inclusions found are alpha and beta
granules, a single contractile vacuole, one nucleus, and one or
more food vacuoles.
22
Preparation of Araebas
General
The most useful method of investigating the cytoplasm of
amebas is undoubtedly by stucty- of the living specimens*
Vital
stains and phase contrast microscopy have both proven extremely
helpful*
However, one can not avoid the use of killed and fixed ma­
terial, since most stains and chemicals which nre of value for
the study of cellular constituents kill the organ!ans*
Some re­
agents, such as Lugol's solution and methyl green, serve as both
killing and fixing agents, and are usually employed with simple
mounts*
However, for most cytochomlcal methods, more complicated
procedures are necessary, including the use of various special re­
agents*
These will be described on a later page*
Many methods have been described for the preliminary handling
in preparation for fixing, staining, etc* of protozoa* (See McClung's
handbook of Microscopical Technique, 3rd ed. p. UJUl et seq,) All
these methods are modifications of four basic techniques or ap­
proaches: (a) handling and staining individual organisms; (b) dried
smears (as in blood preparations); (c) wet smears, as in (b) but
wet film is not allowed todry completely; (d) handling in bulk
(orgazfems are carried in *.ost tubes and re igents are added and re­
moved usually with the aid of centrifugation)*
These four techniques
as used in the present investigation are describod below*
23
Individual organ!ana.
Individual transference of amebas from
and to depression slides with a micropipette and with the aid of
the microscope was attestted.
This procedure, however, was ex­
cessively time consisting and had to be abandoned.
Dried smears. A thin film was prepared by the evaporation of
a drop of culture medium and permitted to clry completely.
Such a
preparation was not completely successful since the amebas would
shrink and become deformed.
If however, the smear was dried quickly
with the aid of dry compressed air (passed through anhydrous calcium
chloride), less shrinkage and distortion occurred.
The number of
amebas lost from such a preparation after fixing, staining, clear­
ing, etc. was never more than one-third.
This method was repeated with the addition of Mayer's albumen
fixative and Haupt's adhesive on the slide before the introduction
of the droplet containing the amebas.
The slide was placed in a
moist chamber for a few hours and in some cases overnight in order
that the amebas might become Imbedded in the albumen film.
But any
advantages in this procedure were outweighed by the fact that the
fixative took up the stain and obstructed the view of the amebas.
Another approach to the preparation of dried anears was at­
tempted.
The anebas were first fixed in omnium tetroxide or formal­
dehyde vapors or in cold Schaudinn solution; then they were washed
and transferred to another slide to eliminate the fixing reagent
and then dried with compressed air.
However, shrinkage and distortion
were not greatly lessen1
^and less than 20% of the amebas adhered to
2U
the slide after subsequent treatment.
Thus
theuse of dried smears
proved to be of little or no value.
Wet swears, Vfet swears were prepared in a variety of ways.
Cover slips with films of Mayer’s albumen or Haupt's fixation were
employed, the films varying frost extremely thin to rather thick.
Other cover-slipa were placed at the bottoms of cultures of amebas
and left there over night or up to three or four days in order that
the amebas might travel onto the surface of the cover-slips and ad­
here to them.
This method proved to be unsuccessful because re­
latively few numbers of amobas migrated onto the cover slips, and
those that did usually did not adhere after subsoquent treatment.
In all c^ses fewer than 10£ of the amebas were found on the finished
slide.
Another method attempted was placing a drop with many amebas
on a slide with either Mayer's or Haupt's fixative.
nesses of film were tried.
Varying thick­
For each fixative, two identical series
of such slides were prepared.
In all cases
thedrop wasallowed to
stand in a moist chamber for two to three hours.
Then in one seria^
the drop was allowed to evaporate until only the thinnest possible
moist film remained.
was poured off.
In the other series the top fluid of the drop
Both series were then fixed, and stained identically.
In both series, only 10-l££ of the amebas adhered to the slide, with
perhaps a slightly larger percentage whore evaporation had been
used.
This technique was repeated with cover-slips instead of
slides, and the results were identical.
2$
Another modification of the wet smear technique was at­
tempted , the slides or cover-slips being kept horizontal at all
times.
The smear was stained, dehydrated, etc,, with the addi­
tion of reagents by means of a pipette.
Reagents were removed
by simple tipping the slide or cover-slip and allowing the fluids
to drain off onto blotting paper.
This technique is similar to
that used in staining bacterial or blood smears.
The final yield
of amebas adhering to the slide or cover-slip was about 1$%.
By-
using a more closely graduated series of alcoholic concentrations
for dehydration, instead of the usual one, the percentage yield of
amebas could be slightly increased.
This indicated that one f actor
tending to reduce the number of amebas left adhering to the slide
or cover-slip is movement caused by diffusion currents.
The use
of dioxan was tried as a substitute for alcohol but without better
results.
Few organisms remain on the slide or cover-clip when xylene
is used as the clearing agent, while cedarwood oil gave much
better results whenever the smear technique was used.
If aqueous stains were used, no attempt was made to dehydrate
the amibas.
Glycering was used as a clearing agent and the smears
were then mounted in glycerine jelly or glycerine alone.
By this
method as many as $0-75% of the amebas remained fixed on the slide
or cover-slip.
Since
cannon dehydrating agents employed in microtechnique
procedures are fat solvents, aqueous mounts were necessary when de-
26
monstrations of lipid material were to be made.
Because of the
greater degree of success, aqueous mounts were used whenever
possible.
Handling In bulk.
for bulk methods.
Very large numbers of amebas are required
One such method is centrifugation followed by
decanting of the supernatant fluid and addition of the next re­
agent.
Another method sometimes employed for larger protozoa is
the use of a finely meshed screen or sieve Which strains out and
thus concentrates for orgaifems.
Most of the amebas do not m o w In
dense enough populations to warrant use of bulk handling, since an
enormous number of cultures would te required in order to produce
the necessary mass of organisms.
make this unnecessary.
27
Other methods are available which
The Parlodlon Trap Technique
The parlodlon trap method used in this study was developed
by Concannon (l?5l) for handling small nematodes.
With slight
modifications, this method was found to be very satisfactory for
handling amebas in preparation for subsequent staining, mounting,
etc.
The Parlodlon solution used consists of 3J grams of par­
lodlon (highly purified cellulose nitrate) dissolved In 1000 ml.
of absolute alcohol and 100 ml. of ether.
The solution should be
kept under refrigeration.
The trap itself is a loop made of lacquer coated wire (about
^30 B St 3 gauge) by taking a piece about three inches long, wrap­
ping it once around some cylindrical object, such as glass tubing,
and then twisting the free ends together.
The circlet is designated
as the trap, and the twisted part the handle.
The trap and handle
are flattened so that both are in the same plane.
The procedure is as follows.
(See Figure 1.)
A drop of medium containing
amebas Is put on a perfectly clean slide and allowed to stand until
only a thin wet film remains.
A drop or two of ab® lute alcohol
is then added to the middle of the film, which forces the water to
the film's periphery.
The alcohol fixes the organisms and most of
them remain in their original location.
removed with filter paper.
The remaining water is then
If some other fixing reagent is used be­
fore the treatment with absolute alcohol, more of the amebas are lsst
for they do not adhere as well to the glass and are thus forced to
28
the periphery to the film with the water.
When almost all of the absolute alcohol has evaporated from
the film, the trap Is placed on the slide so that most or all of
the film containing the amebas will be Inside the loop.
One or
two drops of parlodlon solution are then dropped on the slide with­
in the loop.
Care should be taken to see that too much of the
solution is not added to the loop, since the parlodlon would then
be difficult to de-stain.
When the parlodlon is almost dry, the
slide should be tipped so that excess solution will run down the
handle from the loop.
In this way, a vory thin membrane in which
the amebas are embedded remains in the loop.
The slide, with the trap in place, Is then either flooded or
gentle limnersed in water while kept horizontal.
This is a very im­
portant step, as water must penetrate between the parlodlon and the
glass slide to prevent adhesion between them.
After a few minutes,
the trap may be removed from the slide by grasping the wire handle
with forceps and lifting gently upwards.
If the effort- has been
successful, a very thin membrane of parlodlon is left within the
wire loop, and in this membrane the amebas are embedded.
In a cer­
tain percentage of trials, the parlodium film ruptures, but approxi­
mately 70-80^ of the attempts are successful.
The diameter of the
wire loop should not exceed 12-13 mm., since the membrane ruptures
easily on larger loops.
For the following steps it Is desirable that shallow reagent
and staining dishes be used in order that the wire handle of the
29
trap should not be completely iron rsed in the solutions.
(See
Figure 2.)
If several traps are to be treated identically, the end of the
handle of each may bo bent into a hook.
Then the traps can be
hung from glass rods or thick wire, etc., and in this manner trans­
ferred from solution to solution.
The parlodlon membrane will take up stain along with the amebas
embedded in it, but will lose the 3tain much more readily than the
amebas during washing or passage through alcohol.
Parlodlon is soluble In absolute alcohol, so that it is nec­
essary that the final dehydration in the preparation of permanent
slides be accomplished by the addition of 25 ml. melted carbolic
acid crystals to 75 ml. of xylene.
When the trap is first placed
in this solution from 9$% alcohol, the parlodlon membrane will
appear milky, but it usually becomes clear with a few minutes.
In
any event, the trap should be left in the carbol-xylol long enough
for the membrane to qppear conpletely transparent, for it is not
until then that the water has been completely removed.
30
The trap
is -then transferred to xylene*
After a few minutes the trap Is taken out of a^lene, drained,
to remote the excess and placed onto a drop of mounting medium on
a clean slide.
(Any xylene-soluble mounting medium is satisfactory.)
A drop of the mounting medium is then placed on top of the parlodlon
membrane.
The latter is then freed from the sides of the wire loop
with a sharp dissecting needle or scalpel, and a very thin coverslip (#0 preferred) is placed on the mounting medium*
The parlodion trap technique is not suitable for some cytq|Jchemical tests, because both free lipids and free carbohydrates arm
removed by the organic solvents.
However, for protein reactions
such as Feulgen's and Hillon's and for water insoluble ploysaccharides
visualized by the PAS reaction, material prepared by the parlodlon
trap method is quite adequate*
31
Cytochemlcal Methods
Cytocheatlstry is concerned vith the localisation of chemical
entities in living
and plant cells.
Lison (1936) first
systematlsed the criteria by which one must evaluate cytochemlcal
techniques.
Briefly they may be stated as follows:
a) One must pay attention to the morphology of the cell.
ation must preserve the general morphology.
Fix-
It must not destroy
the structure to be studied, nor must it interfere with the react­
ions that are to be employed.
The fixing reagent must not change
the location of the structures to be studied.
b) The reaction used must be specific for the compounds or
material to be studied.
The chemical reaction must be understood.
Usually the specificity is not for a particular compound, but rather
for a group or radical.
c) Rie product of the reaction must be visible under the
microscope.
d) A specific cytochemical reaction must be differentiated
from other reactions.
It may be permissible to use an empirical
method, if we know that it has a very high degree of specificity
for a single chemical structure.
e) We can use a balance between a rigorous technique and a
less rigorous technique, as long as our faculties for criticism
and self-criticism are still in use.
Since almost
of the cytochemical methods now employed
were originally designed for use with metaaoan tissues, it was
32
often necessary to device modifications In applying these testa to
protosoa.
Such modifications are Incorporated into the descriptions
of these methods in the following pages.
Protein Reactions
Millon1a reaction for tyrosine.
The cytochemical adaptation
of Millon*a reaction was accomplished byBensley and Gerfh (1933).
The procedure, as outlined in Lison (1936), is modified for this
study as followst
1) Amebas mounted in a parlodlon trap are placed in Millon*s
reagent (at room temperature) for about 5 hours or until maximum
color develops.
2) "ihe trap is then immediately placed in l£ nitric acid sol­
ution, and left there for 2-3 minutes.
3) The trap is placed on a slide, and 2-U drops of absolute
alcohol are dropped on the parlodlon film, which dissolves almost
immediately.
The trap is removed and the color-slip is placed on
the alcohol, which now contains all the amebas that were in the film.
More absolute alcohol is added at the edge of the cover-slip as
needed.
Only temporary mounts are usually attempted with this procedure
since the color of the preparation fades within a few days.
Results. A rose or brick color indicates the presence of
tyrosine-containing protein in the cell.
The coloration is due to
the particular aromatic radical found in tyrosine.
33
However, Lison
(1936) points out that a ear tain number of non-protein phenol
compounds may also give this reaction.
Their distribution in
cells, however, is quite limited.
Arginine,
Baker's (19U7) modification of the Sakaguchl
test for arginine was employed.
Reagents and procedures were
modified in the present study as followsi
Reagents:
1) Absoluts alcohol as a fixing agent.
2) Parlodlon, 3*5£* In a mixture of absolute alcohcl and
ether in equal volumes,
3) NaOH, l%t aqueous.
U)
-naphthol, l£, In 70% alcohol,
5) Hypochlorite solution (2 volumes sodium hypociilorlte and
1 volume of 0.05 N NcOH).
6) A mixture of 3 volumes of pyridine with 1 volume of
chloroform.
Procedure:
1) Amebas are mounted in a parlodlon trap.
trap method on page 28.)
(See parlodlon
The trap Is then placed in distilled
water,
2) Then 2 ml, of 1% NaOH are put into an old-fashioned watch
glass and 2 drops of °t -nanhthol solution and U drops of hypochlorite
solution are added.
3) The parlodlon a* ap is now placed in a depression slide,
3U
The mixture just described is then gently shaken and quickly
added to the parlodlon embedded amebas on the trap and allowed
to remain for about 15 minutes.
14) The trap is then transferred to a flat slide and pyridine-chloroform mixture is addod.
This dissolves the film so
that the trap may be removed at once, and a cover-slide is placed
over the mixture with its contained an:bas,
More of this mixture
is added at the edge of the cover-slip as noeded to make up for
evaporation.
Resultst A pink or red color indicates the pr sence of
arginine (free or combined) or some other positively-reacting
guanidine deriviative in cells.
The test is bastd on the develop­
ment of a red color by arginine when <A-naphthol and hypochlorite
react with it in an alkaline medium.
Feuljreji nucleal reaction.
This reaction makes possible the
visualisation of desoxyribonucleic acid (DMA).
Indeed, the Feulgen
reaction is considered to be one of the most specific cytochemical
reactions.
(See Stowell, 19U6.)
The reagents and procedures were
modified from those outlined by Glick (19U9)*
Reagents.
1 ) Fixing reagent-absolute alcohol or formaldehyde-calclum
(10 ml. of full strength formalin, 10 ml. of 10)C calcium chloride
(anhydrous) and (50 ml. of distilled water).
2) HC1# 1 N,
35
3)
Coleman (1936) preparation of Schifffa reagent.
Dissolve
1 gm. of basic fuchsln in 200 ml. of boiling watorj filter, cool,
and add 2 gm. potassium metabisulfite (K232^5) and 10 ml. of 1 N
hydrochloric acid.
Let bleach for 2h hours, and then add 0.5 gm.
of activated carbon (Norit), shake for about one minute, and
filter through coarse paper.
The filtrate should be colorless.
[i) Sulphurous acid solution— add 30 ml. of 1 K sodium bisul­
fite solution and 30 ml. dilute HC1 to 600 ml. of tap wat r.
Procedure:
1) Amebas fixed in either absolute alcohol or formaldehydecalcium are mounted in parlodlon.
(See parlodlon trap technique.)
2) Parlodion traps containing the amebas are then placed for
a minute or two in HC1 solution at room temperature.
3) They are then transferred to HC1 solution at 60 degrees C.
for L-5 minutes.
U) Then they are treated with Schiff's reagent for 1^-3 hours.
5) The trap is then passed through three separate baths of
the sulphurous acid solution and left in each for 1-2 minutes,
agitating frequently.
6 ) Then the trap is washed for about 5 minutes in tap water.
7) The trap is then dehydrated and cleared as has been described
for the parlodlon trap technique, and mounted in balsam.
Results; A purple color indicates the presence of deso^ribonucleic acid.
Acid hydrolysis removes the purine bases and ex­
poses free adhehyde radicals which stain purple when treated with
36
fuchsin-sulfurous acid or Schlff's reagent.
The sulphurous acid
solution removes the excess Schiff's reagent from the cell*
Detection of ribonucleic acid*
Visualisation by ribonucleic
acid (RHA.) in the cell is accomplished cytochemlc ally by the use
of substractlve methods, these being either depolymerization of RNA,
with rlbonuclease or extraction with perchloric acid as compared
with non-treated controls*
Since nucleoproteins are basophilic,
basic dyes such as toluidine blue 0 will reveal their presence in
the cell.
The rlbonuclease technique used in this study was modified
from that recommended by Stotft=ll and Zorzoll (19h7).
Reagents.
1 ) Fixing reagent--formaldehyde.
2) Rlbonuclease (Worthington Biochemical Company, Freehold,
New Jersey) 0.1 mg*/ml. of buffer (Mcllvaine’s citric acid-disodiua
phosphate mixture buffered at pH 6*5)*
3) Toluidine blue 0— 0*5£ in distilled water.
Procedure:
1) Amebas, fixed in formalhehyde, are embedded in parlodlon*
(See parlodlon trap technique.)
2) One group of amebas is then put -nto the rlbonuclease sol­
ution at 50 degrees 0. while the other is put into plain MeIIvain
buffer at 50 degrees 0 .
Both are kept under these conditions for
37
at least 3 hours.
3)
Both groups are quickly washed in separate dishes with dis­
tilled water,
U) Then In separate staining dishes, both groups are treated
for a few minutes with toluidine blue 0.
After another quick rinse
in distilled water, the amebas are mounted in distilled wat^r.
Results: The ribonuclease-treated group is compared with the
non-treatod group, on the basis of the presence and distribution of
basophilic substances.
The absence of basophilia in the treated
anebas as compared to the non-treated controls, indicates RNa .
Perchloric acid technique.
The methods used here was modified
from those of Seshach^r and Flick (I9h9) and Erickson, Sax, and
Ggur, 19U9*.
Reagentst
1) Fixing reagent— acetic acid alcohol mixture (1 volume
acetic acid to 3 volumes of 95% alcohol).
2) Perchloric acid (HCIO^)— 5$ in distilled water.
3) Toluidine blue 0— 0,5% in distilled water.
Procedure;
1) Amebas fixed in acetic acid alcohol mixture are embedded
in parlodlon.
(See parlodlon trap technique.)
2) One group of amebas is then put into the perchloric acid
solution at 70 degrees ^ • while the oth^r is put into plain dis­
tilled water also at 70 degrees C.
38
Both g roups are kept under those
conditions for about 30 minutes*
3)
Both groups are quickly washed in separate dishes with
distilled water*
1*) Then, in separate staining dishes, both groups are treated
for a few minutes with toluidine blue 0*
ifter another quick rinae
in distilled water, the amebas are mounted in distilled water*
Resultsi The perchloric acid treated group is compared with
the non-treated group as to the presence and distribution of baso­
philic substances*
The absence of basophilia in the treated amebas
as compared to the non-treated controls indicates the presence of
RNA*
Carbohydrate Reactions
Lugol's solution (1* gm. iodine, 6 gm* potassium iodide per
100 ml. of solution) was used, as well as iodine (0.3^) solution,
as fixing reagents and as attains for starch*
Periodic acid-Schlif (PAS) reaction for polysaccharides*
McManus (191*6) and Hotchkiss (191*8) have formulated a cytochemical
reaction for the visualisation of polysaccharides such as glycogen,
mucin, mucopro teins and pr sumably hyaluronic acid and chi tin.
The
method of Hotchkiss was modified for this study*
Reagents i
1) Fixing reagent-absolute alcohol*
2) Periodic acid solution (f^lO^) - 0*93* in distilled water*
Take 1*5 ml. of this concentration and add 5 ml* W/$ sodium acetate*
39
3)
Iodide-thiosulfate solution.
Dissolve 1 gm. potassium
iodide and 1 gm. sodium thiosulfate (NagSgO^-^HjO) in 20 ml. of
distilled water and add with stirring 30 nil. of alcohol followed
by 0.5 ml. of 2 N HC1.
A sulfur precipitate forms and settles
out slowly although the solution may be used immediately,
U) Schif^s reagent,
(For preparation, see Feulgen nucleal
reaction.)
5) Sulfite wash solution.
Add 0.5 ml. concentrated HC1 and
2 ml. of 10,£ potassium mctabisulfite to 50 ml. of distilled water.
Proceduret
1 ) Amebas, fixed in absolute alcohol, are embedded in parlodlon.
(See parlodlon trap technique.)
2) The amebas, embedded in.parlodlon, are placed in the per­
iodic acid solution for about 10 minutes.
3) Washed with watr, they are then transferred to the iodidethiosulfate solution for about 10 minutes.
k) Again they are washed with water and placed in Schiff's
reagent for about 30-60 minutes.
5) They are then rinsed in the sulfite warh solution for a
few minutes.
6) The amebas are then dehydrated and mounted in balsam In
the usual manner for the parlodlon trap technique.
Results; A violet color indicates the presence of poly­
saccharides.
Polysaccharides are oxidized under mild conditions
by periodic acid to polyaldehydes.
Uo
Then the iodide thiosulfate
solution removes the periodic acid from the cells.
aldehydes are then colored by Schiff's reagent.
The poly­
Excess Schiff's
reagent is then removed by the sulfite wash solution.
The pen­
toses of nucleic acid are so affected by periodic acid that they
will not react.
Lipid Fteac ♦ions
Lipids may be characterized by their sudanophilic properties,
i.e. they become colored by Sudan reagents.
are not dyes because they are not ionizod.
dissolving in them.
The Sudan substances
They color lipids by
(See Lison, 1936 and Gain, 1950.)
Sudan III, IV and black B were prepared as saturated solutions
in 70% alcohol and filtered twice.
Sudan black B, the most powerful
of the Sudan reagents, was also prepared in ethylene glycol as sug­
gested by Chiffelle and Putt (1951): "0.7 gm. of Sudan black B is
dissolved in 100 ml. of pure ethylene glycol by heating to 100-110
degrees C. and thoroughly stirred for a few minutes.
Care should
be taken not to exceed 110 degrees C., since a useful gelatinous
suspension will result.
Filtering the hot solution through What­
man #2 paper removes most of the excess dye and undissolved im­
purities.
After cooling to room temperature, it is filtered again
through a fritted-glass filter of m.-dium porosity with the aid of
suction.
As an alternative the solution may be filtered through
paper for a second time, but the procedure is long, due to the
vicosity of the glycol.
The glycol solvent should be obtained
Ul
in as nearly pure form as possible, since an appreciable water con­
tent (30-U0JS) will prevent much of the dye going into solution*"
According to Cain (195>0), coloration with Sudan black B depends
ultimately not on unsaturation but on the physical state of the
object tested*
A 1% solution of nlle blue sulfate was used on amebas which
usually were fixed previously in formalin*
Nile blue sulfate has
been used to distinguish neutral f ats (triglycerides) from fatty
acids, the former being dyed red or pink, the latter blue*
Lison
(1936), after a detailed study, concluded that the red c oloration
was characteristic of lipids in general and the blue was nearly that
of a basic dye and therefore totally unspecific.
Cain (I?li7)
maintains that if a substance is known to be a lipid colors
red with nlle blue, it consists of neutral lipidsj if it colors
blue, it may contain these, but acidic lipids (fatty acids, phos­
pholipids and others) may also be present.
The specificity of the extended procedure required by Baker's
(19b6, 19li7) acid hematin test for the recognition of phospholipids
is based on empirical erltera.
In this study it was not possible
to carry out each step of the test exactly as outlined by Baker*
Unless new empirical data were to be collected, results which would
have been obtained by modifying the original test would have bed
no cytochemical meaning*
The dchults method for the demonstration of c holesterol was
used as outlined by Lison (1936).
U2
After the amebas have been placed
in a 2.55f iron alum solution at 37 degrees C. for about 8 hours,
they are transferred to a slide*
Then they are treated with a
few drops of acetic sulphuric mixture (1 part glacial acetic acid
in 1 part concentrated sulfuric acid).
If a blue-green color
develops, then cholesterol is present.
If the color does not
develop then no conclusion can be drawn, since negative results
in the Schultse test do not mean that cholesterol is absent.
(Lison, 1936).
The mordant transforms cholesterol to oxychole-
sterol which in turn becomes a colored substance when acted upon
by the acetic-sulfuric mixture.
Plasmal reaction.
Feulgen and Zoit (I92li) have described
the demonstration of acetal lipids in the cell.
From these acetal
lipids ("plaamalogen"), the "plasmal" (free aldehyde) is unmasked.
The procedure employed in this study for the visualization of the
"plasmal* is that suggested by Hayes (191*9).
Reagents t
1) HgCl2,
aqueous solution.
2) Schlff's reagent.
(For preparation, see Feulgen nucleal
reaction.)
3) Sulfurous acid wash solution.
(For preparation, see Feulgen
nucleal reaction.)
Procedure:
1) One group of amebas is placed in 1£ IigCl2 In a depression
slide for about 3-5 minutes.
2) This group and a control group are transferred into Schiff's
1*3
reagent in two separate depression slides end left for about 10
minutes.
The depression slides must be cohered during this
period.
3)
Both groups are then transferred into depression slides
containing the sulfurous acid wash solution, and left for 2 min­
utes.
This step is repeated three times.
) The amebas are then transferred into d epression slides con­
taining distilled water,
5) Temporary mounts are then prepared in distilled water.
Results: The presence of violet fuchsin color indicates acetal
lipids.
According to Feulgen and Voit (1921:), the brief action of
HgCl2 on the acetal bond of the bound plaamalogen splits it into
a fatty aldehyde (pi aanal) and glycero-phosphoro-ethanol amine .
The pi aanal, being an aldehyde, is then readily colored by Schiff*s
reagent,
Hayes (19U?) maintains thnt the plaemal reaction is higlily
specific for acetal lipids alone, if the time for the
ction of the
HgCl2 is limited to Insure that neither acid hydrolysis nor oxida­
tion takes place, and if controls are carefully checked.
As far as
is known the only acetal lipids which have been demonstrated as
present in cells are phospholipids.
However, other non-phospholipid
acetals might exist.
Other Methods. Fixing Reagents. Stains, and Chemicals
Sulfhydryl test. The nltroprusside test for sulfhydryl groups
in enebas as outlined by Chalkley (1937) was employed in the present
Uh
study.
One drop of 20% sine acetate was placed on a clean depres­
sion slide.
By means of a micropipette, amebas were transferred
into the sine acetate solution.
Care was taken so that only a
minimum of culture medium was transferred with the amebas.
One
drop of a l£ sodium nitroprusside solution was then added,
k rose
red oolor results where sulfhydryl is present.
Since the color
fades in a few hours, observations on the amebas in the depression
slide must be made immediately.
The nitroprusside test has been
severely criticised by Lison (1936) and Hammett and Chapmen (1939)
as being unrell ble as a quantitative reaction and also because
the resulting coloration may diffuse throughout the cell.
Bennett
(19U8) has developed a more accurate method for the cytochemical
determination of sulfhydryl, employing a compound which was synth­
esized specifically for this purpose.
The compound was not avail­
able however, for this study.
Mlsc*T1aneous. k 2% solution of osmium tetroxide (0.5 gm.
osmic acid crystals in 25 ml. distilled water) was employed as a
fixing reagent.
Exposure to the OsOjj vapors of this solution for
30 seconds to 2 minutes was quite satisfactory.
Commercial formalin (hO% formaldehyde) vapors were employed
as well as 10* solutions of formalin.
Formalin-calclum solution
(10 ml. full strength formalin, 10 ml. calcium chloride 10^, an­
hydrous, and 80 ml. distilled water) was also used.
Champy's
fluid (70 ml. of y% potassium bichromate, 70 ml. of 1# chromic
US
acid, liO ml. of 2% osmium tetroxide) was used as a fixing agant
for mitochondria,
Tranee u's fixatire (H2O-6 parts, 9b% alcohol-
3 parts, formalin-1 part) was used as a general fixing reagent.
Schaudlnn's fluid (saturated aqueous solution HgCl2-2 par is, 9S%
alcohol-1 part) was also used as a routine fixing agent,
A 0.02^ solution of ruthenium red (ruthenium oxychloride,
ammonlated, 1*5,OCX)) was employed.
Neutral red was used at the
following concentrations!
1:75,000 (0.0013350, 1 :100,000 (0.001/0,
and 1:150,000 (0.00077O•
Janus green B was used in a concentration
of 1:100,000 (0.0015C).
in
acetic acid.
A 0.2^ solution of methyl green was prepard
Aniline fuchsin for staining mitochondria was
prepared from 10 gm. acid fuchsin in 100 ml. of aniline water (8
ml. amiline oil in 180 ml. of distilled water).
Of the hematoxylin
stains, Delafield's and Heidenhain's iron hematoxylin were used.
A 2% iron alum solution was used as a mordant when staining with
Heidenhain's iron hematoxylin.
Toluidine blue 0 was prepared as
a 0.5-• solution in distilled water as recommended by Lison (193^)•
A stock solution of 35- H2O2 was also used.
Doyle's (1933) preparation for the simultaneous demonstration
of fat and starch was used.
The preparation is as follows:
"Two
stock solutions are prepared, one containing a 0 .3> solution of
iodine in 6d£ alcohol,9 the other a s^aturated
solution of Sudan III
w*
in a mixture containing 605b of alcohol and 1C& of acetone,
ivjual
parts of each solution are mixed in a anall bottle to contain 2h
hours supply.
The stock solutions keep indefinitely, whereas the
U6
mixture may be used as a killing fluid or the organisms may pre­
viously be killed with neutral saline formol^.
Microscopes and Accessories
A fpencer (American Optical Company) research compound bino­
cular microscope with apochromatdc objectives (16, li, and 2 mm,)
and compensating oculars (10, 15, and 20X) was used throughout this
study,
A phase-dii'ference microscope (American Optical Company)
equipped with a 971 B minus contrast - low objective as well as
10, U3, and 97X dark contrast - medium objectives was also used,
A steroscopic binocular (American Optical Company) microscope was
used as an aid in manipulating and iransferring amebas,
A polarizing
microscope (American Optical ComDany) was used to determine ontical
activity of cell particulates.
Measurements were made with the aid
f ocular micrometers.
Each microscope was calibratod for use with a specific ocular micro­
meter.
All drawings were made with the aid of a camera lucida.
hi
RESULTS
The Cell Membrane
The cell membrane or the outermost limiting layer of the
amebaa is a eeewingly unspecialised plasma membrane in Amoeba
guttala and Maarorella bigemma.
In the genua Thee amoeba, there
ia formed an outer relatively thick covering called a pellicle*
Howland (l?2ljc) removed the pellicle from Thecamoeba verrucosa
leaving the plaaaa membrane intact*
Intermediate to a thickened
pellicle and an unspeclallsed plasna membrane la the outer cover­
ing * the plasm alaama* found in Amoeba proteus and Chaos chaos.
Thecamoeba atriata ia characterized by the presence of a
number of delicate longitudinal lines (usually U - 6) on the surface.
These longitudinal lines are present during locomotion or whenever
the ameba is elongate*
When it is rounded up, there are many folds
and indentations on the surface*
lines is about 0.75 micron*
The width of the longitudinal
The thickness of the pellicle is a-
round 0.33 micron if we assume that the longitudinal folds are com­
posed of two layers of pellicle with little* if any* material be­
tween them*
Measurements of the pellicle made at the edi e of the
ameba* whether living or fixed* were approximately the same*
Unless an extreme amount of pressure is applied to the coverslip* Thecamoeba striata will not rupture even in the event of
evaporation of the surrounding medium.
U8
The pellicle does not
stain with neutral red, not does It hamper the entrance of the
stain into the cytoplaara to any great degree.
The time required
for given concentrations (1:75,000, 1:100,000 and 1:150,000) of
neutral red to appear within the cytoplaan of this ameba is about
the same ae in the non-pelliculate amebas studied.
When Amoeba proteue and Chaos chaos are active and have ex­
tended pseudopodia, the pl&amalemma is generally smooth and with­
out folds.
Wrinkling of the plaamalemma occurs only when the cells
are rounded up.
The thickness of the plasmalenvna was found to be
about 0.25 micron.
It should be noted that the plaanalemma rup­
tures much more easily than the pellicle of Thee amoeba striata.
In the other amebas studied, Amoeba guttula and Mayore i1a
bigemma, the thickness of the cell membrane was not ascertained
since it is extremely thin and much less than 0.25 micron.
Cytochamlstry of the cell membrane.
The cytochemical re­
actions hereinafter described were very easily visualized in the
cell membranes of all the amebas studied, both polliculate and
non-pellicula te.
In the extremely thin plasna membranes of Mayor ell a
bigemma and Amoeba guttula. the resul Le were not as pronouncled as in
the other species, but were definite nevertheless.
The cell membrane stained red with 1:5000 ruthenium red
(ruthenium ojqrchloride, ammonia ted).
This metallic pigment has
been employed in microscopic work as a test for pectin in plant
cells (Bonner, 1936, 19^6, 1950).
Its specificity for pectin
alone, however, is greatly in doubt.
U9
Hence no definite conclusions
as to the chemical nature of the cell membrane can be drawn Tram
the use or ruthenium red.
The cell membrane appeared violet after being treated by the
FAS reaction.
As was stated earlier, this reaction Involves the
oxidation of adjacent hydrojqtI groups to aldehydes by means of
periodic acid, and the coloring of the aldehydes with Schiff's
reagent.
Thus the presence of the violet fuchsia color indicated
that the pellicle contains polysaccharides*
Since non-figureA (free) glycogen is water-soluble to sone
degree, and of course, the cell membrane is not, it cannot be
present as an Important constituent of the cell membrane.
How­
ever, glycogen could be present as a figured substance, i.e. com­
bined with or bound to seme other substance and thus not necessarily
water soluble.
By the application of saliva, the pellicle was found
to be ptyalln-resistant, thus showing that it does not contain fig­
ured glycogen.
The Millon reaction (Bensley and Oersh, 1933) for tyrosine
and the Saleaguehi test (Baker, 191*7) for arginine were employed*
Since the vast majority of proteins contain tyrosine and arginine,
these two cytochemical tests are actually good protein indie-tors.
The cell membrane stained red with both tests, indie atint: the pres­
ence of protein.
Thus the cell membrane of the amebas tested con­
tains protein and polysaccharides.
The saliva-resistant, PAS po­
sitive, protein-combined carbohydrates belong to the mnco-polysaccharlde group*
A mucopolysaccharide (mucin) is a compound
50
containing protein and polysaccharide In which the polysaccharide
is predominant.
Some basic dyes stain certain cell components the same color
as that or the dye itself, but stain certain other elements with
a different color.
This is called metachromatlc staining.
Xt
has been explained (Lison, 1936) that muco-polysaccharldes which
stain metachromatlc ally, with toluidine blue, for instance, (us­
ually pink or red) are acid polysaccharides and usually contain
sulfate,
hyaluronic acid is the common acid polysaccharide found
to be present intercellularly in metasoan tissues, although it
does not contain sulfate.
metachromatlc.
The cell membrane was found not to be
That is, it stained blue with toluidine blue, the
same color as the dye.
ks a further chock, several amebas were
treated with hyaluronidase before staining with toluidine Vine.
Those treated with hyaluronidase did not stnin any differently
than the non-treated controls.
Therefore it may be concluded
from these tests that the cell membrane is composed of a neutral
muco-polysaccharide or mucin.
$1
Cytoplasmic Inclusions
The Alpha Granules
The alpha granules are rather abundantly represented in
the amebas studied.
In the unstained living cells they are easily
seen with the phase—difference microscope.
The size of these grrn-
ulesf about 0.25 micron, appears to be the same in all the species
studied.
Due to the fact that the staining reactions were incon­
clusive, nothing could be ascertained concerning the nature of these
granules.
The Beta Granules
The beta granules in the five species of amebas studied are
very similar in else and shape.
They are predominantly spherical
and measure about 1 micron in diameter.
Although Mast and Doyle
(1935a) claim that the beta granules change their shape in Amoeba
proteus. no evidence for this was obtained in the present study.
However, in Thecamoeba striata (and in no other species studied)
coalescence of beta granules has been observed several times both
in stained and unstained amebas.
The coalesced granules appear
as homogeneous spheres, measuring up to 2*6 microns in diameter.
Six to ten such spheres have been found in some individuals.
Clumps
of beta granules have also been observed in this species, but these
granules retain their own shapes in the clump.
52
No direct obser-
ration* ware made a* to whether this clumping of granules proceeds
coalescence; but this seems to be probable*
Coalescence and clump­
ing of beta granules have not been reported previously in amebas.
Bourne (l?5l) reports that clumping and coalescence of mitochondria
in certain mammalian cells often occurs in cases of scurvy.
The
significance o* this phenomenon in Thecamoeba striata is not known.
As was pointed out in the iiistorical He view, the beta gran­
ules may be considered under two headings, those that surround the
contractile vacuole and those th^t do not.
The observations made
in this study corroborate the conclusions of Mast and Andresen that
this division is purely an artificial one, since the only difference
in the granules surrounding the contractile vacuole is thrt they are
more or less flattened as they lie on the surface of this vacuole.
The staining qualities, plasticity, and composition appear to be
the same in all the beta granules, regardless of their distribution
in the cell.
Janus green B has been found to be a specific stain for beta
granules.
(See Mast and Doyle, 1935a, 1935b, Andre sen, l?li2.)
Cytologists have long considered Janus green B a specific stain
for metasoan mitochondria.
(See KcClung, Handbook of Microscopical
Technique, 3 rd. ed. p. 11*0.)
When used in optimum concentration
(1:100,000) the beta granules, and no other structures, appear
distinctly green after about 3 or U hours.
Mast and Doyle (1935a)
declared that in Amoeba proteua only an outer layer of the beta
granules stained with Janus green B indicating a different! ted
53
surface layer.
In the present Investigation the sane results were
at first obtained with Aaoeba proteus and Thecamoeba striata. How­
ever , when the amebas ware left in the Janus green solution for more
than 2h hours, the granules appeared homogeneous.
Observations
made with the phase-difference microscope on unstained amebas also
showed no differentiation of parts.
Hast and Doyle (1935a) applied classical metasoan mitochondrial
staining methods to Aaoeba proteus and found that the beta gran­
ules behaved like the mitochondria of metasoa.
Andre sen (l?Lt2) used
aniline fuchain after Champy-Kull fixati:n and successfully stained
the beta granules of Chaos chaos.
These methods were repeated in
the present study and similar results were obtained.
Since Janu#
green B and the classical mitochondrial staining methods also stnin
these granules, it seems very probable that the beta granules are
actually the mitochondria of amebas.
Hast and Doyle (1935b) state that the beta granules are much
more numerous at the surface of the food vacuoles during the in­
itiation of digestion and toward the end of this process.
Today,
observations on this point are made much easier and more definite
by the use of a phase-difference microscope, which was not avail­
able to Mast mkl Doyle*
Such observations at different phasos of
digestion revealed no beta granules definitely associated with food
vacuoles at any time.
Cytochemistry of beta granules.
The beta granules gave a
positive reaction when treated with Millon's retgent for tyrosine
5U
and Sakaguchl test for arginine.
In order to test for the presence
of lipids in the beta granules, amebas were treated with Sudan III
and also Sudan IV in 70£ alcohol.
The results were negative.
When
Sudan black B was employed, however, they did show a faint bluishblack color.
The affinity of the Sudan black B for the beta gran­
ules was greatly enhanced when ethylene glycol (Chiffelle and Putt,
1951) was used as a solvent Instead of 70^ alcohol.
action for polysaccharides gave negative reaif ts.
The PAS re­
Ribonuclease
and perchloric acid techniques for the presence of ribonucleic acid
also gave negative results.
It seems, therefore, that if ribonu­
cleic acid is present in,these granules it ±3 in minute quantities.
Using the nitroprusside test, negative results were also obtained
for sulfhydryl groups.
The cytochenical tests described above sug­
gest that the beta granules of the amebas studied are lipid and
protein In composition as are raetazoan mitochondria.
The Spherical Refractive Bodies
Numerous spherical refractive bodies varying in size and num­
ber were found in three of the five species studied, (Amoeba proteua. Chaos chaos, and Thecamoeba striata).
to 7 microns in diameter.
They vary fram l.U
At times it was estimated that as many
as 200 were present in Chaos chaos, while Amoeba proteus was found
to have as many as UO and occasionally as Tew as U of these bodies.
The number in Thecamoeba striata varies from as many as 20 to as
55
few as 6,
In Amoeba proteus and Thecamoeba striata these spherical re­
fractive bodies appear homogeneous In unstained individuals, and
stain red, vitally* with neutral red.
However, when various con­
centrations of neutral red (1;75fOOO, 1*100,000 and 1*150,000) were
used, it was found that only the outer or cortical layer of the
bodies was stained.
In many of the smallest ol' the shperical re­
fractive bodies (those measuring 1,1* - 1,8 microns) no such differ­
entiation between inner and outer portions was sliown, and neutral
red stained them homogeneously in the living ameba.
In the larger
spherical refractive bodies, the differentiation of cortial and
inner regions was also observed in untreated living amebas with the
aid of the phase-difference microscope.
The spherical refractive bodies in Chaos chaos did not stain
vitally with neutral red.
of Andre sen (1?U2),
This is in accord with the observations
In one or two living individuals, however, a
few of the larger bodies did stain.
In every other respect, the
spherical refractive bodies were found to be alike qualitatively
in the three species of amebas in which they were present.
Osmium tetroxide vapors blacken the outer portion of the sp­
herical refractive bodies.
peroxide did not bleach it.
Subsequent treatment with
hydrogen
This outer portion stained a faint
grey-blue with Sudan black B, indicating the presence of lipid
material.
Both Hillon's reagent for tyrosine and the Sakaguchi
test (Baker's 191*7 modification) for arginine gave a pink color-
56
ation of the same Intensity as the cytoplaanlc ground substance
indicating the presence of protein in the cortex of the refractive
bodies*
Mast and Doyle (1935a) claim that between the inner and
outer portion of these refractive bodies a layer of carbohydrate
is present*
However, in the present study only negative results
were obtained when the PAS reaction was used to test for the pre­
sence of polysaccharides and Lugol's solution did not demonstrate
any starch in these bodies*
Thus it was found that the outer cortical layer of the spherical
refractive bodies is composed of lipid and protein material, itfiile
the inner, medullary portion contains a fluid of unknown composi­
tion,
The reason for assuming that it is fluid in nature is its
eccentric position in the majority of cases*
The ratio of the outer
lipo-protein layer to the inner portion varies grt atly*
body
When the
larger there is proportionally more of the central fluid
present*
It seems probable that these bodies grow in size by the
increase of the inner fluid portion*
The general staining properties and cytochamical reactions of
the refractive spherical bodies are very similar to those of metasoan Golgl substance*
The probable mode of increase in size of these
bodies, by the progressive accumulation of the central fluid, may be
similar to the condensation function of the metazoan Golgi sub­
stance*
The observations here recorded lend support to the ideas
of previous workers, who claim that the spherical refractive bodies
are the Qolgi bodies of amebas*
57
(See MacLennan, 19U1*)
It ha* been suggested (Wilber, 19U2, 19U5b) that these re­
fractive bodies merely represent reserve food or volutin.
How­
ever, as was first shown by Andre sen (I9ii2) and substantiated in
the present work, they do not diminish greatly in number under
starvation conditions.
When they do so, the remaining bodies
become larger, indicating coalescence rather than utilisation as
reserve food.
The Contractile Vacuoles
The number of contractile vacuoles varies depending on the
species.
Amoeba proteus and Amoeba guttula have only one con­
tractile vacuole.
The contractile vacuole of Amoeba proteus and
related species has undergone a thorough investigation by Mast
(193d) and a few other authors, and there is general agreement as
to the facts.
vacuole.
Chaos chaos gener lly has about 7-10 contractile
Sach of these vacuoles is rather similar in size (80 -
120 microns before systole) and behavior to that of Amoeba proteus.
Mayprella bigemma has h-9 contractile vacuoles, with a dia­
meter of about 15 microns before systole.
Schaeffer (1918) states
that the contractile vacuoles of Mayorella biganma never coalesce.
However in the present study it was found that among the smaller
"growing" vacuoles coalescence was fairly common.
In the larger
vacuoles no coalescence was observed.
There are usually two contractile vacuoles in Thecamoeba
58
striata.
The outstanding and unique characteristic of this ameba
is the curiously shaped larger contractile vacuole.
This vacuole
is almost never spherical; it is irregularly lobed and constantly
changing In shape during locomotion,
Irfhen the ameba is rounded up
and stationary, the contractile vacuole assumes a more regular,
rather oval form,
Penard (1902) suggested that the curiously
lobed appearance or this vacuole is due to the constant formation
of smaller new vacuoles adjacent to it, which coalesce with it.
During the present study, no evidence was obtained for this inter­
pretation.
As stated earlier, there are two varieties of Thecamoeba
striata.
In the larger variety, the two contractile vacuoles
present Include a analler, spherical v acuole at the posterior end
and anterior to it, the larger amorphic vacuole which may at times,
move forward carried by the streaming protoplasm.
The snaller
spherical vacuole sometimes develops into and assumes the position
of the larger lobed vacuole which then disappears,
vacuole then develop a at the posterior end.
the general rule.
A new spherical
This, however, is not
The vacuoles function independently, both empty­
ing their contents to the outside of the cell.
The smaller variety of T, striata usually possesses only the
amorphic contractile vacuole.
The larger vacuole, in Individuals whose length is 80 microns,
sometimes reaches a length of 30 microns but the spherical smaller
vacuole is never larger than 15 microns in diameter,
$9
In the past, investigations have ignored or minimized the
possible effects of metabolic activity on the rate of contraction
of the contractile vacuole,
(See Weatherby, lpidL.)
Hudzinaka
and Chambers (1951) report that the pulsation rate of the con­
tractile vacuole in the sue torian Tokophrya infusiomm accelerated
greatly during an increase in metabolic activity of the organism.
In Thecamoeba striata during locomotion, the time between con­
tractions of the larger lobed vacuole averages 2.5 - 3 minutes.
The smaller posterior vacuole takes almost the same length of
time.
They usually do not contract simultaneously but rather,
alternately.
In individuals that are not moving but are rounded
\ip and with little or no internal protoplasmic streaming, the time
between contractions is about 8 or 10 minutes.
Ho id.and (I92l*a) reported that the contractile vacuole in
Thecamoeba verrucosa is not surrounded by beta granules.
In The-
camoeba striata how ver, such a layer of beta granules is definitely
present.
Those granules are not so densely packed and occasionally
gaps without granules can be found.
As was stated earlier, these
granules are more or less flattened out on the surface of the con­
tractile vacuole} upon the contraction of the vacuole, the granules
become spherical.
Hast (193^) showed that in Amoeba proteus these
granules do not determine the site of formation of the new vacuole.
In the present study, the same results were obtained with fhecamoeba
striata.
It was also observed, using the phas* indifference microscope,
that the layer of cytoplasm about 2 microns thick, surrounding the
60
contractile vacuole and containing the be a granules, appears to
be in the gel state, and differs in this respect from the cytoplasm
surrounding it.
It was found that the beta granules which surround the larger
amorphic vacuole of Thecamoeba striata do not in any given period
between contractions int rchange position with other granules either
during the vacuole's rapid changes of shape or during its movement
with the streaming protoplasm.
The origin of the contractile vacuoles in most of the species
studiod was investigated.
The evidence indicates that the new
vacuole is usually formed de novo.
The site of origin, as stated
earlier, can not be predicted by the position of the beta granules
that surrounded the old vacuole.
However, the new vacuole does form
in the approximate position where the earlier vacuole had undergone
systole.
In Mnyn^Aiia bigemma the formation of a new contractile
vacuole is accomplished by the coalescence of at least 6 or 8 very
small vacuoles.
It was found on car ful observation of the vacuoles of Amoeba
proteus. Chaos chaos and Thecrnoeba striata that occasionally the
vacuole does not contract completely and a minute vacuole, more oval
than spherical, measuring 2 to 2,5 microns remains.
The growth of
the new vacuole is then merely the enlargement of this minute vacuole.
But in a few cases, it was observed that a new vacuole formed adjac­
ent to the minute residual one, and upon subsequent enlargement in­
corporated the latter,
61
Crystals and Crystal Vacuoles
Crystal Inclusions were found in Amoeba proteus, Chaos chaos
and Mayorella bigemma.
Those found in Amoeba proteus and Chaos
ohaos are similar and of two types, plat -like and bipyramidal.
Their sise varies from 2 - 7 microns and they are always found in
vacuoles, which vary in else from 2 . 5 - 9 microns.
The crystal
vacuoles show a great affinity for neutral red, varying from an
orange to a light red and deep red color.
Some were found to be
more alkaline than others, as demonstrated by the color of the neu­
tral red stain.
Aft^r 2h - 36 hours, however, most of the vacuoles
were stained a deep red.
The ratio of the crystal to the vacuole size varies greatly.
(ften a small crystal was found in a large vacuolo and vice versa.
The crystals were usually found to lie eccentrically in the vac­
uoles.
Not more than one crystal was found in any diven crystal
vacuole.
According to Mast and Doyle (1935a) the plat --like crystals
are composed of leucine and the bipyramidal ones consist of a mag­
nesium salt of a substituted glycine.
Mast and Doyle (1935a) described "blebs" adherent to the sur­
face of some of the crystals, and suggested that these blebs re­
present the beginnings of the spherical refractive bodies.
present study such "blebs" were seen only occasionally.
In the
It seems
very unlikely that such crystals would give rise to the lipoprotein-
62
composed spherical refractire bodies.
Furthermore it was found
that the blebs do not reduce OsO^, as do the spherical refractive
bodies.
In no case did the eytoohemical tests indicate the nature
of the fluid within the crystal vacuoles.
Coalescence of crystal
vacuoles was observed only under starvation conditions.
Schaeffer (1918) described in Mayorella bigemma. small dumb­
bell shaped, or hour-glass shaped and occasionally club-shaped
crystals as diagnostic for this species.
With the use of the pol­
arising microscope these crystals were easily seen to be anisotropic
and strongly birefrigent.
Schaeffer reported that these crystals
have a close affinity to "excretion spheres" which are never larger
than 3 microns.
These so-called "excretion spheres" were not found
to be present in any ameba that was studied however.
These crystals
unlike those in Amoeba proteus and Chaos chaos. lie free in the cyto­
plasm and were not found at any time to be enclosed in vacuoles.
These observations were repeatedly checked with vital staining and
with the aid of the phase-difference raicrsocope.
2 microns through their longest axis.
Their size was 1-
Along with Schaeffer, Lotze
(193ftf) also though that this type of crystal was only found in Mayorella bjgeiwaa.
However, Bemheimer (1938a) stat ^s that the supposed
diagnostic crystals are also found in Amoeba dofie ini.
The Food 7acuoles
63
The breakdown of food vacuoles into smaller vacuoles was
traced in all of the species studied except Amoeba guttula.
In
Thecamoeba striata and Mayo rails bigemma it was found that a
newly formed food vacuole very soon ( 1 - 2 hours) breaks down into
two vacuoles.
Within this short time the food organism, Chlamy-
donvonas, loses its definite outlines, and the two food vacuoles
are colored uniformly green by the presence of chlorophyll.
Also
desoxyribonucleic acid (DNA), demonstrable by the Feulgen test
In the nucleus of the food organism, is found diffused in the
resulting two vacuoles.
After subsequent breakdown of the se­
condary food vacuoles into still smaller vacuoles, chlorophyll
is no longer present and DNA can only rarely be demons trated with­
in them.
However, the presence of the latter might be accounted
for in another way wliich will be discussed later on.
It was not possible in any of the species studied to de­
termine the exact number of times that a newly-formed food vacuole
divides into smaller vacuoles.
It is probably variable from
species to qpecles, and probably to a lesser degree from in­
dividual to individual.
In attempting to study the frequency of division of food vac­
uoles, amebas were placed in a medium containing no food organisms,
to avoid confusion arising from the formation of new food vacuoles.
It was found that within 2h hours most of the larger food vacuoles
had disappeared and in 36 hours all of the food vacuoles had dis­
appeared,
6h
During starvation, In those species possessing spherical
refractive bodies, sons of the latter apparently coalesced.
At
about this time, numerous non-con tractile vacuoles appeared,
some of ifilch coalesced.
In those species which normally have
non-contraetile vacuoles (Chaos chaos and Mayorella bigemma) setae
of these vacuoles also coalesced and more appeared.
Previous
investigators (Andresen and Hoi ter, 191*5, and Andre sen, 19U5)
who studied the changes in Chaos chaos and Amoeba proteus during
starvation, recorded similar results.
The s ignlficance of these
changes is not understood.
No spherical refractive bodies, beta granules, or crystals
were found inside the food vacuoles at anytime.
This result is
supported by observations made earlier on Chaos chaos by Andresen
and Bolter (19U2).
In contrast. Mast and Doyle (1935a, 1935b) re­
ported crystals and spherical refractive bodies in the food vacuoles
of Amoeba proteus. and concluded that these inclusions are formed
dlroctly by the food vacuole.
In all the qpecles studied, small food vacuoles containing
undigested food residues accumulate in t he posterior end of the
maeba mid are subsequently egested.
It was observed that two or
three waste food vacuoles were sometimes egested at one time as
if they were one mass, but after their release into the surrounding
medium it was found that they had not coalesced but were still
separate.
The process of egestion was observed in Amoeba proteus.
Thecamoeba striate, and Mayorella bigemma.
65
The Fat Globules
Relatively large numbers of fat globules were found in
of the species of moebaa studied.
1
Fat globules were easily seen
with the phase-difference mlcroecopej but In the unstained ameba,
they could not always be differentiated from the smallest spherical
refractive bodies, In those species possessing the latter.
They
measured 1.2 - 2.5 microns in diameter being somewhat smaller In
the smaller amebas.
They colored red with Sudan III and Sudan IV
and blackish blue with Sudan black B.
Doyle*s (1933) mixture for
the simultaneous visualization of starch and fat proved unsatis­
factory, since some of the fat globules present In the cytoplasm
did not react.
The fat globules stained red with Nile blue sul­
fate, indicating that the globules are composed of neutral fat-s.
Negative results were obtained with the Schultze test for cholesterol.
The number and size of the fat globules varied greatly from
individual to individual in a given culture.
Generally speaking,
however, when an ameba appears to b e well fed, large numbers of
these globules are found, varying in size and scattered through­
out most of the cytoplasm.
vacuoles of any kind.
These globules were never within
Under starvation conditions, the number
of fat globules decreases.
Whenjieath f ram starvation occurs, some
fat globules are still prosent.
An estimate of the amount of fat
present after death showed that there was great variation from
cell to cell.
In the majority of the individuals, around one-
66
third of the original total still remained, while in some, verylittle fat remained.
Contrary to the above results, Andresen
(l9US) found that in Amoeba proteus all microscopically visible
fat had disappeared by the time death occurred from starvation.
However, the present experiments ware conducted on Chaos chaos.
Thecamoeba striata, and Mayorella bigemma as well as Amoeba proteus.
and the results obtained were identical in all four species.
Other Inclusions
Glycogen.
Although glycogen was not visible in the untreated
cells, it was easily demonstrated in all species studied with the
use of Lugol's solution and alcoholic iodine solution.
'When the
amebas had been starved for 36 hours or more, the results obtained
with these iodine solutions were entirely negative.
Permanent vacuoles.
cussed,
Various vacuoles have already been dis­
However, under normal conditions, clear permanent vacuoles
are also present in Chaos chaos and Mayorella bigemma.
These
vacuoles differ from contractile vacuoles in that they do not under­
go systole and are not surrounded by beta granules.
They are rcadly
distinguished from empty crystal vacuoles in Chaos chaos because the
permanent vacuoles do not show any affinity f or neutral red.
In
Chaos chaos permanent vacuoles vary from 2 to 10 microns in diameter.
In Mayorella bigemma they are usually around 6 microns, although a
few have been found as large as 1$ microns in diameter.
67
These
permanent vacuole* never contain any granules or other particulate
matter.
Under normal conditions, they n*?ver coalesce.
During
starvation, however, coalescence is common and the slse of the
vacuoles increases greatly.
Also many new non-contractile vacuoles
are formed at this time, even in Amoeba proteus and Ihecamoeba
striata where they are not ordinarily found, similar vacuoles fre­
quently form under starvation conditions.
Neutral red granules.
Many observations in this study have
been made with the aid of the vital stain, neutral rod.
When amebas
are stained with neutral red (/onwiller, 1913, and many later au­
thors), some of th0 cytoplasmic inclusions show an affinity for
it while othem's do not*
Howe er, certain granules appear which
are not visible previous to the neutral red staining.
ules are generally known as neutral r*;d granules.
These gran­
A tremendous
amount of confusion concerning them has accumulated in the litTature.
(See MacLennen. 19U1.)
\
the facts obtained concerning the neutral red granules were
about the $sane in all the species studied.
A dilute solution (1:
150,000) of neutral rad was found to be the most satisfactory for
this purpose.
About a half-hour after the neutral red solution
was added, numerous small deep red granules about 0.5 to 1 micron
in diameter appeared.
These granules could not be confusod with
the alpha and beta granules, which do not take up neutral red.
In about ft hours, more of these deep red granules appeared and
some had enlarged to about 2.5 or 3 microns in diameter.
66
Inter-
mediate sixes were also found.
In 2h to 1*8 hours a few granules
measures as much as 6 microns In diameter, and no granules smaller
than 1.8 microns were found after a day or two.
The color of all
these granules, whether large or amall was the same deep red.
It
might be pointed out that the maximum sizes reached by these gran­
ules were less in the smaller species than In the larger species,
being about U mic ons in M a y o r U a bigemma and Amoeba guttula.
Extended observations showed no coalescence of neutral red
granules In any of the species studied.
Up to about 2h hours, the
neutral red granules were found free in the cytoplasm.
Later, how»
ever, these granules were found In vacuoles, usually one granules,
3 - 6 microns in diameter, per vacuole.
uoles vas found to b e 5 - 10 microns.
2-3
The diameter of the vac­
A few vacuoles contained
granules, but these wers not common.
These lntravacuolar
neutral red granules accumulate In the posterior portion of the cell
during locomotion.
Three days after the neutral red solution had
been added, the situation had not changed In regard to the number,
size, and color of the neutral red granules.
However, the number
of granules per vacuole had Increased from one to as many as 12.
The diameter of the vacuoles had Increased to 10 - 20 microns, but
the number of vacuoles had decreased.
While coalescence of the v acuoles containing neutral red
granules was observed a few times, coalescence of the granules
themselves was not observed at saRytime.
Therefore the observed
Increase in sise of these granules may be explained as a result of
69
condensation of material but not as the staining of pre—formed in­
clusion bodies.
The Cytoplasmic Ground Substance
No exhaustive study was made especially on the cytoplasmic
ground substance of the amebas.
However, when cytochemlcal tests
were used to determine the distribution of certain compounds in
the cell inclusions, it was observed that some of these compounds
were found only in the cytoplasmic ground substance.
Ribonucleic
acid (RNA) was found in this ground substance and in the nuclei, but
not in any of the cytoplasmic inclusions.
Similar results were ob­
tained by Roskin and Oinsburg (I9l*lia, l?hUb)m
Desoxyribonucleic
acid (DNA.) was demonstrated only in the nucleus and some food
vacuoles.
(See Lucas, 1930, and Chalkley, 1936.)
The results of
Chalkley (1937, 19f>l) showing that sulfhydryl material is found
diffuse in the ground substance, were confirmed in the present
investigation in Amoeba proteus, Chaos chaos, and Thecamoeba striata.
With the aid of the plasmel reaction, plasmalogen was found diffuse
in the ground substance, and not in any cell inclusion.
Only the
three larger species (Amoeba proteus. Chaos chaos, and Thecamoeba
striata) were tested, ^s it was not possible to follow the necessary
procedure with the smaller amibas.
Brachet (19^0a) was not able
to demonstrate pLasmalogens in amebas.
However, it is net possible
to gcraluate these contrary results since he does not tell us what
70
species of m o b a s he tasted or what procedure he followed.
Andresen (I9i«5), and Andresen and Hoi ter (I9li5) state that
during starvation in Aaoeba proteus and Chaos chaos the viscosity
of the cytoplasm decreases.
in this study.
Their observations were corroborated
It was found that the heaviest inclusions, the
spherical refractive bodies, settled to the bottom of the ameba
during starvation, indicating decrease in cytoplasmic viscosity.
71
DISCUSSION
The criteria for separating genera of the Family Amoebldae
are, in general, such gross morphological features as size, num­
ber and shape of pseudopodia, number and form of nuclei, etc*
(See Shaeffer, 1926*)
The five species hero studied represent
at least four different genera*
They vary In size from 20 microns
to 3 millimeters, and the number of nuclei from one to several
hundred, etc*
In these and other respects, these species show
great diversity in their gross structure*
It is therefore sur­
prising to find a remarkable degree of similarity in their finer
structure and cytochemistry.
The differences noted are chiefly
in the presence or absence of certain components rather than
differences in the components themselves*
That is, a given cell
component shows only slight differences in the various species.
This will be brought out in the subsequent discussion*
Cytochemlcal tests indicate that the outermost layer of all the
amebas studied (the "cell membrane"), regardless of thickness, con­
tains neutral musopolysaccharide material*
The tests upon which
this finding Is based have apparently not been applied to other
protozoa*
Carbohydrates and proteins in various relationships, are
known to be essential constituents of the external membranes of
metazo&n cells*(DeRobertis, Nowinski, and Suez, 19U6.}
Alpha granules (about 0,2$ microns) wero found to be present
in all the species studied*
Nothing new was ascertained concerning
the function, structure, and origin of these granules*
72
Beta granules, predominantly spherical and measuring about
one micron In diameter, were found to be present In nil the
species studied.
In the present study, cytochemlcal methods were
applied for the first time to these granules, and confirmed that
they are lipid and protein in composition, as are mtazoan mito­
chondria.
Their staining vitally with Janus green B, as first re­
ported by Mast and Doyle in 1935, was also confirmed.
Hence the
conclusion of these and other authors that the beta granules re­
present mitochondria seems to be Justified.
It was found with the aid of the phasc-contrast microscope
that the beta granules are not directly involved in food vacuole
digestion, as suggested by Horning and others.
Recent Investigations (Bourne, 1950) on liver and other metasoan tissues Indicate that mitochondria contain a high percentage
of those enzymes which play an important part in the aerobic me­
tabolism of the cell.
According to Bourne, many of the enzymes
found in mitochondria are constituents of the Krebs trie arboxylic
acid cycle, the cycle which has been described as a meeting point
of protein, fat, and carbohydrate metabolisms.
This indicates
that mitochondria may be concerned with breakdown of protein, fat,
and carbohydrate, as well as with the synthesis of these three
substances.
Wilber (19-j 5 ) states that in Chaos chaoj*, after the
removal of most of the beta granules, the giant ameba appeared
normal.
This would seem to indicate that the Krebs cycle enzymes
may not be found exclusively in the beta granules, but may be
73
present- In the cytoplasmic ground substance as well.
Spherical refractive bodies (present in three of the five
species studied) are generally considered to be the Golgi sub­
stance, as first proposed by Brown (1930), because of their
staining reactions are similar to those of metasoan Golgi material.
In general the Golgi elements of metasoan cells are oanlophilicj
they stain as a rule with neutral red; and they are believed to act
as secretory centers.
They are believed to be primarily spheres
containing lipid and protein, from which a variety of substances
segregate or condense out.
(Baker,
1 9 U a .)
A
s
already pointed out
the spherical refractive bodies of amebas are always osmiophilic
and generally stain vitally with neutral rt d.
The interpretation
of Brown would thus seem to be a reasonable one.
It has been shown In the present paper that at least the larger
of the spherical refractive bodies In the amebas consist of an outer
osmiophilic portion staining with neutral red and an Inner portion
Which shows these reactions very little or not at all.
Although the
composition of this inner portion was not determined, it might be
a condensation substance.
It may be further pointed out that the
larger sized bodies contain a proportionally greater amount of this
Inner material, indicating a possible condensation of the outer
cortical lip Id-protein layer.
As in the case of Golgi material of
the cell, the amount of spherical refractive material present in
amebas varies from cell to cell and from time to time.
It has
been shown In this and previous (Andre sen and Hoi ter, 19h5) studies
7U
that the variation In numbers of spherical refractive bodies Is
not due to their action as reserve food materials, but rather to
some other, as yet unknown, physiological condition arising In the
ameba,
Wilber (19li5b) claims that these bodies consist of volutin
and contain free aldehydes because they stain with Schiff's reagent
without prior treatment*
criticism.
His methods, however, are open to serious
First, the use of Schiff's reagent on cells without prior
treatment does not give specific results, and second, the presence
in the cell of free aldehydes which have not been uranasked is
highly improbable, (Hayes, 19U9.)
Hast a nd Doylo (1935a) suggested that the origin of the
spherical refractive bodies may be directly from the food vacuoles.
The present observation do not substantiate such an origin since
no spherical refractive bodies were ever found in or near food
vacuoles.
Further it seems highly unlikely that both the process
of break-down of nutrient material and its re-synthesis into cell
components should occur in the food vacuole,
Extende^bservations
on the food vacuole showed a progressive disintegration of parti­
culate food organisms (digestion), and n vcr a differentiation of
this material into anything that would resemble spherical refractive
bodies, crystals, or any other type of cell inclusion.
Therefore
since there is no definite evidence that these bodies arise from the
food vacuoles directly, it may be postulated that they arise from
the cytoplasmic ground substance.
Spherical refractive bodies are not present in Mayorella bigeeana
75
or Amoeba gjuttula* and no other Qolgi-like material has been de­
monstrated In these amebas*
This obviously somewhat weakens the
interpretation of the ref active bodies as Oolgi elements.
An­
other possible difficulty lies in the high refractive Index of
these bodies| the Oolgi material of metasoan cells is notoriously
difficult to differentiate from the surrounding cytoplasm*
It is
of course, conceivable that the function of Oolgi bodies may be
carried out in these species without any demonstrable specialised
inclusion bodies*
The number of contractile vacuoles present in amebas varies
from one to about twelve*
ber is fairly constant*
However, for a given species the num­
It has been found in this study that the
rate of pulsation of the contractile vacuole is directly related
to the degree of activity of the organism.
Similar observations
have boon made on the contractile vacuole of a suctorlean.
(Hudsink a and Chanbenf l<?5l •)
It is now generally agreed that
the contractile vacuole is primarily a hydrostatic organelle,
equalizing the intake and outgo of water*
It is conceivable that
minute quantities of nitrogenous waste products from the cyto­
plasmic ground substance may also be excretod*
(See Weathorby,
19ijl.)
Crystals were found in Amoeba proteus, Chaos chaos* and
Mayorella bigengna.
Those found in A. proteus and C* chaos, mere
similar and of two types, plate-like and bipyramidal.
Their
composition was determined by Hast and Doyle (1935a) to be leucine
76
and a magnesium salt or a substituted glycine, respectively.
Theie crystals are always found enclosed in vacuoles, which show
a great affinity for neutral red.
The crystals found in Mayorella
bigerma, however, are usually dumb-bell shaped and lie free in
the cytoplasm, never enclosed in vacuoles.
In a few Instances certain "blebs** (first described by Mast
and Doyle, 1935a) were observed ©a the crystals of ^rtoeba proteus
and Chaps chaos. According to Hast and Do; le these "blebs" represent
the beginning ©f the Attrition of spherical ref active bodies.
To
the present writer they appear to be merely imperfections or ble­
mishes on this-surface of the crystals.
They do not reduce osmium
t©troxide as do- the refractive bodies.
Msdt ahid Deyle Cl935a) postulated that the crystals originate
directly from the. fpod TUduolcs.
Ho evidence has been ibund to
support this.
In the study of food xaCuoles, it was found that the process
of digestion is usually accompanied by the division of a vacuole
into two or several smaller vacuoles.
The undigested residues
were found to be massed or packed in the posterior of th 1 organism.
Subsequent egestion of these waste vacuoles was observed.
These
observations approximate those maae earlier by Hast (I9d2).
In a few cases, it was noted that in Theeamoeba striata very
small food vacuoles (about 3 microns in cliam ter), which were con­
sidered to be waste vacuoles containing food residues, contained
desoxyribonucleic acid.
It may be postulated that the presence
77
of DNA In these waste vacuole indicates that at times not all of
this compound Is broken down during digestion.
It is possible
however that these vacuoles were not waste vacuoles but rather
newly formed, small fo d vacuoles containing bacteria.
Ingestion
of bacteria alone has not been observed in this species, however.
Egcstlon of food vacuoles under starvation conditions has
been observed In the present ctudy In all the species except Amoeba
gut tula.
Andreeen (19l*-) reported this phenomenon in Chaos chaos.
Mast and 0ahn®rt (1935) slrt-i that food vacuoles in Amot ba proteus
coalesce during starvation conditions.
This was not observed in
the present work.
The fat globules ware found to be c omposod of neutral lipids.
It was found further that when death from s tarvation occurred,
approximately one-third of the original msnbor of fat globules
still remained.
The phenomena wfriich occur when amebas are placed
under starvation conditions shew th?t starvation do«s n*t result in
a simple diminution of available
"cod until none exists.
Hie non-
availabdlitfr of food initiates certain phenomena, such as cgostion
of fo“d vacuoles, vacuolization of the cytoplasm, coalescence and
Vaeuoliaetlon of refractive bodies, etc.
changes is not known.
The significance of these
The process of starvation is a vt ry c omplox
one and the end r suit of that process, death, does not cone about
simply by the lack of available food,
lather, it probably occurs
by the derangement of certain processes, caused by a deficiency of
essential compounds.
76
The permanent vacuoles, nor* ally present only in Mayorell a
blgawaa and Chaos chaos, are distinguished from contractile vacuoles
not only by their non-contractility, but also because they are not
surrounded by a layer of beta granules.
They are further differen­
tiated from empty crystal vacuoles in Chaos chaos by the fact that
they do not stain with neutral red.
not coalesce,
These vacuoles normally do
Nassonov (1921*) thought that the permanent vacuoles
together with the contractile vacuoles make up the Golgi complex of
protosoa.
However, staining properties and behavior of these Vac­
uoles lend no suppojf to this theory.
the permanent vacuoles is not known.
The origin and function of
It is interesting to note
that under starvation conditions coalescence and the appearance of
new permanent vacuoles occur not only in Mayorella M g e w w and
Chaos chaps, where they are normal components, but also in Amoeba
proteus and Thecjttnosba striata, where they are not ordinarily found.
On an earlier page we have described the appearance and en­
largement of neutral red granules during staining with neutral red,
and the subsequent formation of vacuoles around them.
servations were made by Andre sen in 19U5.
Similar ob­
Since the enlargement of
neutral red granules is not brought about by coalescence, and also
since these granules have not been demonstrated in the cytoplasm
prior to the Introduction of neutral red, it is concluded that
these granules are not prefonned.
Probably the action of neutral
red on the cytoplamalc ground substance causes certain elements
which show a great affinity for the dye to condense or precipitate
79
O't and to appear as discrete bodies.
The results obtained in present study on the cytochomical of
the cyt plasmic ground substance (hyaloplasm) lead to the intorpreatation that this cytoplasnic ground substance is more than a
matrix in which the various inclusion bodies are found.
Indeed, it
has been suggest.d that the inclusion bodies to a great degree are
marely evidences of the physiological activity of this ground sub­
stance.
In metazoan cells (Lagarow, 19li3, Claude, 19li3, Brachet, 1950)
two types of submicroscopic particles have been separated by means
of ultracentrifugaticn from the ground substance: particulate
glycogen and the "microsomes".
In the present study glycogen was
found to be microscopically visible only when treated with an
iodine solution.
It seems quite possible, there!ore, that it is
normally present as submicroscopic particles.
The action of iodine
probably causes these particles to precisirate and to stain reddish
brown.
According to Brachet (1950b), the "microsam.es" isolated by
means of ultracentrifugation were found to contain most of the cyto­
plasmic 3NA, sulfhydryl, and plasmologen.
Bayliss (1920) with the
aid of the ultramicroscope described certain particles in ameba v:hich
are not ordinarily microscopically visible.
According to Lazarow
(19h3) these particlrs of Bayliss wore the "microcom s" which hove
been subsequently isolated from metazoan cells.
However, it is not
cl<ar exactly what Bayliss did see as "shimm ring points of light11.
80
Until all the techniques which have been applied to m tasaan cells
are adapted and applied to the amebas, no definite statement can
be made as to the presence or absence of "micro somea".
81
SUMMARY
1,
A comparative study has been made of the structure and
cytochemistry of the cytoplaan of five soecies in the Family
Amoebidae;
Amoeba proteus (Pallas) Leidy, Chaos chaos (Linnaeus),
Thee amoeba striata (Penard) Schaeffer, Mayorella bigenma Schaeffer,
and Amoeba gut tula Dujordin,
2 • A review of the pertinent literature dealing with the
origin, function, and composition of the cytoplasmic components
has been included,
3,
A description is given of the various technique employed,
Including the parlodion trap technique, various kinds of staining,
and cytochemical methods,
U,
The cytoplasmic components studied were:
the cell membrane,
the alpha and beta tqranulos, the spherical refractive bodies, the
contractile vacuoles, the crystals and crystal vacuoles, the food
vacuoles, the fat globules, the permanent vacuoles, the neutral red
granules and the cytoplasmic ground substance or hyaloplasm,
5>.
The five species of amebas studied show great diversity
in their sise and gross structure.
However, on the whole, a re­
markable degree of similarity was found in the finer structure and
cytochemistry of their cell components,
6.
It was found that in all the species studied the out rmost
layer, or cell membrane, contains neutral mucopolysaccharides.
Whether this layer was the seemingly unspecialised plasma-membrane
of Mayorella bjgemma and Amoeba guttula, or whether it was the much
82
thicker, more or less rigid covering (pellicle) of Thecaraoeba
striata. or whether it was the somewhat intermediate "plasmalerama1'
of Amoeba protoua and Chaos chaos, the cytoc hectical results were
the same.
The thickness of the pellicle of Thecamoeba striata
was found to be about 0*33 micron.
In both Araooba proteus and
Chaoa chaoa* the thickness of the plasmalentma is somewhat luss,
or about 0.25 micron.
The cell membrane of Mayorella bigamma and
Amoeba gut tula ia very much thinner, and it was not possible to
measure it*
7.
The alpha granules (0*2$ micron in diameter) were found
to be present in all the species studied*
8*
The beta granules in all the species studied are very
similar in size (about 1 micron in diameter) and in shape (usually
round).
In Theeamoeba striata, and in no other sp-cics studied,
coalescence of beta granules was observed.
In
granules were found to stain with Janus green B.
cases the bota
VJhen cytochenical
tests were applied to them, it was found that these granules are
lipid and protein in composition, as are metasoan mitochondria.
was concluded that these granules are mitochondria*
It
No evidence was
found that they are involved directly with food vacuole digestion
or with the functioning of the contractile v acuole.
9.
The spherical refractive bodies were found in only three
species (Amoeba proteus. Chaos chaos, and Thecamoeba striata) of
the five studied.
They vary in nunber and size.
The larger bo dim
are not homogeneous, but are divided into an out-r cortical layer
83
which stains with neutral red and an inner unstained portion, which
is often eccentric in position*
7 microns in diameter.
These bodies measure from l.li to
The ratio of the outer layer to the inner,
eccentric portion varies greatly.
Vlhen the body is larger, there
is proprtionally more of the central portion present,
Cytochemical
tests visualize the presence of lipid and protoin material in the
outer portion.
certained.
material.
The composition of the inner portion was not as­
It seems probable that ttuso bodies repro3cnt Golgi
These rfractive bodies do not seem to arise from the
food vacuoles, but r ather from the cytoplasmic ground subs bonce*
10.
The number of contractile vacuoles present in amebas is
variable.
However, for a given species the number is more or X cjm
constant.
The shape of these vacuoles is almost always apJncfAbal*
The only exception is the larger of the two v acunl^s lb Thetagoeba
striata. This vacuole is irregularly lobed and constantly changing
in shape.
11.
It has b een found in this study that the rate of pulsation
of the contractile vacuole is directly related to the metabolic
activity of the organism.
Further, it was observed that the beta
granules surrounding the contractile vacuoles do not uetcminc the
site of formation of the new vacuole.
It was f ound that in a few
instances the vacuole does not contract completely.
A nunuto
vacuole (2 - 2.5 microns) remains and is usually the site of forma­
tion of the noxt vacuole.
12.
Crystal inclusions were found in Amoeba proteus, Chaos
8U
chaoa, and Mayorella blgemma.
Those found in Amoeba proteus and
Chaoa chaos are similar, 2 - 7
microns in diameter, and of two
types, plate-like and bipyramldal.
These crystals are found in
small vacuoles (2,5 - 9 microns in diameter).
vacuoles stain readily with neutral red.
The crystal
In a few instances
tiny "blebs" were seen on crystal surfaces.
It is suggested that
these "blebs" do not represent the formation of new refractive
bodies as suggested b y Mast and Doyle (1935), but rath r crystal
imperfections.
The crystals of Mayorella blgcnma are usually dumb-bell
shaped and rather small, measuring 1 - 2
longest axis.
microns through their
Unlike the crystals of Amoeba proteus and Chaos
chaos they are not enclosed in vacuoles, but lie free in the cyto­
plasm,
13.
In the study of food vacuoles,
it was founu that at
least in the four larger species the process of digestion is ac­
companied by the breakdown of the vacuoles Into smaller ones.
tion of waste food vacuoles was observed.
Ege3-
However, under gtirvalion
conditions, egestion of all food vacuoles occurs.
Ik.
Fat globules ware found in al1 the sp-cies studI'd.
The fat globules are composed of neutral fats.
Usually about one-
third of the fat globules are still present after death from
starvation occurs.
During starvation, al1 the free glycogen is
absorbed.
15.
Permanent non-contractile vacuoles are normally present
85
in Mayorella bigemma and Chaos chaos.
These vacuoles differ from
contractile vacuoles in that they do not contract, and are not
surrounded by beta granules.
They are readily distinguished from
empty crystal vacuoles because they do not strin with neutral red.
These p rmanent vacuol *s normally do not coalesce, but under
starvation conditions coalescence is common, and both size and
manber of the vacuoles increases*
In Thee amoeba stria~ a and Amoeba
proteus, permanent vacuol-s appear in the cytoplasm under starva­
tion conditions only*
16,
The appearance, color, and growth of neutral red gran­
ules have been described, as well as the development of vacuoles
around them.
Since the growth in size of neutral red granules is
not brought about by coalescence, and also since these granules
ara not found in the cytoplasm prior to the introduction of neutral
red, it is concluded that these granules are not performed.
It is
thought that they arise by the precipitation of certain material
from the cytoplasmic ground substance by the action of the neutral
red,
17.
Diffused in the cytoplasmic ground substance or hyaloplasm
were found ribonucleic acid, plasmalogen, and sulfhydryl*
pounds were not found in any of the cytoplasmic inclusions.
These com­
The
ground substance, it is concluded, is not merely a matrix in which
various inclusions arc suspended.
On the contrary, the inclusion
bodies may be looked upon as evidence of the physiological activity
of the ground substance itself*
86
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97
PLATE I
Theeamoeba striata (Penard) Schaeffer.
Length, 67*0 microns.
1 - hyaline cap
2 - longitudinal striation or fold
3 - food vacuole
h - nucleus
5
-
amorphic contractile vacuole
6
-
larger spherical r fractive body
7 - smaller spherical refractive body
8
-
beta granules
9 - posterior spherical contractile vacuole
10 - layer of beta granules surrounding the contractile vacuole
u
-
coalesced homogeneous sphere of beta granules
12 - alpha granules
13
_
fat globules
98
3
o -o
:o:;:
,
•
\: o* p. :■•■■■.; o '■.p-.o .■;
'j>y Wyb:.6vt-f'
4
>°
'
.vp ■.6*/ * c •' *'* '
o /
O
* :o-
5
=— >12
°-.0’o',. .o ■;
«
.
o. • •• -O
*.-■
■?!#:
V • P • • -."Cr
**■ ry.mc-.
■ wK.
vO»: o :■
*r
/:
^
/
.*v _ J N —/
6
'‘ • :-
?o ?
5?:’V
3
'
/
6
-II
■7
IO
/
}13
7
8 I
3
9
10
II
PLATE I
99
PLATE II
M a y orell a
blgenuoa (Schaeffer) Schaeffer,
Length, 7£.0 microns.
1 - pseudopodium containing hyaloplasm
2 - permanent vacuole
3 - beta granule
1* - alpha granules
$ - contractile vacuole
6 - layer of beta granules surrounding the contractile vacuole
7 - crystal inclusion
8 - fat globules
9 - food vacuole
10 - nucleus
100
*02
PLATE III
FIGUiiE A
Diagram showing an area of cytoplasm of Amoeba proteus (Pallas)
I*©±dy,
FIGURE B
Amoeba gut.tula ihijardin.
Length, 20.5 microns,
1 - beta granule
2 - smaller spherical refractive body
3 - fat globule
U - larger spherical refractive body
5 - alpha granules
6 - bipyramidal crystal in vacuole
7 - food vacuole
8 - pi ate-like crystal in vacuole
9 - empty crystal vacuole
10 - hyaline cap
U
- pseudopodium containing hyaloplasm
12 - nucleus
13 - contractile vacuole
lit - layer of beta granules surrounding the contractile vacuole
102
o •<-■••
6
,
-7
9
00 fO
i
?
/
r
-
.
:
r
■;
"
8
f
• -*v. v --
jx
-3
4
I’O- - V. o
5
'
'■■
/
FIG A
10
11
13
I£
14
5 V'
7
FIG B
PLATE
111
103
AUTOBIOGRAPHY
I, George Demetrios Pappas, was b o m in Portland, Maine,
on November 26, 1926.
I received my secondary school education
in the public schools of the city of Portland, Maine.
My under­
graduate training was obtained at Bowdoin College, Brunswick,
Maine, from which I received the degree Bachelor of Arts in
19^7.
From The Ohio State University, I received the degree
Master of Science in 19l*6»
In 19U8 I received an appointment
as Graduate Assistant in the Department of Zoology and Ento­
mology of The Ohio Stat_ University.
I held this position while
conqsleting the requirements for the degree Doctor of Philosophy.
10U