PHYCOLOGICAL STUDIES 

V. Comparative studies of the algal genera 
Tetracystis and Chlorococcum 

R. MALCOLM BROWN, JR. AND HAROLD C. BOLD 



Phycological Studies 

V. Comparative studies of the algal genera 
Tetracystis and Chlorococcum 

R. MALCOLM BROWN, JR. AND HAROLD C. BoLD1 
(Immunological studies with the collaboration of Richard N. Lester2 ) 
1 Department of Botany, University of Texas, Austin, Texas. 2 Bolton Institute of Technology, Bolton, Lancashire, England. 

Acknowledgments 
The writers would like to express their appreciation to Professors Wilson S. Stone, C. J. Alexopoulos, Richard C. Starr, and Donald A. Larson for giving fully of their time in reading this manuscript, and for their valuable criticisms and sug­gestions. The writers are particularly indebted to Dr. Richard N. Lester who taught the senior author immunochemical techniques and who critically evaluated the section on lmmunochemistry. The writers are also grateful to Mr. Marion Dodson for his valuable technical assistance throughout the immunochemical investiga­tions, as well as to Miss Lynn Hamilton, Miss Bettty Faulkner, and Mr. Vermell Kyser. The assistance and guidance of Professors W. G. Whaley and Hilton Mol­·lenhauer and of Mr. George Sheng in electron-microscopic aspects of this investi­gation are deeply appreciated. The writers acknowledge with gratitude Dr. Hannah 
T. Croasdale's preparation of the Latin diagnoses. The editorial assistance of Mrs. Frances Denny is acknowledged with gratitude. Finally, the authors wish to express their appreciation for support from the Publications Fund of the Graduate School of The University of Texas. Cost of publication was supported, in part, by funds from NIH Grants AI-06023-02 and Gm-08211-03 and NSF Grant GB-313. 


Table of Contents 
PAGE 
I. General Introduction 	7 
II. Morphology, Taxonomy, and Physiology of Tetracystis Gen. Nov. 8 
A. 	Materials and Methods . 8 
B. 	Generic Characterization 10 Text Figure 1 . 12 
C. Specific Characterization 	13 Chlorosphaerales Chlorosphaeraceae Tetracystis aeria sp. nov. 13 · T etracystis aggregata sp. nov. 15 Tetracystis dissociata sp. nov. 16 Tetracystis excentrica sp. nov. 18 Tetracystis illinoisensis sp. nov. 20 Tetracystis isobilateralis sp. nov. 21 T etracystis pampae sp. nov. 23 Tetracystis pulchra sp. nov. . 24 Tetracystis texensis sp. nov. . 25 Tetracystis aplanosporum comb. nov. 26 Tetracystis intermedium comb. nov. . 27 Tetracystis tetrasporum comb. nov. · . 28 Key to the currently known series of T etracystis Brown and Bold 29 
D. Physiological Attributes in T etracystis 	31 
III. Electron Microscopy of Tetracystis and Certain Other Genera 41 
A. 	Materials and Methods 42 
B. 	R~u~ 45 
1. Fixation images . 	45 
2. Comparative study of cellular organelles in T etracystis 4 7 
( 1 ) The Chloroplast . 	4 7 
( 2) 	The Pyrenoid 49 
(3) 	The Mitochondrion 52 
(4) The Golgi Apparatus 	53 
(5) 	The Cell Wall 55 
(6) 	Miscellaneous Organelles 56 
3. Pyrenoid division . 	56 
4. Cell Division. 	58 Text Figure 2 61 Text Figure 3 63 Text Figure 4 65 
5. Zoosporogenesis in T etracystis and C hlorococcum 67 Text Figure 5 71 
C. Discussion . 	72 
IV. Immunochemical Studies of Tetracystis and Chlorococcum 76 
A. Introduction 	76 
B. Materials and Methods . 	77 
1. Preparation of cells for extraction 	• . 77 
2. Extraction procedures 	79 
3. Protein determinations 	80 
4. Preparation of antisera 	81 
5. Processing of antisera 	83 
6. Absorption of antisera 	83 
7. Preparation of materials for double-diffusion and immuno­electrophoresis 83 
( 1) Preparation of Buffered Agar . 	83 
( 2) Preparation of Glass Plates 	84 
(3) "Running" the plate, Double-diffusion 	84 
(4) "Running" the plate, Immunoelectrophoresis 	84 
(5) Examination of the plates 	85 
C. Results and Discussion · . 	85 
1. Analysis of double-diffusion 	85 
2. Absorption studies 	90 
3. Immunoelectrophoresis analysis (LE.A.) 	93 
( 1) The T. aplanosporum group 	93 
( 2) The T. isobilateralis group 	94 
(3) The T. aeria group 	·94 
( 4) Chlorococcum hypnosporum 	95 
(5) The C. sp. (tetra isolate) group 	95 
(6) The C. perforatum group 	96 
( 7 ) Miscellaneous reactions 	96 
D. Conclusions 	97 
V. General Discussion . 	99 
VI. Summary . 	103 
VII. Literature Cited 	104 
VIII. Illustrations 	108-213 
I. General Introduction 
During the past 5 years, the senior author has been devoting attention to air­borne microorganisms, especially algae, with reference to their abundance and heterogeneity. Since January 1, 1963, he has been sampling the air daily on the the campus of The University of Texas and has been isolating the algae from these samples into axenic culture in view of their probable allergenic properties ( McEl­henny, Bold, Brown, and McGovern, 1963). In addition to the Austin samples, algae have been cultured from air samples taken at various locations in the conti­nental United States ( 21 states), Hawaii,1 and Mexico.2 These procedures have re­vealed a hitherto unexpectedly rich and diversified algal component of the atmos­phere (Brown, Larson, and Bold, 1964) and have brought into clear focus the desperate need for taxonomic and monographic studies of soil and air-borne algae per se and as a prelude to investigations of the allergenic properties of these organ­isms. 
During the course of these investigations; a number of cultures were assembled of a unicellular, chlorophycean alga, frequently present in the air, the alga being characterized by undergoing vegetative cell division ( sensu Herndon, 1958) and clearly unlike any alga previously described. These isolates have been included in the newly proposed genus T etracystis (Chlorosphaeraceae, Chlorosphaerales) to be described in this paper. In addition to the cultures of Tetracystis isolated from air, a number have been isolated from soil samples, soil itself clearly being the source of most air-borne algae. 
From approximately 50 isolates of T etracystis, 10 cultures representing 9 species, have been chosen for intensive study and characterization. In addition, the writers have uncovered evidence which indicates that 3 other algae described earlier as species of the genus C hlorococcum (Chlorococcaceae, Chlorococcales) are prob­ably also members of the chlorosphaeralean genus Tetracystis. According, 13 or­ganisms have been studied intensively and on the basis of these studies, 9 new species3 and 3 new combinations have been proposed. 
It has . become increasingly clear through our expanding knowledge of the soil algal flora (Trainor and Bold, 1953; Starr, 1955; Herndon, 1958b; Arce and Bold, 1958; Deason, 1959; Deason and Bold, 1960; Chantanachat and Bold, 1962; Mattox and Bold, 1962; and Bischoff and Bold, 1963) that there are numerous taxa of nonmotile, spherical unicellular algae with biflagellate, motile. stages. That these organisms cannot be identified by direct inspection in mixed collections and 
1 Courtesy of Dr. and Mrs. Wilson S. Stone, Department of Zoology, The University of Texas, Austin, Texas. 
2 Courtesy of Dr. C. J. Alexopoulos, Department of Botany, The University of Texas, Austin, Texas. 
3 Including two isob;ites of T. aeria. 
Studies of Algal Genera Tetracystis and Chlorococcum 
cultures is conceded by all who have examined such mixtures. Accordingly, the methods of study are of necessity microbiological, with attention to morphological and, increasingly, to physiological attributes of the organisms in axenic culture. 
This methodology has been applied to the algae which form the subject of this report. It has been augmented by including electron-microscopic and immuno­chemical methods. It is the writers' opinion that these enriched and expanded procedures have provided considerable insight into the taxonomy of the T etracystis species under consideration, while at the same time contributing data of broader biological significance and interest. These data will be discussed under 3 major headings, namely: ( 1) Morphology, taxonomy, and physiology of Tetracystis gen. nov.; ( 2) Electron microscopy of T etracystis and certain C hlorococcum species; and (3) Immunochemical studies of Tetracystis and Chlorococcum. Presentation of the data under each of these headings will be preceded by an ac­count of the materials and methods employed. 
II. 
Morphology, Taxonomy, and Physiology of 
Tetracystis Gen. Nov. 


A. 
MATERIALS AND METHODS 


Four of the 13 intensively studied isolates of Tetracystis were collected from the air by exposing Petri dishes ( 100 X 15 mm) of solidified ( 1.6%) "Bold's Basal Medium" ( BBM) (Bischoff and Bold, 1963 ) at various locations for given periods. The remaining 9 isolates from the soil were either provided to the writers in unialgal or axenic condition, or isolated from soil in the following manner: 5 g of a given soil sample were inoculated into a 125-ml Erlenmeyer flask containing 50 ml of sterile liquid BBM. 
"Bold's Basal Medium" (BBM) was prepared as follows: macroelements were supplied in the form of 6 stock solutions by dissolving the indicated weight of the following salts into 400 ml distilled or de-ionized water: 
NaN03 ··--···················------·----·-·-··············--·-··············---·---------­10.0 g KH2P04 -----····························---------------·-·······--······················· 7.0 g K2HP04 ···············---········-·····-···························· ................... . 3.0g MgS04·7H20 -······················ ··----···················-·····················----3.0 g CaCl2·2H20 ·································· ··········· ······---···· ········----------1.0 g NaCl -----·--·············----------····-----········· ·····························----------1.0 g 
Ten ml of each stock were employed for each liter of final solution. 
Minor (trace) elements were supplied in the form of the 4 following stocks: 
EDTA Stock Solution 
50 g EDTA (Ethylenediaminetetraacetic Acid ) and 31 g KOH were diluted to 1 liter with de-ionized or glass-distilled water. 
H-Fe Stock Solution 
· 4.98 g FeS04·7H20 were diluted to 1 liter with acidified water. Acidified water was made by adding 1 ml concentrated H2S04 to 999 ml de-ionized or glass-distilled water. 
H-Boron Stock Solution 
11.42 g H3B03 were diluted to 1 liter with de-ionized or glass-distilled water. 
H-H5 Stock Solution 
8.82 g ZnSo.-7H20 
1.44 g MnCl2·4H20 
0.71 
g Mo03 

1.57 
g CuS04·5H20 


0.49 g Co(N03 ) 2·6H20 were diluted to 1 liter with acidified water (as 
above). 
One ml of each stock solution was added to a liter of the final solution. 

Following exposure to the air for various lengths of time, the Petri dishes with BBM were incubated from 2 to 4 weeks under standard conditions of culture.1 Likewise, the Erlenmeyer flasks containing soil were incubated from 2 to 6 weeks, after which a portion of the supernatant was streaked across 100 X 15 mm Petri dishes of solidified BBM. After 2 weeks' incubation under standard conditions of culture, macroscopically visible colonies were identified and isolated into unialgal culture on agar slants of BBM. 
Other media used for growing fastidious organisms included soil extract agar and protease agar (Starr, 1964). 
The morphology and life-cycle phases of T etracystis were studied by making fresh mounts and hanging-drop preparations from cultures which were growing on agar slants or Petri dishes. Agar slant cultures of T etracystis were maintained as stocks, and when cultures were desired for study, they were transferred into fresh agar slants of BBM. Cultures prepared for study on Petri dishes were inoculated from actively growing agar-slant cultures. Five ml of sterile, liquid BBM were aseptically added to each agar slant culture and the algal material gently removed from the agar surface into the liquid phase by means of a sterile platinum loop. The cells were further dispersed into a homogeneous suspension by a 5-10 sec treatment in an ultrasonic water bath.2Six drops of this homogeneous suspension were trans­ferred aseptically from the tube to the surface of agar in Petri dishes by a disposable Pasteur pipette. The inoculum was then vigorously swirled to disperse the algae evenly, and the Petri dishes were inverted and maintained under standard con­ditions for from 2 to 4 weeks. Ifthe NaNOa concentration was increased to 3 times that of BBM, the Petri dish cultures could be maintained in log phase of growth up to 3 weeks, as compared to 10-14 days on the standard basal medium. 
1 Standard conditions of culture: illumination of 250-300 ft-c intensity; a 12-12 hr diurnal, light-dark cycle; and a temperature range of 19-22° C. 2 Di-sontegrator, System 80; Model G-80Cl; Ultrasonic Industries, Inc., Albertson, New York. 
Tetrad formation occurred in greatest frequency in cultures exp0sed for about 6 hr to light of the 12-12 diurnal dark-light period. Zoospore formation usually could be evoked in actively growing cultures by placing a culture (growing on freshly poured agar) in an uninterrupted dark period of 5-8 hr, followed by a continuous light period of 1-3 hr. However, some species of T etracystis, T. texensis for example, were very fastidious. Here, zoospore formation could be effected only by repeated transfer from solidified BBM to liquid BBM and by manipulating the day-length cycle. 
Cell-wall thickness and the presence or absence of gelatinous matrices were de­termined with India ink and/ or a weak aqueous solution of Methylene blue. Vari­ous concentrations of I2KI were used to determine the presence of starch, the posi­tion of the nucleus, and the number and length of zoospore flagella. 
Limited cytological studies were made by fixing the algae (which had been at­tached to a microscope slide by egg albumin) in a freshly prepared solution of 1 part glacial acetic acid to 3 parts absolute ethanol for 30 min. Then the slides were flooded with acetocarmine prepared according to the method of Cave and Pocock ( 1956) and passed over a low flame until vapors arose from the stain. After the heating process, the slides were drained and observations were made immediately. No permanent preparations were made. 
Various physiological and certain morphological tests require the use of axenic cultures. These were achieved by processing unialgal cultures of Tetracystis through certain physical and chemical treatments which are described in detail by Brown and Bischoff ( 1962) . 
A number of media were employed repeatedly for purity of cultures and for studying comparative growth. These included Protease Agar (Starr, 1964); Difeo Nutrient Agar; Difeo Nutrient Broth; Difeo Thioglycollate Broth, and Yeast Ex­tract Agar. The latter was prepared by adding 5 g of yeast extract powder to 1 liter of distilled or deionized water and 16 g of agar. 
Other media were employed specifically for comparative study of certain physi­ological attributes. Their composition and preparation are described at appropriate sites in the body of the paper. 
Observations of colony characteristics and isolation of single cells or colonies for axenic cultures were made with a Bausch and Lomb stereroscopic binocular micro­scope. Photomicrographs were taken with a 35-mm Zeiss-Winkel camera attached to a Bausch and Lomb microscope with apochromatic objectives. Macroscopic pic­tures were made with a Zeiss Super Contaflex 35-mm single-lens reflex camera. 
B. GENERIC CHARACTERIZATION The fact that many of the 13 algae under consideration at some stage in their life cycle occur as tetrahedral tetrads of vegetative cells (Text-fig. 1) suggested at 
once to the writers that they were species of the little known and infrequently en­countered genus Borodinella (Miller, 1927). However, continuing study of the 
Brown and Bold 
isolates, careful reading of Miller's paper, and examination of his figures made it certain that the algae under consideration could not be assigned to Borodinella. Miller described and figured for the latter an axile chloroplast, while those of the writers' organisms are always parietal, however massive. Although it is not abso­lutely cert~in from Miller's paper (and no living type cultures are available for verification), that the zoospores of Borodinella become spherical upon quiescence, there is evidence from Miller's figures that, in fact, they did so. Borodinella, accord­ingly, would differ from the writer's organisms, not only in the nature of its chloro­plast, but also in the fate of its zoospores, 2 characters shown by Starr ( 1955), and a number of subsequent investigators, to be reliable taxonomic criteria at the generic level. These considerations have impelled the assignment of the algae under discussion to the new genus Tetracystis. 
Tetracystis, like Chlorosarcinopsis, clearly belongs to the order Chlorosphaerales (Herndon, 1958). Herndon proposed this order for those unicellular, nonmotile, 
. zoospore-producing Chlorophyceae with vegetative cell division. This last attribute has been discussed critically by Herndon ( 1958) and later by Deason and Bold ( 1960) . As conceived by Herndon, vegetative cell division involves partitioning of a cell and deposition of new wall material in such fashion that portions of the parent cell wall clothe the daughter protoplasts, at least immediately following cell division. Deason and Bold believed that contiguity of the daughter cell wall with that of the parent was not necessarily essential to the concept of vegetative cell di­vision. Instead, vegetative cell division was conceived by them to involve interven­ing cell-wall deposition between each mitosis and cytokinesis. In contrast with cells dividing non-vegetatively (to form zoospores and aplanospores), rapidly oc­curring nuclear divisions and cytokineses are followed by cell wall deposition only after all nuclear and protoplast divisions have been completed, according to Deason and Bold. 
The writers have found that both Herndon's and Deason and Bold's concepts of vegetative cell division would not include unequivocally all the species of Tetra­cystis,1 yet this genus clearly seemed to belong in the Chlorosphaerales because of the presence of tetrads of daughter vegetative cells (Fig. 43) and complexes of the same (Fig. 22). For this reason, the writers sought more evidence which would aid in the stabilization of taxonomic characteristics for the order Chlorosphaerales and found in T etracystis, along with all presently known chlorosphaeralean algae, an additional precise, ordinal attribute for the Chlorosphaerales, namely, the fol­lowing: intercalated between the motile and vegetative phases in a given cycle (Text-fig. 1), members of the Chlorosphaerales have a nonmotile phase, in which the division products of vegetative cells are neither motile nor potentially motile like aplanospores. These products, daughter vegetative cells, may become dissoci­
1 Some species of T etracystis lose parent and daughter wall contiguity immediately after wall formation; some species form tetrads of daughter cells directly, without intervening diad formation. 

I. 

Text-fig. 1 
Diagrammatic representation of the life cycles of the Chlorosphaerales, exemplified by Tetracystis II and Ill and the Chlorococcales, exemplified by Chlorococcum Ill only). Z = zoospore, YVC = young vege­tative cell, VC = vegetative cell, ZS = zoosporangium, APL = aplanospore, T = tetrads of daughter . vegetative cells, both small and large, and TC = tetrad complexing. The vegetative cells of Chlorococcum have the capacity to form only zoosporangia which, in turn, produce zoospores or aplanospores llll. The vegetative cells of Tetracystis also may form zoospores or aplanospores llll, and, in addition, they may form tetrads of nonmotile, daughter vegetative cells (11 which do not have the capacity for potential motil­ity. These tetrads may be of 2 sizes at maturity. The doughier vegetative cells within the tetrad may form 
zoosporangia which, in turn, form zoospores or aplanospores, or they may continue to produce tetrads 
of nonmotile daughter cells resulting in tetrad complexes (TC). The tetrad complexes may form zoospores 
or aplanospores (not shown>. Daughter vegetative cells may be released from tetrads or tetrad complexes, 
either very soon after their formation, or only very late, depending on the species. 
ated at the diad, tetrad, or octad levels, or they may remain in association to form complexes. The products of the nonmotile pha~e are similar to autospores' in that both are nonmotile, and unlike aplanospores in that the daughter vegetative cells in chlorosphaeralean tetrads have not developed from zoosporic antecedent. 
1 Until autospore formation has been more thoroughly studied electron microscopically than by Murakami, Morimura, and Takamiya ( 1963), no final decision can be made regarding whether the vegetative cells are identical with autospores in their ontogeny. The writers dis­tinguish aplanospores and autospores on the basis that aplanospores arise (in ontogeny) from zoospores or potential zoospores, which seemingly is not true of autospores. 
Brown and Bold 
By contrast, it will be recalled that nonmotile vegetative cells of chlorococcacean algae always reproduce by forming either motile ( zoospores or gametes, or both) cells or nonmotile, but potentially motile, cells ( aplanospores) (Text-fig. 1). 
Tetracystis gen. nov. 
Cellulae vegetativae seiunctae aut binae quaternae, octonae, senae denae, etc., cel­lulis filiabus aggregationum primum associatis, interdum deinde dissociantibus. Cel­lulae chloroplastrum cavum plus minusve solidum, parietalem, per fissuras saepe trans­versum, pyrenoideo praeditum, habentes. 
Reproductio per divisionem in duas-octo cellulas vegetativas, sine potestate mobi­litatis directae, eo modo differans ab aplanosporis quas cellulae quoque efficere possunt. Reproductio asexualis per zoosporas per cellulas omnis aetatis ( cellulis filibus vegetativis tetradis inclusis), formatas, zoosporis admodum quiescentibus sphericis non factis. 
Reproductio sexualis, cum apparet, per gametas biflagellatas. 
Vegetative cells isolated or in groups of 2, 4, 8, or in multiples of 2 or 4, the daughter cells of the groups associated at first, sometimes secondarily dissociating. Cells with a hollow, more or less massive, parietal chloroplast, often transversed by fissures; with a pyrenoid. 
Asexual reproduction by division into 2-8 vegetative cells which lack the po­tentiality of direct motility; thus differing from aplanospores which arise from motile or potentially motile precursors. Asexual reproduction also by zoospores formed by cells of all ages (including the daughter vegetative cells of the tetrad) , the zoospores not becoming spherical immediately upon quiescence. 
Sexual reproduction, when present, by biftagellate gametes. 
Unlike Chlorosarcinopsis and Chlorosarcina, which may form regular aggre­gates of packets, T etracystis is characterized by the presence of tetrads of nonmotile daughter vegetative cells (or diads and octads) which may or may not cohere to form irregular complexes (Text-fig. 1 ; Fig. 80). Furthermore, the zoospores of Tetracystis do not become spherical immediately upon quiescence (Fig. 31) . Un­like Chlorococcum, Tetracystis forms nonmotile daughter vegetative cells (Text­fig. 1 ; Fig. 60) which are not aplanosporic in origin and nature. 
C. 	SPECIFIC CHARACTERIZATION Attributes of the newly named species of Tetracystis follow. 
Tetracytis aeria sp. nov. (C-6) 1 (Fig. 13-18, 89-92 )2 (Type species) 
Cellulae vegetativae iuvenes ellipsoideae ad ovatus, in cellulae vegetativas sphericas 14-16 µ. diam. celeriter maturescentes. Membrana cellulae aliquantulum incrassata (0.5-1.0 µ. in culturis duarum hebdomadum aetate), et magis incrassata (2-4 µ.) in 
1 Designation of isolates in authors' culture collection. 
2 Figures illustrating ultrastructure are not cited here. 

culturis trium mensium aetate. Chloroplastus solidus, aliquot fissuras radiantes non profundas praebens, unicum pyrenoideum sphericum, 4 µ. diam., multis amyli micis circumdatum, habens. Cellulae ftavovirides in incrementi periodo immobili ( tribus mensibus) factae. Vacuolae contractiles in cellulis vegetativis uninucleatis infrequenter observatae. 
Reproductio asexualis per cellulae divisionem diades aut plerumque tetrades cellu­larum vegetativarum directe efficientem, quarum maturarum membranae cellulae parentis membranae conferte associatae. Complexus unius ad aliquot tetrades fre­quentes. Reproductio asexualis etiam per zoosporas aplanosporas. Zoosporae dolio­formes (late cylindricae) 10-12 X 5 µ., nucleum anteriorem, duas vacuolas contractiles anteriores, chloroplastum parietalem, unicum pyrenoideum equatoriale stigma anterius magnum duo flagella longitudine aequa, 1-1/~1-l/2 longi Iongiora quam longi­tudo corporis habentes. 
Reproductio sexualis non observata. 
Culturae duarum hebdomadum aetate in medio "agar" basali modice dilute virides; superficies coloniae levis ad aliquantulum asperam (minute granulosam) magnifi­catione sex et 12 plo; coloniae magnificatione nulla opaco-nitidae ad nitidas. 
Origo: ex acre super locum Pampa, Texas, dictum, b. Jun. 1960. 
Young vegetative cells ellipsoidal to ovoid, rapidly maturing into spherical vege­tative cells 14-16 µ.in diameter. Cell wall slightly thickened (0.5-1.0 µ.in 2-week­old cultures) and more thickened ( 2-4 µ.) in cultures 3 months old. Chloroplast massive, with a few shallow, radiating fissures, containing a single, spherical pyre­noid, 4 µ. in diameter, surrounded by many starch grains. Cells becoming yellow­green in the stationary phase of growth ( 3 months). Contractile vacuoles infre­quently observed in the uninucleate vegetative cells. 
Asexual reproduction by cell division giving rise directly to diads or mostly to tetrads of vegetative cells, the walls of which are closely associated with the parent cell wall at maturity. Complexes of 1 to several tetrads frequent. Asexual reproduc­tion also by zoospores and aplanospores. Zoospores barrel-shaped (broadly cylin­drical), 10-12 X 5 µ. , with an anterior nucleus, 2 anterior contractile vacuoles, a parietal chloroplast, a single, equatorial pyrenoid, a large anterior stigma, and 2 flagella of equal length 1-1/4-1-1/2 times body length. 
Sexual reproduction not observed. 
Two-week-old cultures on basal agar medium light-green; colony surface smooth to slightly rough (minutely granular) at 6 and 12 X magnification; colonies dull­shiny to shiny macroscopically. 
Source: from air over Pampa, Texas, June, 1960. 
Two cultures of T etiacystis (C-6 and Pa-3), which are presumably the same species ( Tetracystis aeria), have been isolated from diverse geographical localities. Isolate C-6 was made from an air collection over Pampa, Texas, 485 miles north of Austin, Texas, the source of the second isolate, Pa-3. The latter was isolated from an air collection from the campus of The University of Texas, approximately 250 ft above ground level. 
It is of interest to note that these 2 isolates are virtually indistinguishable from one another morphologically and physiologically, and when compared ultrastruc­turally and immunochemically, no differences were detected. T etracystis aeria and 
T. aplanosporum are the only 2 presently known species of T etracystis in which the zoospores have anterior nuclei. Tetracystis aeria is very readily distinguishable morphologically from T. aplanosporum by: ( 1) smaller size of mature vegetative cells of 14-16 p.; (2) tetrad coherence to form cellul.ar complexes; ( 3 ) cell wall thickening of 2--4 p. at 3 months on BBM agar; (4) zoospores which have a large and distinct anterior stigma; and ( 5 ) zoospores with flagella longer than the cell body length. 
T etracystis aeria reproduces asexually to form diads or tetrahedral tetrads of daughter cells (i.e., only 3 of the 4 daughter cells can be observed in a given focal plane (Fig. 122, 126). The tetrahedral tetrads are formed directly without inter­vening diads formation, cytokinesis not being initiated until all nuclear divisions have been completed. Diad stages are infrequently present; however, isobilateral tetrads, which originate from diads, have not been observed in T. aeria. 
Tetracystis aggregata sp. nov. ( Pc-1 ) (Fig. 33--40, 83-84) 
Cellulae vegetativae iuvenes ellipsoideae ad ovatas, in cellulas vegetativas sphericas 
15-16 p. diam. celeriter maturescentes. Incrassatio membranae cellulae in culturis 
duarum hebdomadum non notabilis, in culturis trium autem, mensium, aetate a 2 ad 
3 p. varians. Chloroplastus solidus, fissuras radiales latas saepe praebens. Pyrenoideum 
4-6 p. diam., centrale, saepe lobatum, micis amyli multis circumdatum. Cellulae in 
incrementi periodo immobili semper virides. Vacuolae contractiles in cellulis vegetativis 
uninueleatis interdum observatae. 
Reproductio asexualis per cellulae divisionem et diades et tetrades cellularum vege­
tativarum immobilium, maturarum membranae cellulae perentis conferte associatarum, 
directe efficientem. Multae diades tetradesque cohaerentes, complexus magnos for­
mantes. Reproductio asexualis etiam per zoosporas aplanosporasque. Zoosporae ovatae 
4 X 10-12 µ., nucleum posteriorem duas vacuolas contractiles anteriores, chloroplastum 
parietalem, unicum pyrenoideum equatoriale, stigma equatoriale ad paululo anterius 
et duo flagella longitudine aequa, 1-1/ 2 plo longiora quam longitudo corporis cellulae . habentes. 
Reproductio sexualis per gametas isogamicas a zoosporis morphologicaliter indis­
tinguibiles nisi quod saepe minores. Zygotum echinatum, 15-20 p. diam., divisione 
quattuor cellulas vegetativas efficiens. 
Culturae duarum hebdomadum aetate in medio "agar" basali atrovirides; super­
ficies coloniae granulosa (minute botryoidea) magnificatione sex et 12 plo; coloniae 
magnificatione nulla siccae. 
Origo: a collectione in aere ca. 83 metra alt., in campo loci Univ. Texas, Austin, 
dicti, m. Jul. 1960. 
Young vegetative cells ellipsoidal to ovoid, rapidly maturing into spherical vege­tative cells, 15-16 p. in diameter. Cell wall thickening insignificant in 2-week-old cultures but ranging from 2 to 3 p. in cultures 3 months old. Chloroplast massive, with broad, radial fissures often present. Pyrenoid 4-6 p. in diameter, central, often lobed, surrounded by many starch grains. Cells remaining green in stationary growth phase. Contractile vacuoles occasionally observed in uninucleate vegetative cells. 
Asexual reproduction by cell division giving rise directly to both diads and tetrads of nonmotile vegetative cells which are intimately associated with parent cell wall at maturity. Many diads and tetrads coherent, forming large complexes. Asexual reproduction also by zoospores and aplanospores. Zoospores ovoid, 4 X 10-12 P., with a posterior nucleus; 2 anterior contractile vacuoles; parietal chloroplast; a single equatorial pyrenoid; equatorial-to-slightly anterior stigma; and 2 flagella of equal length, 1-1/2 times cell body length. 
Sexual reproduction by isogamous gametes, indistinguishable morphologically from zoospores except often by their smaller size. Zygote echinate, 15-20 p. in diameter, undergoing division giving rise to 4 vegetative cells. 
Two-week-old cultures on basal agar medium, dark green; colony surface gran­ular (minutely botryoid) at 6 and 12 X magnifications; colonies dry macroscop­ically. 
Source: from air collection approximately 250 ft above ground level, The Uni­versity of Texas campus, Austin, July, 1960. 
Tetracystis aggregata has many attributes in common with T. isobilateralis, T. dissociata, and T. illinoisensis; however, it differs significantly from T. isobilateralis, the species which it most clearly resembles, in: ( 1) the presence of a rough granular colony at 2 weeks on BBM agar; ( 2) strong adherence of diads and tetrads to form large cellular complexes; and ( 3) the presence of occasional deep, radial fissuring of the chloroplast. The latter characteristic can sometimes lead to misinterpretation of the chloroplast as axile. However, since the manifestation of deep, broad, radial fissures is not always apparent, and since the chloroplast is most typieally massive, hollow, and cup-like, this organism is herein described as a species of Tetracystis. 
Tetracystis dissociata1 sp. nov. ("Starr") (Fig. 25-32, 87-88) 
Cellulae vegetativae iuvenes ellipsoideae multae, in cellulas vegetativas sphericas ad subsphericas 14-16 p. diam. tarde maturescentes. Incrassationes internales unipolares suque ad 1 p. crass. in culturis trium mensium aetate interdum repertae. Chloroplastus nitido-viridis, parietalis, satis solidus, paucissimas fissuras non profundas praebens, unicum pyrenoideum centrale lobatum 3-4 p. diam., multis amyli micis circumdatum, habens. Culturae virides in incrementi periodo immobili ( tribus mensibus) aerolis albis ( cellulis defunctis) maculosae factae. Vacuolae contractiles in cellulis vegetativis uni­nucleatis nullae. 
Reproductio asexualis per cellulae divisione raro diades, plerumque tetrades cellu­
1 No. 128 in Culture Collection of Algae, Indiana University; isolated by Dr. Vischer and labeled "Borodinella sp." 
!arum vegetativarum, membranae cellulae parent parentis conferte associatarum, directe efficientem. Tetrades diadesve ad complexus formandos numquam associatae, sed ut diades tetradesque manentes. Reproductio asexualis etiam per zoosporas aplano­sporas. Zoosporae cylindricae, 3-4 X 9-10 µ. nucleum posteriorem, duas vacuolas con­tractiles anteriores, chloroplastum parietalem, unicum stigma quatoriale-ad-anterius, et duo flagella longitudine aequa, 1-1/ 2 plo longiora quam longitudo corporis ce!lulae habentes. 
Reproductio sexualis non observata. 
Culturae duarum hebdomadum aetate in medio "agar" basali nitido-virides ; supe~­ficies coloniae levis homogeneaque magnificatione sex et 12 plo, colonia magn:fi::atione nulla opaconitida. 
Origo: e collectione culturarum Algarum in Univ. Indiana #128 ut Borodinella sp. sec. Vischer qui earn seiunxit. 
Young vegetative cells ellipsoidal and numerous, maturing slowly into spherical to subspherical vegetative cells 14-16 µ. in diameter. Occasional internal unipolar wall thickenings up to 1 µ. thick in cultures 3 months old. Chloroplast bright-green, parietal, and rather massive, with very few shallow fissures. Chloroplast with a single, central, lobed pyrenoid, 3-4 µ. in diameter, surrounded by many starch grains. Cultures green, becoming mottled with white areas (dead cells) in the sta­tionary phase of growth ( 3 months). Contractile vacuoles absent in uninucleate vegetative cells. 
Asexual reproduction by cell division only occasionally giving rise directly to diads and moo:;tly to tetrads of vegetative cells which are intimately associated with the parent cell wall. Tetrads or diads never associated to form complexes but re­maining at the diad and tetrad level of association. Asexual reproduction also by zoospores and aplanospores. Zoospores cylindrical, 3-4 X 9-10 µ.,with a posterior nucleus, 2 anterior contractile vacuoles, a parietal chloroplast, a single, equatorial­to-anterior stigma, and 2 flagella of equal length, 1-1/2 times cell body length. 
Sexual reproduction not observed. 
Two-week-old cultures on basal agar medium, bright-green ; colony surface srhooth and homogeneous at 6 and 12 X magnification; colony dull-shiny macro­scopically. 
Source: Culture Collection of Algae at Indiana University No. 128 as "Borodi­nella sp." sec. Vischer, the isolator. 
This organism was obtained for study from the Culture Collection of Algae, Indiana University, at the suggestion of Dr. Richard C. Starr. The alga is clearly not a member of Borodinella as Miller ( 1928) originally described it, because the chloroplasts are not axile with radiating arms, but, instead, hollow and cup-like, with varying degrees of peripheral fissuring; furthermore, the zoospores do not be­come spherical upon quiescence. Accordingly, the writers consider this culture to exemplify organisms in the genus T etracystis, rather than Borodinella. 
Tetracystis dissociata has many attributes in common with T. isobilateralis, T. 
aggregata, and T. illinoisensis but differs significantly from T. illinoisensis, which it most closely resembles, in: ( 1 ) the absence of contractile vacuoles in the vegetative cells; (2) zoospores which have flagella longer than the cellular body length; (3) insignificant cell wall thickening at 3 months on BBM agar; and (4) the apparent absence of sexual reproduction. 
Tetracystis excentrica sp. nov. ("Opera") (Fig. 1-12, 101-102) 
Cellulae vegetativae iuvenes ellipsoideae, in cellulas vegetativas sphericas 16 µ.diam. celeriter maturescentes. Incrassationes, membranae internales unipolares bipolaresque (1-1/2--4 X 3/ 4 µ. ) in culturis duarum habdomadum aetate notabiles. Chloroplastus parietalis, subsolidus, punctatus dilute viridis; fissuris nullis. Areola cytoplasmica inter­nalis magna liquida in cellulis vegetativis maturis observabiles. Chloroplastus unicum chloroplastum parietalem, unicum pyrenoideum equatoriale, stigma maxime anterius, hemisphericis praeditum, continens. Cellulae in incrementi periodo immobili flavo­aurantiae factae. Duae vel multae vacuolae contractiles in cellulis vegetativis maturis uninucleatis. 
Reproductio sexualis per gametas isogameticas a zoosporis morphologicaliter indis­vegetativarum, maturarum a membrana cellulae parentis mox dissociatarum, directe efficientem. Tetrades ad complexus formandos non associatae. Reproductio asexualis etiam per zoosporas aplanosporasque. Zoosporae cylindricae, 3 X 10 µ. postice satis amplificiatae, antice acuminatae; nucleum posteriorem duas vacuolas contractiles, chloroplastum parietalem, unicum, pyrenoideum equatoriale, stigma maxime anterius, minutum lineare et duo flagella longitudine aequa paululo breviora quam longitudo corporis cellulae habentes. 
Reproductio sexualis per gametas isogameticas a zoosporis morphologicaliter indis­tinguibiles. Plasmogamia 3-5 minutis post copulationem perfecta. Zygotum mem­branam levem habens, a cellulis vegetativis indistinguible, fnictus germinationis zygoti ignoti. 
Culturae duarum hebdomadum aetate in medio "agar" basali dilute-virides; super­
ficies coloniae levis ( magnificatione sex et 12 plo) ; coloniae magnificatione nulla humi­
dae ad nitidas. 
Origo: e solo granitico distante 7 milia passuum ab loco Evergreen, Colorado diet~, 
m. Aug. 1961. 
Young vegetative cells ellipsoidal, rapidly maturing into spherical vegetative cells 16 µ.in diameter. Significant, internal unipolar and bi-polar wall thickenings ( 1 ~-4 X 3/4 µ.) in 2-week-old cultures. Chloroplast parietal, somewhat massive, punctate, light-green; fissures absent. Large, clear internal cytoplasmic area observ­able in mature vegetative cells. Chloroplast containing a single, excentric, ellipsoidal pyrenoid (long axis, 3-4 µ.), with 2 hemispherical starch grains. Cells becoming yellow-orange in stationary phase of growth. Two-to-many contractile vacuoles present in mature, uninucleate vegetative cells. 
Asexual reproduction by cell division giving rise directly to tetrads (rarely to 
diads) of vegetative cells which soon dissociate from parent cell wall at maturity. 
Tetrads not associated to form complexes. Asexual reproduction also by zoospores and aplanospores. Zoospores cylindrical, 3 X 10 µ., somewhat broadened pos­teriorly and pointed anteriorly; with a posterior nucleus, 2 anterior contractile vacuoles, a parietal chloroplast, a single equatorial pyrenoid, a very anterior, min­ute, linear stigma, and 2 flagella of equal length, slightly shorter than cell body length. 
Sexual reproduction by isogamous gametes indistinguishable morphologically from zoospores. Plasmogamy completed within 3-5 min after pairing. Zygote smooth-walled, indistinguishable from vegetative cells, the products of zygote germination unknown. 
Two-week-old cultures on basal agar medium, light-green; colony surface smooth ( 6 and 12 X magnification); colonies moist to shiny macroscopically. 
Source: granitic soil 7 miles west of Evergreen, Colorado, August, 1961. 
The soil collection from which this organism was isolated was made by Dr. and Mrs. R. Malcolm Brown of Pampa, Texas, August, 1961. Tetracystis excentrica is closeiy related to 3 other species of Tetracystis, namely T. intermedium, T. texensis, and T. pulchra. All 4 of these species of Tetracystis have in common vege­tative cells with an ellipsoidal pyrenoid surrounded by 2 starch grains. The pyre­noid is also excentric in the cells. 
T etracystis excentrica is characterized by several salient features: ( 1 ) yellow­oi'ange colonies on BBM agar at 3 months; ( 2) 2-to-many contractile vacuoles in the vegetative cells (when more than two are present, they are in the peripheral protoplasm of the cell rather than in the perinuclear region) ; ( 3) daughter cells of the tetrad which dissociate soon after wall formation is complete; and ( 4) a smooth colony surface on BBM at 2 weeks. 
Tetracystis excentrica is the only species in which the product of isogamous plasmogamy (Fig. 4-11) is a smooth-walled zygote, indistinguishable from the vegetative cell, at least during some part of its ontogeny. A diplobiontic cycle may be operative within this species; however, more intensive study will be needed in order to prove this hypothesis. After a culture on BBM agar has grown for about 2-3 weeks, several very large cells ( 20-35 µ. in diameter ) with several pyrenoids have been observed (Fig. 12). It is entirely possible that these cells may represent the zygote in a later phase of its ontogeny. The giant cells are multinucleate. Un­equal, uninucleate segments of the protoplast frequently are formed and break away from the main portion (Fig. 12). Whether or not these uninucleate segments abort or live is unknown at present. 
One of the most beautiful and striking occurrences of sexual reproduction among the Chlorophyta in the writers' experience has been observed in T. excentrica.1 If one takes an actively growing culture and places a loopful of inoculum into a hanging drop culture (in distilled water) in the afternoon of a given day, he will 
1 The complete process, photographed at 5-sec intervals, is shown in Fig. 4-11. , 
find abundant pairing of gametes the following morning. Following pairing, the process of plasmogamy is extremely rapid, once it starts. About 5-15 min after pair­ing, a cytoplasmic bridge will be formed between the 2 gametes. Then, in an in­stant ( 5-30 sec) , the protoplast of one gamete will unite with that of the other, and the gametic walls will be shed. The daughter chloroplasts seem to unite (Fig. 5-9), but the 2 pyrenoids of the zygote have not been observed to fuse. The fate of the zygote is unknown because, as it enlarges, it becomes indistinguishable from the vegetative cells. 
Tetracystis illinoisensis. (R-3-3 ) (Figs. 53-56, 85-86 ) 
Cellulae vegetativae iuvens ellipsoidales ad ovatas, in cellulas vegetativas ovatas ad sphericas 12-14 µ.diam. maturescentes. Incrassationes membranae cellulae in culturis duarum hebdomadum aetate non notabilis, post tres menses, autem, incrassationes ( 5 µ.) internae manifestae unipolases bipolaresque adsunt. Chloroplastus solidus paucis­simas fissuras praebens, unicum pyrenoideum paululum excentricum forma irregulare ( 4-5 µ. diam.) et multas amyli micas continens. Cellulae in incrementi periodo im­mobili ( tribus mensibus) virides. Cellulae vegetativae uninucleatae duas vacuolas contractiles habens. 
Reproductio asexualis per cellulae divisionem in diades tetradesque cellularum­filiarum vegetativarum quae maturae primum in membrana cellulae parentis inclu­dentur et eis adhaerent. Diades tetradesque ad complexus formandos non aggregatae. Reproductio asexualis etiam per zoosporas aplanosporasque. Zoosporae cylindricae 3-4 X 8-10 µ., nucleum posteriorem, duas vacuolas contractiles anteriores, chloroplas­tum parietalem, unicum pyrenoideum equatoriale, stigma equatoriale ad paululo anterius, et duo flagella paululo breviora quam longitudo corporis cellulae habentes. -
Reproductio sexualis non observata. 
Culturae duarum hebdomadum aetate in medio "agar" basali dilute virides. Super­
ficies coloniae homogenea (ad minute granulosa) magnificatione sex et 12 plo; coloniae 
magnificatione nulla opaconitidae. 
Origo : e patina "Petri" medii culturae aeri ex automobili exposita, in loco distante 
30 milia passuum ab oppido Effingham, Ill. dicto, m. Sept. 1962. 
Young vegetative cells ellipsoidal to ovoidal, maturing into ovoidal to spherical 
vegetative cells 12-14 µ. in diameter. Cell wall thickening insignificant in cultures 
2 weeks old, but prominent internal, unipolar and bipolar thickenings ( 5 µ.) present 
at 3 months. Chloroplast massive with very few fissures, containing a single, slightly 
excentric, irregularly shaped pyrenoid ( 4-5 µ. in diameter) with many starch 
grains. Cells green in the stationary phase of growth ( 3 months). U ninucleate 
vegetative cells with 2 contractile vacuoles. 
Asexual reproduction by cell division into diads and tetrads of daughter vegeta­
tive cells which at maturity initially remain enclosed by and adhere to the parent 
cell wall. Diads and tetrads not aggregating to form complexes. Asexual reproduc­
tion also by zoospores and aplanospores. Zoospores cylindrical 3-4 X 8-10 µ.,with 
a posterior nucleus, 2 anterior contractile vacuoles, parietal chloroplast, a single 
equatorial pyrenoid, and equatorial to slightly anterior stigma, and 2 flagella which 
are slightly shorter than cell body length. 
Sexual reproduction not observed. 
Two-week-old cultures on basal agar medium, light green; colony surface homo­
geneous (to minutely granular) at 6 and 12 X magnification; colonies dull-shiny macroscopically. Source: from Petri dish of culture medium exposed to air from an automobile, 30 miles east of Effingham, Illinois, September, 1962. 
This organism was kindly provided to the writers by Misses Pat Walne and Elenor Cox who collected it from the air by exposing a Petri plate to sterile BBM agar from a moving automobile 30 miles east of Effingham, Illinois, September, 1962. 
Tetracystis illinoisensis has many attributes in common with T. isobilateralis, T. aggregata, and T. dissociata; however, it differs significantly from T. dissociata, which it most closely resembles, in: ( 1 ) the presence of 2 contractile vacuoles in the vegetative cell; ( 2) zoospores with flagella slightly shorter than the cellular body length; and (3) cell walls with unipolar and bipolar thickenings (up to 5 µ.) on BBM agar at 3 months. 
Like T. dissociata, T. illinoisensis is characterized by the presence of tetrads (mostly tetrahedral) which never aggregate to form complexes. Unlike T. disso­ciata, T. illinoisensis undergoes a delayed tetrad formation; this accounting for the small population of tetrads in cultures on BBM agar at 2 weeks. 
Tetracystis isohilateralis sp. nov. (AG-2-3) (Fig. 41-48, 81-82) 
Cellulae vegetativae maturae sphericae ad subsphericas, 18-19 µ. diam. Incrassatio membranae cellulae non notabilis in culturis duarum hebdomadum aetate, varians, autem, ab 2 ad 3 µ.in culturis trium mensium aetate. Chloroplastus solidus quasi macu­olosus atque dilute, viridis, totum cellulae lumen, area perinucleari excepta, comp!ens ; fissuris profundis nullis. Chloroplastus unicum pryenoideum centrale magnum 5-7 µ. diam., forma irregulare, multis amyli lamellis circumdatum, habens. Cellulae in in­crementi periodo immobili semper virides. Vacuolae contractiles in cellulis vegetativis uninucleatis nullae. 
Reproductio asexualis per cellulae divisionem diades tetradesque cellularum vege­tativarum, maturarum membranae parentis conferte associatarum, directe effr:ientem. Complexus unius vel aliquot tetradum frequentes. Reproductio asexua.Jis etiam per zoosporas aplanosporas bipartitione successiva protoplasti multinucleati formatas: Zoosporae ovatae 4-6 X 8-10 µ. , nucleum posteriorem, duas vacuolas contractiles an­teriores, chloroplastum parietalem, unum ad aliquot pyrenoidea equatorialia, stigma equatoriale ad paululo anterius et duo flagella longitudine aequa, 1Y2 plo longiora quam longitudo corporis habentes. 
Reproductio sexualis per gametas isogamicas, a zoosporis, nisi quod paululo minor­ibus, morphologicaliter indistinguibiles. Zygotum echinatum, 15-20 µ. diam., di­visionem instantem subiens, quattuor cellulas vegetativas efficiens. 
Culturae duarum hebdomadum aetate in medio "agar" basali dilute virides; super­ficies coloniae aspera (minute scabellata) magnificatione sex et 12 plo; coloniae mag­nificatione nulla opaconitidae aut siccae. 
Origo: e solo a loco Blackland Prairie Region of Williamson County, Texas, dicto, 
m. July 1960. 
Mature vegetative cells spherical to subspherical, 18-19 p. in diameter. Cell wall thickening insignificant in 2-week-old cultures but ranging from 2 to 3 p. in cultures 3 months old. Chloroplast massive, somewhat mottled and light green, filling the entire cell lumen except for the perinuclear area; deep fissures absent. Chloroplast with a single, large, central pyrenoid, 5-7 p. in diameter, irregular shaped and sur­rounded by many starch plates. Cells remaining green in the stationary phase of growth. Contractile vacuoles absent in uninucleate vegetative cells. 
Asexual reproduction by cell division giving rise directly to diads and tetrads of vegetative cells which are intimately associated with the parent wall at maturity. Comp!exes of 1 to several tetrads frequent. Asexual reproduction also by zoospores and aplanospores formed by succt;ssive bipartition of a multinucleate protoplast. Zoospores ovoid 4-6 X 8-10 p.; with a posterior nucleus, 2 anterior contractile vacuoles, a parietal chloroplast, 1-to-several equatorial pyrenoids, equatorial-to­slightly anterior stigma, and 2 flagella of equal length, 1 y2 times body length. 
Sexual reproduction by isogamous gametes, indistinguishable morphologically from the zoospores except for their slightly smaller size. Zygote echinate, 15-20 p. in diameter, undergoing immediate division giving rise to 4 vegetative cells. 
Two-week-old cultures on basal agar medium, light-green; colony surface rough (minutely scabellate) at 6 and 12 X magnification; colonies dull-shiny or dry macroscopically. 
Source: soil from Blackland Prairie Region of Williamson County, Texas, July, 
1960. 
This species was provided to the writer by Mrs. LaVerne Johnston who orig­inally isolated it. This alga is especially striking in the mode of formation of iso­bilateral tetrads of daughter cells (i.e., all 4 daughter cells can be observed in the same focal plane) . 
Most tetrads originate through an intervening diad stage, but tetrads also can be formed directly in T . isobilateralis. The sequence of events leading to the for­mation of tetrads as it com:nonly occurs in T. isobilateralis is shown in Text-fig. 3 (see page 63 ). 
Tetracystis aggregata, T . dissociata, and T . illinoisensis have many attributes in common with T. isobilateralis ; however, the latter is characterized by a larger ma­ture vegetative cell, which ranges from 18-19 p. in ,diameter, and by the more abundant production of isobilateral tetrads. 
T etracystis isobilateralis is particularly well delineated ultrastructurally in that it is the only presently known species of T etracystis with giant, branched mitochon­dria. 
Tetracystis pampaesp. nov. (Pampa) (Fig. 57-64, 105-106) 
Cellulae vegetativae iuvenes ellipsoideae, in cellulas vegetativas sphericas ad sub­sphericas 14 p. diam. maturescentes. Incrassatio membranae cellulae non notabilis in culturis duarum hebdomadum usque ad eas trium mensium aetate. Chloroplastus solidus, amylo impletus, aliquot fissuras non profundas praebens, pyrenoideum vix excentricum ad centrale, forma irregulare ( 4 p. diam. ), multis amyli micis circum­datum, continens. Cellulae in incrementi periodo immobili ( tribus mensibus ) semper nitido-virides. Cellulae vegetativae uninucleatae duas vacuolas contractiles continentes. 
Reproductio asexualis per divisionem cellulae in diades atque plerumque tetrades in periodo incrementi tardo-logarithmico. Diades tetradesque cellularum filiarum ma­turarum membranae cellulae parentis firme adhaerentes. Tetrades complexus saepe formantes. Reproductio asexualis etiam per zoosporas aplanosporasque. Zoosporae cylindricae, 4-8 p., nucleum posteriorem, duas vacuolas contractiles anteriores, chloro­plastum parietalem, unicum pyrenoideum equatoriale stigma maxime anterius et duo flagella longitudine, aequa, quasi aequa longitudine corpori cellulae habentes. 
· Reproductio sexualis non observata. 
Culturae duarum hebdomadum aetate in medio "agar" basali atrovirides; super­ficies coloniae paululum aspera rugosaque magnificatione sex, et 12 plo ; coloniae mag­nificatione nulla siccae. Materia propria brunnea e cellulis in "agar" sub duabus ad tres hebdomades aetate diffusa. 
Origo: e solo a florum areola in loco Pampa, Texas, dicto, m. Apr. 1961. 
Young vegetative cells ellipsoidal, maturing into spherical to subspherical vege­tative cells 14 p. in diameter. Cell wall thickening insignificant in cultures from 2 weeks to 3 months old. Chloroplast massive and starchy, with few shallow fissures. Chloroplast containing a very slightly excentric to central irregularly shaped pyre­noid ( 4 p. in diameter), surrounded by many starch grains. Cells remaining bright green in stationary phase of growth ( 3 months). Uninucleate vegetative cells con­taining 2 contractile vacuoles. 
Asexual reproduction by cell division into diads and mostly tetrads in late log phase of growth. Diads and tetrads of daughter cells at maturity strongly adherent to parent cell wall. Tetrads often forming complexes. Asexual reproduction also by zoospores and aplanospores. Zoospores cylindrical,' 4-8 p., with a posterior nucleus, 2 anterior contractile vacuoles, parietal chloroplast, single equatorial pyrenoid, a very anterior stigma, and 2 flagella of equal length, about equal to the cell body length. 
Sexual reproduction not observed. 
Two-week-old cultures on basal agar medium, dark-green; colony surface slightly rough and rugose at 6 and 12 X magnification; colonies dry macroscop­ically. Characteristic brown substance diffusing from cells into agar by 2-3 weeks. 
Source: soil from flower bed, Pampa, Texas, April, 1961. Tetracystis pampae has several attributes in common with T. aggregata and T. isobilateralis, its closest allies; however, T. pampae is distinct from all species of 
T etracystis in the production-of a characteristic brown substance diffusing from the cells into BBM agar by 2-3 weeks. Tetracystis pampae is distinguished from its 2 closest allies in having: (1) mature vegetative cells, 14-'16 µ.in diameter, which are slightly ellipsoidal; ( 2) delayed tetrad production; (3) a rugose colony surface at 2 weeks on BBM; (4) zoospores with a very anterior stigma; (5) insignificant cell wall thickening at 3 months on BBM agar; and (6) the absence of sexual re­production. 
Tetracystis pampae is distinct from all other species of Tetracystis in the ultra­structure of its pyrenoid and chloroplast as will be discussed in a subsequent section. In addition, certain physiological tests show T. pampae to be a very distinct Tetra­cystis species. 
Tetracystis pulchra sp. nov. (Sweet) Fig. 49-52, 97-98) 
Cellulae sphericae iuvenes ellipsoideae, in cellulas vegetativas paululum ellipsoideas ad sphericas 12-14 µ.diam. maturescentes. Incrassatio bipolaris membranae cellularum (ad 1-3 µ.in culturis duarum hebdomadum aetate ad 5 µ. trium mensium). Chloro­plastus parietalis, paucissimas fissuras praebens; pyrenoideum ellipsoideum axe longo 3-4 µ., duabus amyli micis circumdatum, excentricum. Cellulae nitidoaurantiae in in~ crementi periodo immobili factae. Duae vacuolae contractiles in cellulis vegetativis uninucleatis. 
Reproductio asexualis per cellulae divisionem (plerumque ) tetrades cellularum­filiarum vegetativarum, maturarum membrane cellulae-parentis firme adhaerentium directe efficientem. Tetrades ad complexus satis magnos formandos aggregatae. Re­productio asexualis etiam per zoosporas aplanosporasque. Zoosporae cylindricae, 2 X 7 µ., nucleum posteriorem, duas vacuolas contractiles anteriores, chloroplas~um par:e · talem, unicum pyrenoideum minutum equatoriale, stigma maxime anterius minutis­simum et duo flagella longitudine aequa, longitudine quasi aequa corpori cellulae aut paululo longiora habentes. 
Reproductio sexualis non observata. 
Culturae duarum hebdomadum aetate in medio "agar" basali modice viriles aut paululum aurantias; superficies coloniae homogenea ad minute granulosam, magnifi­catione sex et 12 plo ; coloniae magnificatione nulla siccae. 
Origo: plantae e solo isolatae e loco Austin, Texas, dicto., m. Jul. 1962. 
Young vegetative cells ellipsoidal, maturing into slightly ellipsoidal to spherical vegetative cells 12-14 µ.in diameter. Cells with bipolar wall thickening (to 1-3 µ.in 2-week-old cultures, to 5 µ. in 3-month-old cultures). Chloroplast parietal with very few fissures; pyrenoid ellipsoidal, long axis 3-4 µ., surrounded by 2 starch grains, and excentric. Cells becoming bright-orange in stationary phase of growth. Two contractile vacuoles present in the uninucleate vegetative cells. 
Asexual reproduction by cell division giving rise directly (mostly) to tetrads of daughter vegetative cells strongly adherent to the parent cell wall at maturity. Tetrads aggregated to form moderately large complexes. Asexual reproduction also by zoospores and aplanospores. Zoospores cylindrical, 2 X 7 µ., with a posterior 
Brown and Bold 
nucleus, 2 anterior contractile vacuoles, a parietal chloroplast, a single, minute, equatorial pyrenoid, a very anterior and very minute stigma, and 2 flagella of equal length, about equal to slightly longer than cell body length. 
Sexual reproduction not observed. 
Two-week-old cultures on basal agar medium, green to slightly orange; colony surface homogeneous to minutely granular at 6 and 12 X magnification; colonies dry macroscopically. 
Source: isolated from garden soil, exact origin of which is unknown. Austin, Texas, July, 1962. This organism, herein described as a new species of Tetracystis, was isolated in July, 1962, by Mr. Charles Sweet. 
Tetracystis pulchra is closely related to T. excentrica, to T. texensis, and, more especially, to T. intermedium. Two attributes distinguish T. pulchra from T. inter­medium: (1) the occurrence of a thickened cell wall in cultures of T. pulchra at 3 months on BBM agar; and ( 2) the presence of a homogeneous to minutely granu­lar colony. 
Tetracystis texensis sp. nov. ( Mx2-c) (Fig. 19-24, 95-96) 
Cellulae vegetativae iuvenes ellipsoideae, in cellulas vegetativas sphericas 14-15 µ. diam. celeriter maturescentes. Incrassatio membranae cellulae usque 3-4 µ. in culturis duarum hebdomadum aetate, in culturis vetustioribus non aucta. Chloroplastus parie­talis tenuisque saepe striatus et profunde lobatus, pyrenoideum maxime excen':ricum ellipsoideum (axe longo 3-4 µ.) duabus micis amyli hemisphericis praeditum, conti­nens. Cellulae in incrementi periodo immobili ( tribus mensibus) flavo-brunneae factae. Vacuolae contractiles in cellulis vegetativis uninucleatis nullae. 
Reproductio asexualis per cellulae divisionem tetrades octadesque directe effi'.:ien­tem. Aggregationes tetradum membranis cellulae parentis conferte associatae, octades, autem, minus conferte associatae. Octades, saepius tetrades, ad complexus cellularum multarum formandos cohaerentes. Reproductio asexualis raro quoque per zoosporas aplanosporasque. Zoosporae 3 X 10 µ. nucleum posteriorem, duas vacuolas contractiles anteriores, chloroplastum parietalem, unicum pyrenoideum paululo anterius, stigma anterius et duo flagella longitudine aequa, corpori cellulae aequa aut paululo breviora. 
Reproductio sexualis non observata. 
Culturae duarum hebdomadum aetate in medio basali atro -ad nitido-virides ; super­ficies coloniae irregulariter aspera maculosaque (minute botryoidea) magnificatione sex et 12 plo; coloniae a humidis ad siccas variantes magnificatione nulla. 
Origo: e soli examplo a loco Pilot Knob, Travis County, Texas dicto, m. Mai. 1961. 
Young vegetative cells ellipsoidal, rapidly maturing into spherical vegetative cells 14-15 µ.in diameter. Cell wall thickening up to 3-4 µ.in 2-week-old cultures, thick­ening not increasing in older cultures. Chloroplast parietal and thin, often striate and deeply lobed. Chloroplast containing a very excentric, ellipsoidal pyrenoid (long axis 3-4 µ.),with 2 hemispherical starch grains. Cells becoming yellow-brown 
Studies of Algal Genera Tetracystis and Chlorococcum 
in stationary phase of growth ( 3 months). Contractile vacuoles absent in the uni­nucleate vegetative cells. 
Asexual reproduction by cell division giving rise directly to tetrads or octads. Tetrad aggregates intimately associated with parent cell walls, but octads less inti­mately associated. Octads, and more frequently, tetrads coherent to form com­plexes of many cells. Asexual reproduction infrequently also by zoospores and aplanospores. Zoospores 3 X 10 µ., with a posterior nucleus, 2 anterior contractile vacuoles, a parietal chloroplast, a slightly anterior, single pyrenoid, an anterior stigma, and 2 flagella of equal length, slightly shorter than equal to the cell body length. 
Sexual reproduction not observed. 
Two-week-old cultures on basal medium, dark-to-bright green; colony surface irregularly rough, mottled (minutely botryoid) at 6 and 12 X magnification; colonies range from moist to dry macroscopically. 
Source: soil sample from Pilot Knob, Travis County, Texas, May, 1961. This organism was kindly given to the writers by Dr. Karl Mattox who originally isolated it. 
T etracystis texensis belongs to a group of closely related T etracystis species ( T. excentrica, T. intermedium, and T. pulchra), all of which have similar pyrenoid types as discussed previously. However, T. texensis is readily differentiated from these species by having: ( 1) yellow-brown colonies on BBM agar at 3 months; (2) formation of octads of non-motile daughter cells in addition to tetrads; ( 3) infre­quent and difficult-to-evoke zoospore formation; and (4) in lacking contractile vacuoles in the vegetative cells. In light of these considerations, T. texensis is herein described as a new species of the genus T etracystis. 
As noted in the Introduction, 3 algae previously described by other investigators as species of Chlorococcum have been investigated concurrently and comparatively with the species of T etracystis, inasmuch as the writers hypothesized that the former 3 taxa might more appropriately be included in the genus T etracystis. All the data obtained with the several methodologies used in this research have convinced us that, indeed, these 3 suspect Chlorococcum species should be transferred to Tetra­cystis. Itshould be noted in passing that Deason and Bold ( 1960) earlier had ques­tioned whether these 3 taxa were, in fact, members of Chlorococcum. The amended ~nd augmented characterizations of these taxa now follow. 
Tetracystis aplanosporum (C hlorococcum aplanosporum Arce and Bold) 
comb.nov. (Fig.65-72, 103-104) 

Young vegetative cells ovoidal, maturing into spherical vegetative cells 18-27 µ. in diameter. Cell wall thickening insignificant at 2 weeks but ranging from 2 to 3 µ. in cultures 3 months old. Chloroplast massive with deep fissures and a large, clear, ­unilateral, cytoplasmic sinus observable in actively growing cultures. Chloroplast containing a central irregularly shaped pyrenoid, 5-6 µ.in diameter, surrounded by many starch grains. Cells remaining green in stationary phase of growth ( 3 months). From 2 to 6 large contractile vacuoles always present in actively grow­ing reproductive ( zoospores and aplanospores) and non-motile uninucleate vege­tative cells (Fig. 66, arrow). 
Asexual reproduction by cell division giving rise exclusively, directly to tetrads of daughter vegetative cells which at maturity are loosely associated with parent cell wall. Tetrads not characteristically aggregating to form complexes. Asexual re­production also by zoospores and aplanospores formed by a series of successive bi­partitions. Zoospores broadly cylindrical ( 3-6 X 8-16 µ.) with an anterior nucleus, 2 anterior contractile vacuoles, a parietal chloroplast, a single slightly posterior pyrenoid, equatorial to slightly anterior stigma, and 2 equal flagella approximately the length of the cellular body. 
Sexual reproduction not observed. Two-week-old cultures on basal agar medium, light green; colony surface rough, minutely vermiform at 6 and 12 X magnification; colonies moist macroscopically. Source: Indiana University Culture Collection No. 773 as Chlorococcum aplanosporum Arce and Bold. 
What appear superficially to be tetrads of aplanospores are, in fact, tetrads of non-motile daughter cells which have not originated from zoosporic antecedents. For this reason, Chlorococcum aplanosporum is herein transferred to the genus T etr acystis. 
Tetracystis aplanosporum and T. aeria are the only 2 presently known species of Tetracystis in which the zoospores have anterior nuclei. However, T. aplanosporum is clearly distinguishable from T. aeria by: ( 1) its larger mature vegetative cells, 19-27 µ. in diameter; ( 2) 2-4 large contractile vacuoles in the vegetative cell; ( 3) dissociation of tetrads at the tetrad level; ( 4) zoospores with flagella equal to the cell body length; ( 5) an equatorial-to-slightly anterior stigma in the zoospore; ( 6) thinner cell walls at 2 weeks and 3 months on BBM agar; and ( 7) a rough colony surface which is minutely vermiform on BBM agar at 2 weeks. 
Tetracystis aplanosporum clearly is one of the most distinct species of Tetracystis on the basis of both light microscopy and its physiology. This statement is further supported by ultrastructural and immunochemical evidence which will be pre­sented in later sections. 
Tetracystis intermedium (C hlorococcum intermedium Deason and Bold ) comb.nov. (Fig. 73-78,99-100) 
Young vegetative cells ellipsoidal, maturing into vegetative cells 12-20 µ. in di­ameter; cell wall not markedly thickened even in cultures 2 months old. Chloroplast thin and parietal with a few shallow fissures. Chloroplast containing a single, ex­centric, ellipsoidal pyrenoid 2 p. in diameter, surrounded by 2 starch grains. Cells becoming bright-orange in stationary phase of growth ( 3 months). Uninucleate vegetative cells containing 2 contractile vacuoles. 
Asexual reproduction by cell division giving rise to diads and (mostly) to tetrads of vegetative cells which at maturity are strongly adherent to the parent cell wall; several tetrads aggregating to form simple complexes. Asexual reproduction also by zoospores and aplanospores formed by rapid successive bipartitions. Zoospores 2.5­
9.5 µ., with a posterior nucleus, 2 anterior contractile vacuoles, a parietal chloro­plast, a single equatorial pyrenoid, a very anterior stigma, and 2 flagella which are slightly longer than body length. 
Sexual reproduction not observed. 
Two-week-old cultures on basal medium, green, becoming slightly orange; colony surface rough and granular at 6 and 12 X magnification; colonies moist to dull-shiny macroscopically. 
Source: Indiana University Culture Collection No. 974 Chlorococcum inter­medium Deason and Bold. 
As noted above, this organism, as well as T. aplanosporum and T. tetrasporum, was originally described as a Chlorococcum species. The basis for Deason and Bold considering this alga to be a Chlorococcum species was that this organism did not undergo vegetative cell division sensu Herndon. Deason and Bold considered the timing of cytokinesis and wall formation especially significant. Each cytokinesis fol­lowed by intervening cell-wall deposition was considered by them as true vegetative cell division sensu Herndon. If rapidly occurring divisions resulted in a multi­nucleate protoplast in which cell-wall deposition was delayed until all cytokinesis had been completed, the organism in question was classified by them in the Chloro­coccales. Such divisions of a multinucleate protoplast were considered analagous to those in zoosporogenesis by Deason and Bold. 
In view of the writers' emphasis that in the order Chlorosphaerales, there are 
formed, by cell division, non-motile daughter vegetative cells which lack the po­
tential for motility, Chlorococcum intermedium has been transferred from the 
Chlorococcales to the genus T etracystis of the Chlorosphaerales. In addition, ultra­
structural evidence seems to support the validity of this transfer in that ultrastruc­
tural differences in the ontogeny of zoospore and daughter vegetative cells are 
striking. 
T etracystis intermedium belongs to a group of closely related T etracystis species, namely, T. texensis, T. excentrica, and T. pulchra. Tetracystis intermedium differs from T. pulchra, which it mostly closely resembles, in: ( 1) the absence of cell wall thickenings on BBM at 3 months; and ( 2) in its rough colony surface. 
Tetracystis tetrasporum (Chlorococcum tetrasporum Arce and Bold) comb. nov. (Fig. 79-80,93-94) 
Young vegetative cells ellipsoidal, maturing into spherical vegetative cells 15­
18 µ. in diameter. Cell wall thickening insignificant at 2 weeks and also at 3 months. Chloroplast massive with few shallow fissures. Chloroplast containing a central-to­slightly excentric, almost spherical pyrenoid, 4-5 µ. in diameter, surrounded by many closely packed starch grains. Cells remaining green in stationary phase of growth. Uniculeate vegetative cells contain 2 contractile vacuoles. 
Asexual reproduction by cell division giving rise to tetrads of daughter vegetative cells, only late in growth phase (after 1 month). Tetrads strongly adherent to par­ent cell wall. Several tetrads adhere at maturity to form cellular complexes. Asexual reproduction also by zoospores and aplanospores. Zoospore production prolonged for at least 2 weeks following inoculation in basal agar medium. Zoospores 4 X 10 µ., with a posterior nucleus, 2 anterior contractile vacuoles, a parietal chlo­roplast, a single equatorial pyrenoid, a large anterior stigma, and 2 flagella of equal length which are slightly shorter than the cellular body length. 
Two-week-old cultures on basal medium, green; colony surface smooth and homogeneous at 6 and 12 X magnification; colonies macroscopically shiny. Four­to-six week old cultures on basal medium, green; colony surface rough and granular (due to tetrad production) at 6 and 12 X magnification; colonies macroscopically moist-to-dry. 
Source: Indiana University Culture Collection of Algae No. 780 as Chlorococ­cum tetrasporum Arce and Bold. 
T etracystis tetrasporum can be especially deceptive and difficult to identify as a species of T etracystis because tetrad formation occurs only very late after inocula­tion upon slants of BBM agar (i.e., 4-6 weeks). This very much delayed tetrad production is peculiar to T. tetrasporum among the presently known species of T etracystis. During the initial period of growth ( 2-4 weeks), prolonged zoosporo­genesis occurs. Another closely related species, T. illinoisensis, produces tetrads late after inoculation, not so late, however, as T. tetrasporum. T etracystis illinoisensis, furthermore, is differentiated from T . tetrasporum in that the latter undergoes a prolonged period of zoosporogenesis prior to tetrad formation. 
Therefore, in view of the occurrence of non-motile daughter cells in tetrads, C hlorococcum tetrasporum is herein trans{ erred from C hlorococcum to T etracystis. 
KEY TO THE CURRENTLY KNOWN SPECIES OF TETRACYSTIS 
BROWN AND BOLD  
1. Zoospore with anterior nucleus ....................................................... .... ....... 1. Zoospore with posterior nucleus ................. ................................. .... 2. Mature vegetative cells 18-19 µ. in diameter; 2-to-many con­tractile vacuoles in vegetative cells; tetrad dissociation at the  2 3  

tetrad level, forming no cellular complexes ...... T. aplanosporum comb. nov. 
2. Mature vegetative cells 14-16 µ.in diameter; 2 contractile vacu­oles occasionally present in vegetative cells; tetrads cohering to form cellular complexes ........................................................ T. aeria sp. nov. 
3. Pyrenoid more or less centrally located in vegetative cell and sur­ 
rounded by many starch grains; pyrenoid spherical to irregular ______ ____ _____ _  4  
3. Pyrenoid ellipsoidal, with only 2 starch grains; excentric in the  
vegetative cell ---­-----­---------­----­---­---­--­----------­-­--­-----­----­-­-­---­-----­----­----­--­---­ 9  
4. Colonies on BBM agar smooth and homogeneous at 2 weeks __ ___ __ ___ ______  5  
4. Colonies rough on BBM agar at 2 weeks -------­---­--­--­--­----­---­--­---­-­-----­--- 7  
5. Cell wall thickenings insignificant both at 2 weeks and 3 months  
on BBM agar ----­---­-------­-----­------­-­----­-­-----­---------­---­---­----­--------­-­--------­--­--­ 6  
5. Uni-and bipolar cell wall thickenings (up to 5 µ., present in cul­ 

tures on BBM agar at 3 months ------------------------------------T. illinoisensis sp. nov. 
6. Tetrad production delayed until very late in development (after 1 month on BBM agar) --------------------------------------T. tetrasporum comb. nov. 
6. 
Tetrad production occurring within 1-2 weeks after inoculation 1 on BBM agar_ ----------------------------------------------------------___T. dissociata sp. nov. 

7. 
Mature vegetative cells slightly ellipsoidal, 12-14 µ.; zoospores with a very anterior stigma; colony surface rugose; cell wall thickening insignificant at 3 months on BBM agar; sexual reproduction not observed; delayed tetrad production; a distinct, brown substance secreted into BBM agar at 2-4 weeks----------------------------------T. pampae sp. nov. 


7. 
Mature vegetative cells mostly spherical; zoospores with an equatorial-to-slightly anterior stigma; cell wall thickening 2-4 µ. at 3 months on BBM agar; sexual reproduction present ----------------------------8 

8. 
Mature vegetative cells 15-16 µ.in diameter, with 2 contractile vacuoles; colonies coarsely granular at 2 weeks on BBM agar; deep radiating fissures often present in chloroplast ___ _____ T. aggregata sp. nov. 


8. 
Mature vegetative cells 18-19 µ.in diameter; contracile vacu­oles absent in vegetative cells; colony surface minutely scabellate on BBM agar at 2 weeks; isobilateral tetrads prorriinent at 2 weeks ------------------------------------------------------------------------T. isobilateralis sp. nov. 

9. 
Color of colony bright-orange to red-orange at 3 months on BBM agar -------------------------------------------------------------------------------------------------------------10 


9. 
Color of colony otherwise ----------------------------------------------------------------------------11 

10. 
Colony surface rough on BBM agar at 2 weeks; cell wall thick­


enings insignificant ( 1-2 µ.) at 3 months on BBM agar ____ T. intermedium comb.nov. 
10. 
Colony surface homogeneous to minutely granular on BBM agar at 2 weeks; cell wall thickenings up to 5 µ. at 3 months on BBM agar ----------------------------------------------------------------T. pulchra, sp. nov. 

11. 
Color of colony yellow-orange at 3 months on BBM agar; colony surface smooth; daughter cells in tetrads dissociate early; 2-to­many peripheral, contractile vacuoles in vegetative cell; sexual reproduction observed; zygote smooth-walled __ __ ____ _________ _T. excentrica sp. nov. 


11. Color of colony yellow-brown at 3 months on BBM agar; colony surface rough; numerous octads of non-motile daughter cells pro­duced in addition to tetrads; contractile vacuoles absent from vegetative cells; zoospore formation infrequent and difficult to evoke ------------------------------------------------------------------------------------T. texensis sp. nov. 
D. PHYSIOLOGICAL ATTRIBUTES IN Tetracystis 
_In recent years, unicellular and other chlorophycean algae have been treated taxonomically, in part, on the basis of physiological attributes (Deason and Bold, 1960; Mattox and Bold, 1962; Bold and Parker, 1962; Chantanachat and Bold, 1962; and Bischoff and Bold, 1963). 
Physiological and other, so-called supplementary, attributes have provided ex­tremely useful data whith help determine the taxonomic affinities of certain species and genera. In the writers' experience, as well as that of previous investigators, it has been found indispensable to run duplicate cultures when performing a given physiological test, and to repeat each test at least one or more times. 
Most of the physiological tests were performed before the writers had isolated all but two of the Tetracystis species discussed in this report. Accordingly, data re­garding the last 2 species isolated, namely, T. illinoisensis and T . pulchra, regret­fully will not be included in summaries of several of the physiological tests. Since these 2 species, as well as the other species of Tetracystis, are easily characterized on morphological grounds, the physiological data are not necessary for their differ­entiation but may have served to amplify and to augment our knowledge based on morphological attributes. 
The data on these physiological attributes are summarized in Tables 1-6 and in the sections immediately following. 
Effects of carbon compounds 
Bold and Parker ( 1962), in an investigation of supplementary attributes in classifying species of C hlorococcum, reported taxonomically useful results when the inorganic medium was supplemented with a variety of sugars, acetate, and pyru­vate. It should be noted that in their procedures, the carbon compounds were auto­claved after they had been dissolved in the inorganic medium. In the present investigation, xylose, ribose, fructose, glucose, arabinose, and sodium acetate were separately dissolved in de-ionized water and treated in 2 different ways: in one procedure, these fractions were Seitz-filtered, and in another, they were autoclaved at 15 p.s.i. for exactly 15 min. In both instances, these fractions were added asepti­cally to aliquots of sterile, cooled, liquid BBM in order to make a total concen­tration of 0.75 % for each carbon source. The culture tubes had been rinsed 3 times in distilled water followed by 3 rinses in de-ionized water, plugged, and sterilized. 
Studies of Algal Genera Tetracystis and Chlorococcum 
Axenic cultures of T etracystis were inoculated into quadruplicate culture tubes containing 20 ml of BBM enriched with a given carbon source. A homogeneous inoculum was prepared as previously described (page 9) , and 5 drops were asepti­cally transferred to each tube of the quadruplicate set by sterile Pasteur pipettes. 
One set each of the autoclaved and Seitz-filtered media supplemented with the various carbon sources was placed in the light under standard conditions. A dupli­cate set of each was stored in darkness at the same temperature. After 14 days, cultures in the light were appraised for amount of growth. Cultures placed in dark­ness were removed 2 months after inoculation and examined for evidence of facul­tative heterotrophy. These same cultures were then placed in the light for 2 weeks under standard conditions, and the growth (recovery) in light, following a 2­month dark period, was noted and recorded. Growth was estimated visually in comparison with a set of standards, designated previously as "Excellent", "Good", "Fair", "Trace" and "None". 
TABLE 1. Growth of Tetracystis species in BBM supplemented with various carbon 
sources (at 0.75%) in light under standard conditions for 2 weeks 
(A = autoclaved; B = Seitz-filtered) 

Control 
(BBM) Xylose Ribose Arabinose Glucose Fructose Na-acetate T ype of sterilization A B A B A B A B A B A B A 
T. aeria (C-6)  Ea  E  N  F  F  G  G  E  E  G  G  F  F  
T. aeria (Pa-3 )  G  F  F  F  F  G  G  E  E  G-G  F  F  
T. isobilateralis  E  E  G  F  F  G  G  E  G  G+ G  F  F  
T. aggregata  E  G  F  F  N  G  G  G  G  G+ F  F­ N  
T . dissociata  G  G  G  T  N  F  F  G  G  F  F  F­ N  
T. illinoisensis  G  G  N.D.  T  N.D.  G  N.D.  G  N.D.  G  N.D.  F+N.D.  
T. jJampae  E  E  E  E­ E  E  E  E  E  E  E  E  E  
T. aplanosporum E  E  E  E­ E  E  E  E  E  E  E  F-F  
T. intermedium  E  E  E  G  F  E  E  E  E  E  E  F  F  
T. tetrasporum  E  G  G  G  F  E  G  G  G  E  G  E  E  
T. texensis  T  F  N.D.  N  N.D.  F  N.D.  T  N.D.  T  N.D.  N  N.D.  
T. excentrica  G  G  G  F  F  F  F  G  G  G  G  N  N  
T. pulchra  E  G  N.D.  F  N.D.  G  N.D.  G  N.D.  G  N.D.  F  N.D.  

a E, growth excellent; G, good; F, fair; T, trace; N, no growth; N.D., no data; in this and succeeding tables; these are further qualified with plus and minus signs. 
It is clear from Table 1 that when the several carbon sources are autoclaved in distilled water and added to autoclaved BBM, certain of them are inhibitory to the growth of certain T etracystis species, as compared with the control in organic medium alone. Ribose, of all the carbon sources used, inhibits most T etracystis species, especially when autoclaved. At the other extreme is fructose, in which almost the same results were obtained with autoclaved and Seitz-filtered material, neither inhibitory as compared with the BBM control. 
On the basis of the data summarized in Table 1, the several species of Tetracystis may be divided among 4 groups as follows: Group 1 : Growth not markedly affected by supplementary carbon compounds­
T. pampae, T. aplanosporum, T. intermedium Group 2 : Growth poor, in inorganic medium alone and with supplementary 
carbon sources-T. texensis Group 3 : Growth affected by supplementary carbon compounds but with poor growth in acetate-T. aeria (both C-6 and Pa-3 ) ; T. isobilateralis, T. illinoi­sensis, T. excentrica, T. pulchra, T . dissociata, T. aggregata. Group 4 : Growth affected by supplementary carbon compounds; however, growth 
excellent in acetate-T. tetrasporum 
Furthermore, inspection of cultures in the sugar-and acetate-supplemented media after 2 months in darkness revealed that the only heterotrophism in Tetra­cystis, under conditions of the present experiment, was exhibited by the 2 isolates ( C-6 and Pa-3) of T. aeria with both glucose and fructose and by T. pampae in acetate. No growth of the other organisms occurred in darkness in the control or in media containing xylose, ribose, arabinose, glucose, fructose, or sodium acetate. 
Data on "recovery" in light of the organisms after 2 months in darkness in BBM, with and without the carbon sources, are summarized in Table 2. The patterns of inhibition are similar to those in Table 1, but they are markedly accentuated. 
TABLE 2. Growth of Tetracystis species (r ecovery) in light at 2 weeks following a 2-month period in darkness 
Type of sterilization  BBM A  Xylose B A  Ribose B A  Arabinose B A  Glucose B A  Fructose B A  Na -acetate B A  
T. aeria (C-6)  N  N  N  T  T  T  T  E  E  E  E  N  N  
T. aeria (Pa-3)  N  T  N  T  T  T  T  E  E  E  E  N  N  
T . isobilateralis  N  E  E  E  E  E  E  E  E  E  G  T -T­ 
T. aggregata  N  G  G  F  T ­ G  G  T  G  F  T  T-N  
T. dissociata  N  T  N  T  N  F  N  F  F  F  F  N  N  
T. pampae  N  E  E  E  E  E  E  E  E  E  E  E  E  
T. aplanosporum N  E  E  E  E  E  E  E  E  E  E  T  T  
T. interm edium N  G  F  E  G  E  E  E  E  E  E  T  T  
T. tetrasporum  N  F  T  T  N  E  G  F  F  F  T  N  N  
T. texensis  N  T  N  N  N  N  N  T -N  N  N  N  N  
T. excentrica  N  T  N  N  N  T  N  F+ N  F+ N  N  N  

A = Autoclaved B = Seitz-filtered 
Sensitivity to crystal violet 
Mattox and Bold ( 1962) used tolerance to crystal violet as a criterion in their classification of certain ulotrichacean algae. However, as indicated, the medium must be inoculated immediately after preparation, because the inhibitory efficacy of crystal violet deteriorates markedly with prolonged storage in the light, but less so in darkness (Table 3). 
One gram of Difeo crystal violet powder was dissolved in 100 ml of distilled water to make a 1.0% stock solution. To liquid BBM was added sufficient stock solution to make the following final concentrations of crystal violet: 1 part crystal violet/ 100,000 parts BBM (0.001 % ) ; 1 part crystal violet/50,000 parts BBM (0.002% ); 1 part crystal violet/25,000 parts BBM (0.0045% ); and 1 part crystal violet/ 10,000 parts BBM (0.01 % ) . The crystal violet media were then solidified with Difeo Bacto powdered agar ( 1.6% concentration) and used immediately. 
TABLE 3. Growth of T etracystis species (at 2 weeks) on BBM agar with various 
concentrations of crystal violet (Fig. 107) 
Inoculated  
immediately after preparation  
I I 1 1  
Concentration of er. violet8  100 50 25 10  100Lt.  
T. aeria (C-6)  N N N  E  
T . aeria (Pa-3)  N N N  
T. isobilateralis  T T T N  E  
T. aggregata  T T N N  E  
T. dissociata  T T T N  E  
T. tetrasporum  T F T N  E  
T. excentrica  F F T N  N  
T. intermedium  E E E T  E  
T. texensis  G G F T  E  
T. pampae  E E G T  E  
T. aplanosporum  E E E T  E  

Inoculated 2 weeks after preparation 
1  1  
100 Dk.  50Lt.  50 Dk.b  
ND  E  ND  

ND D ND ND ND E T ND E ND T E T F G T ND N ND E E E G N N ND E ND E E E 
a 1/ 100 = 1 pt. crystal violet to 100,000 parts BBM. 
1 / 50 = 1 pt. crystal violet to 50,000 parts BBM. 
1/25 = 1 pt. crystal violet to 25,000 parts BBM. 
1/ 10 = 1 pt. crystal violet to 10,000 parts BBM. 
b Storage during 2-week period before inoculation. 

The data in Table 3 and Fig. 107 indicate differential sensitivity of the Tetra­cystis isolates to varying concentrations of crystal violet. For example, T . inter­medium, T . texensis, T. pampae, and T. aplanosporum are clearly less sensitive than other isolates to a concentration of crystal violet of 1 : 25000. Other species 
(T. aeria, both strains, T. isobilateralis, T. aggregata, T. dissociata and T. tetra­sporum ) cannot tolerate crystal violet even in as dilute a solution as 1 : 100,000 ! 
Extracellular amylasic activity 
Extracellular amylasic activity of green algae has been explored as a possible taxonomic criterion by Bischoff and Bold ( 1963) and the same criterion has been tested in the several T etracystis species. 
For testing amylasic activity, starch agar was prepared by using a 0.01 o/o con­centration of ACS-grade starch in BBM· solidified with 1.6% Difeo Bacto pow­dered agar. The presence of starch was indicated by I2KI solution which was made by dissolving 10.0 g of KI in 500 ml distilled water and subsequently adding 2.0 g of metallic iodine. 
The results of duplicate tests run twice are presented in Table 4. The degree of activity was appraised by the width of the clear (starch-digested ) zone which was evoked by adding I2KI. From the data in Table 4 and Fig. 108, it is apparent that the species of Tetracystis differ consistently in their extracellular amylasic activity. 
TABLE 4. Amylasic activity of Tetracystis species after growth on BBM supplemented 
with 0.01% starcha and grown for 2 weeks under standard conditions 
(Fig. 108) 

Organism  Amy lasic activity  
T. excentrica  slight  
T. texensis  slight  
T. intermedium  slight  
T. aplanosporum  fair  
T. aeria (C-6)  good  
T. aeria (Pa-3)  good  
T. dissociata  good  
T. isobilateralis  good  
T. aggregata  good  
T. pampae  good  
T. tetrasporum  excellent  

a Potato. 
Growth in several complex media 
Growth in certain complex media, while useful in routine detection of contami­nation, sometimes has also proven to be consistently differential. In the present investigation, differential growth of the T etracystis species was exhibited after 3 weeks in Difeo Nutrient Broth, Difeo Nutrient Agar, 1 % yeast agar,1 and Difeo Thioglycollate medium (Table 5). 
1 5 g Difeo yeast extract dissolved in 1 liter of de-ionized water and solidified with 16 g of agar. 
TABLE 5. Growth of T etracystis species on various complex mediaa 
Growth on Growth on Growth in Growth in Organism nutrient agar yeast agar nutrient broth thioglycollate mediwn 
T. aeria (C-6)  G  G  G  F  
T. aeria (Pa-3)  G  G  G  F  
T. isobilateralis  N  G  N  G  
T. dissociata  N  G  N  F  
T. aggregata  N  G  N  G  
T. excentrica  G  N  N  F  
T. texensis  G  N  N  G  
T. aplanosporum  G  G  N  G  
T. intermedium  G  G  F  G  
T. tetrasporum  G  G  F  G  
T. pampae  G  G  G  G  

a At 3 weeks incubation. 
Sensitivity to antibiotics 
The use of antibiotics for differentiation of algal isolates has been a practice in 
this laboratory for some years (Deason and Bold, 1960; Chantanachat and Bold, 
1962; Mattox and Bold, 1962; Bold and Parker, 1962; Bischoffand Bold, 1963) . 
From more than 30 antibiotic agents which were screened, 9 were chosen for 
further use. 
Selective inhibitory action of certain antibiotics was demonstrated by preparing 
Petri dishes of BBM Ion Agar1 ( 1.0% ) to which an inoculum was added before 
the medium had sodidified. After gelation, paper tabs impregnated with various 
antibiotics2 were aseptically dispensed onto the surface of the agar. The plates were 
then inverted and stored under standard conditions, for 2 weeks, after which they 
were examined and the degree of inhibition of growth; if any, was recorded (as 
measured by a clear zone circumscribing the antibiotic disc.) The data are pre­
sented in Table 6. 
One isolate of T. aeria (Pa-3) was not sensitive to any of the antibiotics used, 
while the other strain of the same species was sensitive only to Polymyxin B. Fur­
thermore, no two of the isolates tested had identical responses of sensitivity, so that 
this is a helpful supplement in classifying the organisms. 
The writers have carefully considered data obtained in each of the tests sum­marized in Tables 1-63 and have constructed species groupings based on responses · of the algae to a given procedure. These species groupings are summarized here­
with. 
1 Oxoid Ion agar No. 2, Consolidated Laboratories, Inc., Chicago Heights, Illinois. 2 Sensi Disc Method, Baltimore Biological Laboratory, Inc., Baltimore 18, Maryland. 3 And other data. 
TABLE 6. R esponse of T etracystis species to certain antibiotic agents (Sensi-disc method) 
CL" FX SM N PB E SD G DS 
T. aeria (C-6 )  _c  
T. aeria (Pa-3)  
T. isobilateralis  +  +  +  +  +  s1a  
T. aggregata  +  +  +  +  +  
T. dissociata  +  +  +  +  +  
T. intermedium  +  +  +  +  
T. texensis  +  +  +  +  ND•  
T. excentrica  +  +  +  +  +  ND  +  
T. pampae  +  +  +  +  +  +  +  
T. aplanosporum  +  SI  +  ND  
T. tetrasporum  +  +  +  +  +  +  +  ND  

a Antibiotics used and their concentrations: 
CL = Coly-Mycin, 10.0 mcg. 
FX 100 = Furoxone, 100.0 mcg. 
SM 1 = Sulfamerazine, 1.0 mg. 
N 30 = Neomycin, 30.0 mcg. 
PB 300 = Polymyxin B, 300.0 units. 
E 15 = Erythromycin, 1'5.0 mcg. 
SD I = Sulfadiazine, 1.0 mg. 
G 2 = Gantrisin, 2.0 mg. 
DS 10 = Dihydrostreptomyocin, I 0.0 mcg. 
b + indicates a definite inhibition. c -indicates no inhibition. 
d SJ indicates a slight inhibition. 
• 
ND = no data (disc fell off agar). 

A. 
From Table 1. Growth on various carbon sources in the light 


Group 1: Group 3 (continued) : 
T. pampae T. illinoisensis 
T. aplanosporum T. excentrica 
T. intermedium T. pulchra 
T. dissociata 
Group2: 
T . aggregata T. texensis 
Group4:
Group 3: 
T. tetrasporum 
T. aeria (both C-6 and Pa-3) 
T. isobilateralis 
B. From Table 2. Growth on various carbon sources in the light following a prolonged dark period (recovery) 
Group 1: 
T. aeria (both C-6 and Pa-3) 
Group2: 
T. aplanosporum 
Group3: 
T.pampae 
Group4: 
T. dissociata 
T. texensis 
T. excentrica 
T. tetrasporum 
Group5: 
T. aggregata 
Group6: 
T. intermedium 
C. From Table 3. Growth in BBM supplemented with crystal violet 
Group 1: 
T. aeria (both C-6 and Pa-3) 
Group2: 
T. isobilateralis 
T. aggregata 
T. dissociata 
T. tetrasporum 
T. excentrica 
D. From Table 4. Amalysic activity 
Group 1: 
T. tetrasporum 
Group 2: 
T. aeria (both C-6 and Pa-3) 
T. dissociata 
T. isobilateralis · 
T. aggregata 
T. pampae 
Group3: 
T. intermedium T.pampae 
T. aplanosporum 
Group4: 
T. texensis 
Group3: 
T. aplanosporum 
Group4: 
T. excentrica 
T. texensis 
T. intermedium 
E. From Table 5. Growth in Thioglycollate Broth 
Group 1: Group 2 (continued) : 
T. aeria (both C-6 and Pa-3) T. pampae 
T. dissociata T. texensis 
T. excentrica T . tetrasporum 
T. intermedium
Group 2: 
T. aplanosporum
T. isobilateralis 
T. aggregata 
Growth in Nutrient Broth (Table 5 ) 
Group 1: Group 3: 
T. aeria (both C-6 and Pa-3) T. isobilateralis 
T. pampae T. aggregata 
T. dissociata
Group 2: 
T. excentrica
T. tetrasporum 
T. texensis
T. intermedium 
T. aplanosporum 
Growth on Nutrient Agar and Yeast Agar (Table 5) 
Group 1: Group 2: 
T. aeria (both C-6 and Pa-3) T. excentrica 
T. aplanosporum T. texensis · 
T. intermedium 
Group 3: 
T. tetrasporum 
T. isobilateralis 
T. pampae 
T. dissociata 
T. aggregata 
F. From Table 6. Response to certain antibiotics 
Group 1: Group4: 
T. aeria (both C-6 and Pa-3) T. aplanosporum 
T. dissociata
Group 2: 
T. intermedium
T. tetrasporum 
Group 3: 
T . isobilateralis 

T. 
aggregata 

T. 
pampae 

T. 
excentrica 

T. 
texensis 


G. Facultative heterotrophy 
Group 1: Group5: 
T. aeria (both C-6 and Pa-3) T. aggregata 
T. isobilateralis
Group2: 
T. dissociataT. aplanosporum 
T. intermedium Group3: T. texensis 
T. tetrasporum T. excentrica 
Group4: 
T. pampae 
H. Growth on BBM supplemented with vitamins 
Group 1 : Group 4: 
T. aeria (both C-6 and Pa-3) T. isobilateralis 
T. pulchra T. illinoisensis 
T. aggregata
Group 2: 
T. texensis Group5: 
T. dissociata T. tetrasporum 
Group3: 
T.pampae 
T. excentrica 
T. aplanosporum 
T. intermedium 
From these data, it is clear that no single species has a unique pattern of response to all of the tests; however, certain species seem to fall into groups which indicate significant relationships. So that the reader may visualize the over-all picture more conveniently, Table 7 has been constructed to correlate the number of different physiological tests (out of a total of 101 ) in which a given species (left hand col­umn) appears in a group by itself, or with 1 additional species, 2 additional species, etc., up to the case of 6 additional species. For example, both isolates of T. aeria appear as the only species comprising the given group in 4 different physiological tests. In only 1 physiological test, however, T. aeria does appear in a group consist­ing of 6 additional species, etc. 
The most distinctive species on the basis of this analysis of physiological attri­butes are: ( 1) T. aeria which stands alone in 4 different physiological tests; and 
(2) T. tetrasporum which is unique in its response to 5 of the 10 experimental pro­cedures. 
1 An experiment adding vitamin supplement to the basal medium, did not prove very differ­ential. This, while not discussed in the text, is in~luded in Table 7. 
TABLE 7. Summary of the number of physiological tests in which a given species is unique or similar to other species 
Species O• 2 3 
T. aeria (C-6)  
and (Pa-3 )  1111"  11  1  
T. isobilateralis  1  11  
T. aggregata  11  
T. dissociata  111  1  
T. excentrica  11  11  
T. texensis  11  11  1  
T. intermedium  1111  
T. pampae  11  11  
T. aplanosporum  11  111  
T. tetrasporum  11111  

4 
11  3  
111  11  11  7  
111  111  7  
11  11  5  
11  11  5  
11  4  
1  3  
111  4  
3  
11  3  

a The number of additional species sharing a given physiological response with the species in the left-hand column. 
"The number of different physiological tests (out of a total of 10) in which a given species (left-hand column ) appears in a grouping by itself or with a number of other species shown across the top of the table. 
Tetracystis isobilateralis, T. aggregata, T. dissociata, and T. excentrica may be considered in a physiological species grouping in that they all form groups with at least 4 additional algae in over half of the 10 physiological tests. (See Roman numerals in column 7, Table 7.) 
Likewise, T. texensis, T. intermedium, T. pampae, and T. aplanosporum may be considered a physiological species grouping in that they all form groups with at least 3-4 additional algae in over half of the 10 physiological tests. (See column 7, Table 7.) 
Finally, it is quite possible to identify and segregate 7 of the isolates of Tetracystis tested on the basis of their pattern of physiological response alone. Thus, a diversity of physiological tests can provide useful data for supplementing the morphological criteria used in classifying T etracystis and other algal species. This will be borne out by the correlations to be made later in this study on the bases of ultrastructural and immunochemical evidence. 
III. 	Electron Microscopy of Tetracystis and Certain Chlorococcum Species In addition to light-microscopic studies of species of the genera T etracystis and Chlorococcum, it became clear upon preliminary examination with the electron 
microscope that ultrastructural differences within and between these genera would provide additional, precise, continuously available criteria of taxonomic signifi­
cance. Furthermore, the exact nature of cell division, especially in Chlorococcum and T etracystis was incompletely known through available light-microscopic studies (Herndon, 1958; Deason and Bold, 1960); and for this reason it seemed worthwhile to investigate with the electron microscope1 the processes ofcytokinesis leading to the formation of both daughter vegetative cells and zoospores, in Tetra­cystis, and to zoospores alone, in Chlorococcum. Tetracystis provided excellent ma­terial for studies of pyrenoid organization and formation. Accordingly, a special section has been devoted to this topic. 
A. MATERIALS AND METHODS 
Unialgal and bacteria-free cultures of 11 species of Tetracystis and of 4 species of Chlorococcum were selected for electron-microscopic investigations (Table 8) . 
TABLE 8. Algae investigated electron microscopically 
T etracystis isobilateralis 
T. aggregata 
T. aeria (C-6 ) (Pa-3) 
T. excentrica 
T. dissociata 
T. texensis 
T. intermedium 
T. aplanosporum T etracystis pampae 
T . illinoisensis 

T. 
pulchra Chlorococcum perforatum 

C. 
echinozygotum 

C. 
multinucleatum 


C. sp. (tetra isolate) 
All the organisms were maintained on Bold's Basal Medium ( BBM) solidified with 1.6% agar. When culturing the algae in preparation for electron-microscopic studies, 10 ml of liquid medium were added aseptically to each agar slant, and the latter was then immersed for about 5 sec in an ultrasonic water bath. Six drops of the resultant homogeneous cell suspension were trans£ erred aseptically by sterile Pasteur pipettes to 15 X 100 mm Petri dishes of BBM agar. The latter were then vigorously swirled to disperse the suspension evenly over the agar surf ace. The Petri dish cultures were maintained upside-down under standard conditions of culture for from 3 days to 3 weeks, depending on the time required to obtain the desired life-cycle stage for study. Growth in late log phase (ca. 12 days after inoculation) under these conditions provided adequate material with most of the desired life cycle stages present; oils, starches, and other reserve metabolites were minimal at this phase. 
The algal material was harvested by scraping from the agar surface with the edge of a microscope slide and trans£ erring the mass to a spatula for immediate immersion in the desired fixative. 
1 An R.C.A. E.M.U. 3 D Electron Microscope operated at 50 kv was used for the examina­tion of all material throughout this study. 
Various fixation procedures were used in order to determine the fixative best suited for demonstration of the several cellular organelles. In terms of penetration, most of the algae studied presented no problems. Occasionally, however, when some of the T etracystis species had entered the stationary phase of growth and undergone wall thickening, the fixatives did not penetrate well. 
A preliminary study was made with 6 different, freshly prepared permanganate fixatives, namely, Ca++, Sr++, Mg++, Na+, Li+, and K+ permanganates (Fig. 109-114) . All permanganates were prepared as a 2 % aqueous solution in deion­ized water. In addition, K+ and Li+ permanganates were prepared and tested at 4% concentration. The schedule for fixation, dyhydration, and embedding of ma­terial treated with these 6 permanganate fixatives is shown in Table 9. The images obtained with these fixatives in the test organism, Tetracystis isobilateralis, are shown in Fig. 109-114 and will be discussed below. 
Later in this study, the effects of glutaraldehyde fixation on material to be treated with Os04 and LiMn04 were investigated. The fixation and post-fixation schedule for this test series, using T etracystis isobilateralis, is shown in Table 10. 
A given volume of 5% glutaraldehyde (diluted from a 25% stock in which the 
TABLE 9. Fixation, dehydration, and embedding schedule of test series with 6 different permanganate fixatives, using T etracystis isobilateralis ( A6-2-3) 
Treatment 	Methods 
1.  Fixation  a.  Six different permanganates at 2% and 4%  
(unbuffered, aqueous) .  
b.  Fixation time : 70 min ( 5 ° C )  

2. 
Rinse 	a. Three times with distilled H 2 0 (5° C) 

3. 	
Dehydration 25 % EtOH 50% EtOH 75 % EtOH 95 % EtOH 


100% EtOH 100% EtOH 10 min (5° C) 10 min (5° C) 20 min ( R. T. ) a 10 min (R. T. ) 10 min (R. T.) 10 min (R. T.) 
4. 
Infiltration Propylene oxide 15 min (R. T. ) Propylene oxide 30 min (R. T .) 

5. 	
Embedding 25% Plastic/ 75% Propylene oxide 1 hr (R. T.) 50% Plastic/50% Propylene oxide 1 hr (R. T. ) 75% Plastic/ 25% Propylene oxide 21 hr (R. T. ) 

100% Plastic 	24 hr (R. T. ) 

6. 
Polymerization 100% Plastic 	24 hr ( 110° C) 


a Room temperature. 
Studies of Algal Genera Tetracystis and Chlorococcum 
TABLE 10. Glutaraldehyde fixation, permanganate and osmium post-fixation, and post-treatments schedule with Tetracystis isobilateralis 
Treatment 

Methods 
l. Fixation (for both LiMn04 2.5% Glutaraldehyde buffered and Osmium, Os04 , fixed 
to pH 7.4 with equal volume materials) of Cacodylate 
a. Two changes of Cacodylateb 
A. Material to followed by: be treated inLiMn0.1 
b. Two changes deionized H 20 
2. Rinse B. Material to 
a. Four changes in Cacodylateb be treated inOsO.J 
A. LiMnO. 
a. 2% aqueous 
3. Post-fixation 
B. Os04 
b. 	1%, buffered to pH 7.4 with Cacodylate 
A. For LiMn04 
a. Four changes of deionized 
4. Post-treatments 
B. For Os0 4 
b. Four changes in Cacodylateb 
Temp. time  
5°C  16 days  
5° C  lOmin each  
5° C 5° C 5° C  lOmin each 2 hr 2 hr  
5° C 5° C  5min each 5min  

each 
I 
a Cacodylate-50 ml of 0.2 M sodium cacodylate plus 2.7 ml 0.2 M HCL diluted to a total of 200 ml with deionized H 20. 
b Cacodylate-0.2 M odium cacodylate only. 
pH must be no higher than 4) was mixed immediately before use with an equal volume of Cacodylate buffer (pH = 7.4). The results of the glutaraldehyde fix­ation series are illustrated photographically in Fig. 115-120. 
During the first phase of this work, propylene oxide was used as the intermediate between the alcohol and plastic series. This procedure worked very well, but be­cause of the expense of the chemical and the possible health hazards involved, 99.9% purified acetone was utilized in place of propylene oxide and worked equally well. The plastic was diluted also with acetone for the embedding pro­cedure. 
The following formulation of plastic mixture # 1, as commonly employed at the Electron Microscope Laboratory at The University of Texas, was used through­out this study: 
Plastic Mixture #11 
15 ml Araldite "M" 25 ml Epon 812 55mlDDSA 2-4 ml dibutyl phthlate 
H. H. Mollenhauer (1964). 
To use: add 1 drop of DMP-30 for each milliliter of 100% plastic as an accelerator 
for hardening (this includes the diluted plastics at the initiation of the embedding 
process). 
Plastic mixture #1 may be prepared separately in 2 parts: Part 1-Araldite "M," 
Epon 812, and dibutyl phthlate; Part 2-DDSA. Combine separate parts imme­
diately before use in the ratio of 9 parts of ( 1 ) and 11 parts of ( 2) . This procedure 
avoids the formation of a very viscous plastic mixture which develops in 1-2 weeks 
if both parts are mixed during initial preparation. 

Embedding was begun by introducing the material into a 25 % plastic mixture diluted with propylene oxide or 99.9% acetone. The material was left in this mix­ture for 1 hr, then was transferred ,to a 50% plastic mixture for 1 hour and then to a 75% plastic mixture for 21 hr (Table 9). In some schedules, the algal material was left in the 75 % plastic mixture for 3 days without harmful effects. Following the 75% dilution, the material was transferred to 100% plastic and immediately afterwards was poured into molds (aluminum cups or "boats," or plastic snap-on bottle caps; the plastic caps were re-usable, and no mold-release was needed when these were used). The material was kept in the molds (which were placed in 100-ml glass Petri dishes to keep out dust and contamination) at room temperature for at least 12 hr before being placed in an oven at 110° C. The plastic mixture poly­merized into a sufficiently hard block after 12--'-l 6 hr at oven temperature. 
Embedding schedules using a dilution series of 33%, 66%, and 100% plastic worked equally well, especially when freshly prepared plastics were used. 
All sectioning was carried out with a diamond knife, Type C (from Ge-Fe-Re: Via Marillima, Frosinone, Italia) on a Porter Blum Microtome. Cut sections were placed on 300-mesh grids which had been hand-polished on 1 side and then dipped in a solution of 5% Formvar in ethylene dichloride. No cleaning of the grids was necessary as long as they were adequately coated with Formvar. Coating of the grids when used in con junction with plastic mixture # 1 was also unnecessary. 
Most permanganate-fixed materials prepared in this study were routinely post­stained after sectioning in Millonig's PbOH (Millonig, 1961) for 5 min. A 5% Ba (OH) 2 solution was employed on occasion as a post-stain to resolve wall struc­ture, Golgi vacuolar products, and metabolic products such as starch, oils, etc. 
Material fixed in the glutaraldehyde series was post-stained in several ways. The Os04-fixed material was post-stained in a saturated solution of Uranyl acetate for 15 min (Karnowvsky, 1961) followed by Reynolds' lead citrate post-stain (5 min) (Reynolds, 1963). LiMn04-fixed material was post-stained for 5 min only in the Reynolds' post-stain. 
B. RESULTS 
1. Fixation images 
Two categories of permanganate-fixation images resulted from the permanga­nate series with T etracystis isobilateralis. Magnesium, calcium, and stronium per­manganates yielded the first type, notably that with swollen chloroplast lamellae (Fig. 112-114). Within this series Sr( Mn04) 2 resulted in a granular configuration of the pyrenoid matrix. The latter was preserved differently from the chloroplast matrix, and a fibrillar appearance was often present (Fig. 114-2) .1 The cytoplasm and its lamellar components appeared essentially similar with calcium, magnesium, and stronium permanganates. 
The second category of fixation image was obtained with sodium, lithium, and · potassium permangantes (Fig. 109-111). Here, there was much less distortion and swelling of the chloroplast lamellar components. The fixation image was most granular with potassium and sodium permanganates (Fig. 110, 111). The best fixation image within this test series was obtained with 2% LiMn04 (Fig. 109). For this reason the latter was used for the majority of species subsequently studied. 
Later in this investigation the writers' attention was called to a glutaraldehyde fixation technique (Sabatini, Bensch, and Barrnett, 1962; Ledbetter and Porter, 1963). The authors' modifications of these techniques for the algal material under investigation a~e presented in Table 10. The results of this study are presented in Fig. 115-120. In glutaraldehyde fixation, followed by Os04 or LiMn04treatment, the preservation was superior ·to that of material in Os04 or LiMn04 without prior glutaraldehyde treatment. 
Lithium permanganate-treated material, first fixed with glutaraldehyde, is simi­lar in many aspects to osmium-fixed material. It is of interest that ribosome-like particles (Fig. 119-6), "osmiophilic particles" (Fig. 116-4), and nucleolar ma­terial (Fig. 116-3) are observable in permanganate-treated preparations fixed with glutaraldehyde. The above mentioned cellular components are not preserved in algal material fixed directly in LiMn04 (Fig. 127-131). ' 
Thus, permanganate fixation may be made to approach more closely that of osmium when the former is used in con junction with glutaraldehyde. This schedule maintains the well-known advantages of permanganate fixation of plant material with respect to preservation of membrane and lamellar systems. 
Osmium-treated material of T etracystis isobilateralis also is preserved much bet­ter when fixed with glutaraldehyde. In the writers' algal material, when 1 % Os04 was employed without glutaraldehyde fixation, the lamellar systems, particularly those of the chloroplast, swelled markedly, as they do with calcium-, magnesium-1 and stronium-permanganate fixation. Such a swelling effect is greatly reduced when glutaraldehyde is employed first (Fig. 115, 117, 118). However, such ma­terial is of very low contrast and is only slightly improved with Reynolds' and Uranyl acetate post-staining procedures. 
Thus, LiMn04 treatment provided the best fixation image of all when prior fixation in glutaraldehyde was instituted. Such a fixation image may indicate a 
1 The number after the dash in each case refers to the numbered arrow in the figure. 
heretofore unrealized potential with permanganate preservation, particularly with reference to algal material. 
2. Comparative study of cellular organelles in Tetracystis 
(1 ) The chloroplast 
The ultrastructure of the chloroplast in all species of T etracystis is essentially similar to that of other chlorophycean algae (Gibbs, 1962c; Lang, 1963) in that the plastids are limited by a double membrane which surrounds a granular stroma (Fig. 125). The latter contains a number of lamellar structures. The unit struc­tures, which are flattened sacs, are termed the discs (Fig. 136-3). Varying amounts of starch are present within the chloroplast stroma (Fig. 132) and are also associ­ated with the pyrenoid (Fig. 143). 
Variations in gross chloroplast form have been used as taxonomic attributes at the generic level in the Chlorococcales and Chlorosphaerales (Starr, 1955; Hern­don, 1958). At the specific level, such chloroplast attributes as presence or absence of perforations, fissuring, mass, and pyrenoid number and position have been found reliable in delineating species. In the present investigation, attention has been devoted to these same attributes electron microscopically. Light-mic~oscopic study has revealed that the several species of Tetracystis may be divided into 3 categories on the basis of chloroplast mass (thin, thick, and intermediate) . In this connection, the electron-microscopic data support those obtained by light microscopy. These categories and their attributes along with the type of fissuring, if present, are sum­marized in Table 11. 
Ultrastructurally, the organization of the chloroplast has been found reliable in segregating species of T etracystis on the basis of lamellae. In 1 species, T etracystis pampae (Fig. 156-158), the discs are always associated in three's. In the remain­ing species, both number and pattern of disc association may vary, or the number alone may vary and the pattern of associated discs may be distinct (Table 12). 
In the latter case, 3 basic patterns of disc association have been observed, and there is some evidence of intergradation. In the first pattern, irregularly alternat­ing groups of 1-2 discs occur with groups of 3~5 (Fig. 155-6). In the second, there are irregularly dispersed groups of greater variability with respect to number of discs associated, these numbers ranging from 2-10 (Fig. 133) . The third pattern of disc association is composed of 2 to 6-8 discs per group (Fig. 121). 
The second and third patterns of disc association may be subdivided further on the basis of presence or absence of reticulations between the groups of associated discs. These reticulations consist of end-to-end association of curving, single lamel­lae, usually near the pyrenoid (Fig. 121-2). Finally, certain Tetracystis species have both variable patterns and numbers of associated discs (Table 12, III) . 
· Other investigators apparently have not reported ultrastructural differences in the chloroplast useful for taxonomic purposes at the species level. Gibbs ( l 962c) found that association of chloroplast discs in the Chlorophyta varied in number 
TABLE 11. Chloroplast organization in Tetracystis as revealed by electron microscopy 
Species  Chloroplast mass• Inter-Thinb Thick• mediated  Fissuring Present Absent  Type of fi ssuring  
T. jJUlchra (Fig. 152 ) T. interm edium (Fig. 144-3) T. texensis (Fig. 147-1) T. excentrica (Fig. 143-7) T. pampae (Fig. 158-4) T. illinoisensis (Fig. 141 ) T. aplanosporum (Fig. 153, 155-7) T. aeria (C-6) (Fig. 121-3) T. aeria (Pa-3) (Fig. 124-3) T. isobilateralis (Fig. 130-2, -3) T. aggregata (Fig. 134-7 ) T. dissociata (Fig. 135, 137 )  + --+ --+ --+ --+ -+ + --+ ----+ --+ -+ --+ --+ - -+ -+ -+ -+ -+ + -+ -+ -+ -+ -+ -+ - -----Deep, broad invagina­tion, external only Deep, narrow fissures, external only Deep, broad invagina­tion, external only Deep, broad invagina­tion, external only Both internal and external fissures Dee:.i, broad invagina­tion, external only Both in ternal and external fissures  

a As seen in optical section. b Thin: chloroplast mostly thin and parietal, slightly thickened in region of pyrenoid (Fig. 144 ) . c Thick: chloroplast massive, thick, almost filling the cell lumen except for nucleus (Fig. 130 ) . d Intermediate: intermediate between band 0 . 
from 2 to 6. That she noted such small variation may be explained by her investiga­tion of only a single species of each of 3 genera. Lang ( 1963 ) reported the most common number of disc associations to be 3 in members of the Volvocaceae and Astrephomenaceae in which she studied 8 different genera and at least 14 different species. She did not state maximum numbers of associated discs but did indicate that it varied from 3 to many in the same chloroplast. Thus, it appears that internal chloroplast diversity does not always accompany species and generic differences within certain families. On the other hand, the investigations of Ueda ( 1961 ) indi­cate a wide range of variation of chloroplast organization within the Chlorophyta. According to Ueda, disc association varied from 2 to 20 in Chlamydomonas, Oedo­gonium, and Tetraspora; from 2 to 40 in Chlorococcum sp.; and from 2 to 80 in Palmella. 
The stability of certain internal chloroplast features may account for consistent · differences reported in different species. The consistency of numbers of disc tom­
TABLE 12. I nternal chloroplast organization of T etracystis 
I. Discs consistently associated in 3's. T etracystis pampae (Fig. 158-6) 
II. 
Distinct patterns, but variable numbers of associated discs. 

A. 
Groups of 1-2 discs irregularly alternating with, and widely spaced from, 


groups of 3-5 discs. 
Tetracystis aplanosporum (Fig. 155-6) 

B. 	Two-10 associated discs : no definite reticulations between disc systems. Tetracystis isobilateralis (Fig. 128-2) 
T. aggregata (Fig. 133-5) 
T. dissociata (Fig. 136-3) 
C. Two-6 or 8 associated discs; definite reticulations present. 
T etracystis aeria (C-6) (Fig. 123-6) 

T. aeria (Pa-3) (Fig. 125-4) 
T. illinoisensis (Fig. 142-5) 
III. Pattern and number of associated discs variable. 
T etracystis intermedium (Fig. 145...:.5) 

T . pulchra (Fig. 151-1) 
T. excentrica (Fig. 143-7) 
T. texensis (Fig. 149-4) 
ponents in an association, as observed in T etracystis, is demonstrated also in the discs or tubules which enter the pyrenoid. For example, the pyrenoid of T. pampae is traversed by triple discs (Fig. 158-5). In other species in which the chloroplast disc system is more variable, as in Tetracystis texensis or T. isobilateralis, the disc associations are reduced to 2 or to 1 within the pyrenoid (Fig. 159, 163). When definite patterns of association occur (Table 12), a greater variability is noted among the T etracystis species. 
Not only gross, but also ultrastructural, features of chloroplast organization, therefore, are of value in distinguishing species of T etracystis. 
(2) The pyrenoid 
The pyrenoid is a region of the chloroplast consisting of a homogeneously granu­lar matrix surrounded by starch plates. Lamellar structures may or may not pene­trate the pyrenoid matrix. 
As far as the writers are aware, there have been no comparative studies of pyre­noid ultrastructure at the species level. Various investigators have described the ultrastructure of the pyrenoid (Albertson and Leyon, 1954; Butterfass, 1957; Chcirdad and Roullar, 195 7; Sager and Palade, 195 7; Hovasse and Joyon, 195 7; Brody and Vatter, 1959; Gibbs, 1960, 1962b; Ueda, 1961 ; Lang, 1963; and Murakami, Morimura, and Takamiya, 1963). These studies have been principally descriptive but include incidental experiments with reference to the occurrence of pyrenoids, their ultrastructure, and the response of pyrenoid ultrastructure to en­vironmental changes. 
The pyrenoids of 11 species of Tetracystis1 accordingly were studied with the electron microscope. Several types of pyrenoids could be differentiated at the ultra­structural level. Criteria include lamellar position, number of lamellar structures which penetrate the pyrenoid, their interconnecting patterns, the number and position of pyrenoid starch grains, and pyrenoid shape and size. 
The lamellar structures associated with the pyrenoid provide useful taxonomic criteria at the species level first with respect to their location within or upon the pyrenoid matrix (Table 13 ). Two alternative arrangements have been observed, namely, pyrenoids with largely "peripheral" and those with "internal" lamellae. Peripheral lamellae lie between the periphery of the pyrenoid matrix and the starch grains (Fig. 163). These are limited, single-disc systems at 1 or more loci along the surface of the pyrenoid matrix (Fig. 163-3,-4). The lamellae show a higher degree of undulation where they are single. 
TABLE 13. Pyrenoid organization among the presently known species of Tetracystis1 
I. Pyrenoid enclosed by a single starch grain. C hlorococcum sp. (tetra) 
II. Pyrenoid surrounded by 2 starch grains. 
T etracystis intermedium 
T. excentrica 
T. texensis 
T. pulchra 
III. 
Pyrenoid surrounded by many starch grains. 

A. 	
Single-disc (tubular) system 


T. aplanosporum 
B. Triple-disc system 
T. pampae 
C. 	Double-disc system 
a. 	
Disc pattern contorted, pyrenoid less than 5 µ.diameter. 

T. aeria (C-6) 
T. aeria (Pa-3) 
T. dissociata 
T. illinoisensis 

b. 	
Disc pattern largely parallel and straight, pyrenoid more than 5 µ. diameter. 


T. isobilateralis 
T. aggregata 
1 And of 1 species of Chlorococcum. 
Brown and Bold 
Species with internal pyrenoid lamellae could be subdivided on the basis of num­ber of associated lamellar discs penetrating the pyrenoid. These lamellar discs were single (Fig. 165), double (Fig. 159, 160), or triple (Fig. 161 ) . Regardless of the condition of the culture and of the fixation method, the number of associated discs within the pyrenoid of a given species of T etracystis is constant. When single ele­ments penetrate the pyrenoid, the individual components may be tubular (Fig. 164-5) or flattened cylinders (Fig. 164-6). In T etracystis aplanosporum, these tubules or flattened cylinders branch and occasionally (and only locally) cohere to form areas of appressed double or triple aggregates. That these lamellar elements penetrating the pyrenoid of T. aplanosporum are flattened cylinders or tubules can best be illustrated by comparison with the lamellae within the pyrenoid of Chloro­coccum mu!tinucleatum in which a tubular, reticulate, network of lamellar com­ponents is found (Fig. 166-8,-9). 
In pyrenoids containing paired lamellae, the latter generally occur as broad sheets or plates which contain pores (Fig. 162-4) . The double-disc systems were either contorted in some species (Fig. 160) or largely straight and parallel in others (Fig. 159). 
Only 1 species of Tetracystis, namely, T. pampae, has a triple-disc system of lamellae penetrating the pyrenoid. Such a system is merely a continuation of the triple-disc lamellar system of the chloroplast itself (Fig. 161-3). That this triple­disc system in both pyrenoid and chloroplast is so far unique, not only within the genus T etracystis, but also among the Chlorophyta, is supported by Gibbs' ( 1962b) statement that "No green algae have been observed yet in which the pyrenoid matrix is traversed by bands containing as many discs as do the bands in the chloro­plast proper." 
The number and position of starch grains about the pyrenoid are consistent and very useful comparative criteria. There may be 1 starch grain (Fig. 165), 2 starch grains (Fig. 163), or many starch grains (Fig. 159, 160) about the pyrenoid. Two starch grains are inevitably associated with ellipsoidal pyrenoids within the genus T etracystis (Fig. 163-2). Furthermore, ellipsoidal pyrenoids always have lamellae restricted to the periphery of the matrix, and they are always reduced to a single­disc system at one or more points. 
Pyrenoids with many starch plates are never ellipsoidal but may be spherical or irregular (Fig. 121, 132 ). Observations of spherical pyrenoids with light micros­copy show little of taxonomic value except for their size and position; however, observations with the electron microscope reveal significant taxonomic differences with respect to internal pyrenoid structure not observable in light microscopy. 
The species of T etracystis may be grouped into 5 categories on the basis of pyre­noid organization. This diversity of pyrenoid structure is greater in T etracystis than among all 7 genera of the Chlorophyta studied by Gibbs ( 1962b). The gross structure of the pyrenoid and the variation of the lamellar structures which pene­trate or associate with it appear to be very reliable and useful supplementary data in elucidating the taxonomy of T etracystis. 
(3 ) The mitochondrion 
It has been well established that mitochondria occur in the cells of green algae (Sager, 1959 ; Lang, 1963, etc.). Comparative study of the 11 species of Tetracystis has revealed considerable variation in the mitochondria, especially with respect to their size and form. 
The mitochondria of the several species range from small, bac_illiform organelles 
0.1 p. in length (Fig. 121-4) to larger, bran_ched or unbranched structures up to 8 p. in length (Fig. 131-5). Furthermore, the mitochondria differ in form. Two basic types have been recognized: (1) cylindrical mitochondria (Fig. 147-2, small; and Fig. 141-2, large), straight or curved (even vermiform) (Fig. 156-2); and (2) compressed, ribbon-like mitochondria, unbranched or branched (Fig. l33-4, 131-5. These 2 categories are mutually exclusive ; that is, species of T etra­cystis with compressed mitochondria seem never to have cylindrical ones. 
Table 14 summarizes the data obtained with respect to mitochondrial organiza­tion in the genus T etracystis. As is clear from the table, T etracystis isobilateralis can be distinguished at once on the basis of electron microscopy from all other known species by its compressed, branched, giant mitochondria (Fig. 130, 131) . These mitochondria seem to be larger than any so far reported in the algae. 
It is clear also from Table 14 that small, simple mitochondria characterize a , 
TABLE 14. Types of mitochondria in the genus Tetracystis 
I. 	Cylindrical T etracystis aeria (C-6) (Fig. 121-4) 
T. aeria (Pa-3) (Fig. 126-5) 
T. excentrica (Fig. 143-8) 
T. texensis (Fig. 147-2) 
T . aplanosporum (Fig. 153-3) 
T. intermedium (Fig. 144-1) 
T. pampae (Fig. 156-2) 
T. pulchra (Fig. 152-5) 
T. illinoisensis (Fig. 141-2) 
II. 
Compressed 

A. 
Rarely branched 


T. aggregata (Fig. 133-4) 
T. dissociata (Fig. 137 ) 
B. Frequently branched 
T. isobilateralis (Fig. 131-5) 
Brown and Bold 
majority of the species of Tetracystis. No significant differences among the cristae of the several types of mitochondria were observed. 
(4) The Golgi apparatus 
Although a number of investigators have reported the presence of Golgi bodies in the cells of green algae (Sager, 1959; Lang, 1963, among others), no special intensive study of these organelles seems to have been undertaken. The availability of material of T etracystis with these organelles especially well preserved impelled the writers to study them carefully and comparatively. 
These comparative studies of 11 species of T etracystis have revealed that 2 basic types of Golgi organization are present, namely, that in which the groups of cis­ternae are not distinctly associated with an extension of the nuclear envelope (Fig. 131--4) and that in which they are so associated (Fig. 143-9). In the latter case, extensions of the outer nuclear membrane protrude and the protrusion always en­compasses the Golgi apparatus. This type of relationship was first observed by Moner and Chapman ( 1960) in Pediastrum, and the nuclear protrusion was des­ignated an "amplexus" by Lang ( 1963). 
The Golgi apparatus itself may be differentiated in that some of its cisternae remain uninflated during all phases of growth. In Golgi aggregates without am-· plexi, the cisternae are characteristically uninflated (Fig. 121, 141 ) . In those with amplexi, however, the cisternae are always differentiated, at least to some degree, into flat and inflated components (Fig. 154, 143). Table 15 summarizes the oc­currence of the several types of Golgi apparatus among the species of Tetracystis and includes references to illustrative figures. 
In the course of this investigation, some indication of the possible origin of the Golgi cisternae was uncovered. The protruding branches of the nuclear membranes, reported above and by others merely to encompass the Golgi apparatus, were ob­served by the writers also to give rise, apparently, to the cisternae themselves (Fig. 175-179). In this sequence of figures, it seems clear that the am plexus is giving rise to cisternae by a sort of budding process. It should be noted in these figures that the cisternae nearest the amplexus, and putatively, those most recently formed, are uninflated and closely packed, while those further away are somewhat inflated and seem to be separating. Furthermore, budding from the amplexus is limited to that portion adjacent to the most recently formed cisternae. These considerations are in harmony with the suggestion that the cisternae are arising from the amplexus by budding and orderly deposition of the products of this budding. No evidence was uncovered regarding the origin of cisternae in Golgi groups lacking distinct amplexi. 
In this connection, Hodge and his co-workers ( 1956) suggested that a periodic continuity between the lamellar structures of the Golgi apparatus and the endo­plasmic reticulum must occur. Whaley, Kephart, and Mollenhauer ( 1959) noted 
TABLE 15. T ypes of Golgi apparatus in T etracystis 
I. 	Distinct amplexi present ( cisternae differentiated) T etracystis pampae (Fig. 157-3) 
T. aplanosporum (Fig. 154-5) 
T. excentrica (Fig. 143-9) 
T. texensis (Fig. 150-6 ) 
T. pulchra (Fig. 152--4) 
T. interm edium (Fig. 146-6) 
II. 
Distinct amplexi absent (i.e., no distinct relationship between the Golgi system and the extension of the outer nuclear envelope). 

A. 	
At least some cisternae inflated 
T etracystis aggregata (Fig. 132-2) 



T. dissociata (Fig. 137-6) 
B. 	Cisternae not inflated 
T etracystis isobilateralis (Fig. 131--4) 

T. aeria (C-6) (Fig. 123-7) 
T. aeria (Pa-3) (Fig. 124-2) 
T. illinoisensis (Fig. 141-3) 
what appeared to be 1 or 2 isolated cisternal structures which might represent early stages in development of the Golgi apparatus. Moore and McAlear ( 1962) re­ported that in the fungus N eobulgaria pura, "the dictyosome1 appears to be formed by a series of vesiculations of the outer membrane of the perinuclear cisternae that align to form a stack of sacs." Rhodin ( 1963) presented evidence on the origin of the Golgi bodies from the rough elements of the endoplasmic reticulum in animal tissue. 
It is clear from these citations that there is some evidence for the origin of Golgi cisternae from the endoplasmic reticulum and from the amplexus, the latter prob­ably representing a segment of the endoplasmic reticulum. One must be extremely cautious in correlating ultrastructure, which is static as viewed by the investigator, with function which is dynamic. However, study of Tetracystis has provided a pos­sible indication of the function of the Golgi apparatus in algae, that of forming vacuoles. This same function has been ascribed to the Golgi apparatus in animal tissue by Rhodin ( 1963 ) and plant tissue by Marinos ( 1963) . The evidence for this in T etracystis is presented in Fig. 180-185 and their legends. In Fig. 183 one observes that the cistemae furthest from the amplexus have enlarged, presum­ably through the accumulation of fluid. In Tetracystis aplanosporum, the cisternae may become greatly enlarged and often separate from the Golgi system in pairs 
(Fig. 181 ) or in chains (Fig. 153-2). In other species of Tetracystis, the enlarged cisternae separate singly as shown in Fig. 179 and 152-4. 
1 Another appellation for the Golgi apparatus. 
Finally, it should be stated that the present study has uncovered no evidence for a role of the Golgi apparatus in cell-plate formation, for cytokinesis in T etracystis does not involve cell plate formation. 
(5 ) The cell wall Electron-microscopic observations of the cell wall layers of Tetracystis confirm the observations with light microscopy. Thus, the electron microscope reveals 2 layers in the wall, an outer, electron-dense layer, and an inner, less electron-dense, granular layer (Fig. 133-3). The outer layer often appears striated or lamellated which may indicate periodic deposition. This will be discussed in more detail under the section on cell division. As the cells age, the outer strata of the electron-dense layer sloughs off (Fig. 138-2). For comparative purposes, the inner and outer wall thicknesses provided use­ful criteria for segregating actively growing T etracystis species into 4 categories as is shown in Table 16. The cell walls of species of Tetracystis appear to be similar to those of Chlamy­domonas (Sager and Palade, 195 7) . These authors described a homogeneous, less electron-dense layer, about 400 A thick, immediately adjacent to the plasma mem­'brane. Exterior to this wall layer, they observed an electron-dense fibrillar layer 
which had a frayed appearance. This layer corresponded to the capsule (sheath) as seen with the light microscope. 
TABLE 16. Thickness of inner and outer wall layers in species of T etracystis in the active growth phase 
I. Outer wall layer thicker than inner 
T. aeria (C-6) (Fig. 121-1) 
T. aeria (Pa-3) (Fig. 124-1) 
II. 
Outer and inner wall layers of equal thickness, but both thin 

T. 
aplanosporum (Fig. 153-1) 


III. 
Outer wall layer thinner than inner 

A. 	
Inner wall layer very much thicker than the outer, forming uni-and 


bipolar thickenings 
T.pulchra (Fig.151 ) 

T. excentrica (Fig. 143-6) 
B. 	Inner wall layer only slightly thicker than the outer; polar thickenings absent 
T. isobilateralis (Fig. 130-1) 
T. aggregata (Fig. 133 ) 
T. dissociata (Fig. 135-2) 
T. illinoisensis (Fig. 141-4) 
T. intermedium (Fig. 144-2) 
T. texensis (Fig. 14 7-3) 
T . pampae (Fig. 156-1) 
Lang ( 1963), however, observed this duality of wall structure only in the zygote of Astrephomene. The vegetative cells of all 8 genera studied by Lang contained little or no material adjacent to the plasma membrane when fixed in KMn04. However, when fixed in Os04, her material showed a fibrous layer with dense granules immediately outside the cytoplasmic membrane. 
Bisalputra and Weier ( 1963) found the inner wall layer adjacent to the cyto­plasmic membrane in Scenedesmus quadricauda to be cellulosic. Outside this layer was a "middle lamella" bounded on both sides by an interface, and outside of the middle lamella was a pectic layer composed of a net of hexagonal configurations. The observations of Bisalputra and Weier of S. quadricauda seem to correlate with the writers' observations of the cell wall of T etracystis, with the exception that pectic networks and a pectic spine are missing in T etracystis species. 
The thickness of the inner and outer wall layers of actively growing Tetracystis cells is a very constant and reliable criterion for species differentiation. Electron­microscopic studies have supported and augmented light-microscopic observations of wall structure. Cytochemical studies of the walls have not been included in the present investigation. 
(6) Miscellaneous organelles 
Other ultrastructural components of the T etracystis cell were observed inci­dentally and are treated briefly below. 
Endoplasmic reticulum (ER) was present in all species of T etracystis. However, no consistent differences in its distribution and morphology could be discerned except, perhaps, for the amplexus which may be thought of as specialized ER. Just preceding cell division, ER abounds in the area immediately adjacent to the cyto­plasmic membranes, particularly those formed during zoosporogenesis (Fig. 207­6) and occasionally during tetrad or diad formation (Fig. 190, 193, 199-4, and 201 ). 
T etracystis aplanosporum provided particularly interesting data with regard to contractile vacuolar organization. As described by Lang ( 1963 ) , the contractile vacuole is delimited by a single membrane, and when fully distended, this layer is quite obvious (Fig. 186-2). When the contractile vacuole is partially (Fig. 187-5, 188-6) or completely collapsed (Fig. 186-1 ) , this single membrane is folded ra­dially. That segment of the contractile vacuole nearest the periphery of the cell has fewer folds in its limiting membrane (Fig. 187-5). Extensions from the outer nu­clear envelope always seem to be associated with or in close proximity to the con­tractile vacuole (Fig. 187-4, 186-3, and 188-7). 
3. Pyrenoid division 
Although it is well known through light microscopy that pyrenoids divide dur­ing cellular replication (Timberlake, 1901; Lutman, 1910; Bold, 1931; Buffaloe, 1958; Bischoff and Bold, 1963), ultrastructural details of this process are not as well known. The pyrenoid has been observed to divide by fission, with light micros­copy, giving rise to 2 or more pyrenoids. Many pyrenoids in division have been seen in electron micrographs. The details of pyrenoid division were observed with the greatest clarity in Tetracystis isobilateralis. 
The lamellar elements of the chloroplast which penetrate the pyrenoid of T. isobilateralis have been studied before, during, and after pyrenoid division. In the non-division stages, the lamellar discs which penetrate the pyrenoid matrix assume a reticulate pattern with elongate interstices (Fig. 159-1). By the onset of di­vision, the pyrenoid has achieved maximum size, and its lamellar discs re-orient and become parallel, extending across the entire pyrenoid matrix (Fig. 167-3). Still later, these parallel lamellae either are broken by elongation of the matrix, or break autonomously in a plane perpendicular to their long axis (Fig. 167-2). These breakages are not simultaneous. 
Following cleavage of the parallel lamellae, 2 separate, double-disc systems within the pyrenoid penetrate perpendicular to the parallel discs and segregate the parallel disc system into 2 parts (Fig. 167-1, 169-8). The separated disc systems are still parallel (Fig. 168, 169). Figures 168 and 169 are from another section through the same pyrenoid and illustrate the contiguity of the intervening, perpen­dicular, double-disc systems with their respective segregated groupings of original parallel lamellae. Note also that the pyrenoid is more ellipsoidal, which may indi­cate a duplication or formation of additional matrix material at this stage before the initiation of fission. 
Multiple stacks of chloroplast lamellae now begin to grow centripetally into the pyrenoid matrix (Fig. 168-5, 170). Starch formation is now initiated in the di­vision zone (Fig. 170). The parallel relationship of the lamellae in the daughter pyrenoids persists (Fig. 170-15). At this stage, an ellipsoidal body has been ob­served several times between the daughter pyrenoids (Fig. 170-11). The sig­nificance of this body has not been resolved. 
The writers consider pyrenoid cleavage to have been completed when the chloro­plast lamellar intrusions have cut the daughter pyrenoids completely in half (Fig. 171-2). Whether or not the 2 sets of invaginating chloroplast lamellae anastomose or intertwine when they meet is unknown at present. When cleavage has been com­pleted, 2 separate pyrenoid matrices can be discerned (Fig. 171 ) , and starch syn­thesis becomes very active on the matrix interfaces of the daughter pyrenoids (Fig. 171 ) . The lamellae in the daughter pyrenoids are still parallel to one another for the most part, but re-orientation soon occurs (Fig. 1 72-6). 
The final stage in division of the pyrenoid consists of a reorientation of the paral­lel lamellae within the pyrenoid matrix to form the reticulate pattern of the "rest­ing" pyrenoid. 
Thus, when observed ultrastructurally, pyrenoid division appears to be more complex than light-microscopic observations have revealed. The role of the lamellae within the pyrenoid matrix before, during, and after pyrenoid division is still un­resolved. However, the characteristic orientations of t):ie lamellae during division suggest that an orderly process, perhaps one analogous to nuclear division, may be involved in the division of the pyrenoid. 
Since the discovery of DNA in the chloroplast (Ris, 1961; Ris and Plaut, 1962) it would seem possible that DNA might occur in the pyrenoid matrix, and, if so, that the pyrenoid may function as the genetic center of the chloroplast. In this lab­oratory, recent fluorescent microscopic studies have indicated that the pyrenoid of 
T. isobilateralis possibly contains RNA. Radio-isotopic studies with labeled cyto­sine and other cytochemical evidence will be required to confirm the suggestion made above. 
4. Cell division 
Since the nature of cell division has been emphasized in the characterization of the C hlorosphaerales (Herndon, 1958; Deason and Bold, 1960), and because so little was known about the nature of wall formation following cytokinesis, the writers employed the electron microscope to aid in the understanding of these processes, using species of the chlorosphaeralean genus T etracystis. 
It has been the writers' experience that it is very difficult to obtain large numbers of actively dividing cells at any given time without special preparation. Further­more, the chance of observing a dividing cell with electron microscopy are limited, because of the relatively small spectrum of cells which can be observed at a given time. When grown on solidified BBM under standard conditions, the log phase of growth is relatively short (a 5-8 day span). Within 5-8 days after inoculation, the rate of cell division slows down, and the cells enter a stationary phase, accumulating large reserves of starch. Deficiency of nitrogen was found to be the primary cause for this. Therefore, the nitrogen concentration of BBM was increased 3-fold. Species of T etracystis grown in the 3N1 BBM remained in log phase up to 17 days following inoculation. When such cultures were harvested for electron-micro­scopic studies of cell division at 2 weeks, many more cells were found to be actively dividing. 
Light plays an important role in determining which pathway will be followed, whether that leading to the formation of a zoosporangium or to a tetrad of non­motile daughter vegetative cells. It will be recalled that cultures of Tetracystis were routinely maintained under a 12-12 hr photoperiod. If one were to examine ac­tively growing cultures 6 hr into the light period, the highest frequency of division leading to the formation of nonmotile tetrads of daughter cells would be observable. However, when cultures are placed in continuo~s light for 1-3 hr, following an uninterrupted dark period of 5-8 hr, vegetative cells divide actively to form zoosporangia. Accordingly, an abundant supply of cells in various division stages could be obtained at will. 
1 3-fold nitrogen increase as compared with BBM. 
The first type of vegetative cell division to be described in Tetracystis involves division of a mature vegetative cell into a tetrad of vegetative cells with intervening diad formation. Tetracystis isobilateralis and T. aggregata most commonly under­go division in this manner. The chronological sequence of events, as observed by light microscopy, leading to the formation of a tetrad are shown diagrammatically in Text-fig. 3. In the second type of vegetative cell division, a diad stage does not intervene, but, instead, cytokinesis forms simultaneously 4 daughter cells (Text-fig. 2). 
It is of interest to examine the ultrastructural events in both types of tetrad for­mation. Five of 12 species of T etracystis will be discussed with reference to the 2 types of vegetative cell division. 
Electron-microscopic observation of vegetative cell division into a tetrad, with intervening diads, will now be described. Examination of the wall structure of a mature vegetative cell of T. aggregata reveals 2 wall layers: an electron-dense, fibrillar, outer layer and an inner, less electron-dense, granular layer (Fig. 133-3). Following division of the pyrenoid, chloroplast, and nucleus (in that order) (Text­fig. 4) ,cytokinesis is initiated from the surface of the cell and progresses centripetal­ly between the nuclei (Fig. 190-2). When the cleavage furrow reaches about mid­way into the cell, the inner wall begins to form in the area where the cleavage furrow had been initiated at the periphery (Fig. 191-4) . The first sign of the presence of an inner wall layer is evidenced by a fine granulation image in LiMnO. fixation with Millonig's post-staining. Inner wall secretion appears to be progres­sive, following the cleavage furrow as it traverses the cell. The apex of the advanc­ing cleavage furrow always seems to be surrounded by endoplasmic reticu~um (Fig. 190, 195, 196). Golgi complexes are frequently observed in the perinuclear area during cleavage (Fig. 195-1, 190-1 ). However, their association seems to be less intimate with the cleavage furrow than is the association of the endoplasmic reticu­lum with the furrow. Figures 195 and 196 show 2 different sections through the same cell in which a cleavage furrow has cut through about 75% of the cell diam­eter. At the apex of the furrow, the Golgi apparatus is in sectional view of one figure (Fig. 195-1 ) and in surface view in the other (Fig. 196-2) . Here no direct role of the Golgi apparatus in wall formation seems apparent. 
It is of particular interest to note the presence of coarse granulation in the parent inner wall of the dividing cell shown in Fig. 191-3. Note that this granulation is present only in the inner wall of the parent cell (arrows) which is undergoing di­vision to form a diad. A zone which separates coarse from fine granulation can be observed in the region in the transverse inner wall where cytokinesis began (Fig. 191-3). Thus, no coarse granulation appears in the transverse inner wall (Fig. 191-4), and no outer wall develops until such coarse granulation is present (Fig. 191). Other evidence for the relationship between coarse· granulation of the inner wall and formation of the outer wall layer comes from examination of the "inter­cellular space" between the mature members of the diads (Fig. 192-5). Prior to the 
Text-fig. 2 Schematic diagram of sequence of events leading to the formation of tetrads directly. I . Mature vege­tative cell; 2. pyrenoid division into 4 segments which may occur by successive bipartition or by direct fragmentation; 3. chloroplast division which may occur by successive bipartition or by direct fragmentation; 
4. nuclear division; 5. cell wall deposition by invagination. 





Text-fig. 3 Schematic representation of events leading to the formation of isobilateral tetrads by the process of intervening diad formation as exemplified by Telracyslis isobilaterialis. 1. mature vegetative cell: n = nucleus, cl = chloroplast, py = pyrenoid, and cw= cell wall; 2. division of the pyrenoid; 3. chloroplast division; 4. nuclear division; 5. cell wall deposition, to form a diad of daughter vegetative cells; 6. pyre­noid division in diad members; 7. nuclear migration and chloraplast division; 8. stages in wall formation to form the isobilateral tetrad. 



Text-fig. 4 Schematic representation of the various stages of wall formation during vegetative cell division. I, wall layers of vegetative cell prior to division; sl = sloughing layer of the electron-dense wall ledl; igl = inner granular layer; pm = plasma membrane. 2, cleavage-furrow formation; no granulation is present in the apex of the furrow. The zone delimiting coarse and fine granulation originally present in parent inner wall is shown by z. 3, later stage in which cleavage furrow has cut across most of cell lbr = ·break which indicates that only a portion of the furrow is shownl. Note that inner wall deposition has begun in the furrow as indicated by the presence of fine granulation. A zone lzl delimits the newly deposited fine granulation of the daughter inner walls from the fine coarse granulation of the inner wall of the parent cell. 4, later stage of wall formation in which the inner walls of the daughter cells have become coarsely granulated; note also initiation of electron-dense wall ledl. An intercullular space is now evident lisl and coarse graulation is reduced here apparently by blockage from the electron-dense wall layer led, newl. Nsl = non-sloughing layer of the parent-cell, electron-dense wall. 5, electron-dense wall formation of daughter cells now complete (edl due to presence of coarse granulation of inner granular layer of daugh­ter cell ligll. Coarse granulation completely absent in intercellular space lisl which accounh for sloughing in inner surface of parent-cell, electron-dense cell lsll. Nsl = non-sloughing layr eof daughter cell, electron­dense wall layer which is in close proximity to coarse granulation from the inner granular wall layer ligll. 6, final stage in vegetative cell division. The electron-dense walls of the parent cell have deteriorated ' to such an extent that they will break lows = outer wall separation) releasing the daughter cells. The electron-dense walls of the daughter cells now slough Isl = sloughing layerl to form an abscission zone lazl which also aids in the separation of the daughter cell. 


pm cf 

i 5 
-.,_,_~""..~~
.·'-"':"<.~~~~ 
~ .: ; ... ·.~ .
..: · -~~ 
.. . :.. 
...... .: . •.·..... ;. .•...:.: ~ . ~ .• .·: ;. .· ·.·...; .. 
' ~ 
..··...·. 

formation of the outer layers of the transverse walls, this intercellular space contains coarse granulation peripherally (Fig. 191-3), and the outer wall of the parent cell is being actively formed. Upon formation of the outer wall layer of the daughter · cells, the coarse granulation disappears in the intercellular space (Fig. 192-5), and the outer wall layer (Fig. 192-6) of the parent wall begins to break down on both externally and internally, presumably because it is no longer being augmented 
(Fig. 192-6). The formation of the new outer wall layers of daughter cells appears, therefore, to block the passage of coarse granulation into the intercellular space, thus terminating augmentation of the outer wall layer of the parent cell in this sec­tor. Accordingly, deterioration of the .outer wall at the intercellular space probably accounts for tetrad dissociation as the cellular complex ages. 
The origin of the wall layers just described has been most completely observed in T. aggregata, but it also occurs in similar fashion in T. texensis and T. dissociata. However, in T. dissociata a different mode of cytokinesis may occur instead of the unilateral invagination seen in T. aggregata. In T. dissociata, a plane of vacuoliza­tion occurs in the area to be occupied by the cleavage furrow (Fig. 197-4). Bi­lateral as well as unilateral cleavage can occur in this species (Fig. 197-3,-5; 198-3). 
In T. excentrica, the number and activity of the Golgi bodies in the perinuclear region increase during cell division (Fig. 200--2). That the amplexus system may be thought of as a complex and unified structure is evidenced in Fig. 200--2 in which 4 Golgi apparatus can be observed to be encased within a common extension of the outer nuclear envelope. In this species, cytokinesis can occur unilaterally or bi­laterally (Fig. 200-4). In T. excentrica, bipolar wall wall thickenings occur and these may possibly be explained by the unequal activity of inner wall formation. 
A modification of the first type of vegetative cell division ( 2-step division to form tetrads) occurs in T. isobilateralis. Like T. aggregata, this species undergoes' di­vision to form a diad and thence proceeds to form a trial or tetrad. However, in T. isobilateralis, the granular component of the inner wall layer appears in the cleav­age furrow as soon as the furrow is formed (Fig. 194). Thus, the outer wall seems to be deposited immediately behind the apex of the advancing furrow (Fig. 193-1). Note also that the intercellular space is devoid of coarse granulation which further supports the views discussed above (Fig. 193-2). · An examination of the newly formed daughter cell outer walls in T. isobilateralis reveals these layers to be very closely appressed, so that the space between their interfaces appears as a middle lamella (Fig. 193-4). This contiguity further sup­ports previously published information (Herndon, 1958) which utilizes the con­tiguity of daughter cells with parent cells as a prime ordinal attribute delimiting the Chlorosphaerales. 
T etracystis isobilateralis may be useful in giving us insight into the origin of coarse granulation and its function in wall formation. The area of cytoplasm in the immediate vicinity of the advancing cleavage furrow (Fig. 194-6, 193-2) contains an abundance of coarse granules as compared to other cytoplasmic areas. The only other areas containing an equal abundance of coarse granulation are the "vacuoles" (Fig. 193-5) which, perhaps, release these granules into the cytoplasm upon rupture. (The possibility of fixation artifact cannot be excluded here.) It is quite possible, therefore, that formation of the outer wall may be indirectly based on Golgi activity, for evidence has been presented earlier in this report that Golgi bodies may give rise to vacuoles. 
The second type of vegetative cell division among T etracystis species involves direct formation of a tetrad without intervening diad formation (Fig. 201). Here, the chloroplast and nuclei undergo 2 successive divisions prior to cytokinesis and subsequent wall formation. The writers have not yet seen enough cell divisions of this type to warrant a complete and detailed description. However, information of some value has been gained from observations of cell divisions in T. aeria (Pa-3). Outer wall formation in T. aeria is similar to that in T. isobilateralis in that the outer wall is formed immediately behind the apex of the advancing cleavage fur­row. One of the major differences in the second type of vegetative cell division is the path the cleavage furrow will take, once it has been initiated, after the cell has become quadrinucleate. If thickness of the newly deposited outer wall layer is a magnification of a time-deposition relationship, then the later-formed layers may be detected. Note in Fig. 201-5,-6 that 2 such thick outer walls are present. Thus, 2 simultaneous cleavages could have arisen from the cell surface (arrows) and de­veloped centripetally (Fig. 201-5,-6) and.thence centrifugally (Fig. 201-7,-8) to the surface of the other pole of the cell (note the thinner outer wall layers in the centrifugal cleavages). 
In view of the foregoing, the type of vegetative cell division which forms tetrads directly may be thought of in terms of a limited progressive cleavage, with a con­siderably more specific pattern than is exhibited in other algae which have many more nuclei per cell (e.g., Protosiphon). 
Vegetative cell division in Tetracystis, then, is effected by cytokinesis followed by the formation of an inner wall in the plane of the cleavage furrow. When coarse granulation is present ( T. aggregata and T. isobilateralis), an outer wall layer is formed. Outer wall formation may be delayed until the cleavage furrow and sub­sequent inner wall deposition have been completed ( T. aggregata), or outer-wall formation may occur simultaneously with cleavage and inner wall formation (T. isobilateralis). In all species, the outer walls of newly formed daughter cells remain closely appressed to the parent cell wall immediately following division, thus sup­porting Hemdon's ( 1958) statements on wall contiguity. However, daughter-cell separation may be early or late, depending on the rapidity of outer wall breakdown at the intercellular spaces of a given species. 
5 Zoosporogenesis in T etracystis and C hlorococcum During the present electron-microscopic investigation of Tetracystis, the oppor­
tunity became available to examine the ultrastructural events leading to the for­mation of zoospores. It will be recalled that Tetracystis may follow 2 pathways of cell division, one leading to the formation of nonmotile daughter cells, and the other leading to the development of zoospores (Text-fig. 1). AB has been discussed previously, the existence of the above-mentioned alternatives in Tetracystis delimits it from the genus Chlorococcum. In Chlorococcum, only 1 of the pathways of cellular division is present, namely, zoosporogensis. Since both T etracystis and Chlorococcum can form zoospores, the writers wished to compare zoosporogenesis ultrastructurally in at least 1 species of these 2 genera representative of the Chloro­sphaerales and Chlorococcales, respectively, and also to compare zoosporogenesis 
·with vegetative cell division. 
The first indication of the initiation of zoosporogenesis in a mature vegetative 
cell, as observed by light microscopy, is the "disappearance" of the pyrenoid, ac­

companied by an increase in density of the chloroplast (Fig. 69). Tetracystis 
aplanosporum was used to demonstrate ultrastructural changes in the pyrenoid 
and chloroplast leading to the formation of zoospores. During interphase and 
growth of the mature vegetative cell, the pyrenoid is a conspicuous body in the 
chloroplast and usually is located in the center of the cell (Fig. 153). Aggregates 
of starch plates surround the pyrenoid matrix during this phase (Fig. 153). 
When a vegetative cell is destined to undergo zoospore ~ormation evoked by the 
environmental conditions described on page 58, the pyrenoid becomes segmented 
by the dividing chloroplast (Fig. 202-205). The furrows which divide the chloro­
plast also appear ultimately to divide the pyrenoid first into hemispheres (Fig. 203), 
and then into a number of segments which foreshadow the number of zoospores 
which will be formed (Fig. 205). In contrast, it will be recalled that pyrenoid di­
vision during vegetative cell division is significantly different (Figs. 173, 174). It 
is of interest that in zoosporogensis, starch synthesis at the periphery of the pyrenoid 
matrix is greatly reduced during pyrenoid division. While pyrenoid starch may be 
reduced during this phase, non-pyrenoidal starch seems to increase. Simultaneous 
reduction of pyrenoid starch and increase of non-pyrenoidal starch may account 
partially for light-microscopic observations of the supposed "disappearance" of the 
pyrenoid d_uring this phase of zoosporogenesis. While light-microscopic observa­
tions have been inadequate to reveal the fate of the pyrenoid, electron microscopy 
indicates that the pyrenoid does not actually disappear during division as Fig. 202­
205 clearly show. Evidence for the presence of the pyrenoid, during and after its 
division, is based upon the characteristic presence of pyrenoid tubules associated 
with pyrenoid stroma (Fig. 204-2). Thus, pyrenoid division during zoosporo­
genesis appears to differ little from that process in vegetative cell division, except for 
the reduction of pyrenoid starch and multiple simultaneous cleavages of the chloro­
plast which divide the pyrenoid into fragments, one destined for each zoospore. 
Figure 205 shows a later stage in zoosporogenesis of T. aplanosporum in which the chloroplast has nearly completed division. At this stage, the lamellar structures of the chloroplast are more numerous and more densely aggregated than in the rest­ing cell. The lamellar proliferation just noted represents a period of maximum chloroplast lamellar synthesis and pigment formation. In support of this state­ment, light-microscopic observations show a deeper coloration and increased den­sity of the chloroplast during early zoosporogenesis. 
A major cytoplasmic change during early zoosporogenesis is an increase in quantity and activity of the Golgi apparatus (Fig. 204-3). The activity of the Golgi system is evidenced by the greater degree of vacuolization of certain cisternal com­ponents in the dividing cell (Fig. 204-3) than in the interphase cell (Fig. 153). 
Following pyrenoid, chloroplast, and nuclear division, events leading to the for­mation of the zoospore wall are initiated and will now be described in detail for Chlorococcum multinucleatum and Chlorococcum sp: (tetra, rough) (Text fig. 5). Here, the endoplasmic reticulum, unlike that in vegetative cell division, appears to play a major role in wall formation leading to zoospore development. Branches of the ER develop at the surface around each protoplasmic segment destined to be­come a zoospore (Fig. 206-2, 207-6). There is continuous cytoplasm between the immature zoospore segments bounded by ER. Later, a common wall layer is secreted peripherally to the endoplasmic reticula of adjacent zoospores (Fig. 206-1). Shortly thereafter, adjacent, electron-dense wall layers, one from each zoospore, are formed, these separating the common wall layer which had been previously deposited (Fig. 207-8). These electron-dense layers are destined to become the outer wall layers of the mature zoospores. Since the common wall layer ceases to be actively deposited outside of the developing electron-dense wall layers, the common wall layer ( IW) breaks down, releasing the individual zoospores shortly thereafter (Fig. 208, 209). Remains of the earlier-deposited common wall ( IW) adhere to the electron-dense walls of the newly-separated zoospores but are soon sloughed off, exposing now only the outer, electron-dense layers. Prior to this loss of the common wall, the electron-dense walls were bounded by distinct interfaces on both surfaces. However, when the common wall has been sloughed off completely, exposing only the outer, electron-dense layers, the latter also begin to slough. The rate of loss of electron-dense wall material is less than the rate of is deposition. 
An interesting feature of zoosporogenesis is the very early formation of flagella and stigma, even before zoospore wall formation has been completed (Fig. 207­4,-5). Impending zoosporogenesis may, therefore, be distinguished from impend­ing division to form tetrads of nonmotile vegetative cells even before cytokinesis and wall formation have been initiated. Other investigators have reported that flagella and stigma: arise de novo in each zoospore (Bold, 1951). 
A most significant, comparative, ultrastructural feature of taxonomic value is the relation of the parent zoosporangial wall to that of the zoospore. It will be recalled that in vegetative cell division, the electron-dense wall layers of the daughter cells 
Text-fig. 5 Diagrammatic representation of cell wall formation during zoosporogenesis in Tetracystis and Ch/oro­coccum. The earlier events begin at the top of the illustration and progress as viewed downward. CY = cytoplasm; IW =common inner, electron-transparent wall layer; ER =endoplasmic reticulum, delimiting protoplasmic segments destined to become zoospores; ED = electron-dense wall layer, deposited, peri­pherally to the inner wall layer; IS = intercellular space, formed by deterioration of common wall layer CIWI; sl IW = soughing inner wall layer remains of the earlier-deposited, common, inner wall layer; sl ED = sloughing electron-dense wall layer which begins to slough only when the remains of the common inner wall layer have eroded away. 
. . . . .... ........ . .

) . 
. .. . . .. . 
. . . . . . . . . : ·• .......: ·..·.. . . . 
. .. ·... 
CY J.:::..;: :····...• 
·. 
. . . . . . . . . . . . . . 
. ' . 
. . . . . . . ... 
. . . . . .
. . 
. . . . . . ... 
. . 
sl sl 

arise adjacent to the equivalent, electron-dense layers of the parent (Fig. 192-7) cell wall. The parent wall is composed only of the electron-dense layer, and ruptur­ing of this outer layer will release the daughter cells. 
By contrast, the zoosporangial (parent) wall is composed of both outer, electron­dense and inner, granular layers (retained from its vegetative precursor) (Fig. '208-1,-2). Here, the outer wall layer of the zoospore is not deposited contiguously to the outer wall layer of the parent cell wall. This fact represents a major difference between the 2 types of cell division which have been described and should be given proper emphasis in delimiting the order Chlorococcales from the Chlorosphaerales. 
Zoosporogenesis is significantly different from vegetative cell division with re­spect to: ( 1) the simultaneous, multiple fragmentation of the pyrenoid; ( 2) re• duction of pyrenoid starch during pyrenoid division; ( 3 ) proliferation of chloro­plast lamellae during division; ( 4) increase of chloroplast starch during division; 
(
5) early presence of stigmata and flagella; ( 6) apparent relation of endoplasmic reticulum in wall formation; ( 7 ) early secretion of the zoospore electron-dense wall layer; ( 8) early separation of division products ( zoospores) as a result of early electron-dense wall secretion and/ or maturation; and (9) lack of association of outer wall layers of the zoospore with the outer wall layer of the zoosporangium. 

C. 
DISCUSSION 


The electron-microscopic studies reported above had as their primary purpose 
elucidation of the nature of chlorosphaeralean (as compared with chlorococcalean) 
cytokinesis and wall organization and formation. As the work progressed, secondary 
comparative data of significance were accumulated with respect to the organiza­
tion of the chloroplast as a whole and of its lamellae; pyrenoid structure and di­
vision; mitochondrial form; possible origin and function of the Golgi apparatus; 
cell wall structure; vegetative cell division, and, finally, zoosporogenesis. 
With respect to vegetative cell division, an inner wall is formed in the plane 
of an advancing cleavage furrow, and an electron-dense, outer wall layer is sub­
sequently deposited. The electron-dense wall of the parent cell is contiguous to the. 
electron-dense wall layer of the daughter cell at the time of formation of the latter, 
thus supporting Hemdon's ( 1958) emphasis of contiguity of the cell wall of the 
division products in vegetative cell division. Cell plate formation was not observed 
in T etracystis. 
Zoosporogenesis was studied comparatively with vegetative cell division and was found to differ significantly, particularly in the mode of wall formation and in the relation of the parent cell wall layers to those of the daughter cells. Zoosporogenesis is the only method of cell division present in the Chlorococcales. Vegetative cells of the Chlorosphaerales may undergo both zoosporogenesis and vegetative cell di­vision. Furthermore, the differences between these 2 orders are brought more sharply into focus by ultrastructural studies of cell division. 
Pyrenoid division during vegetative cell division was studied in detail in Tetra­cystis isobilateralis. As observed by the light microscope, the pyrenoid divides by fission. However, the process is more complex ultrastructurally in that the lamellar strucfores penetrating the pyrenoid matrix assume parallel orientation during di­vision and subrandom orientation during interphase. Final division of the pyre­noid is accomplished by centripetal growth of chloroplast lamellae in the division zone between the daughter pyrenoid matrices. In T etracystic aplanosporum and 
T. aeria (C-6), however, the pyrenoid is cleaved by the chloroplast-limiting mem­brane. 
Electron-microscopic study has revealed that parent cells transmit fragments of the original pyrenoid to the zoospores they form, so that they do not, in fact, arise de novo as someti111es reported on the basis of light microscopy. The stigmata and flagella, on the other hand, clearly do arise de novo. 
The species of T etracystis were studied comparatively for consistent differences in organelle structure. The structure of the chloroplast, as observed electron micro­scopically, greatly augmented light-microscopic observations. The internal struc­ture of the chloroplast was useful in differentiating T etracystis species into groups based on the pattern and number of associated lamellar discs. Tetracystis pampae has a most striking internal chloroplast organization in that the chloroplast lamellar discs are associated always in 3's. Tetracystis aplanosporum also has a distinct pat­tern of 1-2 discs alternating with groups of 3-5 discs. Other species have distinct patterns of lamellar disc stacking, but the number of lamellar discs per stack is somewhat more variable. The remaining species of T etracystis were grouped into a category in which both the pattern and number of associated chloroplast lamellar discs were variable. 
Of all the organelles, the pyrenoid provided the most reliable and striking ultra­structural differences. Pyrenoids of T etracystis are surrounded either by 2 starch grains or by many starch grains. Those pyrenoids with 2 starch grains have no lamellae penetrating into the pyrenoid matrix except for a convoluted single-to­double disc system between the starch grain and the pyrenoid matrix. Pyrenoids which fall into the above category are always ellipsoidal and can be easily dif­ferentiated also by light microscopy with respect to shape. Pyrenoids with many starch grains are of 3 types: ( 1) those with single tubules penetrating the pyrenoid matrix; ( 2) those with a double disc system of lamellae penetrating the matrix; and 
(3) those with a triple disc system of lamellae penetrating the matrix. Pyrenoids with a double disc system can be further subdivided on the basis of the disc pattern within the pyrenoid and the pyrenoid size itself. 
Mitochondrial differences were few among the species of Tetracystis, but when such differences were present, they were striking. Tetracystis isobilateralis and T. aggregata were the only 2 species with compressed mitochondria, while all other species of T etracystis contained mitochondria organized as cylinders of variable dimensions. T etracystis isobilateralis could be easily differentiated from all other species on the basis of frequent branching of the large, compressed mitochondria. 
Cell wall ultrastructure provided useful criteria for augmenting light-micro­scopic observations of cell wall thickness of actively growing cultures, and, in ad­dition, for studying comparatively, thickness of inner and outer wall layers. Both isolates ( C-6 and Pa-3) of T etracystis aeria were distinct from all other species in that the outer, electron-dense wall is thicker than the inner wall layer. In T. aplano­sporum, both inner and outer wall layers are thin and equal in thickness. In T. pulchra and T. excentrica, the inner wall layer is often very much thicker than the outer, electron-dense layer, and this may be observed in light microscopy as internal unipolar and bipolar wall thickenings. The remaining species of T etracystis are more variable in wall thickness and could not be further differentiated except that the inner wall layer is only slightly thicker than the outer. 
The Golgi apparatus was studied comparatively and provided useful data for segregating T etracystis species into 2 categories: ( 1 ) those with Golgi bodies with distinct amplexi, the cistemae of which are often inflated furthermost from the en­compassing element of the amplexus; and ( 2) those with Golgi without distinct amplexi, ~istemae of which were rarely observed to inflate. 
A possible origin of the Golgi apparatus was observed in those species in which the Golgi bodies have amplexi (category 1 above). Here, the Golgi cistemae seem to be formed by a budding process of the associated, encompassing portion of the amplexus (which may be specialized endoplasmic reticulum). Such budding ac­tivity seems to be present in both resting and dividing cells of actively growing cultures. Evidence of origin of Golgi cistemae was not so clear in Golgi apparatus without distinct amplexi. 
Although function cannot always be completely understood from structure, a possible function of the Golgi cistemae in the role of vacuole formation was noted among those species which have distinct amplexi. The cistemae seem to have a period of activity of vacuole formation which is greatest during cell division. Contents of the vacuole are unknown, but the structure of the vacuole in LiMn04 preparations seems to indicate that some substance has been removed by the fix­ation, leaving behind a cavity limited by a single membrane. 
Other organelles were examined comparatively. Endoplasmic reticulum pro­vided no reliable comparative data. However, the presence and orientation of the ER during zoosporogenesis may signify its specialized role in wall formation. Con­tractile vacuoles provided no useful comparative data; their structure and possible association with the nucleus in T etracystis aplanosporum were observed. Stigma and flagella were not studied in this investigation. 
Thus, species of T etracystis can be segregated on the basis of ultrastructural or­ganization. The same species which fit into a certain category on the basis of one organelle system, also may be grouped with respect to similarity of other organelles (Table 17). On this basis, it would be reasonable to assume that the majority of the ultrastructural differences observed are significant and that they characterize a given species or a group of closely related species. 
TABLE 17. Groupings of T etracystis species on the basis of similarity of ultrastructurea 
Organelle Groupings 
M1to­chondrion  y · . iso. T. agg. T. diss.  T. aeria (C-6 & Pa-3) T. excen. T . illin. T. pam.  
Cell wall  T. aeria (C-6 & Pa-3)  T. pul. T. aplano. T. excen.  
\  
Pyrenoid  T . iso. T. agg.  T. aeria (C-6 & Pa-3) T. diss. T. illin.  T. excen. T. aplano. T. pul. T. inter. T. tex.  
Chloroplast mass  T. iso. T. agg. T. diss.  T . aeria (C-6 & Pa-3 )  T . excen. T. aplano. T. pul. T. inter. T. tex. T. illin. T . pam.  

Chloroplast  T. iso.  T. aeria  T. excen.  T. aplano.  
internal  T. agg.  (C-6 &  T. pul.  
T. diss.  Pa-3 )  T. inter.  
T. illin.  T. tex.  
Golgi  T. agg.  T. aeria  T . excen .  
apparatus  T. diss.  (C-6 &  T. pul.  
Pa-3 )  T. inter.  
T. illin.  T. tex.  
T. iso.  T. pam.  
T. aplano.  

T . pam.  T . pu . T . tex. T. aplano. T. inter. T. iso. T. agg. T. diss. T. illin. T. inter. T. tex. T. pam.  
T. pam.  

a This table emphasizes groupings of the species. For details of the organelles of the several species, see text and tables. 
IV. 
lmmunochemical Studies with Tetracystis and Chlorococcum 

A. 
INTRODUCTION 


The science of serology or immunochemistry, with its latest developments in technique, is now firmly grounded in medicine, zoology, and microbiology. Serology has received less attention in many areas of botany because there has been no real need for its use in many instances. This has been particularly true where taxonomy has required no supplementary criteria in the identification process, as is considered by many to be the case in the so-called "higher plants." On the other hand, certain groups, namely, the bacteria, pathogenic fungi, and plant allergens, have received more attention by the serologist either because these organisms are not so well­defined morphologically or because of the medical importance of their patho­genicity to man. It must be added that most botanists have not been trained in serology which is regarded principally as a zoological or microbiological technique. Finally, lack of knowledge and bias against its use by botanists may also account for the slow development of serological techniques in botany. 
Plant serology received considerable attention in the first quarter of this century during which the Konigsberg school of plant serology published many works, culmi­nating in the "Stammbaum" or phylogenetic tree of the plant kingdom ( Mez and Ziegenspeck, 1926). This work was subsequently repeated (using different tech­niques) by the Berlin group. Failure of the Berlin group to achieve the same results as the Konigsberg school led to considerable disagreement. Subsequently, the re­liability of serology as a tool for phylogenetic and systematic studies has been ques­tioned. Only recently, however, have the techniques of serology been applied to plant taxonomy with renewed interest and promise. Among others, the works of Gell, Hawkes and Wright ( 1960) and Lester ( 1964) have shown the value of serology in "higher plant" systematics using the newer techniques of double­diffusion ( Ouchterlony, 1948b) and immunoelectrophoresis ( Grabar, 1959). 
Serological studies of the algae have been fewer than those of angiosperms. Ac­cording to Mintz and Lewin ( 1954) , Rosenblat-Lichtenstein ( 1913) reported serological differences between a green strain of C hlorella protothecoides Kruger and a spontaneous mutant of the same species which had reduced pigmentation. 
In 1928, Mary Elmore was able to distinguish Euglena, Chlorella, and Chlamy­
domonas by complement fixation tests; however, she could not differentiate dif­
ferent strains of Euglena on that basis. In 1935, Mary Elmore Sauer reported fur­
ther work based on 6 strains of Euglena gracilis in which she was able to divide 
them into 2 serological groupings of 3 strains each. These categories were based 
on the loss of motility and complete sedimentation, as against no loss of motility and 
no sedimentation, when the organisms were placed in an antiserum to one or 
more of the strains at dilutions up to 1 : 1000. These serological groupings corre­lated exactly with those in which the growth characteristics had been the criteria for their separation. 
Provasoli, Pintner, and Haskins, ( 1951 ) failed to produce antigenic distinctions between mating types of Chlamydomonas moewusii; however, serological differ­ences between C. moewusii, C. chlamydogama, and C. reinhardti were readily de­tected. No mention was made of the methods employed to distinguish the species. 
Mintz and Lewin ( 1954) prepared flagellar suspensions of the plus and minus wild-type cells and paralyzed mutants of Chlamydomonas moewusii, and by complement fixation, were un.able to distinguish between the 2 wild mating types or between the wild-type and paralyzed mutants. 
Flagellar studies have been made with other members of the Volvocales. Among these, the work of Coleman ( 1963 ) deserves special attention. Coleman studied immobilization, agglutination, and agar precipitation of antibodies to flagella of the mating types of Pandorina. The antisera prepared against Pandorina exhibited syngen specificity, i.e., syngens (sexually isolated populations within the species ) maintained their antigenic specificity, even though they were isolated from diverse geographical localities. However, serological mating-type specificity was not achieved with Pandorina. 
The writers became interested in the immunochemical approach to the taxonomy of chlorococcalean and chlorosphaeralean algae during the summer of 1962 when they provided Mr. Charles Sweet with axenic cultures of Chlorococcum and Tetracystis species for injection into rabbits. The antisera produced by Sweet, how­ever, were weak in precipitating antibodies. 
While most of the recent immunochemical investigations have been concerned with flagellar antigens, the somatic or whole-cell antigens have, for the most part, been little studied. For this reason, the writers used whole-cell antigens because it was considered that they might be more useful for taxonomic purposes, since greater diversity and quantity of antigen types would be obtained. Furthermore, the recent development and perfection of double-diffusion and immunoelectrophoresis have provided analytical tools of incomparable resolution for protein systems. These tools have been used here with considerable success with algae. 
B. MATERIALS AND METHODS 
1. Preparation of cells for extraction 
During the early phase of this investigation, algal material was prepared by grow­ing it in liquid BBM in 125-ml Erlenmeyer flasks under standard conditions of cul­ture. However, the yield of algal cells was too low to provide a quantity sufficient for subsequent breakage and protein extraction. Therefore, another method was introduced for growing algal material. Petri dishes ( 20 X 100 mm) containing 1.6% BBM agar with 3 X nitrogen concentration were inoculated with algae (see page 9 ) and grown under standard conditions for 2-3 weeks, after which the algae were harvested. On the day of harvesting, each plate was carefully screened macroscopically and microscopically for possible fungal and/ or bacterial contami­nation, because all cultures to be used of necessity had to be axenic. The cultures were harvested by gently scraping the algae from the agar surface with the edge of a sterile microscope slide. The algal mass was then trans£ erred either to a beaker for storage in a freezer, or directly into a chilled mortar (-15° C) for immediate processing. 
A number of different methods were tested for breakage of the algal cells. The following techniques were unsuccessful in achieving satisfactory breakage: ( 1 ) motar with sand, 5 ° C; (2) mortar with alumina, 5 °C; (3) Mickle Cell; ( 4) Ribi Cell Fractionator; ( 5) Bransen ultrasonic probe; and (6) tissue homogenizers, both Teflon and glass. 
The method which eventually proved to be the most satisfactory for cell break­age was the acetone-powder method (Stafford and Magaldi, 1954; Colowick and Kaplan, 1955). Early experience with this method gave only mediocre results; however, certain modifications, which will be discussed below, resulted in excellent algal cell breakage. 
The acetone-powder method has several advantages over the other methods described above, namely: ( 1 ) very efficient cell breakage with T etracystis and C hlorococcum; (2) acetone-soluble materials such as chlorophyll, lipids, and water were removed, leaving the dehydrated proteins and cellular debris in the prepara­tion; and (3) the complete process was carried on below 0° C until the prepa­ration was in the form of a dry powder. 
Since the acetone-powder method was so successful in breakage of the algal cells, and because proteins of good antigenicity could be obtained in this manner, a somewhat detailed description of the process will be presented here. A block ( 6" X 6" X 2") of dry ice was placed in an enamelled pan (6" deep) on top of layers of paper towels for insulation, and a Coors' No. D-27 mortar was placed on the block for chilling ( 10--15 min) to about -15° C. The pestle was placed in the mortar so that it too would be chilled. Then the algal mass, either from the frozen pellet of storage material, or the mass scraped from a Petri dish, was added to the chilled mortar and allowed to freeze solid ( 1-2 min). When all of the Petri dishes had been harvested and when the material had become frozen, the algal pellets were gently crushed into a coarse powder. Cheese cloth usually was placed over the mortar in order to prevent loss of flying material during this tedious process. When the material had been ground to a state fine enough that no particles would be knocked from the mortar, the cheese cloth was removed and the particles ground to a fine powder. Enough acetone was then added to form a very thick paste. 
If too much acetone was added, a slurry would be formed, and this would need to be evaporated to a paste before efficient cell breakage would occur. When the proper consistency1 had been achieved, the mass was ground with considerable pressure on the pestle in order to push the mass over the bottom surface of the mortar. A swirling action with the pestle tended to push the mass on the sides of the mortar, and when this occurred, the material was scraped back into the bottom, and the grinding process continued. At this stage, periodic checks for efficiency of cell breakage were made by collecting a small amount of the paste on a chilled spatula and transferring it to a microscope slide for immediate examination. 
When 80-90% of the cells had been broken, 40-50 ml of chilled 99.9% acetone (-15° C) were added to the paste, and, with the action of the pestle, a slurry was forined. In preparation for this slurry, a 6"-disc of Whatman No. 1 filter paper was prepared to fit into a glass funnel, and chilled acetone was added to cool the sys­tem. Then the slurry of algal material was poured onto the filter and placed in a freezer ( -10° C) . Fifty-100 ml of additional chilled acetone were added to fur­ther extract the fat-soluble material, chlorophyll, etc., until a clear filtrate was obtained. Since proteins are insoluble in acetone, they and the cellular debris did not pass through the filter. When most of the acetone had passed through the filter, it was folded, stapled, and placed in a freezer with a frost-clearing system and allowed to dry for 24-48 hr. Then the dry filter paper was brought to room tem­perature, and the powder scraped from the surface. The dry material was then placed in a Coors' No. 520-0 mortar and pestle (which was dry and at room temperature) and lightly ground to a very fine powder. The powder was weighed, placed in an air-tight container, and stored at -10° C until needed for extraction. 
2. Extraction procedures 
The following extraction solution was employed for all extraction procedures, namely, 0.15 M NaCl buffered with a 0.01 M sodium phosphate buffer, pH 7.5. This was prepared as follows: 
NaCl ------------------------·---·-----------·------·-----------------------------------8.7 g NaH2P04·H20 _--------------·--------­0.2208 g Na2HP04·12H20 __ _ -------·----· --· .... 3.0085 g Deionized H20 _ ·----·-··------------·----·------··-1000 ml 
It was discovered only later in this study that the stability of algal proteins as potent antigens decreased upon repeated thawing and re-freezing of the extracted powder ( 8-10 times) and upon prolonged storage ( 2-6 months) while in solution. Therefore, it has been found much more desirable to extract only the amount needed, either for injection or testing purposes, and to maintain the antigen in powdered form at -10° C. 
1 The consistency of the algal mass is just right for efficient cell breakage when the cells will form a homogeneous layer which adheres to the bottom of the mortar when gentle-to­moderate pressure is applied to the moving pestle. 
All extractions were carried out at 5° C from 2 to 24 hr. A ratio of 0.1 g dry powder to 1.0 ml extraction solution was employed for most extractions. The pro­tein concentrations of this ratio in all species tested varied from 2.3 to 17 mg/ ml. Several experiments were performed to determine the precipitin reactivity over a wide range of concentrations (Fig. 213). When .the above ratio was diluted .4-8 times, the antigenic activity, as detected by the number and strength of precipitin lines, fell sharply. At twice the dilution very little difference in the number and strength of precipitin lines was detected, and when the extractions were made up of even higher concentrations of powder, no detectable increase in the number or strength of lines was noted (Fig. 213). 
Ar:·tigens were used, either as whole-powder suspensions, or as particle-free supernatants from centrifugations of the whole-powder suspension. Very little dif­ference in antigenic activity (as described above) could be detected between the whole-powder suspensions and the supernatant extra~ts, except possibly by a bit weaker activity in the latter (Fig. 213). Even for immunoelectrophoresis, the whole-powder suspension was quite satisfactory. 
3. Protein determinations 
Three different methods of protein determination were used at one time or an­other in the course of this investigation. They were: (1) Micro-K jeldahl (Burris and Wilson, 1957); (2) Biuret (Gornall, Bardawill, and David, 1949); and (3) 
TABLE 18. Soluble protein concentration of C hlorococcum and T etracystis species used 
for immunochemical investigations (Falin-phenol method of Lowry; 
Crystalline B.S.A., Armour, used as a standard) 

Protein concentration  Protein concentration  
Chlorococcum  mg/ ml  Tetracystis  mg/ml  
C. diplobionticum  16.3  T. aeria (C-6)  11.0  
C. echinozygotum  9.5  T. aeria (Pa-3)  14.3  
C. ellipsoideum  16.3  T. aggregata  17.0  
C. hypnosporum  11.0  T. aplanosporum  6.5  
C. macrostigmatum  11 .0  T. dissociata  13.0  
C. minutum  11.0  T. excentrica  12.5  
C. multinucleatum  12.5  T. illinoisensis  15.0  
C. oleo/ aciens  15.0  T. intermedium  9.0  
C. perforatum  10.0  T. isobilateralis  11.8  
C. pinguideum  10.5  T. pampae  11.0  
C. punctatum  13.0  T. pulchra  14.3  
C. scabellum  19.0  T. tetrasjJorum  11.0  
C. sp. (tetra isolate )  16.3  T. texensis  11.8  
C. vacuolatum  2.3  
C. wimmeri  12.5  

Folin-phenol (Lowry et al., 1951). Of these, the Folin-phenol method provided the most reliable data. Table 18 lists the species of Chlorococcum and T etracystis investigated and the protein concentrations of their extracts. Bovine serum albumin · was used as a standard. With the exception of C h!orococcum vacuolatum, the range in protein concentration varied from 6.5 mg/ ml, in T. aplanosporum, to 19.0 mg/ml, for C. scabellum. Since one of the most potent antisera was obtained from 
T. aplanosporum, it appears that the proteins of 11 other species, with the possible exception of C. vacuolatum, were of sufficient concentration for effective immuni­zation and testing. In addition, 2 species, namely C. diplobionticum and C. echi­nozygotum, differed by their protein concentration almost 2-fold, yet such a dif­ference was hardly, if at all, detectable on the double-diffusion precipitin tests (Fig. 221). Figure 213 shows an experiment designed to test the useful range of antigen concentration of C. sp. (tetra) for double diffusion and immunoelectrophoresis. Note that sufficient reaction occurs with well Nos. 1 (32.6 mg protein/ ml), 2 ( 16.3 mg protein/ ml), and 3 ( 8.15 mg protein/ ml). Detectable decreases in reactivity are noted at the protein concentrations of 4.7 mg protein/ ml (well No. 4 ) and 2.3 mg protein/ ml (well No. 5 ). 
4. Preparation of antisera 
Three different lots of rabbits were used for antiserum production. As techniques were perfected, the antisera from each succeeding lot of rabbits were improved. Therefore, techniques involving the first 2 lots of rabbits will be outlined only briefly. 
The first lot of rabbits was injected with 2-10% saline suspension of whole cells grown in liquid culture ( BBM). The resulting antisera were very poor in precipitat­ing antibodies, even though agglutination titers as high as 1: 8192 were apparently recorded. 
The second batch of rabbits was injected with acetone-powder suspension and with ultrasonic fractionates (including fat-soluble materials). From this batch, it was concluded that the acetone-powder suspension induced a superior antiserum to that of the sonicated material. Of the acetone-powder antisera, only one was satis­factory for use in the final phase of this study, namely, the antiserum to T. aer:a (C-6). 
A third lot of rabbits was immunized with preparations which were higher in protein content, due to improvement .in culture techniques by increasing the nitro­gen content of the culture medium (see page 9), and also to improvement in the grinding techniques in the acetone-powder method. In addition, the writers were in a better position to choose more carefully those species which would be of mmt value.1 
1 However, it was later found that antisera to certain other additional species also would have been valuable. 
Female New Zealand white rabbits were injected with preparations of 2 species of T etracystis and 2 species of C hlorococcum. Control bleedings prior to immuni­zation revealed no antibodies to algae. The routes of injections as well as the amounts of material injected were varied because of difficulties in obtaining suffi­cient antigen preparations for a complete injection series, and because antibody production did not always appear to proceed as expected. The frequent titer checks were made by double-diffusion against liquid extracts which had been frozen and thawed many times. Apparently poor reactions may have been due to the deter­iorated extracts. Since the schedule was prolonged and complicated, the final pro­duction of potent antisera cannot be attributed to any 1 course of injections. The total series of injections is shown in Table 19. 
TABLE 19. Injection and bleeding schedule used in the production of antisera against C hlorococcum and T etracystis species 
Injection Date (after time 0)' Amount (in ml ) Routeh 
1,7,14 2 x 1.0 IM 30 1.0 IP 32,34,36,39,41,43 0.5 IV 58 4 x 0.1 TP 69 2 x 0.2 IM 76 1.4 SC 98 2 x 0.5 IM 99,101 0. 15 IP 103,104 0.33 IP 107,110 0.5 IP 119,120,122,124 0.2 IV 126 2.5 IM 
a T est bleeding dates: 12, 20, 28, 35, 40, 48, 86, 99, 103, 106, 120, 124, 131. 
Mass-bleeding dates : 112, 133 . 
b Key to immunization route: IM = intramuscular, with Complete Freund's Adjuvant or with Sodium Alginate Adjuvant (COLAB); IP = intraperitoneal; IV = intravenous; SC = subcutaneous; TP = toe pad, with Complete Freund's Adjuvant; these are effective but very painful to the rabbits ( Leskowitz, 1960). 
With reference to algae, it is worthwhile to discuss briefly some of the probable essential points for the production of good anti-serum. These appear to be: ( 1 ) a large supply of acetone powder; ( 2) freshly prepared acetone-powder suspensions (soluble protein content about 2.0% ); (3) the use of Complete Freund's Adju­vant; and (4) sufficient time for antibody production. A suitable course of injec­tions might be: days 1 and 14 intramuscular, 1 ml emulsion with Complete Freund's Adjuvant into 2 sites; day 21, subcutaneous, 0.25 ml emulsion into 4 sites; days 35-49, on 5 days, intraperitoneal injections of 1.0 ml of suspension; or.on 10 days, several intradermal injections of 0.2 ml in many sites. About day 55, bleed 40-60 ml of blood, if the titer is high. Titer checks should be made twice a week. After resting the rabbit for 3-4 weeks, this schedule may be repeated. Further in­formation concerning injection schedules in general is given by Leskowitz ( 1960) and by Crowle ( 1961). 
5. Processing of antisera 
The fresh blood was collected in 40-ml centrifuge tubes and kept at room tem­perature for 1-2 hr to allow clotting, and then maintained for several more hours at 5° C. The serum was then decanted into another centrifuge tube and centri­fuged to clear the solution. One drop of 20% sodium azide was added for every 5 ml of serum which was stored at -10° C until needed. Repeated freezing and thawing of the antiserum did not appear to affect its potency, nor did prolonged storage (up to 1 year) . 
6. Absorption of antisera 
For unequivocably demonstrating antigen specificity of only 1 or 2 groups of species having several antigens in common, it was necessary to remove the com­mon antibodies by absorption. Complete absorption could be obtained by adding 
0.5 ml of antiserum to 0.5 ml of antigen of species of the second group (usually at a concentration of 11-14 mg protein/ ml. This mixture was maintained at room temperature for 2 hr during the absorption process. The absorbed antiserum was cleared by centrifugation and reacted immediately on double-diffusion plates (Fig. 225-228). 
7. Preparation of materials for double-diffusion and immunoelectrophoresis . (general reference: Crowle, 1961) 
(1) Preparation of Buffered Agar 
Ionagar No. 2 ( Oxoid, London, distributed by Consolidated Laboratories, Chi­cago) was employed at a final concentration of 0.6 % when mixed with the buffer. A barbital buffer slightly modified from that given by Crowle ( 1961) was used in conjunction with the Ionagar. The buffered agar is made in 2 parts: 
A,. Agar (concentrate) 
Ionagar __ ·------------------·.------·. ------·· ·----·--··----·-·-·-----6.0 g 
500.0 mlDeionized H20 ------------------------------------------­
B. Barbital buffer (concentrate), ionicity approximately 0.05, pH 8.2 
Sodium barbital ----­--­---------­------­--­------­--­ --------------­--·­ 15.85 g  
Deionized H 20  --­-----------­---­---­-------­----­--­--­-------­ 310.0 ml  
Hydrochloric acid, 0.1 N  ----------­--­-­---------­-------·-­ --­ -­ ---­-­ 190.0 ml  

Studies of Algal Genera Tetracystis and Chlorococcum 
Both parts A and B were compounded separately the day before they were to be combined for pouring as separate aliquots. Part A was allowed to soak for 10-30 min before autoclaving for 15 min. Then it was allowed to cool and kept overnight to "mature." Part B was kept overnight to allow maximum electrolytic dissociation. 
The next day, both A and B were heated separately in the autoclave, and then heated together for 1/2 hr at about 90° C. The hot, buffered agar solution was fil­tered twice through Whatman No. 4 paper in a hot Buchner funnel and dispensed in 16.5-ml aliquots into sterile, screw-capped glass vials, with a Brewer automatic pipetting machine. The agar was allowed to cool and set, and then 2 drops of 20% sodium azide were added. The caps were tightened and the agar was stored at room temperature until preparation of the plates. 
(2) Preparation of glass plates 
Lantern-slide cover glasses ( 3 Y4" X 4" X 1/16111 ) were cleaned with a silk cloth and then sprayed with 0.1 % agar and dried 3 times so as to form a surface to which the agar would stick. The buffered agar aliquot was heated for about 10 min over a steam cone to melt the agar. The molten agar was carefully poured onto a per­fectly leveled glass plate and allowed to solidify in a moist, dust-free atmosphere. The solidified agar plate was kept overnight to "mature" before use. 
The next day, it was placed over a pattern drawn on paper, and the pattern was duplicated on the agar plate with the aid of a cork borer ( 4 mm), scapel and ruler. Trough widths ranged from 2 mm for immunoelectrophoresis to 4 mm for double­diffusion. All cup diameters were 4.0 mm. Cup-trough separation ranged from 0.4 to 0.5 cm. The cups and troughs were cleaned by means of a Pasteur pipette con­nected to a vacuum filter pump. 
(3) "Running" the plate, double-diffusion 
The antigen extracts were pipetted into the cups and the antisera into the troughs (or vice versa), and the plate was allowed to react (develop) for 24-28 hr in a moist, dust-free environment. 
(4) "Running" the plate, immunoelectrophoresis 
The antigen extracts were pipetted into the cups (the agar had been removed only from the cups and not the troughs at this time) . A reference spot of bromo­phenol blue was injected into the agar plate which was then placed in the electro­phoresis apparatus. 2 The plates were run at 45 milliamperes per plate (ca. 4 volts per cm in the agar). Suitable separation of the antigens was achieved by the time 
1 Burke and James, Inc., Chicago, Ill. 
" Shandon Universal Electrophoresis Apparatus, distributed by Consolidated Laboratories, 
with a Beckman Model RD-2 Duostat regulated power supply. 
the bromophenol blue reference spot moved from 4.0 cm (about 1.5 cm/ hr) . After electrophoresis, the troughs were removed by suction, and the antiserum was added. The plates were incubated overnight in a moist, dust-free atmosphere. 
(5) Examination of the plates 
The plates were examined 24-48 hr after adding the antigen and antiserum. Precipitin lines were generally at optimum formation and resolution after 24-36 hr. The plates were photographed by positioning over a fluorescent X-ray viewing light with a black background arranged so that the precipitin lines were visible to the camera. The plates were also washed, dried, and stained with 0.2% Ponceau S (for proteins) and preserved for further analysis (Crow le, 1961 ) . 
C. RESULTS AND DISCUSSION 
I. Analysis of double-diffusion 
Fifteen Chlorococcum species and 12 species of Tetracystis were reacted In double-diffusion experiments with 5 antisera. These antisera were: ( 1) anti-T. aeria (C-6 isolate); (2) anti-T. isobilateralis; (3) anti-T. aplanosporum; (4) anti-C. sp. (tetra isolate) ; and (5) anti-C. perforatum. 
, The double-diffusion plates were allowed to develop for at least 48 hr, after which they were washed, dried, and preserved. During development, they were photographed at 24, 36, and 48 hr. Thus, the analyses were based on .continuous observation over a prolonged period because occasionally, several additional pre­cipitin lines would develop. A comparison of the 24-hr (Fig. 217-220) and 48-hr (Fig. 221-224) plates of Chlorococcum species reveals such changes. 
Table 20 presents data obtained by analysis of the double-diffusion spectra. The results with antisera to T etracystis form 3 vertical columns to the left and those with antisera to Chlorococcum form 2 vertical columns to the right. The horizontal columns represent the number of precipitin lines observed, ranging from 2 to 7. The various Chlorococcum and Tetracystis species are arranged on the basis of the number of precipitin bands they produced with each respective antiserum. Photo­graphs of the double-diffusion tests with Chlorococcum and Tetracystis species are shown in Fig. 214-224. 
Table 20 also is coded so that analyses are easier to follow. All presently desig­nated Chlorococcum species are in bold face type, all T etracystis species Roman type. Those species with an asterisk (including both C hlorococcttm and T etracystis species) produced full spectra with more than 5 lines with at least 1 antiserum. 
The fullest spectra produced by any Tetracystis species were with anti-Tetracystis antisera. These same species gave poor reactions ( 3-4 lines or less) with the anti­C hlorococcum antisera. Conversely, the fullest spectra produced by any Ch!oro­coccum species were with anti-Chlorococcum antisera. Again, these species gave poor reactions ( 3-4 lines or less) with the anti-Tetracystis antisera. 
TABLE 20. Double-diffusion reactions of T etracystis and Chlorococcum 
T. aplano. 
T. aplano.* 
T. aggreg:x-C. oleo. 
C. punct. 
C. multi. 
T. dissoc:x- C. ellip.  
T. illin:X·  C. min.  
T. iso:*  C.w.im.  
T. tetra.  
T. excent.  
T. texen.  
T. pulch.  
T. inter.  

T. ae1·ia (C·G) 
T. aeria·:+ (C-6 ) 
T. aeria·X· (Pa-3) 
T. aggreg:x-C. hypno. 
T. pam. 
T. dissoc.* 
T. ill in. -x-
T. iso:x-
T. excent. C.min. 
C. p ing. 
T. iso. 
T. dissoc:* 
T. iso:* 
T. illin:* 
T. aggreg:x-
T. aeria·x­(C-6 ) 
T. aeria·x-C. wiin. (Pa-3) 
C. ellip. 
C. min. 
C. punct. 
C. multi. 
C. sp. (tetra) 
C. perL J No. of line·
I 
I 7 
C. sp.'~ 
C. scab.':' 
6-7 
C. oleo.* 
C. perf.* 
6 
C. echino.* 
C. diplo.* 
5-6 
C. multi. 
5 
C. perf.*  
C. ellip.  C.wim.  4-5  
C. min.  C. multi.  
C. vacuo.  

T. excent. C. echino.* 
C. diplo.* 
C. wim. 
C. punct. 
C. ping. 
C. oleo.* 
C. ellip. 
C. punct. 
C. vacuo. 
T. aeria'x-C. hypno. (C-6 ) C. scab.* 
T . aeria·* C. ping. (Pa-3 ) C. niacro. 

T. 
pam. 

C. 
sp.':' 

C. 
perf.* 

C. 
echino. 

C. 
diplo.':' 

C. 
vacuo. 


T. tetra.  C. perf.*  
T. inter.  C. echino.*  
C. diplo.''  
C. ellip.  
C.wlm.  

T. texen. C. sp.* 
T. pulch. C. scab.':' 
C. oleo.* 
C. punct. 
C. multi. 
C. macro. 
T. aplano:* C. vacuo. 
C. hypno. 
C. oleo.':' 
C. ping. 
C. vacuo. 
C. macro. 
T. excent. C. echino.* 
T. aplano:* C. sp.'' 
T. tetra. C. scab.* 
T. pulch. C. perf. * 
T. inter. C. diplo.* 
T. pam. 
T. texen. 
T. aeria·x-C. macro. 
(C-6 ) 
T. aeria·x­(Pa-3) 
T. texen. 
T. pulch. 
T. aggreg.'"' 
T. inter. 
T. pam. 
T. dissoc:x-C. hypno. 
T. illin:* 
T. iso:* 
T. tetra. 
T. aplano:x-
C. scab.* C.min. 
C. ping. 
T. aggreg.'" C. sp. * 
T. excent. C. macro. 
T. texen. 
T. aeria·x-C. hypno. 
(C-6 ) 
T. dissoc:x-
T. tetra. 
T. pulch. 
T. inter. 
T. a piano. ,x-
T. illin:* 
T. iso:'' 
T. aeria·X­(Pa-3) 
T. pam. 
3-4 
·--­3 
2 or less 
*Species which produced full spectra with more than 5 lines with at least I antiserum. 
These results indicate inter-generic serological specificity inasmuch as, in gen­eral, any species producing a full spectrum (over 5 lines) did so with an antiserum to a species in the same genus. The same species produced poor spectra (less than 3-4 lines) with all antisera to species in the other genus. However, in many cases such species only gave medium spectra ( 4, 4-5, or 5 lines) with antisera to other species in the same genus. This would indicate specificity not only for the whole genus, but in particular, for individual groups of specie.5 within the genus. 
In addition to an analysis of just the fully reacting species, all species of Tetra­cystis and Chlorococcum were examined for possible intergeneric or group specifi­city. For each antiserum, the number of species in either genus producing full and medium reactions (over 3-4 lines) was compared with the number of species giving poor reactions ( 3-4 lines or less). Reciprocal comparisons were made in each case for the Tetracystis species (T) and the Chlorococcum species ( C). The results ob­tained are shown in Table 21. 
TABLE 21. R eciprocal comparisons for Chlorococcum and T etracystis species in double-diffusion 
Antiserum Antigen, genus over 3-4 lines 3-4 lines or less  T . aplanosporum I T. aeria T c I T c 10 6 8 3 ----3 9 5 12  T. isobilateralis T c 6 5 --7 10  
Antiserum Antigen, genus over 3-4 lines 3-4 lines or less  C. sp. ( tetra ) IT c 1 13 --12 2  C. per/oratum T c 0 9 --13 6  

In Table 21, the abbreviation above each fraction indicates the species of Chloro­coccum (C) or T etracystis (T) reacted against the antiserum listed. The "nu­merator" in all cases, represents the number of species which fell above the division line of 3-4 precipitin lines, while the "denominator" indicates those species falling below the division line in Table 20. Note that in all cases, a total of 13 Tetracystis species and 15 Chlorococcum species were tested. It can be seen, first, that a recip­rocal relationship exists in the reactions of the 2 genera against a given antiserum. Secondly, the pair of fractions for any given antiserum of Tetracystis is the reverse of that for any Chlorococcum antiserum. Thus, when all of the species are con­sidered, they indicate intergeneric specificity in almost every combination tested as shown in Table 21. 
Chlorococcum hypnosporum gave unexpected and very interesting reactions (Table 20) in that it reacted weakly with the 2 Chlorococcum antisera but quite strongly ( 5 precipitin lines) against one of the T etracystis antisera, namely, anti-T. aeria. This reaction may not be considered too suspect because the hypnospores which characterize this Chlorococcum species may, in fact, represent nonmotile daughter vegetative cells and not aplanospores. Therefore, in view of the writers' concepts for the Chlorosphaerales (page 11 ) , a re-examination of C. hypnosporum, using an antiserum against it would provide even more conclusive data with regard to its taxonomic position. 
From Table 20, it is clear that T. pampae is certainly unusual serologically be­cause of the poor reactivity with all of the Chlorococcum and 2 of the Tetracystis antisera. An exception is the 5-line precipitin reactions with the antiserum to T. aeria. This reaction, however, is weak when compared with the 5-line precipitin reactions given by the other 2 species against T. aeria antiserum. The protein con­centrations of the species giving 5-line reactions against T. aeria are the sar:ne in 2 of these species, including T. pampae (Table 18). Therefore, the difference in in­tensity strength of reaction probably indicates that T. pampae is not so similar se­rologically to the other 2 species. 
Thus it may be proper at this point to state that serological similarity or diversity cannot be based only on the number of precipitin lines produced in double-diffusion as in Table 20. The number of lines alone (Table 20) does not indicate antigens which are specific and which are in common to other species; however, this may be shown in the photographs of the double-diffusion tests (Fig. 214-224) . In these photographs, the presence of spurs or arcs denotes those antigens which are not in common with adjacent species. A difference of 1 or 2 in the number of precipitin lines may not be very significant, whereas a spur indicates a significant difference. For this reason, techniques with absorption and immunoelectrophoresis were em­ployed in order to display more fully these reactions and to understand their signi­ficance. It should be emphasized, however, that the number of precipitin lines (Table 20) does provide valuable information and should be given full attention in a first approach to the various modes of serological analysis. 
The antisera to T. aeria and T. isobilateralis appear to show differences among those Tetracystis species which undergo intervening diad formation and those which form tetrads directly. It will be recalled that T. isobilateralis is quite dis­tincti.ve in forming diads which divide to form isobilateral or tetrahedral tetrads. Other species of Tetracystis do not have this same capability in that they appear exclusively to form tetrads directly. Note in Table 20 that those Tetracystis species which undergo intervening diad formation react most strongly ( 6-7 lines) with anti-T. isobilateralis. Note also that these species do not react so strongly ( 4-5 lines) with antiserum to T. aeria which forms tetrads directly. It is apparent that all species of Tetracystis which characteristically form tetrads directly react more strongly to anti-T. aeria than to anti-T. isobilateralis. 
The fully reacting species will now be discussed because, on the basis of double­diffusion, several species-groups could be discerned. When T etracystis aeria isolate C-6 was reacted against its homologous antiserum, a very strong reaction of 6-7 lines was detected (Fig. 214, Table 20). Likewise, another isolate Pa-3, from a different locality, was tested against anti-T. aeria (C-6) and produced an identical spectrum to the homologous reaction ( C-6). It will be recalled that these 2 iso­lates have been demonstrated to be the same species on the basis of their morphol­ogy, ultrastructure, and physiology. Thus, serological evidence also supports all these data. 
The strongest reaction with anti-T. aplanosporum antiserum was produced by the homologous extract from T. aplanosporum. This reaction (7 lines) was unique · in that no other T etracystis species even closely approached it in strength (Fig. 215, Table 20). Thus, T. aplanosporum is immediately segregated as a unique species of Tetracystis. On the basis of morphology, T. aplanosporum and T. aeria are the only 2 presently known species of T etracystis in which the nucleus of the zoospore is anterior. Morphological, physiological, and ultrastructural evidence show that the 2 species are only distantly related, even though they share the same zoospore nuclear position. These 2 species are serologically different from one another as is shown in Fig. 214-215 and Table 20. 
T etracystis isobilateralis, when reacted against its homologous antiserum, gave a very strong reaction of 6-7 lines in double-diffusion. Likewise, T. illinoisensis, T. dissociata, and T. aggregata gave equally strong and identical reactions with anti­
T. isobilateralis antiserum. That these 4 taxa are not morphologically, physiologi­cally, and ultrastructurally identical suggests that they are distinct species; however, they do have many similarities in common on the basis of morphology and ultrastructure. Thus, the serological data seem to support these similarities, while one is unable to distinguish among the 4 species on the basis of double­diffusion (Fig. 214-215, Table20). 
Among the Chlorococcum species, 2 serological groupings of importance were detected by double-diffusion. The first group, which reacts strongly and similarly with anti-C. sp. (tetra) antiserum, consisted of C. sp. (tetra), C. scabellum, and 
C. oleofaciens (Fig. 216, 219, 220; Table 20). 
The second group reacted strongly with anti-C. perforatum antiserum and con­sisted of C. perforatum, C. echinozygotum, and C. diplobionticum. These reactions appeared to be identical on the basis of double-diffusion (Fig. 217, 221; Table 20). 
2. Absorption studies 
The writers were limited by shortages of time and material in performing ab­sorption experiments. However, enough material was available to study more critically 2 serological groups among Tetracystis, namely, the T. aeria (C-6 and Pa-3) group, and the T. isobilateralis group (consisting additionally of T. disso­ciata, T. illinoisensis, and T. aggregata). Figures 225 and 226 show reactions of these 2 species groupings in which the antisera to T. isobilateral,is and T. aeria (C-6) were cross-absorbed reciprocally. The controls (non-absorbed antisera) were also included. Anti-T. isobilateralis absorbed with T. aeria (Pa-3) produced no reaction against T. aeria, suggesting complete absorption. Likewise, anti-T. aeria absorbed with T. isobilateralis reproduced no readion against T. isobilateralis. 
Anti-T. isobilateralis antiserum absorbed with T. aeria (Pa-3) produced no re­action with either Pa-3 or C-6 isolates of T. aeria, thus indicating that isolate C-6 has no more antigens in common with T. isobilateralis than does isolate Pa-3. Anti­
T. aeria antiserum absorbed with T. isobilateralis produced no reaction to T. ag­gregata, T. dissociata, and T. illinoisensis, likewise indicating that these 3 species have no more antigens in common with T. aeria than does T. isobilateralis. 
Extracts of T. isobilateralis, T. aggregata, T. dissociata, and T. illinoisensis re­acted with anti-T. isobilateralis antiserum absorbed with T. aeria all produced identical spectra of 2 lines, indicating complete antigenic uniformity of these 4 species. Likewise, with anti-T. aeria antiserum both isolates, C-6 and Pa-3 pro­duced identical spectra of 4 lines, indicating antigenic identity of these 2 species. 
Chlorococcum wimmeri, which had appeared similar to both Tetracystis groups in double-diffusion tests, was here shown to be very different from both of them in that with either absorbed antisera, it produced no reaction (Fig. 225-226). 
A second absorption experiment was performed in which antisera to T. aeria and 
C. sp. (tetra isolate) were cross-absorbed reciprocally. Chlorococcum and T etra­cystis species selected to be most representative for each genus were reacted against the absorbed antisera (Fig. 217, 218, Table 22). There was evidence for incom­plete absorption with one of the antisera, because anti-C. sp. absorbed with T. aeria (Pa-3) gave a single faint precipitin line when reacted against T. aeria (Pa-3 ). For this reason, the significant reactions to absorbed anti-C. sp. must be considered 1-precipitin-band less than the observed number. 
Anti-T. aeria ab3orbed with C. sp. was completely absorbed; therefore, every precipitin line observed against this absorbed antiserum was considered significant. From this absorption experiment, it is evident that almost all of the Tetracystis species appear to have at least 1 genus-specific antigen to anti-T. aeria (absorbed with C. sp.). Tetracystis pampae is the exception. Most of the Chlorococcum species tested lacked specific antigens to the absorbed T etracystis antiserum. The exceptions were C. hypnosporum and, possibly, C. ellipsoideum. 
Conversely, against absorbed antiserum C. sp. all the Chlorococcum species tested (with the exception of C. hypnosporum and C. ellipsoideum) have at least 1 genus-specific antigen. All of the T etracystis species tested lacked this antigen, as­suming that anti-C. sp. was incompletely absorbed by 1 precipitin band. . Therefore, the absorption data from this experiment seem to indicate an inter­generic specificity with both fully and poorly reacting Chlorococcum and Tetra­cystis species. Even though not all of the Tetracystis species are represented, they nevertheless represent the complete antigenic diversity among all species. Those species of Tetracystis not reacted, belong to T. aeria group and the T. isobilateralis group, each of which was shown to be serologically identical in the first absorption 
TABLE 22. Data from the second absorption experiment with selected Chlorococcum and T etracystis species 

Species (antigen)  Reaction witNo. of lines  h anti-T. aeria (-C. sp) Quality of spectrum  Reaction with o. of linesn  anti-C. sp. (-T. aeria) Quality of spectrum  
T . aeria (Pa-3)  4  very strong  0  
T. isobilateralis  3  weak  0  
C. hypnosporum  3-4  weak  Qb  
T. texensis  ca. 2  weak  0  
T. excentrica  1  weak  0  
T. intermedium  weak  0  
T. aplanosporum  1  weak  0  
T. pampae  0  Qb  
C. ellipsoideum  1  very weak  0  
C. perf oratum  0  1  weak  
C. echinozygo tum  0  weak  
C. diplobionticum  0  weak  
C. pinguideum  0  1  weak  
C. sp. (tetra)  0  4-5  very strong  

a This number represents I minus the observed number = corrected number of incomplete absorption. 
b No lines were present, even before compensation for incomplete absorption. 
study. In the case of the Chlorococcum species, this absorption was not entirely representative of every species of the genus, as in T etracystis. 
Most of the species tested in the second absorption experiment produced a spec­trum of only 1 specific antigen ; however, several species produced more than 1 specific antigen. Of these, T. aeria (Pa-3) had 4 specific antigens, against anti­
T . aeria (-C. sp.) , thus forming a species group by itself with the other isolate of T. aeria (C-6 ). Tetracystis isobilateralis produced a spectrum of 3 specific lines against anti-T. aeria (-C. sp.), thus forming a species group of T . isobilateralis, T. dissociata, T . illinoisensis, and T. aggregata. Against anti-C. sp. (-T. aeria), only 

C. 
sp. formed 5 specific antigens. This reaction represents probably the other 2 closely allied species, C. oleofaciens and C. scabellum. 


Against anti-T. aeria absorbed with C. sp., Chlorococcum hypnosporum pro­duced 3-4 specific lines, while against anti-C. sp. absorbed with T. aeria, no lines were produced. These data further support the double-diffusion experiments which indicated that C. hypnosporum has more antigens specific to T etracystis than to Chlorococcum. The absorption data also show that C. hypnosporum is especially close to the T. aeria and T. isobilateralis groups. Of these groups, C. hypnosporum shares approximately the same number of specific antigens with T. isobilateralis (3-4), while T. aeria has at least 1 additional specific antigen, all ofwhich are stronger in intensity than those of C. hypnosporum and T . isobilateralis. It could not be stated with certainty whether the specific antigens shared by C. hypno­sporum and T. isobilateralis were exactly the same, because these 2 species were not placed side-by-side in the agar. 
Tetracystis pampae has no specific antigens to either the Tetracystis or Chloro­coccum absorebd antisera. Thus, it is only distantly related to T. aeria and C. sp. Further studies with T. pampae antiserum would certainly be of great value in de­termining if this species is as unique serologically as it is in its morphological, phy­siological, and ultrastructural characters. 
It is of interest to note that C hlorococcum ellipsoideum shows a single, weak line with the absorbed T etracystis antiserum and with the incompletely absorbed Chlorococcum antiserum, only 1 line, the latter not significant. Chlorococcum el­lipsoideum, then, appears to be related to T etracystis in a similar, but less striking, manner than Chlorococcum hypnosporum. Further work will be needed in order to test this possibility. 
Although far from complete, these few simple investigations have shown the value of this technique in conjunction with double-diffusion tests. The value of serology is increased yet further with the aid of immunoelectrophoresis which will now be discussed. 
3. lmmunoelectrophoresis analysis (I.E.A.) 
Twenty-four preparations of Tetracystis and Chlorococcum species were analyzed by immunoelectrophoresis with 3 antisera to T etracystis and 2 antisera to Chlorococcum. These reactions are summarized in Table 23. Immunoelectropho­resis was performed in order to aid in the analysis of double-diffusion and ab­sorption data. Immunoelectrophoresis analysis (or LE.A. as it is commonly referred to) has the distinct advantage of resolving many more precipitin bands (Table 23 ) than could be observed by double-diffusion (Table 20). In addition, electropho­resis mobilities of specific proteins may be resolved with this technique. Thus ap­parent identity of spectra in double-diffusion may be proven or disproven by I.E.A. 
Six major species groupings discerned with double-diffusion were more critically analyzed by I.E.A., as were certain other species which were of interest. Reactions with each species group will now be discussed. 
(1) The T. aplanosporum group (Fig. 233, 234) 
It will be recalled that in double-diffusion, the strongest reaction to anti-T. aplanosporum was produced by the homologous extract from T. aplanosporum (7 lines). With LE.A., 1-3 additional precipitin lines (over that of double-diffusion) were .detected with the homologous reaction. In I.E.A. against anti-T. aplano­sporum antiserum, T. aplanosporum was unique in having more lines than any other species, and these were stronger and had different electrophoretic mobilities. 
TABLE 23. I mmunoelectrophoresis reaction performed with number of lines indicated 
Antisera T. aplanosporum T. aeria T. isobilateralis C. sp. (tetra) C. perforatum 

T. aplanosporum  8-10  
T. aeria (C-6 )  5-6  11  5-6  
T. aeria (Pa-3 )  11  5-6  
T. isobilateralis  8-9  8-9  
T. aggregata  6-7  8-9  7-8  
T. illinoisensis  5 or6  8-9  7-8  
T. dissociata  6-7  8-9  7-8  
C. hypnosporum  6-7  8  
C. wimmerii  6-7  5-6  4  5-6  
C. sp. (tetra)  3  10-12  
C. scabellum  9-12  
C. oleofaciens  5-6  9or10  
C. multinucleatum  8-9  6  
C. ellij1soideum  5 or6  4-5  5  7  6-7  
C. perf oratum  6 or 7  8-10  
C. diplobionticum  6 or 7  9-10  
C. echinozygotum  4  9  
C. minutum  4  3-4  3  3-4  2 or 3  
C. vacuolatum  7  5  
C. punctatum  5 or 6  6  
C. pinguidium  3-4  
T. pamf1ae  2 or 3  
T. excentrica  4  3-4  
T. intermedium  4-5  3-4  
T. j;ulchra  4  4  


(2) The T. isobilateralis group (Fig. 229-232) 
Tetracystis aggregata, T. dissociata, T. illinoisensis, and T. isobilateralis gave very similar, if not identical, reactions by double-diffusion. Therefore, it was of in­terest to examine them by LE.A. in order to determine if they were, indeed, identi­cal serologically. Against antisera to T. aplanosporum and T. aeria, the reactions to all species comprising this group were very similar, if not identical, in number, intensity, and electrophoretic mobility. Only T. isobilateralis produced 1 extra pre-. cipitin band when reacted with anti-T. isobilateralis. Thus, all members compris­ing the T. isobilateralis group appear to be serologically virtually identical on the basis of double-diffusion, absorption, and immunoelectrophoresis. 
(3) The T. aeria group (Fig. 229-232) 
According to the double-diffusion and absorption data, both isolates C-6 and Pa-3 of T. aeria are serologically identical. The same holds true when these 2 iso­lates were tested by LE.A. Note that against anti-T. aeria (C-6) both isolates produced identical spectra of 11 lines (Fig. 229), while against anti-T. isobilateralis they both produced identical spectra of 5-6 lines (Fig. 230). It is of interest at this point to note that most of the C-6 and Pa-3 specific proteins (not present in the T. isobilateralis group) formed arcs in the general area of the origin (near the cups). Thus, they were proteins of low electrophoretic mobility, either because of a small net charge or large molecular size. It can be deduced, therefore, that there is greater specificity in the proteins of larger molecular size in this instance. 
Note that in the cross reactions between the T. aeria group and the T. isobilat­eralis group, there were 2-4 lines less than between the homologous reactions, thus indicating that each group has 2-4 specific antigens not shared by the other group. This observation was verified with absorption and even more critically in that the 
T. aeria group shared 4 specific antigens, while the T. isobilateralis groups had 2 specific antigens. 
(4) C. hypnosporum (Fig. 234, 240) 
The double-diffusion and absorption studies indicated that C. hypnosporum was unique among the Chlorococcum species tested in that it more closely re­sembled species in Tetracystis than any in Chlorococcum. In the LE.A. study, C. hypnosporum was reacted against anti-T. aplanosporum and anti-T. aeria. Against anti-T. aplanosporum, C. hypnosporum had a spectrum of similar strength and number of lines, to members of the T. isobilateralis group; however, not all of the precipitin lines of C. hypnosporum were of the same electrophoretic mobilities. Chlorococcum hypnosporum had several additional lines not represented by T. aeria, and in addition, lines with different electrophoretic mobilities were observed between C. hypnosporum and the latter. 
Against anti-T. aeria, C. hypnosporum was outstanding in producing a strong spectrum with about 8 lines, similar in number and strength to those of the T. iso­bilateralis group (Fig. 240). However, there were obvious differences in the rela­tive electrophoretic mobilities of many lines between the T. isobilateralis group and 
C. hypnosporum. From this evidence, it could be postulated that C. hypnosporum is in the general complex with the T. aeria and T. isobilateralis groups. However, on the basis of electrophoretic mobilities, C. hypnosporum is in a group separate from the T. isobilateralis group. It was unfortunate that the writers did not have the time nor material to explore the LE.A. reactions of C. hypnosporum to anti­
T. isobilateralis. 
(5) 
The C. sp. (tetra) group (Fig. 237-239) 

C. 
multinucleatum were quite similar in intensity.and number of lines to anti-C. sp. 


The reactions of C. sp. (tetra), C. scabellum, C. oleofaciens, and possibly also (tetra) on double-diffusion. For this reason they were investigated more critically by immunoelectrophoresis. 
Against anti-C. sp. (tetra), Chlorococcum scabellum and C. sp. (tetra) pro­duced similar numbers of lines with equal intensity and of the same electrophoretic mobilities (Fig. 23 7). However, C hlorococcum oleo/ aciens lacked 2 or 3 lines present to C. sp. (tetra). Chlorococcum multinucleatum had almost as full a spec­trum as C. oleofaciens and was like it in the electrophoretic mobility of the precipi­tin lines present, and in the lack of 2-3 lines which were present with C. sp. (tetra) and C. scabellum. 
Thus, all 4 species form a general complex, and within this complex, C. sp. (tetra) and C. scabellum appear to have identical reactions, while C. oleofaciens and C. multinucleatum (Fig. 238), although not identical, are both different from the C. sp. (tetra )-C. scabellum group. 
(6) The C. perforatum group (Fig. 235-236) 
Three species of Chlorococcum, namely, C. perforatum, C. echinozygotum, and 

C. diplobionticum gave almost indistinguishable reactions with double-diffusion; however, when examined with LE.A., several differences among these species were detected. Against anti-C. perforatum, Chlorococcum perforatum and C. echinozy­gotum gave similar spectra in terms of number, intensity, and electrophoretic mo­bility. In contrast, some lines were absent with C. diplobionticum (Fig. 236), and some present had different electrophoretic mobilities. 
All 3 species of the C. perforatum group produced different spectra against anti­
c. sp. (tetra) antiserum. 
Thus, in double-diffusion all 3 species of the C. perforatum group gave indistin­guishable reactions, but LE.A. showed that within this complex, C. perforatum and C. echinozygotum form a group of closely related species, while C. diplobion­ticum is more different. 
(7) Miscellaneous reactions (Fig. 235-236; 238-240) 
In addition to the species in the clear-cut groups displayed by double-diffusion, several other species were studied with LE.A. in order to try to resolve their re­lationship to the better defined groups. 
It will be recalled that Chlorococcum ellipsoideum gave between 3 and 5 pre­cipitin bands with all antisera on double-diffusion. Against anti-T. aplanosporum, 
C. ellipsoideum produced 5 or 6 lines similar to those of T. aeria (C-6) (Fig. 233) ; however, they were of different electrophoretic mobility. Against anti-T. aeria, C. ellipsoideum had about 3 of the 5 total lines similar to T. excentrica, T. inter­medium, and T. pulchra. Against anti-T. isobilateralis, C. ellipsoideum produced spectra more similar to T. excentrica than to T. pulchra or T. intermedium. There was no particular affinity of spectra with C. minutum, even though the number of lines against this antiserum was about the same. Against anti-C. sp. (tetra), C. ellipsoideum produced more lines than against all other antisera, and showed a greater similarity to C. scabellum and C. diplobionticum than to other species tested. This relation should be regarded as rather weak, however, since there were several major line differences. Chlorococcum ellipsoideum reacted weakly with anti-C. perforatum but showed more similarly to C. perforatum and C. echinozy­gotum than any other species tested with this antiserum. 
Therefore, in LE.A. Chlorococcum ellipsoideum shows, as in double-diffusion, similarities to all of the antisera tested. Thus, this species may represent a link be-. tween the 2 genera. The absorption data seem to indicate that C. ellipsoideum may have, (in one case), a specific antigen to Tetracystis not present in Chlorococcum when reacted against absorbed T. aeria (-C. sp.). An antiserum to C. ellipsoideum wou.ld be, indeed, very useful, for no further conclusions can be made on the present basis. Likewise, other groups tested which gave very weak reactions in LE.A. should be re-examined using an antiserum against one or more of these species, for no definite conclusions, other than those obtained by double-diffusion and absorption, could be discerned with LE.A. 
D. CONCLUSIONS 
In conclusion, the writers were able successfully to immunize rabbits to 5 dif­ferent species of algae representing 2 different genera, Chlorococcum of the Chloro­coccales, and T etracystis of the Chlorosphaerales. The reactions of 27 antigens sug­gested that the techniques of double-diffusion, absorption, and immunoelectro­phoresis could be successfully employed to gain valuable knowledge regarding proper disposition of taxa at the species and generic level. There was some evidence that most members of each genus have at least 1 specific antigen which is lacking in the other genus, suggesting an intergeneric, serological specificity. Even though at least 1 genus-specific antigen may be present, there are at least 2-3 common anti­gens shared by most species of both genera. This is rather unique serologically in that Tetracystis and Chlorococcum presently are classified in 2 different orders. Such common antigenicity rarely, if ever, occurs at the ordinal level in the angio­sperms. In view of this situation, it is possible that the present basis of ordinal dis­tinction between the Chlorococcales and Chlorosphaerales may represent a more artificial classification than heretofore believed. On the other hand, the taxonomic significance of the antigens which both genera share in common may have been overestimated, because only recently, a common antigen (protein fraction 1 ) has been shown to be present among all major groups of the green plants, from the angiosperms to the Chlorophyta (Dorwer, Kahn, and Wildman, 1958). 
Finally, the data from double-diffusion, absorption, and immunoelectrophoretic studies indicate that there are species groupings in these unicellular algae. Al­though these are probably natural assemblages, they would be useful, even if arti­ficial, in the identification of species. The species groupings which are based on serological criteria, coincide, in general, with the groupings suggested by morpho­logical (both light and electron-microscopic) and physiological attributes. This last consideration inspires some confidence that these morphological and physiological criteria of themselves, in fact, make it possible to classify these microalgae phylo­genetically. 
V. General Discussion 
The data representing the morphology, physiology, and immunochemistry have been considered independently and are herein presented in summary form based on the assignment of the same letters for identical response to a given test. These data are summarized in Tables 24, 25 and 26. 
T ABL E 24. Electron-microscopic data (based on the comparative study of organellar types) 
U ltrastructure  1"  3  4  6  
Organism  
T. aeria (C-6 )  Cb  A  B  B  B  B  
T. aeria (Pa-3 )  c  A  B  B  B  B  
T. aggregata  B  D  A  A  A  A  
T. aplanosporum  c  c  D  D  D  c  
T. dissociata  c  D  B  A  A  A  
T. excentrica  c  B  c  c  c  c  
T. illinoisensis  c  D  B  c  B  B  
T. intermedium  c  D  c  c  c  c  
T. isobilateralis  A  D  A  A  A  B  
T. pampae  c  D  E  c  E  c  
T. jJUlchra  c  B  c  c  c  c  
T. tetrasporum  xc  x  x  x  x  x  
T. texensis  c  D  c  c  c  c  

a 1 = mitochondrion; 2 = cell wall; 3 = pyrenoid; 4 = chloroplast mass; 5 = chloroplast 
internal; 6 = Golgi apparatus. 
" Letters A through E are based on the groupings in Table 17. 
c X = no data. 

Table 25 is based on the physiological data. For example, in the physiological test for growth in various carbon sources (Table 1), both isolates T. aeria fall into group 3 as shown in the species groupings (page 37) . To these isolates and to other species of T etracystis which fall into group 3 for this physiological test are ascribed the letter C in Table 25. Likewise, T. pampae, T . aplanosporum, and T. inter­medium, fall into group No. 1 and thus have ascribed to them the letter A in Table 25. 
In similar fashion, serological and ultrastructural data have been collated and 
· are presented in Tables 24 and 26. Thus, when reading Tables 24-26 horizontally, the sequence of letters indicates the degree of relationship of the taxa. For example, it will be seen that both isolates of T. aeria have the same sequence of letters (CABBBB) on the basis of ultrastructural data, ( CAAABAAAAA) for physiologi­cal data, and (D JHBD) for serological data. On this basis, a summation table has 
TABLE  25. Physiological data ( based on species groupings)  
Physiological tes ts  '].  3  4  6  8  9  10  
Organism  
T. aeria (C-6 )  ca  A  A  A  B  A  A  A  A  A  
T. aeria (Pa-3 )  c  s  s  s  B  A  A  A  A  A  
T. aggregata  c  E  E  B  B  B  c  c  D  c  
T. aplanosporum  A  B  B  c  c  B  c  A  c  D  
T. dissociata  c  E  D  B  B  A  c  c  B  D  
T. excentrica  c  E  D  B  D  s  c  B  c  c  
T. illinoisensis  c  x  x  x  x  x  x  x  D  x  
T. intermedium  A  E  F  c  D  B  B  A  c  D  
T. isobilateralis  c  E  B  B  B  B  c  c  D  c  
T. pampae  A  D  c  c  B  B  A  A  c  c  
T. pulchra  c  x  x  x  x  x  x  x  A  x  
T. tetrasporum  D  c  D  B  A  B  B  A  E  B  
T. texensis  B  E  D  D  D  B  c  B  B  c  

a Group numbers based on data from species groupings (pp. 3 7-40). 
Group 1 = A 
Group 2 = B 
Group 3 = C 
Group 4 = D 
Group 5 = E 
Group 6 = F 
X = no data. 
been prepared to indicate the over-all relationship of morphological, physiological, ultrastructural, and serological characteristics (Table 27). 
In Table 27, species groupings indicating general relationship of species to one another are indicated by those species which fall within a given square for each category of study (i.e., morphology, electron microscopy, etc. ) . The degree of re-. lationship to one another of the species within each square is indicated by various symbols placed after the specific names. 
From this summary table, it is clear that the ultrastructural, physiological, and serological data correlate quite well with the morphological data. It should be em­phasized that the morphological groups as presented here were derived independ­ently, even before ultrastructural, physiological, and serological data had been ob­tained. It is of special interest to note the striking degree of identity of both isolates of T. aeria in all tests performed, this suggesting further that these 2 isolates are, indeed, the same species. 
On the basis of morphology, T . isobilateralis, T. dissociata, T. illinoisensis, and 
T. aggregata form a group of closely related species; however, T. illinoisensis shows some degree of dissimilarity on the basis of ultrastructure and physiology, while T. aggregata lacks a very close relationship to the other members of this group on the 
T t' lracyslis Clorococcu m 
TABLE 26. Serological data (based on the number of lines observed by d ouble-diffusion ) 

Antisera  1n  2  3  4  
Organism  
T. aeria  (C-6 )  D"  J  H  D  B  
T. aeria (Pa-3 )  D  J  G  D  A  
T. aggregata  F  G  J  c  c  
T. aplanosporum  K  A  B  A  A  
T. dissociata  E  F  J  B  B  
T. excentrica  E  E  c  E  c  
T. illinoisensis  E  F  J  B  A  
T. int erm edium  E  D  B  c  B  
T . isobilateralis  E  F  J  B  B  
T. pampae  D  G  A  c  A  
T. pulchra  E  c  B  D  B  
T. tetrasporum  E  D  B  B  B  
T. tex ensis  E  c  A  D  c  
a Antisera  " Number of precipitin lines  
1 = T. aplanosporum  A =  2 or less  G = 5  
2 = T. aeria  B =  2-3  H =  5-6  
3 = T. iso bilateralis  C =  3  I =6  
4 =  C. per/ oratum  D =  3-4  J =  6-7  
5 =  C. sp. (tetra )  E =  4  K =  7  
F =4-5  

basis of serology. Such variation is to be expected because the organisms in this group differ significantly in morphology, but not sufficiently to warrant a species description for each taxon. 
Two species, namely, T. aplanosporum and T. pampae, are quite distinct from any other species of Tetracystis on the basis of morphology. They are also distinct on the basis of ultrastructure and serology; however, they show some degree of re­lationship to one another and to T. intermedium on the basis of physiology. 
It is of interest to note that T. tetrasporum, although not unequivocally unique among the species of Tetracystis on the basis of morphology and serology, in fact, is so on the basis of physiology. 
While T. excentrica, T. texensis, T. pulchra, and T. intermedium show some degree of morphological relationship to one another, it should be noted that T. pulchra and T. intermedium are very closely related morphologically, while T. texensis and T. excentrica are more distinct from the former 2 species. On the basis of ultrastructure, these 4 species cannot be differentiated. Serologically, T. pulchra and T . texensis are closely related, while T. excentrica is more closely related to T. 
Morphology  U ltrastructure  Physiology  Serology  
T. aeria (C-6) §  T. aeria (C-6) §  T. aeria (C-6) §  T. aeria (C-6 ) §  
T. aeria (Pa-3 ) §  T. aeria (Pa-3) §  T. aeria (Pa-3) §  T.aeria (Pa-3) §  
T. isobilateralist  T. isobilateralist  T. isobilateralist  T. isobilateralist  
T. dissociata+  T. dissociata+  T. dissociata+  T. dissociata+  
T. illinoisensis+  T. illinoisensist  T. illinoisensist  T. illinoisensis+  
T. aggregata+  T. aggregata+  T. aggregata+  T. aggregatat  
T. aplanosporum·X­ T. aplanosporum'*  T. aplanosporumt  T. aplanosporum·X­ 
T. pampae*  T. pampae'*  T. pampaet  T. pampae*  
T. intermediumt  
T. tetrasporumt to'*  T. tetrasporum1]"  T. tetrasporum·x- T. tetrasporum§  
T. intermedium§  
T. excentricat to +  
T. excentricat  T. excentrica+  T. excentrica+  
T. texensist  T. texensist  T. texensist  T. texensist  
T. pulchra+  T. pulchra+  T. pulchra+  
T. intermedium+  T. intermedium+  
T. pulchra1]"  

* = distinct (very little relation) 
t = some degree of relation 
t = very close relation 
§ = identical 
nno data 

-
intermedium and T. tetrasporum than to T. texensis or T. pulchra. Thus, serologi­cal and ultrastructural data may be of value in showing the way in which the 4 species of this group may be related, possibly through a serological similarity to T. 
excentrica. 
VI. Summary 
This paper summarizes a comparative investigation of taxa in the chlorophycean genera Tetracystis and Chlorococcum using morphological (light and electron­microscopic), physiological, and serological techniques. The following are salient results and conclusions: 
1. A new chlorosphaeralean genus Tetracystic, typified by T. aeria ( C-6), has been erected. Eight additional species listed below have also been described: 
T. aggregata sp. nov. T. isohilateralis sp. nov. 
T. dissociata sp. nov. T. pampae sp. nov. 
T. excentrica sp. nov. T. pulchra sp. nov. 
T. illinoisensis sp. nov. T. texensis sp. nov. Herbarium specimens of these taxa have been deposited in the Chicago Natural 
History Museum and cultures of the living organisms have been sent to the Culture Collection of Algae, Indiana University, Bloomington, Indiana. 
2. Three taxa, formerly treated by other investigators as species of the genus Chlorococcum, have been transferred to the genus Tetracystis on the basis of new data obtained in this investigation. These are: 
Tetracystis aplanosporum (Arce and Bold) Brown and Bold comb. nov. 
T. intermedium (Deason and Bold) Brown and Bold comb. nov. 
T. tetrasporum (Arce and Bold) Brown and Bold comb. nov. 
3. 
A key to these 12 species of Tetracystis has been prepared. 

4. 
Electron-microscopic studies have yielded comparative data which are of taxonomic value in distinguishing species of T etracystis. These data include differ­ences in organization of the chloroplast, pyrenoid, mitochondria, Golgi apparatus, and cell wall. 

5. 
The electron-microscopic studies have provided insight into the course and mechanism of pyrenoid division and the behavior of the pyrenoid during zoosporo­genesis and vegetative cell division. Furthermore, electron microscopy has provided a firm basisfor distinguishing the phenomena related to vegetative cell division and zoosporogenesis, as these occur in the Chlorosphaerales and Chlorococcales. 

6. 
Serological investigations have supported taxonomic groupings originally based on morphological and physiological criteria alone. In addition, these .c:lata have revealed serological intergeneric specificity as well as common antigeni<;:ity be­tween C hlorococcum and T etracystis. . 

7. 
On the . basis of the investigations herein reported, which employed diverse techniques; it is concluded that the orders Chlorosphaerales, as exemplified by Tetracystis, and Chlorococcales, as represented by Chlorococcum, are probably validly segregated. 

8. 
A summary of species groupings based on morphological, ultrastructural, and immunochemical data is presented in Table 27 . 


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Fig. 1-8. Tetracystis excentrica.-Fig. 1. Mature vegetative cells; note excentric pyrenoid.-Fig. 2. Mature vegetative cells, some of which are undergoing vegetative cell division.-Fig. 3. Mature tetrad of daughter vegetative cells. Note loose association of parent wall with the daughter cells.-Fig. 4-Sa. Sexual reproduction. Photographs made at 5-sec intervals. Note spherical cells (young zygotesl. All X 1300. 
a See also Fig. 9-11. 

Fig. 9-12. Tetracyslis excenlrica.-Fig. 9-11 . Sexual reproduction, continued.-Fig. 12. Giant cells, pos­sibly zygotes. Note unequal cleavage. Also note young vegetative cells. 
Fig. 13-16.Tetracyslis aeria (C-61.-Fig. 13. Young vegetative cells.-Fig. 14. Mature vegelative cell; note nucleus.-Fig. 15. Mature vegetative cells and tetrahedral tetrads.-Fig. 16. Mature vegetative cell (at right) during early zoosporogenesis. 
All photos, with the exception of Fig. 14, X 1300; Fig. 14, X 2000. 

Fig. 17-18. Telrocystis aeria (continuedl.-Fig. 17. Loter zoosporogenesis.-Fig. 18. Zoosporangium, prior to release of zoosporees. 
Fig. 19-24. Tetracystis lexensis.-Fig. 19. Young and mature vegetative cells.-Fig. 20. Early octad lleftl and tetrod (right) vegetative cells.-Fig. 21. Mature vegetative cell (bottom) and early letrad of cells.-Fig. 22. Tetrad complex.-Fig. 23. Zoosporongium with 8 zoospores.-Fig. 24. Aplonosporangium lleftl and telrad lrightl. All X 1300. 

Fig. 25-32. Tetracystis dissociafa.-Fig. 25. Young and mature vegetative cells.-Fig. 26. Mature · vegetative cells. Note irregularly shaped central pyrenoid and radial fissures in chloroplast.-Fig. 27. Early. died stage.-Fig. 28. Diad undergoing division to form isobilateral tetrad. Fig. 29. Tetrahedral tetrad complexes.-Flg. 30. Zoosporangium with 16 zoospores.-Fig. 31 . Zoospore immediately upon quiescence. -Fig. 32. Aplanosporangium about to release aplanospores. All X 1300. 

Fig. 33-40. Tetracysfis aggregafa.-Fig. 33. Young vegetative cells.-Fig. 34. Mature vegetative cell and young tetrads.-Fig. 35. Complexes of isobilateral and tetrahedral tetrads.-Fig. 36. Early zoo­sporogenesis.-Fig. 37. Daughter cells of the tetrad undergoing zoospore formation.-Fig. 38. Zoospore at quiescence. Note flagella which are longer than cell body length, and posterior nucleus.-Fig. 39. Aplanosporangium.-Fig. 40. Echinate zygotes among young and mature vegetative cells. All X 1300. 

Fig. 41-48. Tetracyslis isobilateralis.-Fig. 41 . Young vegetative cells.-Fig. 42. Mature vegetative cells; no:e massive chloroplast.-Fig. 43. Complex of 2 isobiloteral telrads.-Fi(l. 44. Tetrahedral tetrad undergoing further ve3etative cell division.-Fig. 45. A tetrad of zoosporangia.-Fig. 46. Zoospore at quiescence. Nole flagellum llonger than cell body in length), parietal chloroplast, and posterior nucleus.­Fig. 47. Echinate zygote.-Fig. 48. Zygote germination. All X 1300. 

Fig. 49-S2. Tetracystis pulchra.-Fig. 49. Young and mature vegelative cells.-Fig. SO. Tetrads of vegetative cells and zoosporangium.-Fig. S 1. Mature vegetative cells. Note polar wall thickenings.-Fig. S2. Mature vegetative cell (upper right) with contractile vacuole. 
Fig. S3-S6. Tetracystis illinoisensis.-Fig. S3. Young and mature vegetative cells.-Fig. S4. Early diad formation.-Fig. SS. Mature diads.-Fig. S6. Zoosporangium. All X 1300. 

.: .... .. 
Fig. 57-64. Telracyslis pampae.-Fig. 57. Mature, ellipsoidal, vegetative cells.-Fig. 58. Tetrads of vegetative cells.-Fig. 59. Tetrads of cells in early zoosporogenesis.-Fig. 60, Tetrahedral tetrads viewed from 2 different 'positions.-Fig. 61. Early zoosporogenesis of mature vegetative cells.-Fig. 62. Aplano­speorongium containing zoosporangia with 2 zoospores each.-Fig. 63. Young vegetative. cells and singl9 zoosporangia with 2 zoospores each.-Fig. 64. Aplanosporangia with 2 aplanospores in each. All X 1300. 

Fig. 65-72. Tetracyslis ap/anosporum.-Fig. 65. Young vegetative cells.-Fig. 66. Mature vegetative cells. Note contractile vacuoles (arrowl.-Fig. 67 Early tetrad forrnation.-Fig. 68. Tetrads of vegetative cells.-Fig. 69. Very early zoosporogenesis in cell at left in which the pyrenoid has "disappeared"; chloroplast and cytoplasmic furrowing· are in progress in the cell at the right.--Fig. 70. Later zoosporo­genesis.-Fig. 71. Zoosporangium.-Fig. 72. Aplanosporangium. All X 1300. 

Fig. 73-78. Tetracystis intermedium.-Fig. 73. Young and mature vegetative cells.-Fig. 74. Mature vegetative cell in process of forming a diad al left of photo.-Fig. 75. Tetrads of vegetative cells.-Fig. 
76. Complex of tetrads.-Fig. 77. Zoosporangium.-Fig. 78. Young and mature vegetative cells; aplano­sporangium at right of photo. 
Fig. 79-80. Tetrocystis tetrasporum.-Fig. 79. Young and mature vegetative cells. Note contractile vacuole in mature vegetative cell.-Fig. 80. Tetrads of vegetative cells. All X 1 300. 
Fig. 81-94. Telracystis species at 1 and 2 weeks on BBM agar.-Fig. 81. T. isobilalerJlis, 2 weeks.­Fig. 82. T. isobilaleralis, 1 week.-Fig. 83. T. aggregafa, 2 weeks.-Fig. 84. T. aggregafa, 1 week.­Fig. 85. T. illinoisensis, 2 weeks.-Fig. 86. T. illinoisensis, 1 week.-Fig. 87. T. dissociata, 2 weeks.-Fig. 88. T. dissociata, 1 week-Fig. 89. T. aeria CC-6), 2 week.-Fig. 90. T. aeria CC-6), 1 week.-Fig. 91 . 
T. aeria !Po-3), 2 weeks.-Fig. 92. T. aeria !Po-3), 1 week.-Fig. 93. T. tefrosporum, 2 weeks.-Fig. 94. 
T. lelrasporum, 1 wee'.<. 2 weeks, X 45; 1 week, X 65. 
Fig. 95-106. Telracyslis species at 1 and 2 weeks an BBM agar (cantinuedl.-Fig. 95. T. texensis, 2 weeks.-Fig. 96. T. lexensis, 1 week.-Fig. 97. T. pu/chra, 2 weeks.-Fig. 98. T. pulchra, 1 week.­Fig. 99. T inlermedium, 2 weeks.-Fig. 100. T. inlermedium, l week.-Fig. 101. T. excentrica, 2 weeks. -Fig. l 02. T. excenlrica, l week.-Fig. 103. T. aplanosporum, 2 weeks.-Fig. l 04. T. ap/anosporum, 1 week.-Fig. 105. T. pampae, 2 weeks.-Fig. 106. T. pampae, l week. 2 weeks, X45; 1 week, X65. 
Fig. 107. Growth of Telrocyslis species lot 2 weeks) on BBM agar with crystal violet at a concentration of 1/ 100,000. No. 1, T. lexensis; No. 2, T. oggregolo; No. 3, T. aerie (Pa-3); No. 4, T. isobifaleralls; No. 5, T. excenlrico; No. 6, T. aeria (C-6l; No. 7, T. dissociolo; No. 8, T. pampae; No. 9, unidentified species of Trebouxio; No. 10, T. lelrosporum; No. 11 , T. op/onosporum; No .12, T. inlermedium. 
Fig. 1OB. Amylasic activity of Telrocyslis species on BBM supplemented with 0.01 % soluble starch and grown for 2 weeks under standard conditions. No. 1, T. isobi/alero/is; No. 2, T. pampae; No. 3, T. oggre­golo; No. 4, T. lelrosporum; No. 5, T. excenlrico; No. 6, T. oerio (C-6l; No. 7, T. aeria (Pa-3); No. 8, 
T. lexensis; No. 9, T. dissociolo; No. 10, un!<nown species of Trebouxio; No. 11, T. op/onosporum; No. 12, 
T. inlermedium. 
Fig. 109-114a. Various permanganate fixation images of the pyrenoid and surrounding area of Tetra­cystis isobilaleralis. All material fixed for 1 hr, 10 min al 5° C.-Fig. 109. 2 % LiMn04, X28,000.­Fig. 110. 2% KMn0, X24,200.-Fig. 111. 2% NaMn04, X24,200.-Fig. 112 2% Ca1Mn012, X
44
17,500. Arrow 1; note the swollen pyrenoid lamellae, peculiar to the divalent cation permanganate fix­atives used.-Fig. 113. 2% Mg!Mn0412, Xll,400.-Fig. 114. 2% Sr(Mn041v X16,400. Arrow 2 indicates the granular nature of the pyrenoid matrix, obtained only with 2 % Sr!MnO 412• 
a Unless otherwise indicated, all material for electron-microscopy was fixed at 2 % LiMnO 4 (aqueous) at 5° c for 2 hr. 
Fig. 115-116. Glutaraldehyde fixation study of Tetracystis isobi/atera/is.-Fig. 115. Material fixed lri · glutaraldehyde for 16 days, then treated with 1 o/o Os04• Arrows indicate the nucleolus and "osmio­philic granules" of the chloroplast, X 26,000. -Fig. 116. Material from same glutaraldehyde treatment a.s above but subsequently treated with 2 o/o LIMn04• Arrows indicate nucleolus and "osmiophilic granules'' of the chloroplast not preserved with 2 o/o LiMnO4 alone, X 16,200. 
Studies of Algal Genera T etracystis and Chlorococcum 
Fig. 117-120. Glutaraldehyde fixation study of Tetracystis isobilatera/is (continue:ll.-Fig. 117. Post­treatment with 1 % OsO 1• Arrow 1 shows the lad< of ribosomes on the side of the endoplasmic reticulum facing the Golgi apparatus. Arrow 2 indicates electron-dense material preserved in the vacuole. Arrow 3 shows ribosomes of the ground cytoplasm. Arrow 4 indicates Golgi cisternal elements, X 24,600. Fig. 118. Post-treatment with 1 % Os0 .1 showing general fixation image of the chloroplast. Arrow 5 indicates pyre­noid lamellae, X 24.600.-Fig. 119. Post-treatment with 2 % LiMnO.-Arrow 6 shows ribosome-like particles of the ground cytoplasm. Arrow 7 indicates electron-dense staining material in the vacuole, not so sl·:::in~d in mater:al fixed in LiMn0 1 alone, X 28,900.-Fig. 120. Post-trealment wiih 2 % LiMn0 .
1 
showing general fixation image of the chloroplast. Arrow 8 indicates pyrenoid lamel!ae. Compare with !he p~sl-treatment w ith OsO 1 (Fig. 118) and note the '.)realer contrast between matrix ond lamellae in Fig. 120, X 23,800. 
Fig. 121-123. Telracyslis aeria IC-61.-Fig. 121. Mature vegetative cell. Note invagination of ch!oro­plast (arrow 3), thick outer wall (arrow 1 l, reticulations of the chloroplast lamellae (arrow 21, and cylln­drical mitochondrion (arrow 4), X I 0,300.-Fig. 122. Tetrahedrad tetrad. Note lack of distinct Golgi amplexi !arrow 51, X 5,700.-Fig. 123. Portion of a telrad. Note variable stacking (from 2 to 6 to 8! of lamellae in chloroplast (arrow 6) and undifferentiated clemenls of the Golgi cisterna4! (arrow 7), X l5,400. 
Fig. 124-126. Tetracystis aeria IPa-3).-fig. 124. Vegetative cell. Arrow 1 indicates thic:<, electron­dense wall layer. Arrow 2 shows non-inflated Golgi apparatus which lacks dislinct amplexus. ·Arrow 3 refers to typical chloroplast invaginations, X 10,400.-Fig. 125. Porlion of chloroplast showing lamellar stacking Iarrow 41, X 21,200.-Fig. 126. Early-formed tetrad of daughter vegetative cells. Compare thickness of electron-dense layer of daughter cells with that of parenl. Nole cylindrical mitochondrion (arrow 5) and reticulations of chloroplast !arrow 6), X 5600. 
Fig. 127-129. Tetracystis iso&i/ateralis.-Fig. 127. Mature isobilateral tetrad of daughter vegetative· . cells. Note the wall relation of the daughter cells (arrow 11 which indicates that Intervening dlad fonnaflon.'; has occurred, X 7000.-Fig. 12 8. Portion of vegetative cell showing chloroplast lamellar stacking (ai'fo\,y 21 and thick, inner, granular cell wall l~yer, X 13,000.-Fig. 129. Unknown organelle (arrow 41 often present in this species; chloroplast showing variable stacking of lamellar components (arrow 31, X 15,000; 
,, ,,... , 
Fig. 130-131. Tetracystis isobilateralis (continuedl.-Fig. 130. Mature vegetative cell with thick, inner wall layer (arrow 1 ), massive chloroplast with centrifugal (arrow 2) and centripetal (arrow 31 fissuring, X 8,000.-Fig. 131 . Diad of daughter vegetative cells with giant, branched, compressed mitochondrion (arrow 51 and Golgi apparatus without distinct amplexi, cisternal elements of which are uninflated (arrow 4l, X 7,800. 

Fig. 132-134. Tetracystis aggregala.-Fig. 132. Young tetrad of cells with thin outer walls and thick · inner walls of the newly deposited daughter cells (a.rrow 1). The Golgl clsternae of this species are fre· quently inflated (arrow 21, X 7,350.-Fig. 133. Portion of vegetative cell showing thick gra.nular layer of · the inner cell well (arrow 31, compressed, unbranched mitochondrion (arrow 41, and variablit sta.cking cif the chloroplast lamellae (arrow 51, X 16,200.-Fig. 134. Mature tetrad of daughter vegetative cells. Com­pare walls (arrow 61 with those of the young telrad (arrow 1, Fig. 1321. Note deep, broad, radial fissuring of chloroplast common in this species (arrow 71, X 7,560. 
Fig. 135-137. Tetracystis dissociata.-Fig. 135. Vegetative cell. Note peripheral fissuring of the chloroplast (arrow 11 and the thick, inner wall layer (arrow 21, X 8,700.-Fig. 136. Portion of vegetative cell show;ng variable stacking of chloroplast lamellae (arrows 3 and 51. Arrow 4 indicates a tangential section across the chloroplast-limiting membrane, X 16,300.-Fig. 137. Typical ellipsoidal diad stag·• with Golgi elements (arrow 61 which have indistinct amplexi and characteristically lack cisternal inflation, X l0,550. 
Fig. 138-140. Telracysfis dissociata (continuedl.-Fig. 138. Mature tetrad of daughter cells which are about to be released by rupture of the weakening electron-dense wall layer o,.the parent (arrow 2. Arrow 1 denotes a dividing pyrenoid, X 6,750.-Fig. 139. Mature zoosporangium, X 6,300.-Fig. 140. Zoospore with very thin, electron-dense wall layer (arrow 31 and a posterior nucleus (arrow 41. Note parietal chloro: plast and equatorial pyrenoid, X 15,700. 
Fig. 141-142. Telrocyslis illinoisenis.-Fig. 141. Mature vegetative cell with cylindrical mitochon­drion (arrow 21; Golgi elements with indistinct amplexi, cisternal elements of which are not Inflated (arrow 31; thick inner wall layer (arrow 41; and lack of peripheral invagination (arrow 1 I of the thin, parietal choloroplast. Note fissuring, X 8,300.-Fig. 142. Portion of vegetative cell showing stacking of chloroplast lamellae (arrow 51 and pyrenoid to right in photograph, X 22,800. 
Fig. 143. Tetracystis excentrico. Mature vegetative cell with marked polar thickening of inner wall layer (arrow 61, lack of peripheral invagination of chloroplast (arrow 71 as well as of fissuring; cylindrical mitp­chondrion (arrow 81; Golgi elements with distinct amplexus, cisternal elements of the Golgi Inflated (arrow 91; and a single, excentric, ellipsodial pyrenoid with 2 starch grains, X 14,500. 
Fig. 144-146. Telracyslis intermedium.-Fig. 144. Mature vegetative cell with thick inner wall layer (arrow 2), cylindrical mitochondrion (arrow 1 l, and chloroplast with few peripheral invaglnations and fissures (arrow 3l, X 11,700.-Fig. 145. Portion of vegetative cell showing chloroplast with variable lamellar association (arrow Sl and unidentified structure (arrow 4), X22,800.-Fig. 146. Tetrad of daughter vegetative cells with Golgi elements which have distinct amplexi, cistemal elements of which are inflated (arrow 6). Note granulation in the inner wall layers. X 1 S,900. 
Fig. 147-150. Te1racystis texensis.-Fig. 147. Mature vegetative cell with cylindrical mitochondrion (arrow 21, thin outer electron-dense wall (arrow 31, and chloroplast lacking peripheral invaginations (arrow 11, X 9,000.-Fig. 148. Portion of mature daughter vegetative cell showing Golgi apparatus with distinct amplexi, their cisternal elements characteristically inflated, X 14,300.-Fig. 149. Portion of mature vegetative cell showing variable, associated lamellar components of the choloroplasl (arrow 41, X 19,000. -Fig. 150. Portion of an octad of daughter vegetative cells, peculiar only to this species. Note the lack of peripheral invagination of the chloroplast (arrow 51, variable thickening of the inner granular wall layers, and the amplexus of the Golgi apparatus with inflated cisternae (arrow 61, X 9,750. 
Fig. 151-152. Tetracystis pulchra.-Fig. 151. Mature vegetative cell with ellipsodial pyrenoid sur­rounded by 2 starch grains. Note chloroplast lamellae with variable stacking (arrow 1 l and thickening of the inner wall layers, X 26,400.-Fig. 152. Tetrad of daughter cells showing chloroplast which lacks peripheral invaginations (arrow 31 and which is thin and parietal. Note cylindrical mitochondrion (arrow 51, Golgi apparatus with distinct amplexus, the cisternae characteristically inflated (arrow 41; note also thickened inner wall layer of the daughter cell (arrow 21, X 12,800. 
Studies of Algal Genera Tetracystis and Chlorococcum 
Fig. 153-155. Tetracystis ap/anosporum.-Fig. 153. Mature vegetative cell showing thin inner and outer wall layers (arrow 1 ), cylindrical mitochondrion (arrow 31, and chain of "vacuoles" (arrow 2) which repesent an inflated stack of Golgi cisternae, X 9,200.-Fig. 154. Mature tetrad of daughter cells. Note the loosely associated parent wall (arrow 4) and the Golgi apparati with distinct amplexi and inflated cisternal elements (arrow 51. A contractile vacuole is present just above the nucleus, X 13,600.-Fig. 155. Detail of chloroplast of mature vegetative cell showing stacks of 1-2 Jamellar discs alternating irregularly with 3-5 discs (arrow 61 . Note the peripheral invagination of the massive chloroplast (arrow 7), X 34,400. 
Fig. 156-158. Tetracystis pampae.-Fig. 156. Young vegetative cells with cylindrical mitochondrion (arrow 2) and thinner, outer wall layer (arrow 1l. Note parietal chloroplast, X 8,250.-Fig. 157. Golgi apparatus with distinct amplexus and cisternal elements which are characteristically inflated, though not shown to a great degree here (arrow 31. Pyrenoid is to rig!it in photograph, X 20,500.-Fig. 158. Mature vegetative cell. Note the triple-disc lamellar system of the chloroplast (arrow 6) and of the pyrenoid (arrow 51. The chloroplast does not have paripheral in1a3inations (arrow 4), X 16,200. 
Fig. 159-162. Pyrenoid types among Tetracyslis.-Fig. 159. Pyrenoid showing double-disc lamellar system penetrating it (arrow 1l. Note parallel orientation of pyrenoid lamellae which is characteristic of this type IT. isobilatera/isl, X 17,800.-Fig. 160. Pyrenoid of T. aeria IC-61 showing contorted, double­disc system penetrating it (arrow 2), and characteristic reticulations of the chloroplast which usually lie near the pyrenoid, X 14,200.-Fig. 161 . Pyrenoid of T. pampae showing tripledisc lamellar system penetrating its matrix (arrow 31, X 21,800.-Fig. 162. Pyrenoid of T. isobilateralis showing the flattened or "disc"-like nature of the lamellar components which penetrate it. Note especially the perforations (arrow 4) in thees lamellae which are absent in lamellae situated in the chloroplast proper (arrow SJ, X 16,100. 
Fig. 163-166. Pyrenoid types in Tetracystis (continuedl.-Fig. 163. Pyrenoid of T. texensis with 2 starch grains (arrows 1 and 21. Note the pyrenoid lamellae which lie internal to the starch (arrow 31. These lamellae become reduced from a double-disc system (arrow 4) to a single, convoluted disc system !arrow 3) at 1 or more loci along the pyrenoid matrix, X 31 ,000.-Fig. 164. Pyrenoid of T. aplanosporum showing single tubular lamellar elements which penetrate the matrix (arrow 5) and which are occasionally associated in pairs Carrow 6), X 23,800.-Fig. 165. Pyrenoid of Ch/orococcum sp. (tetra isolate) showing tubular elements (arrow 7) which penetrate through a single, surrounding starch grain, X 21,800.-Fig. 
·166. Pyrenoid of C. multinucleatum. Note the lack of starch synthesis on the periphery of the matrix and 
the presence of reticulated tubules Carrow 9) which penetrate the pyrenoid matrix. Arrow 8 indicates a 
tubule in cross-section, X 15,600. 
Fig. 167-170. Stages of pyrenoid division in Tetracystis isobilatera/is.-Fig. 167. Early stage of pyrenoid division in which the pyrenoid lamellae have become parallel (arrow 31 and have cleaved in a plane (arrow 21 perpendicular to that of the lamellar zone. Note the invaginating pyrenoid lamella !arrow 11, X 16,700.-Fig. 168-169. Different sections through the same pyrenoid at a later stage in division. Note invaginating pyrenoid lamellae in half !arrow 81. Arrows 4, 6, 7, and 9 indicate continuity of parallel pyrenoid lamellae to invaginating pyrenoid lamellae. The chloroplast lamellae are beginning to invaginate at this stage [arrow 51. Fig. 168, X 13,000; Fig. 169, X 14,000.-Fig. 170. Later stage in pyrenoid division in which the chloroplast lamellae (arrow 121 have nearly cleaved the pyrenoid (arrows 13 and 141. Note that the pyrenoid lamellae are still parallel [arrow 151 and that the invaginating chloro· plast lamellae appear to split the pyrenoid lamellae [arrow 101. Arrow 11 indicates an unknown structure, X 15,000. 
Fig. 171-172. Pyrenoid division in Tetrocystis isobiloterolis lcontinuedl.-Fig. 171. later stage of pyrenoid divison in which the chloroplast lamellae have completely split the pyrenoids in half (arrow 2). Note starch synthesis which is now becoming active on the interfaces of the daughter pyrenoids (arrow 1). Note the persistence of parallel lamellae of the daughter pyrenoids (arrow 3), X 17,000.-Fig. 172. Final stage in pyrenoid division in which the separated pyrenoid matrices have become discernible (arrow 5), and in which the pyrenoid lamellae have re-oriented (arrow 6). Note also that the invaginating chloro­plast lamellae are more profuse (arrow 41. Pre-fixed in glutaraldehyde, then post-treated with 2 % LiMn04 , X 23,600. 
Fig. 173. Pyrenoid division during vegetative cell division in Telrocystis aplanosporum in which the chloroplast-limiting membrane splits the pyrenoid matrix (arrow 81 and the pyrenoid tubules (arrow 71, X lS,600. 
Fig. 174. Pyrenoid division of Tetrocystis oerio CC-61 in which the chloroplast-limiting membrane sep­arated the pyrenoids (arrow l 01. Note the probable splitting of the pyrenoid lamellar discs (arrow 71 in the division zone, X 19,000. 
Fig. 175-179. Formation of Golgi cisternae in Tetracystis.-Fig. 175. Amplexus (arrows 1 and 21 with multiple Golgi systems in T. texensis, X 25,000.-Fig. 176. Amplexus system of T. excentrica. Note the close proximity of the several Golgi apparatus to the nucleus in which anticlinical sections of the amplexi show a characteristic spur (arrow 31 and an area along the amplexus which appears to be budding to form the cisternal elements of the Golgi (arrow 4), X 1 8,000.-Fig. 177. Tangential section through a Golgi amplexus system of T. lexensis showing budding elements (arrows 4, 5 and 61 of the amplexus and tubular hexagonal network on the periphery of the Golgi (arrow 81 elements, X 40,000.­Fig. 178. Golgi apparatus of T. lexensis surrounded by budding amplexus (arrows 9, 101. Arrows 12 and 13 show a continuity between the amplexus and the Golgi cisternae. Arrow 14 shows a portion of the amplexus which extends to make contact with the nucleus lout of picture, below), X 39,000.-Fig. 179. Budding amplexus system of T. excenlrica showing attachment to nucleus (arrow 16 indicating nuclear pore) and proliferation of lamellar elements of the amplexus in close proximity to the Golgi cisternae. Note that the Golgi cisternae nearest the encompassing portion of the amplexus are always non-inflated and more closely packed than those away from the am plexus, X 32,500. 
Fig. 180-185. Possible vacuole development from Golg i cisternae in Tetracystis.-Fig. 180. Various stages of cisternal inflation (arrows 1, 2, and 31 in T. ap/anosporum. Arrow 4 indicates a contractile vacuole, X 16,400.-Fig. 181 . Probable greatly inflated cisternal elements which were deposited in pairs. CT. ap/anosporum) Carrow 6). Arrow 5 shows what may be adjacent cistemal membranes which remained together as the cisternae were deposited in the cytoplasm, X 1 5,400.-Fig. 182. Inflated cisternal ele­ments (arrows 8, 9) of T. aplanosporum (fixed in OsO4 ). Arrow 9 shows uninflated cisternae nearest the amplexus, X 31,500.-Fig. 183. Inflated Golgi elements of T. excentrica showing undirectional inflation always opposite the encompassing amplexus, X 34,100.-Fig. 184. Tangential section of Golgi apparatus with inflated cisternae (arrow 10) and uninflated portion of another Golgi apparatus (arrow 11 ), X 26,900. -Fig. 185. Golgi apparati of T. excentrica post-stained wtih 5 % of Ba(Mn04l2• Note the starch (arrow 12) which was densely stained by this process; the inflated Golgi cisternae (arrow 13) were not so stained, this indicating that a soluble product forming the vacuolar elements was probably leeched from the Golgi during the fixation process, X 22,800. 
Fig. 186-189. Contractile vacuoles in Tetracystis ap/anosporum.-Fig. 186. One contractile vacuole completely closed (arrow 1 ). Note the radial, tubular elements of the vacuolar membrane. The other con­tractile vacuole is completely open (arrow 2). Note the granular strings in the vacuole itself, X 24,100.­Fig. 187. Partially closed contractile vacuole (arrow 5) with associated endoplasmic reticulum (arrow 4). Note the absence of radial proliferations on the peripheral side of the vacuole (arrow 5), X 20,900.­Fig. 188. Contractile vacuole caught in the process of contracting (arrow 6). Arrow 7 indicates associated endoplasmic reticulum. Arrow 8 shows a tangential section through a closed contractile vacuole, X 27,900. -Fig. 189. Contractile vacuole of T. ap/anosporum fixed with 1 % Os04 (arrow 9). Note lack of ribo­somes in close proximity lo the radial proliferations of the vacuolar membrane, X 58,000. 
Studies of Algal Genera Tetracystis and Chlorococcum 
Fig. 190-192. Vegetative cell division in Tetracystis oggregato.-Fig. 190. Mature vegetative cell undergoing furrow formation (arrow 2). Note the close proximity of the endoplasmic reticulum and Golgi apparatus (arrow l l to the advancing cleavage furrow (arrow 2l, X 13,500.-Fig. 191 . Portion of cleav­age furrow which hos cut through most of the cell. Note the "intercellular space" (arrow 4) and the zone which separates the fine granulation from the coarse granulated inner-wall layer of the parent cell (arrow 3l. Note also that inner wall deposition (as evidenced by fine granulation) has not occurred in the area of the cleavage furrow at the bottom of the photograph, X 16,500.-Fig. 192. later stage of the same area when cell division has been completed. Note the presence of coarse granulation in the inner layers common to the parent and daughter cell. Note the electron-dense wall layer deposited around the daugh­ter cells (arrow 7l which is contiguous with the electron-dense layer of the parent cell (arrow 6). Note the absence of coarse granulation (arrow 5l in the intercellular space which appears to be blocked by the electron-dense walls of the daughter cells. When such coarse granulation is absent, no further deposition of the perent electron-dense wall occurs, and this layer deteriorates (arrow 6), X 22,800. 
Fig . 193-194. Vegetative cell division in Telracyslis isobilaleralis.-Fig. 193. Diad undergoing vegeta­tive cell division to form a triad of daughter cells. Electron-dense wall layer is deposited immediately be­hind advancing cleavage furrow (arrow l). Arrow 3 shows intercellular space devoid of coarse granu­lation. Arrow 5 indicates probably origin of coarse granulation from vacuoles in the cytoplasm. The granules appear to migrate through the cytoplasm (arrow 2) to the area of wall formation and thence into the inner wall layers to form the electron-dense wall layer (arrow 4), X 11,400.-Fig. 194. Detail of cytoplasmic localization of coarse granulation (arrow 6) near node of advancing cleavage furrow, X 23,600. 
Fig. 195-196. Two sections through same cleavage furrow of Tetracystis aggregata showing the Golgi apparatus in section (arrow 1 l and out of section (arrow 2), X 14,150.-Fig. 196, X 17,600. 
Fig. 197. Early vegetative cell division in T. texensis showing initiation of cleavage furrows (arrows 4, 5) between divided chloroplasts and zone of vacuolization (arrow 41 between the nuclei. Note the close association of outer nuclear envelopes (arrow 4) X 14,200. 
Fig. 198-199. Later stages of vegetative cell division in T. fexensis (continuedl.-Fig. 198. Cleavage furrows (arrows 1, 3) progressing centripetally. Note chromatin of newly formed interphase nucleus (arrow 21, X 13,600.-Fig. 199. Cleavage complete, the furrows having cut around some cytoplasm (arrow 51 between the nuclei. Associated nuclear envelopes appear to have been cleaved by the furrows (arrows 4, 6), X 12,300. 
Fig. 200. Vegetative cell division in T. excentrica. Bilateral cleavage furrows have almost cur through cell (arrow 41. Arrow 1 indicates the outer, electron-dense layer of parent wall. Note the increased activity of the Golgi apparati, 4 of which are shown encompassed by 1 amplexus (arrow 21. Tangential section of nucleus (arrow 3), X 10,000. 
Fig. 201 . Vegetative cell division in T. aeria IC-61. Arrows 5 and 6 show possible pathways rhe cleavage furrows first take, followed by centrifugal deposition (arrows 7, 8), X 6,000. 
Fig. 202-205. Early zoosporogensis in Tetracystis aplanosporum.-Fig. 202. Early chloroplast cleavage (all arrows 1 I in pyrenoid region, as exemplified by the tubular elements of the pyrenoid matrix (all arrows 21. Note the increased activity of the Golgi elements (arrow 31 and the chloroplast lamellar pro­liferation and increase in chloroplast slarch (arrow 41, X 12,700.-Fig. 203. Another early stage of zoo­sporogenesis in which the pyrenoid, as indicated by !he presence of pyrenoid tubules !arrows 2), is being cleaved by the dividing chloroplast at a number of loci (arrows l l, X 12,500.-Fig. 204. A still later stage of zoosporogenesis. Note the absence of starch synthesis on pyrenoid surfaces. Pyrenoid tubules clearly delimit pyrenoid region (arrows 21 Golgi cisternae are greatly inflated during this stage of zoosporogenesis (arrow 31, X 15,200.-Fig. 205. Zoosporogenesis near the completion of pyrenoid fragmentation, yet the pyrenoids can still be discerned by ·the presence of their tubules (arrows 21 even though they are inactive in starch synthesis. Arrow l shows the terminating phase of chloroplast cleavage, X 12,500. 
Fig. 206-207. Cell wall formation during zoosporogenesis.-fig. 206. Early wall development in Chlorococcum sp. (tetra). Note common inner wall layer (arrow l ), endoplasmic reticulum (arrow 2), and splitting of common wall layer (arrow 3), X 18,000.-Fig. 207. later stage of wall development in Chlorococcum multinucleatum. Nole endoplasmic reticulum (arrow 6), plasma membrane (arrow 7), electron-dense wall layer (arrow 8), and splitting of common wall layer (arrow 8). Note also that even before wall formation is complete, the flagella (arrow Sl and stigma (arrow 41 are completely developed, x 16,300. 
Fig. 208-209. Late zoosporogenesis.-Fig. 208. Chlorococcum mullinuc/eatum. Note the separation of zoospore wall from both the outer, electron-dense layer (arrow 1 l and inner wall layer (arrow 2l of parent cell, X 9,350.-Fig. 209. Chlorococcum sp. (tetral. Late zoosporogenesis, just prior to zoospore re­lease, X 6,000. 

.. 

., 
Code to oil ontisero and antigens used in the immunochemical study and which appear in the illustra­tions to follow: 
ANTl5ERA ANTIGENS: 
A = onti-Tetracysfis aeria 1.C-6) = T. aeria !C-61 15 = C. diplobionficum B = anti-Chlorococcum perforatum 2 = T. aeria !Po-31 16 = C. echinozygotum C = anti-Tefrocysfis isobilaferalis 3 = T. dissociato 17 = C. ellipsoideum D = anti-Tefracysfis aplanosporum 4 = T. isobilateralis 18 = C. hypnosporum E = anti-Chlorococcum sp. (tetra isolate) 5 = T. aggregata 19 = C. macrostigmalum 
6 = T. illinoisensis 20 = C. minulum 7 = T. aplanosporum 21 = C. multinuclealum 8 = T. pampae 22 = C. sp. !tetra isolate) 9 = T. infermedium 23 = C. oleofociens 
10 = T. excentrica 24 = C. scabellum 11 = T. texensis 25 = C. pinguidium 12 = T. pulchra 26 = C. punclalum 1 3 = T. fetrasporum 27 = C. vacuo/alum 14 = C. perforafum 28 = C. wimmeri 
Fig. 21 0-213. Experiments establishing proper conditions for reaction of algal antigens upon double­diffusion and immunoelectrophoresis.-Fig. 210. Experiment to detennine the best cup-to-trough distances for reaction. No. 1 = 11 /2 mm; No. 2 = 9/3 mm; No. 3 = 7/5 mm; N.o 4 = 5/7 mm; No. 5 = 3/9 mm; No. 6 = 21 11 mm. Based on this experiment, a distance of 4 mm was chosen for all subsequent double­diffusion and absorption studies.-Fig. 211. Experiment to determine the best cup-to-cup distances for re­action and to compare w ith cup-to-trough reactions. No. 1 = 15 mm; No. 2 = 2 I 11 mm. No. 3 = 3/9 mm; No. 4 = 517 mm; No. 5 = 7 I 5 mm. No. 6 = 913 mm; No. 7 = 11 /2 mm; and No. 8 = 15 mm. Note that cup-to-trough gave better reactions even though the antigens were exactly the some concen­tration 18.15 mg protein/ ml for C. sp.I. In both Figures 210 and 211, antiserum C. sp. was used and both plates were photographed at 24 hr development.-Fig. 212. Experiment to determine the position effects of reactions in proximity with adjacent reactions, and position of cups and troughs on the agar plate. Antigen used: C. sp.-Fig. 213. Testing various concentrations and extraction procedures of Chlorococcum sp. (tetra) against antiserum C. sp. !El 
No. 1 = Whole powder, at protein cone. of 32.6 mg/ ml 
No. 2= " 16.3 
No. 3= 8.15 
No. 4= 4.7 
No. 5= 2.35 
No. 5= 2.35 
No. 6 = 1st extract supernatant (10 min) from whole powder at protein cone. of 8.15 mg/ ml 

No. 7 = 2nd 

No. 8 = 3rd (overnight)" 
No. 9 = Plug of whole cells from which the 3rd extract was taken. 

Plate photographed at 24 hr development. 

Fig. 214-216. Double-diffusion reactions of all Tetracystis species. Plate was photographed at 24 hr development. 
Fig. 214. No. 1 = T. aeria (C-6) 
No. 2 = T. aeria (Pa-3) 
No. 3 = T. dissociata 
No. 4 = T. isobilateralis 
No. S = T. aggregata 

No. 21 S. No. 7 = T. aplonosporum 
No. 8 = T. pompae 
No. 9 = T. intermedium 
No. 6 = T. illinoisensis 
No. 7 = T. ap/onosporum 

Fig. 216. No. 1 0 = T .. excenlrica 
No. 11 = T. texensis 
No. 1 2 = T. pu/chra 
No. 22 = C. sp. (tetra) 
No. 1 3 = T. lelrasporum 

Studies of Algal Genera Tetracystis and Chlorococcum 
Fig. 217-220. Double-diffusion reactions of Chlorococcum species. Plale was pholographed at 24 hr development. 
Fig. 217. No. 7 = T. ap/anosporum 
No. 15 = C. dip/obionlicum 
No. 14 = C. perloralum 
No. 16 = C. echinozygolum 
No. 17 = C. ellipsoideum 

Fig. 218. No. 18 = C. hypnosporum 
No. 9 = T. inlermedium 
No. 14 = C. perloralum 
No. 19 = C. macrosligmalum 
No. 20 = C. minutum 

Fig. 219. No. 21 = C. multinuclealum 
No. 23 = C. o/eolociens 
No. 14 = C. perlorolum 
No. 25 = C. pinguidium 
No. 26 = C. punclatum 

Fig. 220. No. 24 = C. scabellum 
No. 22 = C. sp. (tetral 
No. 14 = C. perloralum 
No. 27 = C. vacuo/alum 
No. 28 = C. wimmeri 

Fig. 221-224. Double-diffusion reactions of Ch/orococcum species with the same plates as in Fig. 222­225 but photographed at 48 hr development. 
Fig. 221. No. 7 = T. aplanosporum 
No. 15 = No. 14 = No. 16 = No. 17 = 
Fig. 222. No. 18 = No. 9 = No. 14 = No. 19 = No. 20 = 
C. diplobionlicum 
C. perforalum 
C. echinozygolum 
C. ellipsoideum 
C. hypnosporum 
T. inlermedium 
C. perforalum 
C. macrosligmalum 
C. minulum 
Fig. 223. No. 21 = C. multinuclealum No. 23 = C. oleofaciens No. 14 = C. perforalum No. 25 = C. pinguidium 
No. 26 = 
Fig. 224. No. 24 = No. 22 = No. 14 = No. 27 = No. 28 = 
C. punclalum 
C. scabellum 
C. sp. !tetra} 
C. perforalum 
C. vacuo/alum 
C. wimmeri 
Fig. 225-226. First-absorption study with the T. aerie and T. isobilatera/is groups. 
Fig. 225. Upper trough contains anliserum to T. isobilateralis, absorbed with T. aerie !Pa-3l. Lower trough contains unabsorbed antiserum lo T. isobilateralis. 
No. 28 = C. wimmeri 
No. 1 = T. aerie (C-61 
No. 3 = T. dissociate 
No. 4 = T. isob:lateralis 
No. 2 = T. aerie !Pa-31 
N >. 5 = T. uggregala 
No. 6 = T. i//inoisensis 
Fig. 226. Upper trough contains antiserum lo T. aerie (C-61 absorbed wilh T. isobilateralis. Lower trough contains unabsorbed antiserum lo T. aerie !C-61. 
No. 28 = C. wimmeri 
No. 1 = T. aerie (C-61 
No. 3 = T. dissociate 
No. 4 = T. isobilateralis 
No. 2 = T. aerie IPA-31 
No. 5 = i'. aggregafa 
No. 6 = T. i//inoisensis 
Fig. 227-228. Second-absorption study with selected species of Ch/orococcum and Telracyslis. 
Fig. 227. Upper trough contains antiserum to T. aeria CC-6) absorbed with C. sp. ltetral. Lower trough contains antiserum lo C. sp. (tetra) absorbed with T. aeria <Pa-3>. 
No. 2 = T. aeria CPA-31 
No. 4 = T. isobi/alera/is 
No. 8 = T. pampae 
No. 1 0 = T. excenlrica 
No. 11 = T. lexensis 
No. 9 = T. inlermedium 
No. 7 = T. ap/anosporum 
Fig. 228. Upper trough contains antiserum lo T. aeria (C-6) absorbed with C. sp. (tetra>. Lower trough contains antiserum to C. sp. (tetra) absorbed with T. aeria (Pa-3). 
No. 22 = C. sp. (tetra) 
No. 14 = C. perforalum 
No. 17 = C. el/ipsoideum 
No. 16 = C. echinozygolum 
No. 1 5 = C. diplobionlicum 
No. 25 = C. pinguidium 
No. 18 = C. hypnosporum 
Fig. 229-232. Reactions of the T. aeria and T. isobilaleralis groups in immunoelectrophoresis (photo­graphed 24 hr in development>. 
Fig. 229. with antiserum to T. aoria (C-61 . (Al 
Antigens: No. 3 = T. dissociala 
No. 1 = T. aeria (C-61 
No. 2 = T. aeria (Pa-3) 

Fig. 230. With anliserum lo T. isobi/oleralis. (Cl 
Antigens: No. 3 = T. dissociala 
No. 1 = T. aeria (C-6) 
N:>. 2 = T. aeria (Pa-31 

Fig. 231. Wah antiserum to T. oeria (C-6}. (Al 
Anligens: No. 4 = T. isobilareralis 
No. 5 = T. aggregota 
No. 6 = T. illinoisensis 

Fig. 232. With antiserum to T. isobilateralis. (Cl 
Antigens: No. 4 = T. isobilaleralis 
No. 5 = T. aggregala 
No. 6 = T. illinoisensis 

Fig. 233-236. Reactions of selected species of Chlorococcum and Tefracysfis in immunoelectrophoresis (photographed 24 hr in development!. 
Fig. 233. With antiserum to T. aplanosporum. (DJ 
Antigens: No. 5 = T. aggregata 
No. 7 = T. oplonosporum 
No. 17 = C. ellipsoideum 

Fig. 234. With antiserum to T. aplonosporum. (DI 
Antigens: 	No. 6 = T. illinoisensis 
No. 18 = C. hypnosporum 
No. 3 = T. dissociata 

Fig. 235. With antiserum to C. perforalum. (8) 
Antigens: No. 16 = C. echinozygofum 
No. 28 = C. wimmeri 
No. 17 = C. ellipsoideum 

Fig. 236. With antiserum to C. perforalum. (8) 
Antigens: No. 15 = C. diplobionficum 
No. 20 = C. minulum 
No. 14 = C. perforalum 

Studies of Algal Genera Tetracystis and Chlorncoccum 
Fig. 237-240. Reactions of selected species of Chlorococcum and Tetracystis in immunoelectrophoresis (photographed 24 hr in development). 
Fig. 237. With antiserum to C. sp. ltetral. (El 
Antigens: No. 24 = C. scabe//um 
No. 22 = C. sp. Oetra) 
No. 23 = C. o/eofaciens 

Fig. 238. With antiserum to C. sp. (tetra). (El 
Antigens: No. 21 = C. multinuc/eatum 
No. 26 = C. punctatum 
No. 27 = C. vacuo/alum 

Fig. 239. With antiserum to C. sp. Oetral. (El 
Antigens: No. 15 = C. dip/obionticum 
No. 20 = C. minutum 
No. l 4 = C. per/oratum 

Fig. 240. With antiserum to T. aeria IC-6l. IA> 
Antigens: 	No. 18 = C. hypnosporum 
No. 25 = C. pinguidium 
No. 8 = T. pampae