AN INVESTIGATION OF FACTORS WHICH AFFECT COLONY FORM AND GROWTH

IN GONIUM PECTORALE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

lynn Boyd Graves, Jr., B.A., M.Sc.

The Ohio State University 1959

Approved by

0 4 ^ d .

Adviser Department of Zoology and Entomology ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation to Professor

W* J* Kostir for suggesting this problem and for his helpful advice and criticism throughout the course of this study, and to Dr* A. C* Broad for his help in the design and construction of the constant temperature illuminated water bath used in this investigation*

ii TABLE OF CONTENTS

Page

INTRODUCTION...... 1

The Organism Studied...... I The Form of the Colony in Culture...... 3 Past Investigations of Changes in Form in Gonlun pectorale . • it The Scope of the Present Investigation ...... 5

MATERIALS AND METHODS...... 7

Source of the Organism Studied ...... 7 General Methods of Culture •••.•• ...... 7 The Constant Temperature Water Bath...... 12 Method of Sampling the Culture ....♦...... 13 Statistical Method ••••...... 15

A DESCRIPTION OF THE CHANGES IN THE FORM AND IN THE RATE OF GROWTH OF THE COLONY THAT ACCOMPANY AGING OF CULTURES IN 0.05 PER CENT KNOP SOLUTION...... 17

INVESTIGATIONS OF THE POSSIBILITY THAT A DEFICIENCY OF SOME SUBSTANCE OR SUBSTANCES IS RESPONSIBLE FOR THE FORMATION OF ABNORMAL COLONIES AND FOR THE RETARDATION OF GROWTH...... 19

The Influence of Different Coneaitratione of Carbonate on the Form of the Colony and on the Rate of Growth. • • • • 19 The Influence of Vitamin B12 on the Form of the Colony and on the Rate of Growth. ••••••.•• ...... 22 The Influence of Iron on the Form of the Colony and on the Rate of Growth 2h The Influence of the Calcium Ion Concentration on the Form of the Colony and on the Rate of Growth ...... 26 The Influence of the Concentration of Knop Solution on the Form of the Colony and on the Rate of G r o w t h ...... 29 The Influence of the Depletion of the Inorganic Carbon on the Form of the Colony and on the Rate of Growth. • • • • 30

INVESTIGATIONS OF THE POSSIBILITY THAT GONIUM PECTORALE PRODUCES AUTOTOXIC SUBSTANCES, WHICH INFLUENCE"HW15tSffi tftfOCRM OF THE COLONY AND THE RATE OF GROWTH...... 32

The Influence of Inocula Containing Fluid from Aged Cultures on the Form of the Colony and on the Rate of Growth. ••••.••••••••••••• ...... 32

iii iv

Page

The Influence of the Dilution of an Aged Culture on the Form of the Colony and on the Rate of Growth•••••••• 3U The Influence of Filtrates from Aged Cultures on Young Cultures ...... 35 The Relation of the pH of theMedium to theForm of the Colony and to Motility...... * ...... 38

DISCUSSION...... hO

SUMMARY...... Uli

BIBLIOGRAPHY...... 16

AUTOBIOGRAPHY...... 81 LIST OF TABLES t a b l e p a g e

1 The Percentage of the Total Number of Cells which is Represented by the Number of Cells in Each of the Differently-Celled Forms in an Eight Day Old Culture in 0.0!? Per Cent Knop Solution...... 50

2 Changes in the Form of the Colony and in the Growth that Accompany Aging of Cultures in 0*05 Per Cent Knop Solution* • • « ...... * * . • ...... 51

3 The Influence of Different Concentrations of Carbonate on the Form of the Colony and on the Rate of Growth. • • 52

I* The Influence of Vitamin on the Form of the Colony and on the Rate of Growth...... 53

5 The Influence of Iron on the Form of the Colony and on the Rate of Growth ...... 5ii

6 The Influence of the Calcium Ion Concentration on the Form of the Colony and on the Rate of Growth ..•••• 55

7 The Influence of the Concentration of Knop Solution on the Form of the Colony and on the Rate of Growth .... 57

8 The Influence of the Depletion of the Inorganic Carbon on the Form of the Colony and on the Rate of Growth* * * 58

9 The Influence of Inocula Containing Fluid from Aged Cultures on the Form of the Colony and on the Rate of Growth* * ...... 59

10 The Influence of Inocula Containing Fluid from Aged Cultures on the F o m of the Colony and on the Rate of Growth. A Repetition ...... 60

11 The Influence of the Dilution of an Aged Culture on the Form of the Colony and on the Rate of Growth • • • • 61

12 The Influence of Filtrates from Aged Cultures on Young Cultures ...... * ...... 63

13 The Relation of the pH of the Medium to the Form of the Colony and to the Motility ...... 66

v U S T OF FIGURES

PAGE FIGURE

1 Normal and Abnormal Colonies of Goniuin pectorale ...•••« 67

2 The Constant Temperature Illuminated Water Bath...... 68

3 Changes in the Form of the Colony and in the Rate of Growth of an Aging Culture...... 69

U The Influence of Different Concentrations of Carbonate on the Rate of Growth...... 70

5 The Influence of Vitamin Bqjj on the Rate of Growth...... 71

6 The Influence of Iron on the Rate of Growth. 72

7 The Influence of the Calcium Ion Concentration onthe Rate of Growth ...... 73

8 The Influence of the Calcium Ion Concentration on the Form of the Colony ...... 7li

9 The Influence of the Concentration of Knop Solution on the Rate of Growth ...... 75

10 The Influence of the Rate of Depletion of the Inorganic Carbon on the Rate of Growth ...... *••••• 76

11 The Influence of Inocula Containing Fluid from Aged Cultures on the Rate of Growth ...... 77

12 The Influence of Dilution of Aged Cultures on the Rate of Growth ...... 73

13 The Influence of Filtrates from Aged Cultures on the Rate of Growth of YoungCultures 79

lit The Influence of Filtrates from Aged Cultures on the Form of the Colony of Young Cultures 80

vi INTRODUCTION

The Organism Studied

Gonlum pectorale is a colonial protozoan which is frequently en­ countered in many bodies of fresh water. I have found it in rivers, lakes, farm ponds, road-side ditches, and water-filled ruts in dirt roads. Pocock (1955), who has collected Gonlum over large areas of

North America, believes that G. pectorale is the most common of all the

colonial Fhytomonadida* She found it in temporary fresh water pools,

ponds in pasture lands, lakes, and in cultures of soil collected as much

as four years previously. Crow (1927) observed the species in a rain barrel and Kostir (personal conmunication) observed a rich growth of

Gonlum in a discarded pan which had previously been used for storing

frogs in an ice box.

Gonium pectorale was originally described by 0. F. ifiller in 1773.

The 16-celled colony forms a nearly square plate which is usually bent back slightly along the two diagonals. The center of the colony is bounded by four of the sixteen cells, while the remaining twelve cells

are distributed, three to a side, around these central four. The colo­

nies studied ranged in size from approximately 2h to lOOyu in diameter,

with each cell being about 7 ** 13^1 wide to 7 - 1 Iji long. The pellicle

of each cell adheres to the pellicles of the adjacent cells. When -the

colony is quite small the pellicle of each cell fits tightly against the

1 2 plasma membrane of the cell and shows no distortion where it adheres to other cells* But as the colony grows, the space between the plasma mem­ brane and the pellicle widens, and there is a distortion of the pellicle to form short protuberances in the area in which one cell adheres to another* Each cell of the colony produces a gelatinous material which unites with that produced by the other cells of the colony and forms a common matrix about the entire colony*

Each cell contains one vase-shaped chloroplast which has one or two large pyrenoids in its base* There are also two flagella, two contrac­ tile vacuoles, and a large eye-spot to each cell* It has been reported that delicate cytoplasmic processes connect the cells to one another

(Ehreriberg, 18385 Harper, 1912; Mast, 1916; Bock, 1926; Crow, 1927;

Tilden, 193$; Prescott et al*, 19ii9) • Stein (19$8b) was unable to ob­ serve such processes in either stained or unstained living material or

in fixed material using either the ordinary light microscope or the phase microscope* The observations made in the present study agree with those

of Stein*

Pascher (1927) lists two species of Gonlum which possess sixteen n cells, G, formosua Pascher, and G, pectorale 0* F* Muller* The main dif­

ference between these two species is that the cells in G* formosum are widely separated and pear-shaped, while the cells in 0. pectorale are

close together and oval* Prescott (19U2) described a 32-celled species,

G. dlacoidemn* that rarely produces l6 -celled forms* Its cells were ir­

regularly pyriform with two basal pyrenoids* Pocock (1955) described an

8-ceUed species, Q* octonariun, which always has two center cells and six outer cells, an arrangement never found in G, pectorale* She also described a species of 8, 16, or 32 cells, G* milticoccum, whose cells may contain many pyrenoids while the cells of G, pectorale contain only one pyrenoid which divides into two Just before the cell divides* She also described a new variety of G, pectorale, G* pectorale var. praecox.

The mature colonies of this variety are comparatively small, usually rather pale green and slightly oblong in shape* The cells are somewhat elongated, ovoid in side view, with the apex usually wider than the base, and contain a single pyrenoid. The apical region is colorless and larger and more conspicuous than in var* pectorale. Also, daughter colony forma­ tion often begins in quite small colonies in var, praecox. while only large, mature colonies of var* pectorale reproduce. The organisms used in this study belonged definitely to var, pectorale*

The Form of the Colony in Culture

Although 8- and U-celled colonies of G* pectorale have been observed in nature (as by Harper, 1912 and Pascher, 1927), any person who has attempted to maintain Qoniua in the laboratory will be familiar with these and the many other non-16-celled forms which occur under such condi­ tions* A culture which is several weeks old may contain forms with every number of cells from the l6 -celled to the 1-celled form. Photographs of various forms are shown in Figure 1, and the relative abundance of these forms in an aged culture is shown in Table 1,

The production of abnormal colonies is not an uncommon occurrence in the colonial Phytamonadida• Hartmann (19210 discussed the formation of abnormal colonies of Eudorina slogans, Pocock (1955) described u colonies with fewer than the normal number of cells in all the species and varieties of Qonlum she studied* Stein (19!>8a) stated that the colonies in older cultures of Astrephomene gubernaculifera tend to fall apart, and that the individual cells with their gelatinous matrices may be seen "spinning aimlessly at the bottom of the container*"

Past Investigations of Changes in Form in Qonium pectorale

Harper (1912) studied G. pectorale which he collected in open pools*

He noted that the 16-celled forms began to disappear from a pool in which they had been numerous and that 8- and U-celled forms became relatively more abundant* He observed that some of the 16-celled colonies split in half to form two 8-celled colonies each* He believed that the colonies were under considerable tension, because certain sudden changes in the surrounding medium would often cause the colony to "fly apart with a suc­ cession of sudden Jerks*"

Hartmann C1921*) maintained the same species in culture in the labo­ ratory* He found that when he maintained it in Knop solution (Hartmann,

1928) each culture at the outset consisted of both 8- and 16-celled forms. As the culture grew older, the colonies tended to degenerate to

8- and U-celled forms, and even to 1-celled forms* He found that this degeneration occurred more rapidly in concentrations of Knop solution that were much stronger than the 0*0$% that he originally used* When he maintained Qonium in Benecke solution (Hartmann, 1928) he found that the

16-celled forms increased in relative abundance until 8-celled forma were

found only occasionally* The cultures in this medium, however, rapidly 5 died at the end of about two weeks. Hartmann believed that an accumula­ tion of toxic substances was responsible for both the degeneration in

Knop solution and the death in Benecke solution.

Crew (1927) studied the production ef abnormal colonies of G. pectorale in an evaporating culture in Knop solution. He observed the break-up of many-celled aggregates into fewer-celled groups, and even to the 1-celled forms, which became predominant by the end of two weeks.

Crow believed that the increasing concentration of the solution, and especially the accumulating organic material from the decay of many colo­ nies, caused the progressive degeneration of the colonies.

Previously I had found that the addition of vitamin to cultures of G. pectorale increased the relative abundance of the 16-celled colo­ nies (Graves, 1957)* I also found that by passing 1# carbon dioxide through a culture for fifteen minutes each day, the relative abundance of the normal, 16-celled colonies was greatly increased and that a much richer growth was obtained than in those cultures through which carbon dioxide was not passed. Cultures combining the use of vitamin and carbon dioxide produced still more favorable results. I believed that the slower growth and the progressive degeneration that was characteristic of an aging culture was related to the crowded condition of the culture, and the accompanying depletion of its carbon dioxide supply.

The Scope of the Present Investigation

It will be seen from the preceding discussion that Hartmann and Crow both believed that a progressive accumulation of substances in the 6 culture medium was responsible for the abnormalities which accompanied aging of the cultures* On the other hand, I believed, then, that a de­ pletion of the inorganic carbon source and a deficiency of vitamin B^2 were chiefly responsible for the abnormalities# The possibility that toxic substances might also be responsible in part for the abnormalities was not completely eliminated, nor was the possibility of a depletion of other nutrient substances besides the inorganic carbon appreciably in­ vestigated. When Hartmann rejected the idea that depletion of one or more of the nutrient salts might be responsible for the abnormalities, he did so because cultures maintained in stronger solutions, as 0.1/6

Knop solution, showed the abnormalities sooner than those maintained in

0.0536 Knop solution. However, he did not consider the possibility that the stronger solutions might themselves be injurious to the colonies*

The main purpose of this work has been to investigate further the possibility that the abnormalities which accompany aging of a culture of

Gonlum pectorale are caused either by an accumulation of toxic substances

or by the depletion or deficiency of some nutrient substance or sub­

stances, or perhaps by a combination of both. MATERIALS AND METHODS

Source of the Organism Studied

The organisms used in this study were from a clone which was iso­ lated frcsn a culture supplied by Carolina Biological Supply Company, Elon

College, North Carolina. The original 16-celled colony from which the clone was established was freed from all other organisms except bacteria by repeated isolation and transfer to sterile culture medium with the aid of a stereoscopic microscope and micropipettes. The same clone was used in my previous study (Graves, 1957)* Other clones of G. pectorale var. pectorale have been established from collections made in and around

Franklin County, Ohio, but these show no observable differences from each other or from the clone used.

General Methods of Culture

Volumetric glassware was used in the preparation of all solutions.

Knop solution, as described by Hartmann (1928), was the basic cul­ ture medium used. The salts were dissolved in distilled water in the following proportions: four parts Ca(ND^)2*one part MgSO^THgOione part

KHgPO^sone part KNO^* A trace of iron was used} see page 9 for a dis­ cussion. The concentrations used, 0 . 0 f & and 0. 0 2 5 5 b , were calculated as the percentage of solid in the solution.

In order to facilitate the measurement of small quantities of the different mineral salts, separate stock solutions of these various salts were prepared in the following concentrations, using a slight

7 8 modification of the method described by Hartmann (1928)i 1056 solution of

CatNO^g, 9/6 solution of KNO^, 9^ solution of KHgPOj^, and a 256 solution

of MgSO^yHgO. The nutrient solutions Here then made up by adding to the

proper amount of redistilled water that volume of each of the separate

salt solutions which was calculated to give the proper amount of that

salt in the culture medium. The concentration of each salt, and the num­

ber of milliliters of each stock solution necessary to make 100 ml of a

0.0936 Knop solution is listed below.

g salt/1 0 0 ml ml stock solution of 0,09)6 Knop solution in 100 ml of 0.09/6 Knop

0.0286 g Ca(N0^)g 0.286

0,0071 g M g S O ^ H g O 0.357

0.0071 g KHgFOk 0.11*3

0.0071 g KNO^ 0.11*3

Trace FeCl^ One drop of a 156 soln, to 200 ml culture medium

Pyrex-distilled water

The other solution that was used throughout a major portion of this

study was the same concentration of Knop solution to which the following

carbonate buffer (after Osterlind, 191*9) had been added. 9

ml stock soln. In 100 ml g/1 0 0 ml stock soln. 0,05% Knop soln.

K2CO3 1.570 5.00 NagtX^ 1.205 5.00

HC1______2.000______2.50______

Three different iron salts were used during the course of this study. The first used was the ferric chloride suggested by Hartmann.

This proved unsatisfactory because the hygroscopic nature of the salt made accurate weighing of it difficult, and its solutions turned readily to an unabsorbable form of colloidal ferric hydroxide. The second salt used was ferric sulfate (Chu, 19U2), but this proved difficult to dis­ solve, and its solutions were also unstable. Rodhe (191*8) discussed a similar problem with the iron salts he used and recommended the use of a ferric citrate-citric acid solution, where the citric acid acted as a chelating agent to keep the iron in solution. This combination was used for most of the experiments in this study. A stock solution containing both the citric acid and its iron salt was prepared in such a concentra­ tion that when five milliliters of the stock solution was part of a liter of the culture medium, there would be 2 .7 milligrams of the salt and 2 .7 milligrams of the acid in the solution.

The culture vessels used were either 250 ml or 500 ml Erlenmeyer flasks fitted with 2h/h0 standard taper ground glass female Joints at the tops. Qas inlet tubes, Corning No. ?8l00 (Corning catalog LG 1, 1958), 10 were fitted into these# If plain Knop solution waa used as the culture medium, the inlet and exit tubes of the tops were left unstoppered# How­ ever, if the carbonate buffer was used, the inlet tube was stoppered with a rubber stopper and the rubber bulb of a medicine dropper waa used to close the outlet tube# Standard tapered "Teflon” sleeves were used on the male joints so that there would be no contamination of the culture fluid from stopcock grease# These flasks and tops were washed in a common de­ tergent solution (Dreft), rinsed in tap water, and drained# They were rinsed again in freshly distilled water just before use# More redis­ tilled water was then placed in each flask, the tops were fitted on, and the flasks were placed on a hot plate# After the water had boiled vig­ orously for several minutes, they were taken off the hot plate, the water drained off, and the tops stoppered until the culture medium was intro­ duced into the flasks#

The redistilled water was prepared in a special all pyrex glass still in which each connection was made with a ground glass joint# A distilling tube, with suction side arm, Corning No# 9l*20, was placed be­ tween the Graham condenser and the collecting flask to equalize changes in gas pressures during the distillations# The side arm was connected to a system of flasks so that, after the distillation, the air sucked back into the still as it cooled would pass through a cotton filter# The con­ tamination of the redistilled water was kept to a minimum in this manner#

Each of the stock solutions was made up with freshly redistilled water, and then each of the solutions was brought to a boil, except those of the carbonate, hydrochloric acid, ferric chloride and ferric sulfate# n

All the pipets were rinsed in boiling, distilled water immediately be- foreeach use. As only redistilled water, in addition to the mineral salts, was used in preparing the culture medium, no further sterilization of the medium or of the flasks was used than that listed above.

The completely inorganic culture medium used was not sufficient to support any appreciable bacterial growth, and frequent microscopical examinations of the living cultures showed only negligible contamination.

In only one case, when medium from a two week old culture was used as the culture medium, was any appreciable contamination noted, A number of gm*n ciliates of a single species— the species was not identified— were found in the cultures. On examination of the culture from which the in­ oculum was taken, a few cysts were found which were probably those of the ciliate; however, no trophic form of the ciliate was seen. It is assumed that the dying of colonies in the old fluid, and mechanical breakage of cells during the process of filtering the fluid through a millipore fil­ ter, contributed enough organic material to support the growth of the bacteria upon which the ciliates seemed to be feeding.

One hundred milliliters of culture medium were placed in each 2$0 ml flask or two hundred milliliters in each 500 ml flask. The inoculum for one experiment was taken from the control of the preceding experiment,

A one milliliter inoculum was used for each one hundred milliliters of culture fluid, unless it is stated differently at the beginning of an experiment. After the flasks were inoculated they were clamped in a constant temperature, illuminated water bath which was kept at 20°± 0,1° C. 12

A light intensity of 600 foot candles was recorded at the top of the seven inch water level of the bath* The bottoms of the 250 ml flasks were four inches below the top of the water* A photoperiod of twelve hours light and twelve hours darkness was used*

The Constant Temperature Water Bath

A water bath (Figure 2), to maintain the cultures at a constant temperature, was built in the following manner* A box, without top or bottom, was constructed by gluing and nailing two-by-two lumber together*

It was sixteen inches high and its inside dimentions were two feet by four feet* The top and bottom boards were two-by-sixes*

The inside was waterproofed with a quarter-inch layer of the poly­ ester resin used in fiberglass; the resin was pigmented white* Two monel metal rods were placed to run the length of the box* They were spaced evenly, six Inches apart, one on either side of the center, and five inches down from the top of the box* Two plate glass sheets one-quarter inch thick were set one inch apart to form the bottom of the box, leav­ ing a working space of 12 3/U inches within the box* A four inch layer of fiberglass insulation was wrapped around the box, which was then covered with masonite*

A top was constructed by fastening one-by-three boards around the edge of a piece of three-quarter inch plywood* A three inch layer of fiberglass insulation was placed within, and a sheet of one-half inch plywood was used to cover this* The top was hinged to tiie box with a piano hinge* All exposed wood and masonite surfaces were painted with white enamel* 13

The water bath was placed on a base, thirty five inches high, con­ structed with six-by-six legs and a two-by-six frame* Eight Uo watt, standard warm white General Electric fluorescent lamps were fastened to a frame two feet by four feet constructed of one inch angle iron* The transformers for the lights were mounted on a table so that the heat produced by them would not be directly under the water bath* A "Tork

Master No* 9U8a" automatic timer controlled the photoperiod* With the aid of adjustable, pilaster shelving strips, the lights could be placed from four and three-quarter inches below the top of the inside plate glass to thirty six and one-quarter inches from the glass.

Water from a fifty gallon, constant temperature water bath, a

"Tempmobile" made by Labline, Inc* of Chicago, Illinois, was circulated through the illuminated water bath* The bath was maintained at 20° C, and the variation of temperature within the illuminated bath was less than 0*02° C at any given time* However, there was a variation of 0*1° C between the temperature of the bath when the lights were on and the tem­ perature when they were off*

Method of Sampling the Culture

When a sample was to be taken from a given flask, the flask was re­ moved from the water bath and swirled vigorously to insure a random dis­ tribution of the organisms* The top of the flask was lifted enough to admit a two milliliter volumetric pipet which had been rinsed in boiling water immediately beforehand* The pipet was rinsed in the culture, and then a two milliliter sample was withdrawn and placed in a clean pyrex lh test tube* A numbered cork stopper was losely placed on the test tube, and the test tube was placed In a beaker of slowly boiling water* After the test tube had been in the boiling water for several minutes, it was removed and securely stoppered* When the test tube and its contents had cooled, the contents of the test tube were stirred, and a one-millillter sample was withdrawn and placed in a Sedgwick-R after counting cell*

Fifty random fields were c ounted from each slide*

The samples were taken at approximately the same time each day, usually about the middle of the morning, but never after the middle of the afternoon* This variation in the time of sampling the cultures did not appreciably affect the correlation of one day’s growth with the next day’s growth, because this clone divided only at night, usually starting at about 10:30 P.M. Pocock (1955) noted a similar phenomenon with the forms with which she worked, but she discovered that different popula­ tions of Qonium divided at different times of the day* The time of the day at which a given population divided remained relatively constant*

In order to know the amount of error incurred in sampling in this way, four samples (Xj.) were taken from a single flask on each of two separate days, and the number of cells per cubic millimeter was determined on each sample* The samples and the percentage deviation from the aver- age (?) of each day's sampling is listed on the following page* 15

Xi - X lay Samples x i ------x X

1 1 7.0U -2.76

2 7.86 8.56

3 7.18 —0.83

U 6 .8 8 -a.97

2 1 10.76 -1 .6 0 2 11.52 5.3a 3 10.82 -l.oa a 10.6 U -2.70

As can be seen, the deviations from the average are within plus or minus 10^ of the average. The time necessary to count a much larger sample becomes prohibitive as the number of cells increases with growth.

Photometric measurements, which have been used extensively in determin­ ing populations, are useless in determining the relative abundance of normal 16-celled colonies. Therefore, only direct microscopic examina­ tion of the samples can give the desired information* The method used seemed to be the best compromise between speed and accuracy.

Statistical Method

It was desirable to know, in a given experiment, whether the dif­ ferent treatments affected the rotes of growth of the organisms. In order to compare visually the rate of growth of various cultures of a given experiment, a plot was made of cell density, as cells per cubic millimeter, against days on semilog paper* Because a comparison of rates by such a method is largely subjective in nature, a more objective method was also used* This consisted of a statistical comparison of the slopes by the method outlined in Section 6*11 of Ostle (195U)* As only

straight line functions may be tested by this method, comparisons were made using only the logarithmic phase of growth portions of the plots*

One of the conditions necessary for this test is that the values used for the abscissa be measured without error* In the present stu

samples were taken* Since all samples were taken at approximately the

same time each morning, and since this strain of Qonium divides only at night (see page lli), it is believed that this condition is fulfilled* A DESCRIPTION OF THE CHANGES IN THE FORM AND IN THE RATE OF GROWTH

OF THE COLON! THAT ACCOMPANY AGING OF CULTURES

IN 0.0^ PER CENT KNOP SOLUTION

It seemed desirable to know the changes in the rate of growth and in the relative abundance of noraal 16-celled colonies in relation to aging of a culture under the more nearly standardised conditions obtainable with the illuminated water bath. Therefore, each of two flasks, contain­ ing one hundred milliliters of 0.05?6 Knop solution, were inoculated each with one-half a milliliter of a Gonlum culture and placed within the water bath. This experiment will be referred to as Experiment 1.

Table 2 shows the changes in the relative abundance of the 16-celled colonies and in the rate of growth. This information is also shown in graphic form in Figure 3. It will be noted that the relative abundance of 16-celled colonies increased rapidly until the third day of growth when cells in 16-celled colonies represented more than $9% of the total number of cells in the culture. Thereafter the relative abundance of

16-celled forms dropped off rapidly, even though the culture was still in its logarithmic phase of growth.

It would seem from this that if some substance required for the formation of the normal colonies is being depleted from the culture me­ dium, either it is not required for growth, or its requirement for growth

17 is much smaller than its requirement for normal colony formation* Or some toxin is responsible for the decrease in relative numbers of l6—celled forms, its presence in the culture medium does not seem to affect the ability of the cells to grow and divide* INVESTIGATIONS OF THE POSSIBILITY THAT A DEFICIENCY OF SOME SUBSTANCE OR

SUBSTANCES IS RESPONSIBLE FOR THE FORMATION OF ABNORMAL COIONIES

AND FOR THE RETARDATION OF GROWTH

The Influence of Different Concentrations of Carbonate on the Form of the Colony and on the kate of Groirth

Steemann Nielsen (1955) and Steemann Nielsen and Jensen (195$) dis­ cuss carbon dioxide as a carbon source* I discussed the influence of carbon dioxide on the stimulation of cell division, and on the production of the gelatinous matrix of Gonlum (Graves, 1957)* I found that passing carbon dioxide, as expired air, through a growing culture of Gonlum greatly increased the relative abundance of 16-celled colonies and pro­ duced a much richer culture*

Ssterlind (19U8, 191*9, 1950a, 1950b, 195la, 1951b, 1952a, 1952b) studied the relation of carbon dioxide and carbonate to groirth and photo­ synthesis in Scenedesinus quadrlcauda and Chlorella pyrenoidosa* He found that in experiments in which poorly buffered media were used, growth was extremely erratic* When he made pH determinations of the various test tube cultures, he found a wide variation of pH* He concluded that in these experiments, it was an accident when a given culture reached a pH value high enough for growth* On other experiments, using pure air as a carbon source, he found that at low pH values (below 5*5) the concentra­ tion of bicarbonate was too low for growth and the concentration of free carbon dioxide in the solution, corresponding to the 0*03£ of carbon dioxide in the air, was so low that Sc enede smus showed hardly

19 20

any growth* In the pH interval of - 6*2, the bicarbonate concentra­

tion was found to be high enough for good growth*

During some preliminary experiments, I found that the growth of

Gonium in the ordinary 0*05£ Knop solution was erratic and unpredictable j

considerable differences appearing among cultures supposedly identical*

Measurements of the pH of these cultures showed that most values were be­

low the pH 5*5 level that Ssterlind (19h9) had found to be critical in

the growth of Scenedesmua in media without added carbonate* Therefore,

in order to obtain more predictable growth of Gonium, I decided to fol­

low his example and add carbonate to the culture medium.

In order to test the influence of different concentrations of car­ bonate on the rate of growth and on the form of the colony in Gonium, a

series of four flasks and their duplicates were prepared* The equimolar

mixture of sodium carbonate and potassium carbonate, which was described

voider general culture methods, was used* The basic culture medium used

in all flasks was 0*0££ Knop solution* Various concentrations of carbon­

ate were added to this basic medium in the different flasks* In Flasks

1A and IB, the carbonate concentration was l/Ui U; in Flasks 2A and 2B,

1/88 l(; in Flasks 3A and 3B, l/bliO Mj and in Flasks IjA and I|BS no carbon­

ate was added* All flasks were inoculated with a one milliliter inocu­

lum, and placed in the illuminated water bath at 20° C* Flask 2jA was

broken midway through the experiment, and therefore no data for it are

shown in the table* This experiment will be referred to as Experiment 2*

The changes in the relative number of normal colonies and in the

number of cells per cubic millimeter are shown in Table 3* The change 21 in the number of cells with the passage of time is shown In graphic form in Figure lu

Examination of Figure h shows that the growth seems to be slightly slower in the l/Ui M carbonate solutions than in the other solutions.

An analysis of the data for slope differences (see page 1$) was made, and it was found that no significant difference was shown, as can be seen by the following analysis*

The analysis was to test the null hypothesis that all the slopes were equal.

Source of variations Df MS F F.Oj? due to deviations about four parallel lines 0.0ii620 16 due to deviations about four separate lines 0.02738 1^ 0.00211 due to slope differences 0.01882 3 0.00627 2.972 3.U1

On the basis of this test, the null hypothesis is accepted that all slopes are equal.

From Table 3, however, it will be noted that there is an appreciable difference in the relative abundance of the 16-celled colonies. The high

carbonate concentration seems to have a detrimental affect, a trend

shown, but not statistically significant, in the comparison of the rates

of growth. The flasks containing the 1/88 H carbonate have approximately

70$ of their cells in normal 16-celled colonies, while the l/UUO M 22 carbonate has about 6C$. The l/Liii M carbonate and the Knop solution have 50% and h$% of their cells, respectively, in the 16-celled state.

Thus, 1/88 U carbonate seems to provide enough inorganic carbon for growth, and is sufficient for Gonium to produce a high number of normal colonies. The l/UhO It carbonate, though sufficient to maintain the same rate of growth, over the period tested, does not seem to have a suffi­ cient concentration of carbonate to allow as much normal colony forma­ tion to occur. This holds true, also, for the 0*0$% Knop solution without added carbonate. On the other hand, the l/hlx M carbonate seems to be too concentrated, and seems to produce some detrimental effects on both the growth and the form of the colony*

The Influence of Vitamin B-i? on the Form of the Colony and on ifoe Rate of flrowth

Harper (1912) states that during asexual reproduction each cell of a Gonium colony forms a new 16-celled colony through a series of four divisions. Harper believed that a large part of the 8-celled forms ob­ served resulted from a failure of that fourth division to take place.

The shortage of some growth factor might result in the failure of the fourth division to take place in Gonium. Hutner and Provasoli

(19E>1, 1955) and Hutner et al. (1953) have shown vitamin to be a growth factor for a number of phytoflagellates, including some members of the same order to which Gonium belongs, the Phytomonadida. The re­ quirement for maximum growth in Euglena (Hutner et al., 1950) is 0.01$ micro gram per one hundred milliliters of culture medium. 23

I found that Gonium grew more rapidly, and had a higher relative number of 16-celled colonies when vitamin was added to the culture medium (Graves, 1957)* I found the optimum concentration to be between

0*01 microgram and 0*02 microgram per one hundred milliliters of culture medium* I also found that a much richer growth and a much higher rela­

tive number of 16-celled colonies were produced if vitamin B^2 added

to a culture that had I# carbon dioxide passed through it for fifteen

minutes each day*

It was decided, therefore, to investigate the influence of vita­

min B^2 on the form of the colony and on the rate of growth in a culture

medium to which inorganic carbon had been added in the form of carbonate*

This experiment will be referred to as Experiment 3* The basic culture

medium that was used was 0*02556 Knop solution with 1/88 M carbonate addect

Ferric citrate was the iron used* Flasks 1A and IB contained one hundred

milliliters of the basic culture medium only* Flasks 2A and 2B contained

0*01 microgram of vitamin each, in addition to the basic culture me­

dium* The inoculum was centrifuged and washed in freshly distilled water

to eliminate the possibility of introducing any vitamin that might

have been produced in the culture from which the inoculum had been taken*

The results of this experiment are given in tabular form in Table U

and in graphic form in Figure 5* From Table U it can be seen that the

cultures to which no vitamin B ^ was added tend to start growing more

slowly, and the relative number of 16-celled forms increases more slowly

than it does in the flasks to which vitamin Bj2 had been added* However,

by the fourth day of growth, the relative number of 16-celled forms 2k starts to decrease in the flasks to which vitamin Bj_2 had been added, while it continues to increase in those to which no vitamin B^2 had been added. Also, though the growth of Gonium lags slightly at the start in the flasks to which no vitamin was added, after growth starts, it proceeds at the same rate as it does in the other flasks; this can be seen in Figure $*

It is not known why the results of this experiment show almost no beneficial effects of vitamin when the results obtained in my pre­ vious study (Graves, 19$7) showed such obvious beneficial effects* If a different strain of Gonium had been used for this present experiment, the results might have been more understandable, for the various strains show differences in their requirement of vitamin B12 for growth (Stein, personal communication)*

One possible explanation is that in the previous experiment (Graves,

19f>7)» either the light intensity might have been too low, 200 foot- candles compared with 600 foot-candles, or the carbon dioxide might have been insufficient, and the additional vitamin was utilised* With the more intense light and more abundant carbon supply of the present ex­ periment, the organism may have been able to produce sufficient vitamin

& 1 2 , and hence the addition of more vitamin had no stimulating ef­ fect* This, however, is only a tentative explanation which will have to be verified by further experimental work*

The Influence of Iron on the Form of the Colony and on the lUte of flrowth

Ketchum (1?5U) states that the requirement of iron for the growth 25 of the algae is well substantiated. Rodhe (19U8) discusses the problem of the utilization of iron by the algae, and found that the organisms he tested could not utilise colloidal iron. He recommends the use of fer­ ric citrate, which stayed in a form available to the algae much longer than did the other iron salts he tested* Pringsheim (19U6) attributes some of the success of his soil-water medium to the ability of humic acids to maintain iron in an available form*

Hewitt (1951) discusses the role of iron in plant mineral nutri­ tion. All the known enzymatic systems depending on iron, as catalase, peroxidase, and the cytochromes, involve porphyrin systems. There was some evidence that iron played some role in chlorophyll production* n Osterlind (19U9) found that Scenedesmus seemed to produce very little chlorophyll in solutions which were deficient in iron*

The effect of iron deficiency on the form of the colony and on the rate of growth of Gonium was examined in the following manner: Two pairs of flasks were used* The basic culture medium used in all flasks was

0.025* Knop solution with l/88 M carbonate added* Flasks 1A and IB had

0*0018 gram per liter FeCl-j concentration, while no iron was put into

Flasks 2A and 2B* A one milliliter inoculum, which had been centrifuged

and washed in distilled water, was used* The flasks were placed in the

illuminated water bath at 20° G* This experiment will be referred to as

Experiment U*

The results of the experiment are tabulated in Table 5* and shown

in graphic form in Figure 6* Some iron must have been present as an im­

purity in Flasks 2A and 2B because there is appreciable growth shown in 26 both flasks* However, the rate of growth was much greater in the flasks to which iron had been added* In addition, the number of 16-celled forms was relatively more abundant in the flasks to which iron had been added*

An analysis of the slope differences of the logarithmic portion of the two growth curves shows that these differences are statistically signifi­ cant* Again, the null hypothesis tested was that there was no difference in the slopes*

Source of variations S2 Df US F F.0 5 due to deviations about two parallel lines 0.15315 17 due to deviations about two separate lines 0.11051* 16 0*0071*1 due to slope differences 0.031*61 1 0.031*61 ML M2

The null hypothesis that the slopes are equal is, therefore, rejected at the

The Influence of the Calcium Ion Concentration on the Form oi the Colony and 'on the Rate oF6rowth

Ketchum (1951*) discusses the calcium requirement for growth in algae, and states that it has been shown to be not essential for growth in

Chlorella* Rodhe (191*8) found that Ankjstrodesmus grew well in a cal­ cium concentration as low as 0*001 milligram of calcium per liter, a con­ centration that Bsterlind (19i*9) found to give good growth in

Scenedesmus* Both authors, however, found that the optimum growth for 27 these organisms occurred in concentrations between 1.0 and 5.0 milli­

grams per liter.

Calcium might not only be a requirement for growth in Gonium, but

also for normal colony formation. Robertson (19111) stressed the im­

portance of calcium in promoting the stability of intercellular matrices,

and Spiegel (195b) stated that for normal adhesion of cells to each

other, either the surface antigen-antibody bond is used, or the calcium

bridge. Using 1ft to symbolize a surface antigen and m as an antibody, the

two types of bonds would be c ell-lft-m-cell or cell-U-Ca-M-cell.

As I had found some evidence of a calcium requirement for both

growth and normal colony formation in Gonium (Graves, 1957), it was de­

cided to examine more fully the influence of the calcium ion concentra­

tion on the form of the colony and on the rate of growth in Gonium. The

basic culture medium, minus the calcium nitrate, was 0»0$% Knop solution

with 1/88 11 carbonate added. A series of six pairs of flasks was pre­

pared with one hundred milliliters of the basic culture medium in each

flask. A different amount of calcium, as calcium nitrate^ was added to

each flask as followsi 1A and XB, no calcium; 2A and 2B, 0.000007 g Caj

3A and 3B, 0.00007 g Ca; I|A and l*B, 0.0007 g Caj 5A and 5&, 0.0035 g Caj

and to 6A and 6B, 0.0070 g Ca— the amount normally in 0*0$$ Knop solu­

tion. The nitrate level was kept constant by adding enough potassium

nitrate to provide an equivalent amount of nitrate for that which would

have been present in the unaltered 0*0$% Knop solution. A one milli­

liter inoculum, which had been centrifuged and washed twice in freshly

distilled water, was used in each flask. Flask 2A was dropped into the 28 water bath on the second day, and an unknown volume of water from the bath entered the flask. Therefore, no data are given for this flask.

The experiment described on page 27 will be referred to as Experiment 5.

The results of this experiment are shown in Table 6, and the number

of cells per cubic millimeter for each pair of flasks is shown plotted

against the number of days of growth in Figure 7. Figure 8 shows a plot

of the relative number of 16-celled colonies versus the log of the cal­

cium ion concentration tines 10^.

There was no growth in the flasks which contained no calcium. The

rates of growth in all the other flasks were similar* An analysis of

the growth of all flasks containing added calcium gave the following

results, based on the null hypothesis that all the slopes were equal*

Source of variations S2 Df US F F,o5

due to deviations about five parallel lines 0.14811*3 39

due to deviations about five separate lines 0*1*2783 3$ 0*01222

due to slope differences 0 .0S360 I* 0.0131*2 1.022 2.63

Therefore, the hypothesis that all slopes are equal is accepted.

However, when one examines the forms of the colonies produced in

the various concentrations, it is apparent that there are considerable

differences according to the concentrations* The only multicellular

forms found in Flasks 1A and IB were a few 2-celled forms* The relative 29

number of 16-celled colonies Increases in direct proportion to the log

of the calcium ion concentration#

Thus, it can be said that the calcium ion is very probably neces-

11 sary for growth in Gonium# Osterlind found a concentration of 0#001 milligram per liter of calcium gave good growth in Scenedesmus# In the

present study, the lowest concentration used, 0#0007 milligram per liter,

supported almost as good a growth of Gonium as did a concentration 1000

times greater# It would seem unlikely that some trace element present

in the salt as a contamination would be effective in such a dilute con­

centration, especially since the iron citrate-citric acid used has a

tendency to chelate metal Ions, removing them from use by the organism

(Rodhe, 191*8 and Hutner et al#, 1950)#

The same reasoning, however, would not hold true for the relation

of calcium to the form of the colony# While it is quite probable that

the calcium ion itself is responsible for the results observed, the pos­

sibility is not removed that some contamination in the calcium salt is

responsible for these results# Only further experimentation with more

highly purified salts will be able to answer this question#

The Influence of the Concentration of Knop Solution on the Form ot the Colony and on the Rate of Growth

Little difference was found in the rate of growth and in the form of

the colony when Gonium was cultured In concentrations of Knop solution

ranging from 0«0l£ to 0.09# (Graves, 1957)# However, the results from

Experiment 5 on the influence of the calcium ion concentration, reported

above, would lead one to believe that if the concentration of Knop were 30 halved, halving the calcium ion concentration also, the relative abun­ dance of 16-celled colonies should decrease*

An experiment, Experiment 6, mas performed in which one pair of flaBks contained 0*0556 Knop solution with 1/88 X carbonate added; another pair contained 0*025£ Knop solution} also with the l/88 U carbonate added* A one BdlUllter inoculum was used for both*

The results of this experiment are recorded in Table 7, and shown in graphic form in Figure 9 • There seems to be no appreciable dif­ ference in either the rate of growth or the form of the colony* The re­ sults of this experiment, therefore, do not correspond with those from the calcium experiment. The discrepancy might be explained by assuming that some other ion competes with the calcium ion for active sites within the cell, or on its surface membrane* Scott (19W O showed that the cal­

cium ion content of cells of Chlorella pyrenoidosa was largely replaced by magnesium and partially by potassium and sodium when the cells were washed in distilled water solutions of the chlorides of these ions*

Hence, not only the concentration of the calcium ion, but also the ratio

of calcium to the competing ion would be important* Again, only further

investigation will clarify this question*

The Influence of the Depletion of the Inorganic Carbon on the Form of ike Colony antf on the itate ot Growth

In Experiment 2, it seemed that the addition of carbonate to a cul­

ture of Gonium in 0 « 0 & Knop solution did not increase the rate of

growth of the organism (pp* 19-20)* However, I had noted that the addi­

tion of carbon dioxide to a culture produced a much richer growth of 31

Gonium than was attained in cultures which contained no added carbon source (Graves, 1957)•

Therefore, it was desirable to compare the growth of Gonium in a culture containing carbonate with a culture which contained no added carbonate over a longer period of time than was done in Experiment 2*

Such a comparison is shown graphically in Figure 10, and the data from which the graph was drawn are shown in Table 8* The data for the flasks in 0*0$% Knop solution were taken from Flasks 1A and IB of Experi­ ment 10 which will be reported on page 3U* The data for the flasks containing 0*0$% Knop solution and l/88 U carbonate were taken from Ex­ periment 11 which will be reported on page 35*

The rate of growth for the two sets of flasks is the same until a concentration of almost fifty cells per cubic millimeter is reached* At that point the rate of growth in the flasks without the carbonate drops off rapidly, while the rate of growth in the flasks with the caxbonate continues at the same rate until there is a concentration of almost four- hundred cells per cubic millimeter* It is assumed from this that the carbon supply is a limiting factor in growth in the 0*0$% Knop solution*

The relative number of l6**celled forms in the 0*0$% Knop solution without added carbonate reached its nunrimm on the fifth day, after the rate of growth had started to decline* On the other hand, in the medium to which caxbonate had been added, the rate of growth had not yet started to decline when the relative number of 16-celled forms started to dimin­ ish* Other aspects of the comparison of these two sets of flasks will be discussed after the next experimental section* INVESTIGATIONS OF THE POSSIBILITI THAT GONIUM PECTORALS PRODUCES

AUTOTOXIC SUBSTANCES, WHICH INFLUENCE AD VS? SELT THE

FORM OF THE COLONY AND THE RATE OF GROWTH

Pratt (19UO) found growth inhibiting substances in cultures of

Chlorella vulgarist and studied the nature and effect of these autotaxic substances (Pratt and Fong, 19b0j Pratt, 19b2, 19b3a» 19b3b, 19bb) •

Swanson (19U3) also studied the effects of autotoxins on the respiration of Chlorella vulgaris. Jorgensen (1956) found that Nitsschia palea,

Scenedesaus quadricauda, and Chlorella pyrenoldosa all produced auto­ toxic substances. He also found that Chlorella and Nitaschia produce substances which stimulate the growth of Scenedesmus. Filtrates of

Scenedesaus cultures, on the other hand, inhibited growth of both

Chlorella and Nitaschia. Nitaschia formed substances which inhibited growth of Asterionella formosa, while filtrates of Asterionella sometimes inhibited the growth of Nitaschia and sometimes stimulated its growth.

Hartmann (192li) and Crow (1927) believed that toxic substances were produced in Gonium cultures, and that these substances were responsible for the abnormalities which they encountered in aging cultures. There­ fore, several experiments were performed to investigate further the possibility of autotoxin production by Gonium pectorale.

The Influence of Inocula Containing Fluid from Aged, Cultures on ihe frorm of the Colony and on tne Rate of Growth

In order to test the possible presence of an autotoxin in inocula from an aged culture, the following experiment was performed. Thirty

32 33 milliliters of a Gonium culture was distributed evenly between two centrifuge tubes and centrifuged to settle the cells* The supernatent fluid of one tube was replaced with fresh 0*05/6 Knop solution, and the cells were resuspended in this fluid* The cells in the other tube were resuspended in the original medium* One milliliter of the suspension in fresh Knop solution was introduced into each of two flasks containing

100 ml of fresh 0*0$% Knop solution buffered with 1/88 M carbonate* One milliliter of the suspension in aged fluid was added to each of two other flasks which also contained 100 ml each of the buffered Knop solu­ tion* This experiment will be referred to as Experiment 8*

The results of this experiment are shown in Table 9, and the rates of growth are shown in graphic form in Figure 11* The cells which were centrifuged and resuspended in fresh Knop solution seemed to grow at a

slightly faster rate than those which were resuspended in the original fluid* The difference, however, was not of statistical significance*

Also, it can be seen that there was almost no difference in the relative number of normal colonies*

Experiment 9, a repetition of Experiment 8, showed a slight lag in the production of 16-celled colonies and in the rate of growth in the

cultures to which had been added the inocula containing old culture

fluid* The results are shown in Table 10* This lag, however, was over­

come by the end of the fourth day of growth*

The results of these two experiments suggest the possibility that

something is present in the medium of an aged culture which inhibits

normal colony formation and growth of Gonium* These results, however, 3k are far from conclusive, and show that if a toxin is introduced in the inoculum, its effects are not long apparent*

The Influence of the Dilution of an Aged Culture on the Form of the Colony and on the Rate of Growth

It has been shown (Graves, 1957) that, in cultures which contain no added carbon source, the concentration of the carbon dioxide in equilib­ rium with the culture fluid is a factor which limits the growth of

Gonium* Removal of half of the cells of an aged culture should then make the carbon source proportionally more available to the remaining cells in the medium, thereby making more rapid growth possible*

On the other hand, if a toxin was responsible for the diminished growth and break-up of the colonies, halving the number of cells in the medium would not appreciably influence the growth of the remaining cells*

However, diluting the medium with fresh fluid should decrease the con­ centration of the toxin and thereby increase the rate of grcsrth of the remaining cells*

Therefore, a series of three pairs of flasks was prepared* One hun­

dred milliliters of 0*05# Knop solution was introduced into each flask*

Each flask was then inoculated with one milliliter of a Gonium culture*

On the seventh day after samples haul been taken, the entire contents of

Flasks 2A, 2B, 3A, and 3B were poured, each separately, into a clean

graduate cylinder, and the volume of each culture was measured* The

cylinder was shaken to insure a random distribution of the cells, and then half the culture was returned to its original flask* In the case of

Flasks 2A and 2B, the other half of the volume was replaced with sterile 35

0*0$% NaCl solution. In the case of Flasks 3A and JBr the other half of the culture fluid was centrifuged and then replaced in the cultures after the removal of its organisms* All flasks were then returned to the water bath, and samples were taken from them for two more days. This experiment will be referred to as Experiment 10*

The results of this experiment are shown in Table 11 and a graph of the number of cells per cubic millimeter plotted against time in days is shown in Figure 12# (In the table the number of cells per cubic milli­ meter in Flasks 2A and 2B, 3A and 3B has been doubled so that the growth in these flasks may be compared with the growth in Flasks 11 and IB*)

The rate of growth increased slightly in both sets of flasks from which cells had been removed, as compared with the rate in the unaltered medium* This increase was not statistically significant, however*

When the relative numbers of 16-celled colonies are compared for all three sets of flasks, it can be seen that they are not very dif­ ferent; although there was a slight increase shown in Series 2 and a de­ crease in Series 3*

The Influence of Filtrates from Aged Cultures on Young Cultures

If toxins are produced in aging cultures of Gonium* as Hartmann

(192U) suggests, then filtrates from cultures of different ages should contain toxins in concentrations which bear some relationship to the age of the culture from which they were taken* To test this hypothesis, the following experiment, Experiment 11, was performed*

It was desirable to prepare inocula as free as possible from any toxin which might have accumulated in the source culture* Therefore, twenty milliliters of a Gonium culture was centrifuged} the supernatant

fluid was drawn off and replaced with fresh culture medium. The cells were resuspended in this fresh fluid* One milliliter aliquots from this

fluid were used as inocula in six pairs of flasks, each of which con­

tained one hundred milliliters of 0.0f$ Knop solution buffered with

l/83 M carbonate. These cultures, when they were in their logarithmic

phase of growth, were to be used for the experiment described below.

On the fourth day, ten milliliters of fluid was removed from each

of five cultures of different ages, filtered separately through a milli-

pore filter; and half of each ten milliliters of fluid was added to one

of a pair of flasks as follows: to Flasks 2A and 2B, fluid from Flask

1A; to Flasks 3A and 3B, fluid from a one and one-half week old culture;

to Flasks liA and l*B, fluid from a two and one-half week old culture; to

Flasks $k and SlB, fluid from a three and one-half week old culture; and

to Flasks 6a and 6B, fluid from a four and one-half week old culture*

The results of this experiment are shown in Table 12, and the num­

ber of cells per cubic millimeter of each set is plotted against time in

days in Figure 13* The relative number of 16-celled forms is plotted

against time in days in Figure llu

As shown in Figure 13, the rate of growth drops off in those flasks

to which filtrates have been added from cultures older than themselves.

This difference in rate of growth is significant. The following is a

summary of the analysis testing the hypothesis that all slopes are equal: 37

Source of variations______Df______MS F______F.05 due to deviations about six parallel lines 0.22969 bl due to deviations about six separate lines 0.161*73 £ 0*001*57$ due to slope differences 0.061*96 s 0*012992 2.81*0 2.1*8

The hypothesis that all slopes were equal was rejected on the basis of this analysis at the 0(9: *0$ level*

A comparison of the relative abundance of the 16-celled colonies also shows that those cultures which had been treated with filtrates from cultures older than themselves were harmed by the filtrates* Though the 16-celled colonies continued to increase in relative abundance for two days after the filtrates were added, they did not increase as rapidly in flask pairs 3-6 as they did in the control flasks* Further, when the

16-celled colonies started to decrease in relative abundance, they de­ creased much more rapidly in those cultures containing filtrates from cultures older than themselves than they did in the other cultures*

This effect was in approximate proportion to the age of the culture from which the added filtrate had been taken* These relationships are shown strikingly in Figure Uj*

The pH of all flasks was taken on the last day of the experiment* 38

The values were as follows:

Flask 1A— IB— 33C— SB— 31— 3B— U— SB— 31 5B 5E ®

pH 9,18 9.01 9.23 9 .US 9.SI 9.38 9.U. 9.31 9.52 9.35 9.50 9.59

There seems to be some relation between the pH of the medium and the amount of Inhibition of growth and the amount of break-up of the colo­ nies. This is not absolute, however.

It seems obvious, therefore, that some substance has accumulated in the culture medium of aging cultures which retards the growth of the culture, and causes a more rapid degeneration of the colonies. The sub­ stance may influence the pH of the solution.

The Relation of the pH of the Medium to the Form of ihe Colony and to Motility

There was some reason to suspect that the toxicity of aged culture media might be related to the pH of the media. For this reason the pH of the culture medium of Flasks 1A and IB of Experiment 11 was taken each day of the experiment; a record of these daily pH values may be found in Table 6. Also, the pH of the culture medium of all flasks in

Experiment 11 was taken on the final day of the experiment, and is listed above. There seems to be some relationship between these final pH values and the deleterious effects of the filtrates.

Xt was desirable, therefore, to know what effect the pH of the cul­

ture medium had on the general well being of the organism. For this purpose, a series of seven flasks was prepared, each flask containing 3 9

100 milliliters of 0*0$% Knop solution buffered with l/88 M carbonate*

The solution of each flask was then titrated with 0»$h M HC1 until the desired pH was obtained; the initial pH of Flask 1 was about li*l, that of each succeeding flask about 1 pH unit above that of its predecessor, with the pH of Flask 7 being about 10*5* The pH values were determined with a Model G Beckman pH meter* Each flask was inoculated with $ milli­ liters of a Gonium culture, and then all flasks were placed in the water bath, A $ milliliter sample was withdrawn from each flask immediately after inoculation; these samples were observed with a dissecting micro­

scope, and notes were made on the motility and on the form of the colo­ nies, This procedure was repeated three hours later and again twenty-

one hours later. Also, twenty-one hours after inoculation, the pH of the medium of each flask was determined* The initial pH, the final pH, and notes on colony form and on motility for each flask are shown in Table 12. From this table it can be seen that Gonium remains both normal in

form and motile at pH values as low as U.l and up to values of about 7*0,

At about pH 8,0 Gonium swims very little and begins to break up* The

organisms were not motile at pH values above 8.2, and were colorless at

pH 10*7* This colorless condition probably indicates that the cells were

dead. DISCUSSION

The main concern of this study has been to find the factor or fac­ tors which promote normal colony formation in Gonium pectorals# It ■was believed that a knowledge of the factors which interfered with normal colony formation would provide information about conditions which are necessary for the growth of normal colonies*

Some of the abnormal colonies observed in this study seem to have been produced by the break-up or malformation of other colonies* Harper

(1912) and Crow (1927) reported having observed the break-up of numerous colonies, and I have also seen this process frequently*

Other colonies, notably the 8-celled ones, seem to have been the result of what Harper (1912) called "dwarfed development in reproduc­ tion," A 16-celled colony is formed from a single cell by a series of four divisions* All divisions are in the same plane, but each division axis is at a right angle to that of the previous division* The first division forms two cells; the second division, four, which are then in the form of a square* The third division forms a rectangle of eight cells, and the fourth division again forms a square, this time of six­ teen cells* Harper noted that this fourth division frequently did not occur, and found that the 8-celled forma of this type were different In shape from the 8-celled forms which resulted from the splitting in half of a 16-celled colony* I have found that the 8-celled forms resulting from this "dwarfed development" are the most common non-16-celled forms throughout most of the life of a culture*

hO Ul

All this indicates that those factors which influence cell reproduc­ tion also directly influence colony formation. One of the most important of these factors is the carbon supply. I would interpret data similar to those shown in Table 8 and In Figure 10 as indicating that whenever the carbon supply is sufficient for maximum growth, the majority of the di­ viding cells seem to form 16-celled colonies. But, as the carbon supply becomes relatively less available for each cell, an increasing number of the dividing cells stop dividing at the end of the third division, and

sometimes at the end of the second or the first division. Under the conditions used in this study, the vast majority of the abnormal colo­ nies formed during the first week or so of growth of Gonium in 0.0!#

Knop solution with no added carbonate seemed to be colonies resulting from a "dwarfed development" caused by an insufficient carbon supply.

Further, a deficiency of some of the mineral constituents, such as

iron, may cause abnormalities, which I believe are also the result of

"dwarfed development." A deficiency of several nutrient requirements,

such as iron, magnesium, and phosphate, can regularly occur in a growing

culture because of the pH changes which accompany growth. The medium be­

comes increasingly alkaline with the aging of a culture, as Table 8

shows; and iron hydroxide and magnesium phosphate are insoluble in alka­

line media, especially above pH 8.

A deficiency of calcium, on the other hand, does not seem to affect

cell division. It does, however, affect the ability of cells to adhere

to each other, as can be seen in Table 6 or Figure 8. It seems probable,

therefore, that the intercellular cement of Gonium cells is mainly of the calcium salt bridge type* Calcium shortages can, and do, commonly occur in aging cultures due to the precipitation of calcium salts from the medium, especially salts of phosphate and carbonate* Autoclaving the culture medium also causes the precipitation of calcium salts* These flocculent precipitates can be seen in the bottom of flasks of aged or autoclaved cultures* Because of the extreme insolubility of calcium phosphate, it is possible that the calcium in the Gonium cells is re­ moved* The calcium ions could be replaced by such ions as the sodium and potassium ion* (See Scott, 19hh, for a discussion of the replacement of calcium by other ions*) The precipitation of calcium also contributes to a deficiency of phosphate in the medium*

The tests for toxins did not produce such clear answers. Table 12 and Figure 13 show that something accumulated in aging cultures which is detrimental to both the form of the colony and to the rate of growth*

These results may be due to a toxin, but they may also be due to pH changes* Table 8 shows that the organisms ceased to grow at about pH 8, and Table 13 shows that they seem to start dying at a somewhat higher pH*

From Table 13 it appears that Gonium is injured by values above pH 8, and that death definitely occurs above pH 10. On Page 38 it is shown that the final pH of the solutions depended, to some extent, upon the age of the culture from which the filtrate was taken* The main influence of the filtrates, then, may have been only to make the medium more alka­ line, so that the toxic pH range was reached sooner*

On the basis of this study, several tentative explanations are pro­ posed for the abnormalities which accompany growth of Gonium pectorals in laboratory cultures* First, a deficiency of the carbon and iron source causes the formation of abnormal colonies by impairing normal cell re­ production* Secondly, a shortage of calcium, especially by the precipita­ tion of calcium phosphate in alkaline media, prevents the adherence of cells to each other and causes the break-up of colonies already existing*

And thirdly, the alkalinity of aged culture medium seems to be the main cause of its toxicity. I

SUMMARY

1, When Gonium pectorale is found in abundance in nature, almost all the colonies are normal 16-celled colonies* However, when the or­

ganism is cultured under ordinary laboratory conditions, many of the colonies contain fewer than 16 cells even during the first few days* As the cultures grow older, the number of norH-16-celled forms increases,

and by the end of a month, little but 1-celled forms remain* A subcul­

ture made at any stage, including one consisting chiefly of 1-celled

forms, goes through the same stages as did the original.

2* It is obvious, then, that the conditions which obtain in ordinaiy

laboratory cultures of this species are not, even at the outset, adequate

for the normal development of all the colonies in a culture, and become

less adequate with the passage of time* Various possible explanations

have been considered, and experiments were performed to test some of the

factors that might be involved*

3* It was found that the addition of up to 1/88 M carbonate to the

culture medium increased the relative abundance of 16-celled colonies,

but did not alter the initial rate of growth, when compared with cul­

tures which had less or no carbonate added* After a concentration of

fifty cells per cubic millimeter was reached, the carbon supply in the

medium with no added carbonate became insufficient to support growth at

the initial rate* The culture containing the added carbonate was able

to divide at the initial rate for a much longer period* Concentrations

hh of carbonate stronger than l/88 M seemed harmful to the form of the colony and to the rate of growth.

ii. The presence of traces of iron in the culture medium was found necessary for both normal colony formation and for normal cell growth.

5. The relative abundance of normal colonies was found to be in direct proportion to the logarithm of the calcium ion concentration.

Growth was not possible in the absence of the calcium ion, but 0.0007 milligram of calcium per liter of solution was sufficient for normal ceil growth.

6. Filtrates of aged cultures had a deleterious effect on the form

of the colony and on the rate of growth in proportion to the age of the

culture from which the filtrate was obtained.

7* Normal colony formation, growth, and motility seemed unimpaired within pH values ranging from Ii.O to 7.7. Colonies started to degen­

erate and growth ceased at pH values above 8.0. Gonium seemed to be

killed at about pH 10.5•

8. Deficiencies of inorganic carbon and of iron produce abnormal

colonies and lower the rate of growth of a culture, presumably through

an impairment of cell reproduction* However, a deficiency of calcium

lessens the ability of cells to adhere to each other. Therefore, sister

cells formed in cell division often do not adhere to each other, and al­

ready formed colonies break up more easily. The form of the colony

tends to become disrupted at pH values above 8.0, and growth and repro­

duction cease. It is presumed that the main toxic effect of filtrates

of aged cultures is due to the alkalinity of the fluid. BIBLIOGRAPHY

Bock, F. 1926. Experiraentelle Untersuchungen an koloniebildenden Volvocaceen. Arch, Protistenk. 96*321-396.

Crow, W. B. 1927* Abnormal forms of Gonium* Ann, & Mag, Nat, Hist, 19 (111*) *996-6 0 1 .

Ehrenberg, C. G. 1838, Die Infusionsthierchen als vollkommene Organismen. Leipzig.

Florell, C. 1957, Calcium, mitochondria, and anion uptake. Physiol. Plantarum 10:781-790.

Graves, L. B., Jr. 1997. Some chemical factprs which influence the form of the colony in Gonium pectorale Muller, 1773* Master's thesis, The Ohio State University.

Harper, R. A. 1912. The structure and development of the colony in Gonium. Trans. Amer. Micros. Soc. ^1*69-83.

ti tt Hartmann, M. 1921*. Uber die Veranderung der Koloniebildung vop Eudorina elegans und Gonium pectorale unterMdem Einfluss ausserer fiedingungen. IV. Mitt, der Untersuchungen uber die Morphologie und Physiologie des Formwechsels der Phytomonadinen (Volvocales). Arch. Protistenk. 1*9*379-399.

11 Hartmann, M, 1928. Praktikum der Protozoologie. Funfte Auflage. Gusrtav Fischer, Jena.

Hewitt, E. J. 1991. The role of the mineral elements in plant nutrition. In* Annual Review of Plant Physiology. Vol. II. D. I. Arnon and L. Machlis, Editors. Annual Reviews, Inc., Stanford.

Hutner, S. H, and Provasoli, L. 1991, The phytoflagellates. In* Biochemistry and Physiology of Protozoa. A. Iaroff, Editor. Academic Press, New York.

Hutner, S. H. and Provasoli, L. 1999* Comparative biochemistry of flagellates. In* Biochemistry and Physiology of Protozoa. Vol. II. S. H. Hutner and A. Lwoff, Editors. Academic Press, New York.

Hutner, S, H., Provasoli, L, and Filfus, J. 1993. Nutrition of some phagotrophic fresh-water Chrysomonads. Ann. BUT. Acad. Scl. 96:892-862.

1*6 1*7

Hutner, S* H*, Provasoli, L*, Schatz, A* and Haskins, C* P* 1950* Some approaches to the study of the role of metals in the metabolism of microorganisms* Proc* Am* Phil* Soc* JgU* 152—170.

Jorgensen, E* G* 1956. Growth inhibiting substances formed by Algae* Physiol* Plantarum 2*712-726*

Ketchum, B, H* 195U. Mineral nutrition of Phytoplankton. Ini Annual Review of Plant Physiology* Vol* V* D. I* Arnon and L* Machlis, Editors* Annual Reviews, Inc., Stanford.

Mast, S. 0. 1916. The process of orientation in the colonial organism, Gonium pectorale, and a study of the structure and function of the eyespot. Jour. Exp* Zool* 2011-17*

Muller, 0* F* 1773* Vermium terrest* et fluviatil* seu animal, infusor*, etc*, Leipzig*

Ssterlind, S* 19U8. The retarding effect of high concentrations of carbon dioxide and carbonate ions on the growth of a green alga. Physiol* Plantarum li 170-175* it . Osterlind, S* 19L9* Growth conditions of the alga Scenedesaus quadricauda, with special reference to the inorganic carbon sources* Symb* Bot* Ups* 10 (3)il-lljl* fisterlind, S. 1950a* Inorganic carbon sources of green algaet I* Growth experiments with Scenedesmus quadricauda and Chlorella pyrenoidosa. Physiol* Plantarum 3*353-360* n Osterlind, S* 1950b* Inorganic carbon sources of green algaet II* Carbonic anhydrase in Scenedesmus quadricauda and Chlorella pyrenoidosa. Physiol* Plantarum 3*u30-U3k* H Osterlind, S* 1951a* Inorganic carbon sources of green algaet III* measurements of photosynthesis in Scenedesmus quadricauda and Chlorella pyrenoidosa. Physiol* Plantarum I^i 2u5-^5li* it Osterlind, S* 1951b* Inorganic carbon sources of green algaet IV. Photoactivation of some factors necessary for bicarbonate assimilation* Physiol* Plantarum U* Slhr$3k» ti Osterlind, S* 1952a* Inorganic carbon sources of green algae: V* Inhibition of photosynthesis by cyanide. Physiol* Plantarum £*372-378. « Osterlind, S* 1952b* Inorganic carbon sources of green algae* VI. Further experiments concerning photoactivation of bicarbonate assimilation* Physiol* Plantarum 5*U03-U08* Ostle, B. 1951« Statistics in Research, Iowa State College Press, Ames, Iowa.

Paschep, A* 1927, Heft 1. VolvocalesaPhytomonadinae. In: Die Susswasser-Flora Deutschlands, Osterreichs und der Schweiz, A, Pascher, Editor, Gustav Fischer, Jena.

Pocock, M. A, 19!?!?* Studies in North American Volvocales. I. The genus Gonium, Madrona 13:19-61.

Pratt, R, 19l0. Influence of the size of the inoculum on the growth of Chlorella vulgaris in freshly prepared culture medium. Amer. Jour. Bot. 27:"j?2.

Pratt, R, 1912. Some properties of the growth-inhib itor formed by Chlorella cells, Amer. Jour. Bot, 29:112.

Pratt, R, I9l3a. Retardation of photosynthesis by a growth-inhibiting substance from Chlorella vulgaris. Amer. Jour. Bot. 30:32.

Pratt, R. 19l3b. Influence of the age of the culture on the rates of photosynthesis and respiration. Amer. Jour. Bot. 30:101;.

Pratt, R. 1911* Influence on the growth of Chlorella of continuous removal of chlorellin from the culture solution. Amer. Jour. Bot, 31:118.

Pratt, R. and Fong, J. 19l0, Further evidence that Chlorella cells form a growth-inhibiting substance. Amer. Jour.~fTot. 27:131*

Prescott, G. Yf. 19l2. The fresh-water algae of Southern United States. II. The algae of Louisiana, with descriptions of some new forms and notes on distribution. Trans. Amer. Microsc. Soc. 61:109-119»

Prescott, G. W., Silva, H., and Wade, V/. E. 19l9# New or otherwise interesting fresh-water algae from North America. Hydrobiol. 2:81-93.

Pringsheim, E. C. 1916. Pure Cultures of Algae, Their Preparation and Maintenance. Cambridge University Press, London.

Robertson, D., Jr. 19H* The function and metabolism of calcium in the Invertebrata. Cambridge Philosophical Society. Biological Reviews 16:106-133.

Rodhe, W. 1918. Environmental requirements of fresh-water plankton algae. Symb. Bot. Ups. 10 (l)*l-ll9* 149

Scott, G. T. 191+1+. Cation exchanges in Chlorella pyrenoidosa. J. Cellular Comp. Physiol. 23*1+7-58.

Spiegel, M. 1951+* The role of specific surface antigens in cell adhesion. Part I. The reaggregation of sponge cells. Biol. Bull. 107*130-11*8.

Steemann Nielsen, E. 1955. Carbon dioxide as carbon source and narcotic in photosynthesis and growth of Chlorella pyrenoidosa. Physiol. Plantarum 8*317-335.

Steemann Nielsen, E. and Jensen, P. K. 1958. Concentration of carbon dioxide and rate of photosynthesis in Chlorella pyrenoidosa. Physiol. Plantarum 11*170-180.

Stein, J. R. 1958a. A morphological study of Astrephomene gubernaculifera and Volvulina steinii. Amer. Jour. Bot. 1+5*388-397.

Stein, J. R. 1958b. A morphologic and genetic study of Gonium pectorale. Amer. Jour. Bot. l+5*661+-672.

Swanson, C. A. 191+3. The effect of culture filtrates on respiration in Chlorella vulgaris. Amer. Jour. Bot. 30*8-11.

Tilden, J. E. 1935* The algae and their life relations, Univ. of Minnesota Press, Minneapolis. TABLE 1

THE PERCENTAGE OF THE TOTAL NUMBER OF CELLS WHICH IS REPRESENTED

BT THE NUMBER OF CELLS IN EACH OF THE DIFFERENTLY-CELLED FORMS

IN AN EIGHT DAY OLD CULTURE IN 0 . 0 $ PER CENT KNOP SOLUTION

Percentage of the total number of cells which Number of cells is represented by cells In the form In form

Sirteen-celled form U2.1*l

Fifteen-celled form 2.1*3

Fourteen-celled form 0.76

Thirteen-celled form 0.35

Twelve-celled form 0.97

Eleven-celled form 0.89

Ten-celled form 0.27

Nine-celled form 0.2U

Eight-celled form 38.08

Seven-celled form 2.08

Six-celled form 1.95

Five-celled form 0 . &

Four-celled form 3 .Hi

Three-celled form . 0*87

Two-celled form 1.19

One-celled form 3.79

50 TABLE 2

CHANGES IN THE FORM OF THE COLONY AND IN THE GROWTH

THAT ACCOMPANY AGING OF CULTURES

IN 0 . 0 5 FBR CENT O O P SOLUTION

Per Cent of total cells as Total No* of Days 16-celled forma cells per cu. mm

1 0.00 3.35

2 2iu62 2.07

3 30.96 3.88

U 59.38 15.95

5 50.53 2li.li3

6 50.19 29.80

7 U3.91 U7.22

8 29.53 71.78

51 TABLE 3

THE INFLUENCE OF DIFFERENT CONCENTRATIONS OF CARBONATE

ON THE FORM OF THE COLONY AND ON THE RATE OF GROWTH

Per cent of Average of total cells as Total No* of flasks No* Treatment Days 16-celled forms cells per cu* mm

1 7.0li li.55

1A it IB 1Alt M 2 28*25 lii .20 carbonate 3 51.69 36.61*

1 7.21 l*.i*i*

2A & 2B 1/88 U 2 1*1*.31 16.26 carbonate 3 71.15 1*9.75

1 12.92 5.87

3A & 3B l/l*l*0 M 2 52.81 15.1*5 carbonate 3 58.15 50.1*6

1 0.00 3.12

IiB no 2 39.25 10.60 carbonate 3 U5.20 21.21*

52 TABLE h

THE INFLUENCE OF VITAMIN B12 ON THE FORM OF THE COIDNT

AND ON THE RATE OF GROWTH

Per cent of Average of total cells as Total No. of flasks No. Treatment Days 16-celled forms cells per cu. ara

1 7.05 ii.58

2 10.51 U.72

1A & IB No vitamin 3 U5.17 U.68 b 12 h 80.99 17.08

5 89.68 h1.22

l 6.11* 5.23

2 16.75 U.73 O.Ol 2A 4c 2B microgram 3 1*8.91 5.82 vitamin B^2 U 86.00 21.06

5 82.03 53.66

53 TABLE $

THE INFLUENCE OF IRON ON THE FORM OF THE COLON!

AND ON THE RATE OF GROWTH

Per cent of Average of total cells as Total No. of flasks No* Treatment Days l6-celled forms cells per cu. ran

1 3.19 h.99*

2 11.78 $.U9

1A it IB 0.0018 g 3 U2.US 7.$8 FeCl3 h 80.98 19.98

5 82.39 38.8$

6 7$.U$ 8U.20

1 3.U2 IwOO*

2 $.76 3.$3

2A it 2B No FeCl3 3 30.18 U.77

h $1.36 8.10

$ 6$.03 21.68

6 61.2$ 26.66

Values not used In the statistical analysis*

$U TABLE 6

THE INFLUENCE OF THE CALCIUM ION CONCENTRATION ON THE

FORM OF THE COLONY AND ON THE RATE OF GROWTH

Per cent of Average of total cells as Total No. of flasks No* Treatment Days l6-celled forms cells per cu. mm

1 29.10 2.77

2 0.00 2.12

1A & IB No calcium 3 0.00 2.31

h 0.00 2.U3

5 0.00 2.29

1 29*36 2.18

2 0.00 2.20

2B O.OOOOO? g 3 0.00 6.52 Ca/lOO ml U 0.00 18.m

5 0.00 2U.92

1 28.51 2.10

2 0.00 U.U3

3A & 3B 0.00007 g 3 8.01 7.97 Ca/lOO ml U 7.13 21.68

5 3.59 26.86

55 TABLE 6 (continued)

Per cent of Average of total cella as Total No. of flasks No* Treatment Days 16-celled forms cells per cu. mm

1 20.25 2.35

2 9.51 1*.98

UA & I4B 0*0007 g 3 22.77 12.76 Ca/lOO ml 1* l*7.l*l 30.16

5 53.13 56.52

1 11.93 2.98

2 28.71 6.99

5A & SB 0.0035 g 3 36.88 15.20 Ca/lOO ml 1* 62.93 37.85

5 65.71 66.71

1 36.1*8 3.98

2 15.70 3.87

6A & 6B 0.0070 g 3 1*0.35 9.78 Ca/lOO ml 1* 76.11 1*1.21*

5 79.87 61.30

56 TABLE 7

THE INFLUENCE OF THE CONCENTRATION OF KNOP SOLUTION ON THE

FORM OF THE COLONY AND ON THE RATE OF GROWTH

Per cent of Average of total cells as Total No. of flasks No* Treatment Days 16-celled forms cells per cu. mm

1 0.00 U.23

1A & IB 0 . 0 2 # 2 1*6.03 111.21 Knop soln. 3 73.77 1*6.67

1 7.21 li.lili

2A & 2B C.O# 2 U*.31 16.26 Knop soln. 3 71.15 1*9.75

57 TABLE 8

THE INFLUENCE OF THE DEPLETION OF THE INORGANIC CARBON ON THE

FORM OF THE COLON! AND ON THE RATE OF GROWTH

Average of Per cent of Total flasks total cells as No. of cells No. Treatment Days pH 16-celled farms per cu. mm

1 5.1*1* 0.00 5.29

2 5.51* 1.1*5 8.71

Expt. 10 No 3 5.52 16.19 23.1*8 1A it IB carbonate added to 1* 5.65 16.99 1*6.81* 0.05* Knop soln. 5 5.97 18.67 51.11

6 6.17 16.55 59.05

7 6.30 17.1*1* 72,36

8 6.50 9.85 10U.13

1 7.1*1* 18.1*8 6.01*

2 7.23 8.88 9.01

Expt. U Carbonate 3 7.33 7.37 21.73 1A it IB added to 0,05* 1* 7.1*3 11.29 1*2.51* Knop soln. 5 7.58 27.81* 81.10

6 7.80 57.59 21*2.21*

7 8.30 51.01* 386.1*6

8 9.09 2*1.11 397.1*0

58 TABLE 9

THE INFLUENCE OF INOCULA CONTAINING FLUID FROM AGED CULTURES

ON THE FORM OF THE COLONY AND ON THE RATE OF GROWTH

Per cent of Average of total cells as Total No. of flasks No* Treatment Days 16-celled forms cells per cu* nan

1 76*09 1.16

1A & IB Centrifuged, 2 90.01* 1.60 resuspended in 3 90.1*6 5.11t original liquid U 90.69 12.71

5 88.83 39.63

l 76.52 1.05

2A & 2B Centrifuged, 2 89.7U 1.25 resuspended in fresh 3 88.91 6.1*8 0*05$ Knop solution k 90.77 21.1*7

5 87.63 53.02

59 TABLE 10

THE INFLUENCE OF INOCULA CONTAINING FLUID FROM AGED CULTURES

ON THE FORM OF THE COLON! AND ON THE RATE OF GROWTH

A REPETITION

Per cent of Average of total cells as Total No. of flasks No* Treatment Days 16-celled forms cells per cu. na

1 Hi.7U 5.U7

7A & ?B Centrifuged, 2 3.37 8.7U resuspended In 3 2.61 18.37 original liquid k 8.58 Ui.58

5 31.21 8U.U8

l 11.88 5.98

1A Centrifuged, 2 6.59 8.12 Through resuspended 6B In fresh 3 6.71 21.21 plus 0+0%% Knop 8A & 8B solution ii 111. 28 1|8.30

5 31.96 82.65

60 TABLE II

THE INFLUENCE OF THE DILUTION OF AN AGED CULTURE ON THE

FORM OF THE COLONY AND ON THE RATE OF GROWTH

Per cent of Average of total cells as Total No. of flasks No* Treatment Days 16-celled forms cells per cu. mm

1 0.00 $.29

2 i.l*$ 8.71

3 16.19 23.1*8

1* 16.99 1*6.61*

1A St IB Control $ 18.67 $1.11

6 16.5$ $9.0$

7 17.1*1* 72.36

8 9.6$ 10l*.13

9 $.20 107.73

10 $.$U 127.80

1 0.00 U.82

2 1.86 8.1$

3 18.99 22.71*

1* 20. $9 1*6.73

2A Sc 2B Half of $ 2$.28 62.31 fluid replaced 6 19.61* $6.33 with 0.0$* NaCl 7 3U.09 7l*.87

61 TABLE U (continued)

Per cent of Average of total cells as Total No. of flasks No* Treatment Days 16-c ailed forms cells per cu. mm

2A & 2B Half of 8 10.61 103.23 fluid replaced 9 6*91 61.59 x 2=; 123.18 with 0 . 0 # NaCl 10 7.65 87.08 x 2 = 17U.16

1 0.00 U.51

2 0.00 7.03

3 17.UU 26.)i3

3A & 3B Half of h 17.96 U9.83 liquid centrifuged 5 16.014 58.07 to remove cells 6 15.57 62.22 and then replaced 7 11.75 65.33

8 10J 42 98.86

9 10.11 53.Wt x 2 x 106.88

10 5.21 86.38 x 2= 172.76

62 TABLE 12

THE INFLUENCE OF FILTRATES FRCU AGED CULTURES

ON XOUNG CULTURES

Per cent of Average of total cells as Total No. of flasks No. Treatment Days 16-celled forms cells per cu. ma

1 18.1*8 6.0l**

2 8.88 9.01* # U & IB No 3 7.37 21.73 filtrate added 1* 11.29 1*2.51*

5 27.81* 81.10

6 57.99 21*2.2!*

7 51.01* 386.1*6

8 1*1.11 397.1*0*

1 9.73 6.21*

2 6.00 8.09*

2A & 2B Filtrate 3 7*89 20.29* from culture 1A I* 12.33 1*6.68 added 32.66 79.16

6 60.15 182.12

7 52.85 391.30

8 1*2.77 376.90*

63 TABLE 12 (continued)

Per cent of Average of total cells as Total No. of flasks No. Treatment Days 16-celled forms cells per cu. mm

1 7.77 6.33*

2 5.01 9.58*

3A & 3B Filtrate 3 7.5U 21.15* from one meek U 16.22 1*5.76 old culture 5 3l*.12 85.90 added 6 56.16 225.38

7 1*7.56 316.1*5

8 23.11* 322.00*

1 12.68 6.57*

2 2.18 7.U1**

1*A & 1*B Filtrate 3 7.87 20.35* from two week 1* 11*. 71 52.02 old culture 5 29.1*3 77.66 added 6 U**73 251.1*1*

7 31.80 370.20

8 9.1*0 308.70*

61* TABLE 12 (continued)

Per cent of Average of total cells as Total Mo. of flasks No. Treatment Days 16-celled forms cells per cu. mm

1 16.51 6.10*

2 2.28 6.58*

5a & 5B Filtrate 3 6.9 6 20.95* from three week U U*.07 52.62 old culture 5 35.51 87.1*0 added 6 1*5.92 169.70

7 32.66 279.00

8 7.88 271.70*

1 9.13 5.18*

2 11.75 8.17*

6A & 6B Filtrate 3 I*.61* 20.36* from four week 1* 17.39 1*7.92 old culture 5 31.75 73.60 added 6 1*6 .9 6 171.08

7 22.96 21*5.10

8 10.37 287.85*

^Values not used In the statistical analysis*

65 TABLE 13

THE RELATION OF THE pH OF THE MEDIUM TO THE

FORM OF THE COLONY AND TO THE MOTILITY

Condition Condition Condition immediately 3 hours 21 hours Flask Initial after after after Final No. pH Inoculation inoculation inoculation pH

1 iul slowly motile,, motile, motile, 5.2 normal normal normal

2 5.2 motile, motile, motile, 6.0 normal normal normal

3 6.2 motile, motile, motile, 7.0 normal normal normal

h 7.3 motile, motile, slight 7.95- normal normal motility, 8.0 slight break-up

5 8.2 not motile, not motile, not motile, 8.6 normal slight extensive break-up break-up

6 9.2 not motile, not motile, not motile, 9.U slight slight normal break-up break-up

•»t 10.5 not motile, not motile, not motile, 10.7 normal slight slight break-up break-up, cells colorless

66 FIGUR E I

NORMAL AND ABNORMAL COLONIES OF SONIUM PECTORALE

A. NORMAL IS-CELLED COLONY

B. 4 -CELLED COLONY , DIVIDING

C-P ABNORMAL COLONICS

ST FIGURE 2

THE C0N8TANT TEMPERATURE ILLUMINATED WATER BATH

6 B CELLS SCR CMS 1C MILLIMETER O B SO TO 46 SB B S 0 4 SO •0 n U IB 10 0 I M NE I TC OM P H 00LN N I TE AE P SROWTH RATE THE OP LONY INCMANSES 0 0 AND PORM THE IN THC OP RSN A I-OLE FORMS IS- OELLED AS PRESENT EL PR UI MILLIMETER CUBIC PER CELLS 1 P N SIS OULTURC AS ANINS OP XEIET I EXPERIMENT IU E 3 FIGURE DAYS 4 S T 69 H IFUNE F IFRN CNETAIN O CARBONATE OF CONCENTRATIONS DIFFERENT OF INFLUENCE THE

CELLS PER CUBIC MILLIMETER S « 0 4 a 6 5 7 3 4 7 I N H RT O QROWTH OF RATE THE ON XEIET 2 EXPERIMENT FIGURE 4 DAYS • 4$ M CARBONATE Mq $ /4 1 • A ■ 44 M CARBONATE M 4 /4 1 ■ O N ADD CARBONATE ADDED NO ■ • 1/440 M CARBONATE M 0 4 4 / 1 •

0 7 H IFUNE F IAI », O TE AE F IROWTH OF RATE THE ON ,| » VITAMIN OF INFLUENCETHE

CELLS PER CUBIC MILLIMETER S t B 7 4 ■ t S 4 3 7 I I ment n e im r e p x e FI SURE 5 DAY 8 3 a 4 * O DE ■ ADDED NO * o .1 S i100 M MEDIUM ML 0 0 .i/1 S MS 0.01 B 71 'I ro

THE INFLUENCE OF IRON ON THE RATE OF IR O W TH « • H• m o o • CELL* K R CUaiC iLLI METER M

DAYS H IFUNE F H CLIM O CNETAIN N THE ON CONCENTRATION ION CALCIUM THE OF INFLUENCE THE

CELLS PER CUBIC MILLIMETER I - AE F GROWTH OF RATE XEIET 8 EXPERIMENT IUE 7 FIGURE DAYS a A • ■ 0 A 00380 8 3 0 .0 0 . 0070 7 0 .0 0 . .00007 0 0 0 0.00 . - 0 00007 0 - 00007 0 0 0 0 . N CALCIUM NO . a AIO ML CA/IOO Q a • ADDED AIO ML CA/IOO CA/IOO ML CA/IOO ML CA/IOO CA/IOO ML CA/IOO 3 7 or total cells as is celled rou sts SO to 0 4 SO SO TO SO O SO 0 H INFLUENCE THE O. ACU IN OCNRTO X 6 0 I X CONCENTRATION ION CALCIUM LOG. f O H CLIM O CNETAIN N H FORM THE ON CONCENTRATION ION CALCIUM THE XEIET B EXPERIMENT f H OOLONY THE Of IUE 8 FIGURE T. DAY TH. S 4 0 4 74 H IFUNE F H CNETAIN F NP OUIN N THE ON SOLUTION KNOP OF CONCENTRATION THE OF INFLUENCE THE

CELLS PER CUBIC MILLIMETER L- 3 S 9 4 S 7 7 I AE F BROWTH OF RATE XEIET • EXPERIMENT IUE 9 FIGURE DAYS B 3 . .E% NP SOLUTION KNOP O.OE5% . O - 05% KO SOLUTION KNOP % 5 0 0 - A 75 CCLLS PE* CUSIC MILLIMETER ft t ft t 4 4 8 ft 7 I t f 4 B B 7 B 8 4 ft t I H IfUME f M f T O OPEIN f H IOtAI CAfttON INOftSANIC THE Of OEPLETION ft Of ATI TMI Of INfLUEMOE THI N H RT O BROWTH Of RATI THI ON XItMN 7 CXSIfttMCNT IUE IO FIQURE 0 AYS - CARBONATE ADDED M M / l - A N CROAE ADDED CARBONATE NO - O 6 7

H IFUNE F NCL CNANN FUD RM SD CULTURES ASCD FROM FLUID CONTAlNlNt INOCULA OF INFLUENCE THE

CELLS PER CUSIC MILLIMETER S S 4 7 ■ S ft 3 4 7 • a I « N H RT O SROWTH OF RATe THE ON XEIET • EXPERIMENT IUE II FIGURE DAYS 3 4 NCL NT WASHED NOT INOCULA NCL WSE WITH WASHED INOCULA RS MEDIUM FRESH s 77 CELLS PER CUSIC MILLIMETER 4 S S 8 T C • I t t I E H T N O S E R U T L U C D E O A F O N O I T U L l O F O E C N E U L F N I E H T 10 8 8 7 8 6 4 8 0 1 T N E M I R E P X E H T W 0 R 8 F O E T A R figure DAYS : 12 D E S L U O F A I N R T N X E 8 0 C . 0 H T M H I T W U I I D W D E T E D U M E L I T D U L I D D E T U L I D T O N 78

U C D E S A M O R F S E T A R T L I F F O E C N E U L F N I E H T

CELLS PER CUOIC MILLIMETER S 7 s S • 4 I S E R U T L U C S N U O Y F O It T N E M I R E P X E IUE 13 FIGURE 0 AYS 6 D E D D A D I U L F O N H I K S A L F M O R F D E D D A D I U L F C C B ADDED FROM 4^ K E E W ^ 4 M O R F K D E E E D W life D M D A I O U R L F F D E D D O A D O I B U L F 0 0 8 ADDED FROM lf K E E W life M O R t F M O D R E F D D D A D E I D U D L D F A I U L F C C B C C S 8 '4 WEEK E E W

E R U T L U O O L O E R U T L U C D L O E R U T L U O O L O E R U T L U O D L O M T W O 79

% OF TOTAL CELL8 A8 1 6 -CELLED FORMS DAYS N O * E R U T L U O D M A M O N P D E T A R T L I F F > * E R U T L U O * N U O V P O V N O L O O I H T P O T N E M I R E P X E IU E 14FIGURE □ Q A ■ A • 8CC C 8 - 800 0 8 - eoc o e . 800 0 8 . 800 0 8 - NO N . II D I U L F D I U L F D E D D A D I U L F D I U L F ■w D I U L F r c 5 D E O D A D E D D A K E E W M O N P O E D D A D E D D A O E D D A K E E W 4 * 8 M O R P M O R F P N O M F L A S K 1 H 1 K S A L F M O N P WEEK OLD L O K E E W 4 * 4 M O R P

K E E W e f i l E R U T L U O D L O D L O D L O E R U T L U O E R U T L U C E R U T L U O 80 AUTOBIOGRAPHY

I, Lynn Boyd Graves, Jr*, was born in Elgin, Illinois, October 5,

1928* I received my secondary school education in the public schools of the cities of Compton and Long Beach, California* My undergraduate training was obtained at Ohio Wesleyan University, Delaware, Ohio, from which I received the degree Bachelor of Arts in 1953* Prom The Ohio

State University, I received the degree Master of Science in 1957* Tn

1953 I received an appointment as a graduate assistant in the Department of Zoology and Entomology* I held this position until 1957, when I received an appointment as assistant in the same department* I held this position while completing the requirements for the degree Doctor of

Philosophy*

81