THE ACCSMULATEON. UTILIZATtON, AND SEGNiFICANC§ 0F
WLY’SACC-WEDE 2N 'FREYMCHOMGNAS AUGUS'E‘A

Thesis for tha Degree of M‘ 5.
MECHEGAN HATE QRNERSWY
Vince-n? A. Svmingrubo
?=?-§4

mam

     
   

  

LIBRARY

Michigan State
University

THE ACCUMULATION, UTILIZATION, AND SIGNIFICANCE OF
POLXSACCHARIDE IN TRITRICHOMONAS AUGUSTA

By.)

w ' .
VINCENT A1 STEINGRUBE

A THESIS
Submitted to

Michigan State University
in partial fulfillment of the requirements

for the degree of
MASTER OF SCIENCE

Department of Microbiology and Public Health

19oh

ACKNOWLEDGMENT

The author wishes to take this opportunity to express
his appreciation for the conscientious guidance and con-
structive criticism during this study extended by Dr. Donald

W. Twohy of the Department of Microbiology and Public Health,
Michigan State University.

ii

TABLE OF CONTENTS

 

Page
IIqTRODUC TION 0 O O 0 O O Q 0 O O O O O O O O O 1
LITERATURE SURVEY . . . . . . . . . . . . . . h
MATERIALS AND METHODS . . . .’; . . . . . . . 13
RESULTS _ 17
Experiment I - A Comparison of Glycogen Con-
tent with the Cultural Age of Tritrichomonas
augusta in the presence of Limiting and Non-
Limiting Concentrations of Glucose 17

Experiment II - A Comparison of Cell Volume
with the Cultural Age of Tritrichomonas au sta
in the presence of Limiting and Non-31miting
Concentrations of Glucose 2h

Experiment III - The Effect of Glycogen Reserves
on the Survival of Tritrichomonas augusta in
Culture medium I? 28

Experiment IV - The Effect of Glycogen Reserves
on the Survival of Tritrichomonas augusta under

 

Non-Nutrient Conditions 31
DISCUSSION AND CONCLUSIONS . . . . . . . . . . 3h
swans! .. .. .. .. .. ;. .. .. .. . us
LITERATURE CITED . . . . . . . . . . . . . . . M9

iii

LIST OF FIGURES
Figure - Page

1. Growth of Trit. augusta in Diamond'S
medium with u.mM glucose . . . . . . . . 2i

2. Variations in glycogen in Trit.
augusta during culture in Diamond's
medium with h mM glucose . . . . . . . . 21

3. Growth of Trit. augusta in Diamond's
medium with 20 mM glucose . . . . . . . 22

 

h. variations in glycogen in Trit.
augusta during culture in Diamond's
medium with 20 mM glucose . . . . . . . 22

5. Total glycogen in Trit. augusta per
ml of culture employing Diamond's
medium with h‘mM glucose . . . . . . . . 23

6. Total glycogen in Trit. agggsta per
ml of culture employing Diamond's
medium with 20 mM glucose . . . . . . . 23

7. Cell Volume frequency of Trit.

augusta grown in Diamond's medium 26

O
O
O

8. Mean cell volumes of Trit. augusta
in Diamond's medium with H'mM
glucose . . . . . . . . . . . . . . . . 27

9. Mean cell volumes of Trit. au usta
in Diamond's medium with mM
glucose................ 27

10. Glycogen content and motility of
Trit. augusta after varied periods
of time in aging conditions following
growth in 2 mM glucose . . . . . . . . . 30

 

'iv

ll. Glycogen content and motility of
Trit. au usta after varied periods
of time In aging conditions
following growth in 20 mM glucose . . . . 3O

12. Glycogen content and.motility of
Trit. gggusta after varied periodS»
of time in non-nutrient conditions
following growth in 2 mM glucose . . . . 33

13. Glycogen content and motility of
Trit. au ta after varied periods
of time in non-nutrient conditions
following growth in 20 mM glucose . . . . 33

 

 

INTRODUCTION

Tritrichomonas augusta is a pyriform protozoan flagel-

 

late about lS—27u long with three unequal anterior flagella
and a fourth posteriorly directed flagellum which forms
part of an undulating membrane. A thick axostyle containing
dark staining granules protrudes posteriorly. This organism
is a common commensal of the colon of frogs and toads.
Because its hosts are poikilothermic, this organism
would encounter a highly variable environment with regard
to temperature and the availability of nutrients. In
general this environment would provide nearly complete
anaerobiasis, a slightly alkaline pH, and an intermittent
source of nutrients. Although the rate of growth and
metabolism of this trichomonad is reduced at lowered
temperatures, 2223- augusta could be expected to require
a reserve carbon and energy source in order to survive
during extended periods of over-winter hibernation of its
host.
Since it does not appear to form a "cyst-like" stage
during its life cycle, 3:;3. agggsta would be expected to
be ESpecially susceptible to the hazards of its host's
external environment during the process of transmission.
The aquatic habitat, which all amphibians frequent during
part of all of their life cycles, would appear to be the most
Opportune route for tranémiSSion of this organism. However,
such an environment probably lacks essential nutrients, and
may possess a high or low pH and possibly high oxygen tensions:
-1-

'Zf
all conditions unfavorable for the survival of this tricho-
monad. 2:;3, augusta is believed to follow an oral route
in infecting a new amfixibian host. Thus, this trichomonad must
be able to resist the low pH encountered during its passage
through the stomach of a prOSpective host. In order to
survive all the adversities listed above long enough to

encounter an Optimal location in a new host, Trit. augusta

 

must be capable of considerable resistance to unfavorable
environmental conditions. Fer this resistance it certainly
must require a reserve carbon and energy source such as
glycogen.

Polysaccharides have been found to occur in varying
amounts in the cytoplasm of nearly all microorganisms. Most
of the investigations of carbohydrate storage have been attempts
at characterization of the pattern of accumulation and util-
ization of reserve polysaccharides by organismmiig,z;§gg.
These reserve compounds were accumulated by microbial cells
when the exogenous carbon and energy source was in excess
of metabolic requirements. When the exogenous energy source
was limiting, the cells utilized previously stored polysac-
charides. Although the data on these basic aSpects of carbon
and energy storage are stgadily increasing, no information
is available which explains theregulatory mechanisms for
the accumulation or utilization of storage polysaccharides,
if such mechanisms exist. lNeidhardt(1963) made this quite

clear When he stated, "To be brutally exact, not a single

 

-3-

mechanism has been established as physiologically respon-
sible for the overall rate of synthesis of one class of
macromolecules with another-..."

The stored carbohydrate occurring in a closely related
trichomonad, Tritrichomonas foetus, has been identified as
an alpha-l,u-glucosan which is quite similar to animal
glycogen. The storage polysaccharide of Eggs, agggpta has
been reported to give a reddish-brown color reaction.with
iodine solutions and appears to be a glycogen-like poly-
saccharide. For the sake of brevity and clarity the carbo-
hydrate found in.T£i£, augggta has been referred to as glycogen
throughout this manuscript.

The purpose of this investigation on the stored poly-
saccharide of Tgitg augusta may be stated as follows:
1) to demonstrate the accumulation and utilization of this
glycogen—like polysaccharide as a carbon and energy reserve,
2) to determine how the pattern of glycogen storage is
affected by cultural age and the availability of glucose,
and 3) to determine the significance of the polysaccharide

reserve in the.life cycle of this organism.

LITERATURE SURVEY
Accumulation and Utilization g; Polysaccharides ig;Micro-
organisms:

Wilkinson (1959) suggested that the basic requirements
which stored compounds must fulfill in order to serve the
cell as functional carbon and energy reserves are as follows:

"a) The compound is accumulated under conditions

when the supply of energy from exogenous sources

is in excess of that required by the cell for growth

and related processes at that particular moment of

time, b) the compound is utilized when the supply

of energy from exogenous sources is insufficient

for the Optimal maintenance of viability and other

processes, and c) that the compound islbroken down

to produce energy in a form utilizable by the cell,

and that it is, in fact, utilized for some purpose

which gives the Cell a biological advantage in the

struggle for existance over those cells which do

not have a comparable compound."
He stated that, in general, storage compounds in bacterial
cells were of high molecular weight, thereby enabling the
cell to accumulate large quantities of these materials
without increasing the internal osmotic pressure. The three
major groups of storage compounds preposed by Wilkinson
(1959) were: volutin, lipid, and polysaccharide.

The most intensive investigations on the influence of
environmental conditions upon the accumulation and utiliza-
tion of storage polysaccharides have involved bacteria
belonging to the Enterobacteriaceae, especially Escherichia
0011 (Wilkinson and Duguid, 1960). Polysaccharide
accumulation was found to reach a maximum of 20-50% of the

total dry cell weight. These maximum.polysaccharide levels

coincided with the attainment of maximal populations in

-u-

-5- ‘gg
cultures of a wide variety of enteric bacteria (Lankard
et al., 1951; Levine et al., 1953; and Duguid and Wilkinson,
1953). The polysaccharide stored by these bacteria during
early phases of culture was utilized in late phases of
culture. Lankford et al. (1951) noted that addition of
glucose to aging cultures of Salmonella oranienburg_and E,
Egg; deficient in glucose resulted in the resumption of
polysaccharide accumulation. Storage of polysaccharide in
a variety of enteric bacteria was found to vary with tem-
Perature by LeVine ct al..(l953). They noted that
a greater accumulation of stored carbohydrate occurred in
organisms incubated at 150-2000 than in organisms incubated
at 37°C.

Palmstierna (1955 & 1956) reported on the pattern of
accumulation of a glycogen-like polymer in cells of E, ngi
B. Polysaccharide storage occurred during the first 30
minutes of culture, which corresponded to the lag phase;
and reached a maximum just prior to the first cell division.
Palmstierna (1955) stated that the stored polysaccharide
appeared to be utilized during the logarithmic phase, since
the concentration dropped to an intermediate level. The
concentration of stored polysaccharide compound remained
constant during the logarithmic phase of growth. When the
rate of growth decreased polysaccharide accumulated, reaching
a maximum level during the maximum stationary phase of growth.

On the basis of these data as well as results from radioactive

-6-
labeling experiments, Palmstierna (1956) proposed: 1) that
the small residual polysaccharide which was always present
in the cells of E, ggli.B, behaved as a primer for the
synthesis of this glycogen—like polymer, 2) that in regard
to the energetics of the cells, the lag phase could be
described as endergonic while the logarithmic phase appeared
to be exergonic in nature, and 3) that during the break
down of reserve polysaccharide, some of the carbon of the
resultant intermediates was incorporated into protein
synthesis. E, ggli_and Aerobacter aerggenes accumulated
more polysaccharide when the ammonium chloride nitrogen
source was deleted from a chemical defined medium than when
the medium was complete. Glucose was in excess of require-
ments under both conditions. (Holme and Palmstierna, 1956
a, b, c, and d; Holme and Cedergren, 1961; and Duguid and
Wilkinson, 1953). Furthermore, the addition of NHuCl to
cultures lacking a nitrogen source resulted in a decline
in the level of glycogen reserves and a concomitant resump-
tion in the growth of g. p_o_l_i_ B (Holme and Palmstierna,
1956b). Following the exhaustion of the added nitrogen
source, cell growth and division ceased and the level of
glycogen reserves again began to rise. Holme and Palm-
stierna proposed that the increased amounts of polysaccharide
stored by E, ggl; B in a medium lacking a nitrogen source
was an indication that both the stored polysaccharide and
the exogenous glucose were utilized as a source of carbon

and energy for protein synthesis and growth. These authors

-7-
concluded that, an "inversely coupled relationship" existed
between synthesis of reserve polysaccharide and synthesis
of protein components required for growth. This conclusion
is supported by 8013 (1961) presentation of a.metabolic
scheme involving separate enzymatic pathways for the synthesis
and degradation of glycogen.

Knowledge of the carbon and energy reserves of proto-
zoa has accrued at a much slower pace than that of the
bacteria. Fennell and Marzke (195u) compared the ratios
of stored glycogen to total dry cell weight of Tetrahymena
gelleii during various phases of ip_zi£rg growth. The
percentages of stored glycogen followed the same general
pattern described for E, ggli by Palmstierna (1955 & 1956)--
high concentrations in the lag phase, diminiShed but constant
concentrations in the logarithmic phase, and increasing
concentrations during the phase of maximal growthm annell
and Marzke (195A) also noticed that the glycogen content.
of organisms from 288 hour cultures decreased nearly h0%
within 30 minutes after organisms were suspended in saline.
Armer (l9uu) demonstrated.that, during periods of starvation
of its host, the paraglycogen reserves in Nyctotherus ovalis
decreased to a base level within h weeks. Hungate (19h3),
Oxford (1951), Masson and Oxford (1951), and Sugden and
Oxford (1952) demonstrated rapid increases of stored poly-
saccharide in the rumen ciliates’of sheep immediately following
feeding of the host. Six to nine hours after host feeding,

or after a similar period of suspension in saline and rumen

-8—
juice, this reserve polysaccharide disappeared.

A cytochemical study on the reserve glycogen in the
various life cycle stages of the coccidian Eimeria tgggllg
was reported by Gill and Ray (l95u). They demonstrated the
presence of glycogen in all the life cycle stages except
the sporozoites. The highest glycogen reserve was found
in the gametocytic stages. The authors suggested that this
high level of storage was required for Sporulation and
prolonged survival outside the host. Gill and Ray found
that the relative amount of glycogen stored by the various
life cycle stages of this parasite varied according to
requirements for the successful transition from one stage
to the next duringthe life cycle. Thus they concluded that
the glycogen of g, tenella played a significant role in
the perpetuation of its parasitic existance.

Changes _i__n_ 931; Volume 2;" Microorganisms:

Neidhardt (1903) suggested two basic groups of environ-
mental conditions Which influenced the size of bacterial
cells as; l) "deleterious agents or conditions . . ."; and
2) "a general ... and simple relation of size to the growth-
supporting ability of the chemical environment, . . ."

In their studies on E, coli, Salmonella gallinarum and
Is. pullorum grown in a medium with excess glucose, Huntington
and Winslow (1937) found that mean cell volume increased
during the lag phase of growth but then decreased upon
entering the logarithmic phase. The mean cell volume then

increased again during late phases of culture. Ormsbee (1942)

-9-
found that the dry weight and the length and width of
individual cells of Tetrahymena galleii varied considerably
during the lag phase of growth. However, the cells showed
less variation during the logarithmic phase When the average
dry weight, length and width was less. At the onset of the
maximal stationary growth phase, the dry weight and dimen-
sions of the organism began to increase. From these data
Ormsbee prOposed cell size to be directly related to cul-
tural age. Loefer (1952) and Summers (1963) also reported
that the mean cell volume of 2,.pyriformis, calculated from
average length and width determinations, increased tea near
maximum during the late logarithmic phase. The cell volume
then remained constant or increased slightly during the
maximal stationary phase. Scherbaum (1956) reported that
the calculated cell volume of T, pyrifqgmis during the log-
arithmic phase decreased to 50% of that during the lag phase
and followed a normal distribution curve. Harding (1937)
reported that when a species of Glaucoma (Tetrahymena) was
subjected to starvation, the cells assumed a slender form
which became more pronounced as long as cell division con-
tinued. Upon cessation of division the organisms assumed
a more spherical form. This author proposed that "rounding-
up" after extended periods of starvation was the result of
a decrease in cellular surface area due to the utilization
of pellicular substances for energy metabolism. Mast and
Hahnert (1935) reported that Amoeba proteus decreased in

volume by 95% when subjected to starvation conditions.

-10-
Movement became progressively more sluggish and pseudOpods
became fewer and smaller as starvation progressed. The
amoebae became spherical just prior to death. Kimball et
al. (1959) reported that starved cells of Paramecium aurelia
continued to divide and decreased in size. These authors
PrOposed that the production of more organisms at the ex-
pense of less nutrient was a survival mechanism. They
further noted that the return of 2, aurelia to excess nutrient
resulted in a rapid increase in cell size as they continued
their normal division rate.
Carbon and Energy Reserve ig Trichomonads:

The trichomonad flagellates parasitic in homeothermic
vertebrates utilize a wide variety of hexose sugars (Lwoff
1951; von Brand, 1952; Read, 1955 a 1957; and Lindblom,
1961). Likewise, Tritrichomonas augusta a commensal of
poikilothermic vertebrates, has a similar ability to utilize
hexose sugars with the resultant production of H2, 002 and
acid end products (Twohy, 1959 & 1961; and Concannon, 1959).
In addition to nutritional studies on carbohydrate utiliz-
ation, a number of investigations have demonstrated the
occurrence of nearly all the enzymes and intermediate com-
pounds of the Enbden-M’eyerhof glycolytic pathway in M-

omon§§_vaginalis and Trit. foetus (Ryley, 1955; Wirtschafter

 

and Jahn, 1956; and Wellerson and Kupferberg, 1962). An
examination of the available data on the acid end products
of carbohydrate metabolism and evolution of carbon dioxide

and hydrogen suggests that this enzymatic pathway can be the

-11-
main route of carbohydrate metabolism in trichomonads
(Suzuoki and Suzuoki, 1951 a & b; Ninomiya and Suzuoki,
1952; Doran, 1958 & 1959; and Wellerson et al., 1959).

The terminal carbohydrate metabolism of trichomonads fol?
lows anaerobic pathways and most forms investigated survive
and multiply only under anaerobic conditions (Bishop, 193H3
Johnson, 19h2; Lwoff, 1951; Hutner and Provasoli, 1955;
Ryley, 1955; and Wellerson et al., 1959). To date glycogen-
like polySaccharides have been the only form of reserve
carbon and energy compounds considered in investigations

on trichomonads. The knowledge of the composition and
occurrence of lipids in trichomonads is extremely limited.
However, in a recent repert on the chemical analysis of
lipids extracted from cells of Trit. foetus, Halevy (1963)
found no evidence that compounds of this nature were stored
in the cytOplasm in quantities sufficient to serve as energy
reserves.

Stewart (1938) and Lyford (19h1) proposed the presence
of glycogen reserves in Trit. foetus on the basis of the
reddish-brown color reaction of the stored polysaccharide
in organisms stained with DdAntoni's iodine solution.
Wantland et a1. (1962) cited the occurrence of a "glycogen
vacuole" in T, tgggg Which was demonstrable by cytochemdcal
techniques. Feinberg and Morgan (1953) isolated and char-
acterized a reserve carbohydrate from Trit. foetus which
they considered to be glycogen. On the basis of chemical

analyses and enzymatic degradation, Manners and Ryley (1955)

-12-
found that a purified polysaccharide isolated from T213:
foetus and T. gallinae resembled but was not identical to
animal glycogen.

Stewart (1938) and Lyford (19h1) reported the pattern
of glycogen storage by Trit. foetus. Glycogen content was
determined by the color reaction obtained When organisms
from various cultural phases were stained with D'Antoni's
iodine solution and expressed as the percentage of organisms
containing glycogen. These authors found that the accumu-
lation of glycogen reached a.maximum along with attainment
of the maximal population. As the cultures aged the level
of glycogen reserves decreased steadily. Stewart (1938)
stated that "young" organisms low in glycogen reserves were
first to die in culture. Ryley (1955) reported that Trit.
foetus appeared to maintain.motility and.metabolism through
fermentation of its glycogen reserves When.maintained
under non-nutrient conditions. Thus far no other aspects
of trichomonad physiology have been studied in reSpect to

possible correlation with the glycogen reserves.~

MATERIALS AND METHODS

Strain T-12l of Tritrichomonas augusta was used
throughout this investigation. A clone of the parent strain
(Clone 5b) was used for most of the experiments involved in
this work. Since there were no consistent differences
between the parent strain and the clone with respect to the
phenomena studied, the results for both have been combined
in this presentation.

All cultures of this organism were maintained at 29°C
in Diamond's (1957) medium modified with the addition of
10% 0.11 M NaZHPoLL to give a pH of 6.6 and by the substi-
tution of either 2 mM, H mM or 20 mM glucose for maltose.

Inocula for all experimental cultures were obtained
from steady state continuous-flow cultures described by
TWOhy (1961). In each experiment the inoculwm was adjusted
to give an initial population of approximately 1 x 105
organisms per ml.

The experimental cultures to be used for growth studies
were maintained in 500 ml "Tissue Culture Spinner Flasks"2
under a nitrogen atmosphere. Cultures used for survival
studies were maintained in 10 ml of culture medium contained
in 15 x 150 mm screwcap tubes.

Samples to be used for glycogen analysis, size deterj

minations, and population counts were taken at varying time

 

1Isolated by Dr. D. H. Twohy from Rana catesbiana on November
12, 1959, at Oklahoma State University.

2Belco Glass, Inc., Vineland, New Jersey.
-13-

-1u-
intervals after inoculation. All samples were withdrawn
with a sterile syringe attached to a 3—inch, 22—gauge needle
which was inserted through a rubber serum bottle stopper
fitted in the sidearm of the flask. The organisms in all
samples were killed and preserved in a final concentration
of 1% formalin, added as either a 2% or 5% solution in 80%
Krebs-Ringer solution (Umbriet et al., 1957).

Preserved samples to be counted were diluted ten-fold.
with 80% Krebs-Ringer solution. POpulation counts were
made on a Model-A Coulter.Counter3 fitted with a 100u aper-
ture and set at an aperture current setting of 2 and a
threshold value of 15. The counts were corrected for coin-
cidental passage of more than one organism at a time through
the aperture prior to the computation of the total number'
of organisms per ml of sample (Mattern, et al., 1957).

The sizes of the organisms were determined on the
Coulter Counter according to the methods described by
Mattern et a1. (1957) from samples prepared in a manner
similar to that used for population counts. Threshold values
were converted to cubic micron volumes by multiplying by a
constant derived empirically by comparing average threshold
values and packed cell volumes on each of several samples.

Organisms to be used for glycogen analyses were col-
lected in 15 m1 centrifuge tubes, killed and preserved as

described for population samples, and stored at hOC until

 

3Coulter Electronics, 5227 N. Kenmore, Chicago, Illinois.

-15-
analyzed. In preparation for analysis the samples were
centrifuged twice, once to free the organisms of medium
and again to wash the organisms after suSpension in 80%
Krebs-Ringer solution. After each centrifugation, the
supernatant was discarded. Glycogen was extracted from the
washed organisms by the method of Good et a1. (1933) and
assayed directly by the anthrone method for blood sugar

described by Roe (1955). Glycogen values were expressed
S

as micrograms per 10 organisms based on population counts
taken with each series of samples.

To test the survival of Trit. augusta in both aging
cultures and under non-nutrient conditions a series of 15
x 150 mm growth tubes containing 10 m1 of modified Diamond's
(1957) medium with either 2 mM or 20 mM glucose were used.
At each sampling interval after peak populations were
attained, three wet mount preparations from each of four
tubes were examined microscopically. Fifty organisms per
wet mount were examined for motility as evidenced'by movement
of either the flagella or undulating membrane. Survival
was expressed as the average per cent of motile forms from
the twelve preparations.

The organisms in each set of four tubes were then killed
and preserved. A p0pu1ation count was made and glycogen
was analyzed. Population counts were corrected by use of the

previously determined survival percentages in order to

express the amount of glycogen in terms of viable cells.

-16-
Calculation of the glycogen reserves on the basis of viable
organisms involved the assumption that: l) glycogen
reserves in dead organisms were nearly negligible and, 2)
the fragile, dead organisms would rupture and any remaining
glycogen would be removed in the supernatant while separating
organisms from medium by centrifugation.

Survival of Trit. augusta under non-nutrient conditions

 

was tested using organisms from h8 hour tube cultures
similar to those used as a source of organism in the pre-
vious survival experiment. The organisms in all samples
were centrifuged free of medium, and.the organisms in each
tube were resuspended in 10 ml of 80% Krebs-Ringer phos-
phate solution per tube (umbriet et al., 1957). At each
sampling interval the organisms in each of u tubes were
examined for motility and the population and glycogen were
determined‘by the methods previously described for aging
cultures. ‘

An approximation of the mass of organisms was obtained
by multiplying mean cell volumes by 1.038; the value reported
by Wichterman (1953) for the specific gravity of live 3335:
mecium species. Use of this value for specific gravity
was based upon the assumption that there is a close simil-

arity in the Specific gravities of protozoa.

RESULTS
I. A Comparison of Glycogen Content with the Cultural
Age of Tritrichomonas augggta in the presence of Limiting
and Non-Limiting Concentrations of Glucose.

The growth curves and corresponding intracellular
glycogen concentrations for Trit. aggusta are shown graph-
ically in Figures 1 through a. Figures 1 through 3 show
that the 33.11332 growth of 2533. aggusta followed a class-
ical growth curve. Due to the impossibility of disting-
uishing between live and dead organisms by the method of
counting employed in this investigation, growth curves were
followed only as far as the maximum stationary phase.
Separate experiments were run for each of two concentrations
of glucose. A comparison of Figures 1 and 3 shows that the
h,mM glucose concentration did not reduce the maximal popu-
lations below that attained with 20 mM glucose.

Glycogen constituted between C.8 and 7.3% of the total
wet cell weight. The respective nitrogen contents were
3.9 and 3.0%. Although dry weights were not determined,
glycogen probably constitutes a high percentage of the non-
aqueous constituents of the cells.

The results of the series of glycogen analyses of
organisms in various phases of culture are given in Figures
2 and u, During the lag and early logarithmic phases of
growth in.h.liglucose, glycogen increased quite rapidly
to a 35- to lOO-fold increase over the low glycogen content
of organisms used as the inoculum (Figure 2).» The level of
glycogen reserves began to decrease during.the remainder of

-17-

-18-
the logarithmic phase. The decrease in the level of reserve
glycogen was more pronounced during the deceleration phase
and continued through the maximal stationary phase of growth.

In the presence of excess glucose (20 mMJ the organisms
showed a similar glycogen increase during the lag and early
logarithmic phases of culture, followed by a rapid dr0p to
an intermediate level during the middle of the logarithmic
growth phase. The drop in glycogen reserves was more
pronounced with growth in the high glucose concentration
than in the low glucose concentration. The concentration
of stored glycogen per organism either continued to decrease
or remained at an intermediate level until the decelerating
growth phase. At that time it again began to increase until
the peak glycogen concentration equaled or exceeded the
amount accumulated during the lag phase.

Figures 5 and 6 Show the pattern of total glycogen
buildup in cultures oflggig. augggta in the presence offia
u mM.and a 20 mM glucose concentration. The rate of total
glycogen synthesis in both concentrations of glucose was
greatest in the first 15 hours, during which time the
organisms had passed through the lag phase and were begin-
ning the logarithmic phase of growth. During the remainder
of the logarithmic growth phase, glycogen accumulated at a
slightly slower rate. No significant differences in the
pattern of total glycogen synthesis were observed in cultures
with different glucose concentrations during the first 25-30

hours after inoculation. However, the maximum.level of

-19-
glycogen reserves attained during early growth varied
between experiments When a h mM glucose concentration was
used, indicating that other factors must influence the
maximum level of glycogen under these conditions.

In late phases of cultural growth in medium with the
h mM glucose concentration glycogen synthesis ceased, and
there was a decline in the level of total glycogen reserves
during the decelerating growth phase and the maximal station-
ary growth phase reSpectively (Figure 5). On the other hand,
Figure 6 shows a continued increase in the level of total
glycogen during the phase of decelerating growth when glu-
cose was in excess in the medium, During the maximal
stationary growth phase the total amount of glycogen reaChed
its maximum level which was maintained for the remainder of
this phase.

In summary, a comparison of growth and glycogen storage
in the presence of a.mM and 20 mM glucose showed that in both
types of culture the glycogen reserves reached a peak during
the lag and early logarithmic phase of growth. The level
of stored glycogen decreased during the logarithmic phase.

In the presence of 20 mM glucose glycogen again began to
increase during the decelerating growth phase and reached

a maximum when cultural growth ceased. In the presence

of h‘mM glucose glycogen reserves continued to decrease until
they reached a very low level during the maximum stationary
growth phase. However, the peak population attained by"

organisms with low glycogen reserves in the presence of the

-20-
limited glucose concentration was the same as that attained
with excess sugar.

Comparison of the total glycogen reserves in these two
types of culture indicates that glchgen was accumulated in
all phases of culture in the presence of excess glucose.
However, in the presence of limited glucose the level of

total glycogen decreased during late phases of culture.

Organisms x IDS/ml.

Glycogen (7)/IO5 Organisms

v

 

 

 

 

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s in glycogen in Trit. august
Il’ur— in JlflmOHd‘S medium with
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Jotal glycogen in Trit. an usta per m1 of
ul"ure employing uiamond's medium with a UM

 

 

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Total glycogen in Trit. auxusta per ml of

culture employing Diamond‘s medium with 20

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II. A Comparison of Cell Volume with the Cultural Age of
Tritrichomonas augusta in the presence of Limiting and Non-
Limiting Concentrations of Glucose.

There was a considerable range in size, expressed as
cell volume, of individual forms of Trit. augusta at all
stages of culture. Size frequency curves for samples.
taken from cultures at varying time intervals after inocula-
tion are given in Figure 7. Figure 7a. shows the charact-
eristic preponderance of very small organisms present in
the inocula for all experiments.

The mean cell volume of organisms raised in.medium
containing a h mM concentration of glucose was found to
increase during early phases of growth (Figure 8). This
was followed by a plateau or decrease in.mean cell volume
during the logarithmic growth phase. During the deceler-
ating and maximum stationary growth phases the mean cell
volume steadily decreased until the organisms attained a
mean volume very similar to thattof the organisms used in
the inoculum (Figures 7h and 8). Mean cell volumes and
glycogen content followed similar increases and decreases
over the course of the growth curve in.mediumcontaining
h mM glucose (Figure 2 and 8). The lower concentration
of glucose appears to have exerted a limiting influence
’ on both the glycogen storage and mean cell volume of Tait,
augusta during late stages of culture.

The pattern of variation of the mean cell volume of
organisms raised in 20 mM glucose differed among experi-

ments (Figure 9), and showed few common tendencies after the

-21,

-25-
initial increase in size during the lag phase. However,
there was a general tendency toward maintenance of larger
cell volumes during late phases of culture.

In summary, the u mM concentration of glucose appears
to be insufficient to meet the metabolic requirements of

Trit, augusta during late phases of culture. The result

 

was a decrease in cell volume. Conversely, the availability
of excess glucose in the medium enabled Trit. augusta to
maintain large cell volumes during late phases of culture.
The similarity of the fluctuations in mean cell volume
and glycogen content suggested a.more meaningful inter-
pretation of glycogen content might be obtained by basing
glycogen concentrations on a unit of cell volume. However,
a comparison of glycogen levels per unit of cell volume at
various phases of culture resulted in essentially the same

patterns as those based on the population (Figures 2 & h).

-26...

 

 

 

 

 

 

 

 

 

 

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Fig. 7a - Common Inoculum Fig. 7e - flax. Stationary
Phase
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10 10.0

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r1 0 iii—TT‘Tv—es

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Fig. 7b - ag :nase Fig. 7f - ~ogarithmic Phase
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15.0.. 15.0.1
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Fig. 7d - Doceleration Fig. 7h - Max. Statioaar
Fhase Phase
Figure 7. Cell volume frequency of Trit.9u1us:a grown

 

in Diamc d‘s leu“. “lgalce (7b) through
(7e) are from glcow'h in 20 mm glucose and
figures (7f) through (7h) in no mh glucose.

-27-

 

 

 

210a_,
1950 .
1800
,. 1650-.
m
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V '1
9 1500-
5
'5 1350..
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750
1 . 1 1 i 1 1 I ,1, 1
o 5 10 1.5 2.0 25 3035 to 45 50 55 e0

Hours

Figure 6. Mean cell volumes of Trit. augusta in
Diamond's medium with—h mM glucose.

Cell Volume (u3)
H
(n
U1
0

 

 

 

1 1 . 1 d
p 10 lb 20 29 30

Figure 9. Mean cell volumes of Trit. augusta in
Diamond‘s medium with 20 mM glucose.

 

III. The Effect of Glycogen Reserves on the Survival of
Tritrichomonas augusta in Culture Medium.

 

A series of MB hour cultures of Trit. augusta in tubes
of Diamond's (1957) medium with 2 mM glucose were used as
a source of organisms containing a low level of glycogen
reserves during the maximal stationary phase of growth.
Organisms high in glycogen content were obtained from
similar h8 hour cultures containing 20 mM glucose.

In the presence of 2 mM glucose the percentage of.
motile organisms gradually began to decrease after approxi-
mately 62 hours in culture. When the last sample was
examined at 168 hours after inoculation, hh% of the organisms
were found to be motile (Figure 10). All microscOpic exam-
inations made during this time interval revealed these
organisms to be small, slender and active. The glycogen
reserves of the organisms in this series of cultures were
minimal, remaining at a level which approached the limit-
ations of the method of analysis.

The percentage of motile organisms in cultures with
excess (20 mM) glucose declined sharply following the 71
'hour sample. Less than 9% of the organisms remained
motile 122 hours after inoculation (Figure 11). Microscopic
observations showed these organisms to be Spherical in
form throughout this period. Although the level of glycogen
reserves was high in MB hour cultures, the amount of gly-
cogen continued to increase until approximately 71 hours
after inoculation. After 71 hours, the glycogen reserves
decreased rapidly. Upon the termination of this experiment

-28;

-29-
122 hours after inoculation, a small reserve of glycogen
still remained in the few motile organisms present.

Results of the previous experiments showed that in
medium containing excess glucose 2313. aususta stored large
quantities of glycogen. Synthesis continued during the
maximum stationary growth phase. At a given point (approx-
imately 71 hours of culture in this investigation) both
glycogen reserves‘and motility started a rapid parallel
decline. On the other hand, organisms maintained in a'
medium with a limiting concentration of glycose contained
very low glycogen reserves during the maximum stationary
phase of growth. After this period a large percentage of

these organisms remained motile in Spite of their low level

of glycogen reserves.

Percent Motile Organisms

T“
‘

Percent Motile Organisms

 

80.? 8.0
I"
71.‘ 1.7.0
50—; .6.0
50.1 .5.0
bro: __I.1.0
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igure lO.

 

 

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L18 5::

Figure 11.

- — - = glycogen / lO5

 

‘5 _ I L I,“ I, I, l L,

oo 90' 1.05 11‘0‘12'0‘ 1361118 136168
Hours

Glycogen content and motility of Trit.

augusta after varied periods of time in

aging conditions following growth in

2 mM glucose. = per cent motility.

/ lO5 organisms.

 

— - - = glycogen

r_8.o
*_.7.o'
__a.o
..S'.o
,_.21.0
_3.0
.2.0
' __1.0

o
.l_ 1 [I l—l_‘l.l l l_l H
88 78 88 98108118 281381115158100

Hours
Glycogen content'and motility of Trit.

 

augusta after varied periods of time in

aging conditions following growth in
20 mM glucose. = per cent motility.
organisms.

 

Organisms

5

Glycogen (7')/10

Glycogen (7’)/IOS Organisms

IV. The Effect of Glycogen Reserves on the Survival of
Tritrichomonas augusta under Non-Nutrient Conditions.

 

Organisms possessing very low glycogen reserves were
obtained from AS hour tube cultures grown in a medium with
2 mM glucose as described in the previous section. Following
suspension of these organisms in 80% Krebs-Ringer saline,
the percentage of motile forms decreased rapidly (Figure 12).
Microsc0pic examinations 12 hours later revealed only 26%
of the organisms were motile. These surviving organisms
were slender and quite active. The very few organisms
remaining motile after 2n hours under these conditions were
spherical and exhibited very sluggish movement.

Organisms possessing relatively high glycogen reserves
were obtained from MB hour tube cultures containing 20 mM
glucose in the medium. After 2h hours under non-nutrient
conditions 96% of these organisms were still motile (Figure
13). Microscopic examination revealed these organisms to
be very active and spherical to moderately slender in form.
During the succeeding 2h hour period the percentage of
motile forms declined to 32% after MB hours under non-nutrient
conditions. The relative size and.motility of the surviving
individual organisms appeared to decrease progressively.
At 48 hours the remaining viable organisms were small and
quite sluggish, The dead organisms were usually smaller
than the living organisms. The intracellular glycogen
content declined in a linear fashion during the first 2h
hours to What may be considered a base level at this time.
The level of glycogen reserves u8 hours after suspension in

-31-

-32-
saline did not differ from the 2M hour values.

A comparison of Figures 12 and 13 shows a correlation
between the level of glycogen reserves in T313, augusta
and survival under non-nutrient conditions. Nearly 100%
of the organisms possessing low glycogen reserves died
in 2h hoursm and the majority were dead in 12.hours.-
However, organisms with a high glycogen content Showed
substantial survival after MB hours under non-nutrient
conditions.'

The data of this section present a survival pattern
which is the reverse of that of the preceding section.
Organisms possessing low glycogen reserves appear to have
been adapted to survival in old cultures, but died rapidly
when subjected to non-nutrient conditions. Conversely,
the organisms containing high glycogen reserves survived
for a much shorter period in old cultures, but utilized
their glycogen and survived under non-nutrient conditions

over twice as long as those possessing low glycogen reserves.

 

-33..

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Glycogen content and motility of Trit.
augusta after varied periods of time in

non-nutrient conditions following growth
2 mM glucose. =Hper cent motility.

 

- - - = glycogen / 109

 

 

organisms.

 

100_3

90
m 80_ .0 is:
5 . 'E
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Hours

 

Glycogen content and motility of Trit.
augusta after varied periods of time in
non-nutrient conditions following growth in
per cent motility.

 

20 mM glucose.
- - - = g1ycogen./ lOS

organisms.

DISCUSSION AND CONCLUSIONS

A polysaccharide assumed to be glycogen has been
extracted from cells of Tritrichomongs augusta by a
technique Specific for high molecular weight polysac-
charides. Two possible explanations for the presence of
this carbohydrate must be considered (Wilkinson, 1959).

A storage polysaccharide may be accumulated as either

an excess by-product which serves no function other than
as a reserve energy source, or as a functional endogenous
energy reserve which is utilized by the cell. With regard
to the glycogen stored by w, augusta, the latter explan-
ation, that this storage polysaccharide serves as a
functional carbon.and energy source, appears to be more
correct in view of the results of the experiments and the
interpretation of these results to follow.

According to Wilkinson (1959), the intracellular
accumulation of a carbon and energy reserve compound.must
occur at a time when the exogenous carbon and energy sources
arein.excess of the immediate metabolic requirements of the
cell. When 22£E° auggéta was grown in a.medium.containing
excess glucose, the environmental conditions necessary for
the accumulation of a reserve polysaccharide were present
throughout the experiment. Under these conditions, the
organisms responded by accumulating glycogen during all
phases of growth. This can be seen by comparing the total
glycogen per unit volume of culture with cultural age in
the presence of 20 mM glucose (Figure 6). Although the rate

-34-

-35-
of total glycogen accumulation varied, showing the most
rapid increases in the lag and logarithmic phases of growth,
there was no period when glycogen synthesis ceased or when
the net glycogen reserves were being utilized for growth.
Thus Wilkinson's first criterion for the existence of a
functional carbon and energy reserve has been satisfied by
considering the culture _i_._r_1. _t_g_1_3_9_.

The above discussion does not take into consideration
the glycogen concentration per organism. The glycogen con-
tent per 105 organisms in Figure u shows considerable
variation in glycogen levels at different phases of culture
with excess glucose. Glycogen reserves were highest during
the lag, decelerating, and.maximum stationary growth phases—-
the periods of less than optimal rates of growth. During
the logarithmic growth phase the glycogen content drOpped
to an intermediate level betWeen that of organisms in the
inoculum and the decelerating and maximum stationary growth
phases.

As organisms increase in number the proportion of
enzymes or sites for glycogen synthesis also increases and
the ratio of these enzymes or sites to the total mass of
protoplasm can be aSsumed to be constant regardless of the
size of the population. Thus, theoretically, the potential
rate of glycogen synthesis per unit of protoplasm would
be equal at all stages of cultural growth and consequently
the total potential for glycogen synthesis would increase 1

at the same relative rate as the increase in total protoplasm.

~36-

However, other incompletely known factors probably limit
the actual rate of synthesis so that it frequently falls
short of this potential. If the rate of glycogen synthesis
were proportional to the rate of growth, the rate of gly-
’ cogen accumulation in culture would equal the rate of proto-
plasmic increase in culture. In this case the glycogen per
organism (or more exactly the glycogen per unit of proto-
plasmic mass that most ideally represents growth) would
remain constant and the curves for the total population
plotted against time would be similar. If, on the other
hand, glycogen synthesis ceased and growth continued, the
amount of total glycogen in culture would remain unchanged;
but the concentration per organism would decrease. Conversely,
if glycogen synthesis occurred at-a constant rate but growth
ceased, glycogen would accumulate and the total glycogen
in culture as well as the amount per organism would increase.
The glycogen concentration is clearly dependent on both the
rate of glycogen synthesis and the rate of growth.

From the above assumptions a comparison of the growth
curve in Figure 3 with the curve for total glycogen in
Figure 6 allows a prediction of the effect of the rate of
growth upon the level of glycogen reserves. Initially
during the lag phase of growth, the rate of glycogen syn-
thesis was high while the rate of growth was Slow to moderate.
Glycogen should accumulate in the cells during this period.

As the organisms entered the logarithmic phase of growth,

-37-

the rate of glycogen synthesis decreased as depicted by
the slepe of the curve for total glycogen values (Figure 6).
Glycogen concentrations should then gradually decrease as
the excess glycogen is diluted to a level that can be main-
tained by the new rate of Synthesis. During the deceler-
ation phase of growth, which probably started shortly after
the 31 hour samples, glycogen synthesis seemed to continue
at the same rate, but the rate of growth decreased. The
result would be an increase in glycogen concentration.
These predictions follow very closely the observed changes
in glycogen concentration shown in Figure u.

The pattern of variation in glycogen concentration at
various cultural ages (Figure u) resembles that reported

for Escherichia coli B by Palmstierna (1955 & 1956) and for

 

Tetrahymena gglleii by Fennell and Marzke (195h). However,
Palmstierna's (1956) statement that glycogen is utilized
by g. 993:}; B during the logarithmic growth phase, based on
a decrease in total glycogen during that phase, did not
hold true for Tait. a ggsta.

The rate of glycogen synthesis showed some variation
over the course of cultural growth (Figure 6). The factors
that influence the rate of synthesis are not known (Neidhardt,
1963). Above minimal levels the concentration of glucose
in the medium is not usually a limiting factor. TWohy (1963)
has shown that the rate of growth of Tait. augusta is inde-

pendent of the concentration of glucose in the medium and

thus it would seem unreasonable to assume that the glucose

-38-
concentration would influence the rate of glucose uptake
or glycogen synthesis. It is quite possible, however,
that the rate of glucose uptake may have some upper limit
set by the transport mechanisms, enzyme capacity or ATP
pool of the cells. If most of the glucose is channeled
into energy metabolism essential to the growth of the
organism, less of the sugar may be available for glycogen
synthesis. Therefore, if the amount of glucose utilized
is directly related to the rate of growth as proposed by
Monod and Tessier (1936), Mbnod (l9u2 & 1950) and Nevick
and Szilard (19SO), and shown to be generally true for '
many species of bacteria if small energy requirements for
cell maintenance were discounted (Herbert et al., 1959),
the amount of growth would serve as an index of the amount
of glucose utilized as an energy and carbon source. Thus
the lower rate of total glycogen accumulation during the
logarithmic phase of culture may reflect a limiting rate of
glucose uptake and the conversion of mest of the assimilated
glucose into energy, but it would not account for the
continuation of the lower rate of g1yCogen synthesis that
seems to occur during the decelerating phase of growth. The
data of this investigation are insufficient, however, for
the calculation of rates of glucose uptake. In summary,
polysaccharide accumulation appears to be effected by the
rate of growth and the rate of glycogen synthesis and both

are shown to vary over the course of cultural growth.

-39-

Fulfillment of Wilkinson's second requirement that
"the compound is utilized when the supply of energy'from
exogenous sources is insufficient for the optimal maintenance
of viability ..." has been tested by following the levels
of glycogen reserves in organisms grown in cultures having
limiting concentrations of glucose and in organisms held
under non-nutrient conditions. When the exogenous supply
of glucOse became limiting in cultures containing a.mM
glucose both the mean glycogen concentration per cell and
the total glycogen in the population decreased showing
that the glycogen reserves were being utilized faster than
they were being synthesized (Figures 2 & 5). Thus the
organisms appeared to continue to multiply under these
conditions atishe expense of their reserves. The amount-
of total protOplasm may not have increased, however, to
the same extent as the number of organisms since there was
a decrease in mean cell volume during this period (Figures
7h and 8). Calculating the glycogen per unit of cell volume,
however, did not change the general tendency for a decrease
in glycogen reserves (Figure 2). The ability to use glyco—
gen was also shown When the organisms were suspended in
non-nutrient buffered saline. Theglycogen reserves
decreased during the period that the organisms remained viable
(Figure 13).

No attempt was made to obtain direct evidence for the
breakdown of glycogen to produce energy according to Wilkinp

son's third requirement, "that the compound is broken down

-uo-

to produce energy in a form utilizable by the cell, and
that it is, in fact, utilized for some purpose which gives
the cell a biological advantage in the struggle for existance
over those cells which do not have a comparable compound,...".
However, the glycogen of Trit. augusta does appear to pro-
vide the organisms with a utilizable form of energy. This
was observed When organisms with different levels of stored
glycogen were subjected to starvation conditions (Figures
12 & 13). Organisms with high levels of stored glycogen
remained motileand survived for a considerably longer period
of time than did organisms containing lower levels of reserve
glycogen. As previously pointed out,'high glycogen reserves‘
were steadily depleted during this period, indicating that
organisms rich in glycogen reserves derive a distinct
"biological advantage" in survival over organisms nearly
devoid of stored glycogen. Ryley (1955) observed that Tait.
foetus maintained motility and metabolism under non-nutrient
conditions through fermentation of intracellular glycogen.

In view of the foregoing discussion, the glycogen stored
by cells of Trit. augusta may be considered to serve as a
functional carbon and energy reserve. However, the data
obtained thus far does not rule out the possibility of
another form of carbon and energy reserve in this organism
(e.g. lipid or volutin). It was found that when aging
cultures were examined, the organisms grown in excess
glucose and possessing relatively high glycogen reserves died

off rapidly after peak populations were attained. On the

-hl-

other hand, organisms from cultures low in glucose, and
thus containing minimal amounts of glycogen, remained
viable,for a considerably longer period of time (Figures
10 & 11). An attempt to explain these results necessitates
a separate consideration of; 1) how organisms containing
minimal glycogen reserves survived for considerable periods
of time in the near absence of known energy sources, and
2) Why organisms possessing relatively high glycogen
reserves died off rapidly following the attainment of peak
population.w

EXplanation of the first phenomenon.may involve any
one or a combination of three possible survival mechanisms:
l)(the utilization of a major reServe energy source other
than glycogen, 2) the limited utilization of some other
component of the medium as an energy source besides glucose
at a ratejjust sufficient for survival, and 3) a.more econo-
mical utilization of minimal reserves or structural components
at a reduced rate of metabolism just sufficient for survival.
The first of these possibilities could easily involve stored
lipid compounds. Halevy (1963) was unable to find evidence
that any compounds of this nature were stored in quantities
essential for energy reserves in the cytoplasm of the closely
related trichomonad, Trit. foetus. The limited utilization
of other compounds in the medium as exogenous carbon and
energy sources remains to be demonstrated. The observations
that these organisms were rather small and slender in form,

as compared to the large spherical form of those with high

-t2-
levels of reserve glycogen, does suggest that Tait.
augusta is capable of utilizing structural cell components
as sources of carbon and energy for survival and possibly
very limited reproduction as has been proposed to occur
in Paramecium aurelia and species of Glaucome.(Tetrahymena)
(Harding, et al., 1937; and Kimball, et al., 1959). This
phenomenon also was observed to occur in Amoeba proteus
by Mast and Hahnert (1935) according to their description
of morphological changes accompanying starvation of this
organism. A more detailed analysis of cell components
before and after periods of time in glucose free medium
might clarify this observation.

The relatively rapid die off of organisms possessing
high levels of glycogen reserves in cultures with excess
glucose is probably the result of factors not related to
glucose uptake or the glycogen reserves. They may die from
accumulation of toxic wastes in the medium or from a critical
deficiency of a nutrient or nutrients other than glucose.
The decrease in the glycogen reserves of these organisms
may have been the result of abnormal and.unregulated cata-
bolic reactions occurring in moribund cells.

The mean cell volumes_of 2332: aggusta were investi-
gated at various cultural ages and with different levels of
glycogen reserves in the hope of detecting the existence
of more than one phSiologicallydistinguishable cell type.

The mean cell volume and frequency of larger cell volumes

 

of Trit. augusta increased during the lag and logarithmic

-u3-

phases of growth in the presence of both the h mM and
the 20 mM glucose concentrations, and the large size was
maintained during late phases of culture in the presence of
the 20 mM concentrations of glucose (Figures 7b'8: 8 & 9).
However, in medium with a h.mM.concentration of glucose the
mean cell volume decreased and the frequency of small forms
increased during the decelerating and maximal stationary
growth phases (Figures 7h & 8). Therefore, the availability
of excess glucose during the late phases of culture appears
to enable Tgit, auggsta to maintain increased cell volumes.
These observations are in agreement with those of Huntington
and Winslow (1937), Ormsbee (19h2), Loefer (1952), Scherbaum
(1956), and Summers (1963) who used several Species of
bacteria as well as by Tetrahymena gelleii and T, pyriformis.

The glycogen concentration was calculated to be between
O.8-7.3% of the total protoplasmic weight.) The specific
gravity of glycogen is greater than.that of water so the
actual contribution to cytoplasmic volume would be less than
the weight of the glycogen and certainly much less than the
variation encountered in cell volume. This conclusion is
based upon the assumption that glycogen is stored as a pool
neither associated with additional cell Structure nor
combined with other compounds which would significantly
increase the volume of the organism.‘ These general consid-
erations seem to rule out the volume of glycogen as the
cause of increases in cell volume since the extremes of

cell volumes represent a two-fold difference and glycogen

-1717- ‘
contributions to calculated cell weight was only 7.3%.
These observations suggest that simultaneous increases and
decreases in glycogen content and cell volume are the result
of the influence of the particular environmental conditions
upon two independent aspects of the physiology of this
trichomonad.

Interest in the_factors influencing the survival of

Trit. augusta was stimulated by consideration of the nec-

 

essity of its survival outside the host during the course
of oral transmission in amphibia reported by Bishop (1930
& 193A). In order to infect a new host via the oral route,
Tait. augusta must be able to endure unfavorable conditions
outside its host, as well as the low pH of the stomach of
a prospective host, without the protective advantage of a
cyst-like stage (Kofoid and Swezy, 1915; Bishop, 1930 &
193h; Rosenberg, 1936; and Buttrey, 195h). Therefore, an
attempt to determine if'a physiologically more hardy stage
exists in a population of basically delicate organisms
seemed worth While.

Although at present the significance of the considerable
range in cell sizes attainable-by a given species of micro-
organism is poorly understood (Neidhardt, 1963), two features
observed in this investigation.seem worthy of comment. A
population of predominantly small organisms containing
minimal glycogen reserves was produced in an environment
nearly devoid of known carbon and energy sources. These

organisms were capable of surviving for a considerable

-u5-
period of time in such an environment, however, they died
rapidly upon suspension in an environment deficient in
all organic nutrients. These observations suggest that
this type of pepulation is achieved automatically in the
absence of glucose and may be capable of surviving during
extended periods of hibernation of the hoSt if carbohydrate
were the limiting nutrient.) 0n.the other hand, the avail-
ability of excess glucose in the medium enabled this organism
to accumulate high glycogen reserves and maintain large
cell volume. Unlike the previous population these organisms
died rapidly in culture following attainment of maximum
populations, but were capable of surviving under non-nutrient
conditions for a moderate period of time through the fermene'
tation of glycogen reserves. These organisms would be
particularly well adapted for surviving the adverSities
encountered during transmission to another amphibian via

the oral route.

SUMMARY
1. Population, polysaccharide content, and cell
volume were determined over the course of growth of Trit-

ichomonas augusta in a medium containing a limiting and a

non-limiting concentration of glucose. Polysaccharide
content, motility and survival time of organisms with
radically different initial levels of stored polysaccharide
were measured and compared during extended periods in aging
cultures and under non-nutrient conditions.

2. In the presence of an excess concentration of
glucose the level of total polysaccharide in culture con;
tinued to increase throughout all phases of growth. How-
ever, two peaks were observed in the polysaccharide con-
centration per organism; one during the lag and early
logarithmic phases of growth, and a second during the
decelerating and maximum stationary phases of growth.
During the logarithmic growth phase, glycogen reserves
decreased to a level intermediate between that in the
organisms of the inoculum and those containing maximal
reserves. Following an initial increase, EEiE- augusta
maintained large cell volumes through all phases of growth.

3. Organisms grown in.medium with a limiting con-
centration of glucose continued to increase in numbers,
but decreased in cell Volume during the decelerating and
maximum stationary phases of growth. They appeared to

utiliZe their reserve polysaccharide as a source of energy

-ue-

-u7-
for survival and limited reproduction, as observed from a
decrease in the total polysaccharide in culture during the
above growth phases.

A. The maximal level of polysaccharide in Trit. augusta
accounted for only 7.3% of the total cell weight of large
organisms. Thus the volume of accumulated polysaccharide
was not considered to be the cause of increased cell volumes
in this organism.

5. When survival and the level of glycogen reserVes
were compared during phases of population decline, organisms
possessing high polysaccharide reserves and large cell vol—
umes from growth in excess glucose died off rapidly. This
was believed to be the result of adverse environmental
conditions. Organisms containing minimal polysaccharide
reserves and small cell volumes as a result of growth in a
medium nearly devoid of known energy sources remained viable
in culture for a considerably extended period of time. This
observation seemed to indicate that polysaccharide storage
is not the only survival mechanism available to 3533.
augusta.

6. When organisms possessing high levels of poly—
saccharide reserves were suspended in an inorganic buffered
saline, they remained viable for moderate periods of time
and polysaccharide reserves declined gradually. However,
small organisms containing minimal polysaccharide reserves
died off rapidly under these conditions.

7. The polysaccharide was accumulated by Trit. augusta

-u8_
during periods When exogenous sources of energy were insuf-
ficient to meet the metabolic needs of the cell, and was
broken down to yield energy in a,form utilized by the cells,
giving them a distinct "biological advantage" over cells
without accumulated polysaccharide reserves. These obser-
vations indicated that the polysaccharide in‘ggit. agggsta
meets the requirements of a functional carbon and energy
reserve.

8. The population of small organisms containing
minimal glycogen reserves was achieved automatically in the
near absence of glucose. These organisms may be expected
to be capable of surviving similar environmental conditions
during extended periods of hibernation of the host. The
pOpulation of large organisms possessing high polysaccharide
reserves was capable of surviving moderate periods of time
in the absence of all organic nutrients.' These organisms
may be particularly well adapted to surviving the adver-
sities encountered during oral transmissiOn to another '

amphibian host.

LITERATURE CITED

Armor, J. M. l9uh. Influence of the diet of Blattidae
on some of their intestinal protozoa. J. Parasit.

39: 131-1un.

Bishop, A. 1930. The action of hydrochloric acid upon
cultures of Trichomonas. Parasitology 22: 230-2ul.

. 193E. The experimental infection of Amphibia with
cultures of Trichomonas. Parasitology 2Q: 26-33.

Buttrey, B. W. 195u. Morphological variations in Trit-‘
richomonas au usta (Alexeieff) from Amphibia. .
Morphol. 9 : 12 -163.

 

Concannon, J. N. 1959. Carbohydrate utilization patterns
of ten axenic isolates of Tritrichomonas augusta.
J. Protozool. §.(Suppl): 23.

Diamond, L. S. 1957. The establishment of various tricho-
monads of animals and man in axenic culture. J.

Parasit. 42; EBB-A90.

Doran, D. J. 1958. Studies on trichomonads. II. The
metabolism of a Trichomonas batrachomm-type flagellate
from the cecum of swine. J. ProtozooI. 5; 89-93.

. 1959. Studies on trichomonads. III. Inhibitors,
acid production, and substrate utilization by four

strains of Tritrichomonas foetus. J. Protozool. é;
177-182.

Duguid, J. P., and J. F. Wilkinson. 1953. The influence
of cultural conditions on polysaccharide production
b Aerobacter aerggenes. J. Gen. Microbiol. 9; 17h-
l 9. T T ,

Feinberg, J. G., and W. T. J. Morgan. 1953. The isolation
of a specific substance and a glycogen-like polysacchah
ride from Trichomonas foetus (var. Manley)» Brit. J.
Expt. Path.. 3E: IUH- .

Fennell, R. A., and F. O. Maere. 195n. ‘The relation between
vitamins, inorganic salts, and the histochemical char-
acteristics of Tetrahymena gelleii. .W. J. Morphol.

Gill, B. S., and H. N. Ray. 195u. Glycogen and its probable

significance in Eimeria tenella Railliet and Lucet, 1891
Ind. J. vet. Sci. and AnimaI Hush. gg; 223-228.

-49-

 

 

-50-

GOOd, Co Ac, Ho Kramer, and Me,Som08yj.o 19330 The deter-
mination of glycogen. J. Biol. Chem. 100: u85-h91.

Halevy, S. 1963. Lipid composition and.metabolism of
Trichomonas foetus (28272). Proc. Soc. Exptl. Biol.

Harding, J. P. 1937.1 Quantitative studies on the ciliate
Glaucoma. II. The effects of starvation. J. Exptl.

Biol. lg; u3o;u39.

Herbert, D. 1959. Some principles of continuous culture.
Rec. Prog. Microbiol., Symp. VII Intern. Congr. ,2“
for Microbiology, Stockholm. 381-396. E

Holme, T., and H. Pahmstierna. 1956a. 0n.the synthesis !
and breakdown of a glycogen-like polysaccharide in i
Esdherichia coli B. Acta. Chem. Scand. 19; 155.

“A ‘

7 . 1956b. Changes in glycogen and nitrogen~containing
compounds in_Ehcherichia_cgli B during growth in
deficient media. I. Nitro en and carbon starvation.

Acta. Chem. Scand. ‘19: ‘57 -586.

. 1956c.- Changes in glyco en and nitrogenpcontaining
compounds in Esdherichia c‘ i B during growth in
deficient media. II. >Phosphorous and sulfur star-
vation. Acta. Chem. Scand. 19; 1553-1556.

. 1956d. On the glycogen in Escherichia coli B; its
synthesis and breakdown and its specific IaEeling with
th. Acta. Chem. Scand. g9; .1557-1561. .

Holme, T. and.B. Cedergren. 1961. Demonstration of intra-
cellular polysaccharide in Escherichia coli by electron
microscOpy and by cytochemical methods. Acta. Path.

MicrObiOle 3(3me 21...: 179‘1860

Hunsate. R. E. l9h3. Further experiments on the cellulose
digestion by the protozoa in the rumen of cattle. Biol.

B1111. @A: 157“].630

Huntington, E., and C. E. A. Winslow. 1937. Cell size and
metabolic activity at various phases of the bacterial
cultural cycle. J. Bacteriol. 33; 123-143.

Hutner, S. H., and L. Provasoli. 1955. Comparative biochem-
istry of flagellates. Biochemistry and Physiology of
Protozoa. ed. 8. H.'Hutner and A. Lwoff-Acad. Press,

1110., New York, V01. E: 17-MO

-51-

Johnson, G. l9u2. Physiology of bacteria-free culture of
Trichomonas vaginalis. IV. Effect of hydrogen ion
concentratiofifand oxygen tension on population. J.
Parasit. '28: 369-379.

Kimball, R. F., T. O. CaSperson, G. Svenson, and L. Carlson.
1959. Quantitative cytochemical studies on Paramecium
aurelia. I. Growth in total dry weight measured by
the scanning interference microscope and X-Ray absorp-
tion methods. ExptloCell. Res. 11; 160-172.

Kofoid, C. A., and O. Swezy. 1915. Mitosis and.multiple
fission in trichomonad flagellates. Procs Am. Acad.
Arts and Sci. ‘51: 289-37 .

Lankford, D. E., H. Hoyd, and J. F. Lutteringer. 1951.
Intracellular polysaccharide of Enterobacteriaceae.
J. Bacteriol. 62: 621-626.

Levine, 3., H. J. R. Stevenson, E. C. Tabor, R. H. Bordner,
and L. A. Chambers. 1953. Glycogen of enteric bacteria.
J. Bacteriol. 66; 664-670.

Lindblom, G. P. 1961. Carbohydrate metabolism of tricho-
monads: growth, respiration, and enzyme activity_in
four species. Protozool. 8; 139-150.

Loefer,J. B. 1952. Some observations on the size of
Tetrahymena. J. Morphol. 29; nO7-uln.

Lwoff, M. 1951. The nutrition of parasitic flagellates
(Trypanosomidae, Trichomonadinae). Biochemistry and
Physiology of Protozoa ed. A. Lwoff. Acad. Press
Inc., New York, vol. I} 129-173.

Lyford, H. S. 19ul. Some reactions of a pathogenic flag-
ellate, Trichomonas foetus, to environmental changes
in bacteria-free cultures. Am. J. Hyg. 33: 69-87.

Manners, D. J., and J. F. Ryley. 1955. Studies on the
metabolism of the protozoa. 6. The glycogen of the
parasitic flagellates Trichomonas foetus and Tricho-
monas gallinae. Biochem. 32 52; 369-372.

Masson, F. M., and A. E. Oxford. 1951. The action of the
ciliates of the sheep's rumen upon various water-soluble
carbohydrates, including polysaccharides. J. Gen.
Microbiol. 5; 66u-672.

Mast, S. 0., and W. F. Hahnert. 1935. Feeding, digestion,
and starvation in Amoeba proteus_(Leidy). Physiol.
2001. Q; 255-272.

 

-52-

Mattern, C. F., F. S. Brackett, and B. J. Olsen. 1957.
Determination of number and size of particles by
electrical gating: blood cells. J. Appl. Physiol.
E: 56-700 .

Monad, J. 19u2. Recherches sur la croissance des cultures
bacteriennes. Hemann., Paris, France. pp. 210.

. 1950. La technique de culture continue; theoric
et applications. Ann. Inst. Pasteur 12; 390-u10.

., and G. Tessier. 1936. La concentration de 1'aliment,
facteur quantitatif de l'accroissement des pepulations
d'infusories. Compt. Rend. 202: 162-16u.

Neidhardt, F. C. 1963. Effects of environment on the com-
Eosggion of bacterial cells. Ann. Rev. Microbiol. 11;
1" Q -

Ninomiya, H., and Z. Suzouki. 1952. The metabolism of
' Trichomonas va inalis, with comparative aSpects of
trichomonads. 3. Biol. Chem. 32; 321-331.- -

 

Novick, A., and L. Szilard. 1950. Experiments with the
chemostat on spontaneous mutations of bacteria. Proc.
Nat. Acad. Sci., wash. 39; 708-719.

Ormsbee, R. A. 1942. The normal growth and respiration of
Tetrahymena gelleii. Biol. Bull. @2; h23-n37.

Oxford, A. E. 1951. The conversion of certain soluble
sugars to a glucosan by holotrich ciliates in the rumen
of sheep. J. Gen. Microbiol. 5; 83-90. .

Palmstierna, H. 1955. The content of polyglucose of glyco-
genic nature during the first hours of growth in Escher-
ichia coli B. Acta. Chem. Scand. 23 195-196-

. 1956. Glycogen-like polyglucose in Escherichia coli
B during the first hours of growth.'lActa. Chem. Scand.

19.: 567-577.

Read, 0. P. 1955. Comparative studies on the physiology
cg trichomonad flagellates. J. Parasit. gg; (Suppl)
1 .

. 1957. Comparative studies on the physiology of
trichomonad protozoa. J. Parasit. Q3; 385-39h-

Roe, J. H. 1955. The determination of sugar in blood and
spinal fluid with anthrone reagent. J. Biol. Chem.

3§E§= BBS-3&3.

-53-

Rosenberg, L. E. 1936. On the viability of Tritrichomonas
augusta. Trans. Am. Micr. Soc. 55} 313-3lh.

Ryley, F. J. 1955. Studies on the metabolism of protozoa..
5. Metabolism of the parasitic flagellate Trichomonas
foetus. Biochem. J. ‘52: 361-369.

Scherbaum, D. 1956. Cell growth in normal and synchronou61y
dividing mass cultures of Tetrahymena pyriformis. Exptl.
Cell Res. 11; ten-A76. _F'

8013, A. 1961. Carbohydrate metabolism. Ann. Rev. Biochem.
3_; 213-238.

Stewart, H. M. 1938. Glycogen content of a flagellate of
cattle, Trichomonas foetus. Am. J. Hyg. 28: 80—8u.

Sugden, B., and A. E. Oxford. 1952. Some cultural studies
with holotrich protozoa of the sheep's rumen. J.
Gen. Microbiol. .1: 1&5-153.

Summers, L. G. 1963. Variation of cell and nuclear volume
of Tetrahymena pyriformis with three parameters of
growth: age of culture, age of cell, and generation.
J. Protozool. ‘19: 288- 293.

Suzouki, Z., and T. Suzouki. 1951a. Carbohydrate meta-
bolism of Trichomonas foetus. J. Biol. Chem. 38:

237-25H-

. 1951b. Hydrogen evolution b Trichomonas foetus.
Nature 168: 610.

Twohy, D. W. 1959. The physiology and nutrtion of intest-
inal flagellates. (Unpublished)

. 1959. Carbohydrates supporting the growth of Trit-
richomonas augusta. J. Protozool. 6; (Suppl. ) 25.

. 1961. A continuous flow culture system for Trit-
richomonas augusta. J. Protozool. §3 (Suppl.) 5.

., and P. A. Tucker. 1961. Characteristics of Trit-
richomonas augusta from a continuous flow culture.
J. Protozool. Q; (Suppl.) 5.

. 1963. (Unpublished data)

Umbreit, W. W., R. H. Burris, and J. F. Stauffer. 1957.
Manometric Techniques, Burgess Pub., Mineapolis, Minesota,

3rd. ed., 338 pp.

 

-gu-

von Brand, T. 1952. Chemical Physiology of Endoparasitic
Animals. Acad. Press., Inc. New York, 339 pp.

Wantland, w. E., E. M. Wantland, and T. A. Weidman. 1962.,
C tochemical studies on Trichomonas tenax. J. Parasit.

L: 305.

Wellerson, R., G. Doscher, and A. B. Kupferberg. 1959.
Metabolic studies on Trichomonas vaginalis. Ann.
New York Acad. Sci. §35 253-258. ' .

Wellerson, H., and A. B. Kupferberg. 1962. On glycolysis
in Trichomonas vaginalis. J. Protozool. 2; R18-

LLZLL.

Wflchterman, R. 1953. The biology of Paramecium, The
Balkiston Co., Inc., New Ybrk, 527 pp.

Wilkinson, J. F. 1959. The problem of energy-storage
compounds in bacteria. Expt. Cell Res., Suppl. 1;
111-130 9,

Wilkinson, J. F., and J. P. Duguid. 1960. The influence
of cultural conditions on bacterial cytology. Intern.
Rev. CytOlo 2: 1'76.

Wirtschafter, S. M. and T. L. Jahn. 1956. The metabolism)
of Trichomonas va inalis: The glycoglytic pathway.
J. Protozool. 3; 83-35.

 

 

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