ELECTRON MICROSCOPY OF ULTRATHIN SECTIONS OFSCHIZOSACCHAROMYCES OCTOSPORUS

II. MORPHOLOGICAL AND CYTOLOGICAL CHANGES PRECEDING ASCOSPORE FORMATION

S. F. CONTI' AND H. B. NAYLORLaboratory of Bacteriology, Department of Dairy Industry, Cornell University, Ithaca, New York2

Received for publication July 23, 1959

Conjugation (plasmogamy) in yeast was firstdescribed by Shionning (1895). He observedthat ascus formation in Schizosaccharomycesoctosporus was preceded by fusion of two sistercells. Further studies by Hoffmeister (1900)indicated that nuclear fusion accompanied cellfusion. Guilliermond (1901) clearly demonstratedthat nuclear fusion occurred after cell fusion, andthat ascus formation is preceded by a true isoga-mous conjugation. Further studies of sporo-genesis, utilizing the iron-hematoxylin techniqueswere carried out by Guilliermond (1917). Recentstudies by Widra and DeLamater (1955), de-scribe the occurrence of a typical meiotic cyclein S. octosporus after the haploid cells have united.Lindegren (1951) described the involvement ofthe vacuole during copulation of Saccharomycesbayanus, whereas Widra and DeLamater (1955)tend to confirm Guilliermond's belief that thevacuolar contents are ergastic in nature.

Although general agreement appears to belacking, these various studies revealed the se-quence, and nature of some of the cytologicalchanges which occur during plasmogamy, kary-ogamy, and subsequent nuclear division. Theprocess of conjugation in S. octosporus was there-fore studied by electron microscopy in the hopethat the observations of other investigators bylight microscopy could be correlated with our ob-servations by electron microscopy, thereby lead-ing to a more refined and accurate descriptionof the cytological changes which precede asco-spore formation in S. octosporus.

1 Present address: Department of Biology,Brookhaven National Laboratories, Upton, LongIsland, New York.

2 Electron microscopy was done in the ElectronMicroscope Laboratory, Department of Engineer-ing Physics, Cornell University, Ithaca, NewYork.

MATERIALS AND METHODS

The culture used in these studies was S. octo-sporus strain NRRL Y-854, obtained from Dr.L. J. Wickerham. Cells were routinely grown ina glucose-yeast extract medium (glucose, 1.0 percent; yeast extract, 2.0 per cent; peptone, 0.5 percent; KH2PO4, 0.1 per cent and MgSO4, 0.05per cent). The pH of the medium was adjustedto 7.0 before autoclaving, and 1.5 per cent agaradded when a solid medium was required. Con-jugating cells were obtained by inoculating 24-hr-old cultures onto YM agar plates (glucose1.0 per cent; malt extract, 0.3 per cent; yeastextract 0.3 per cent; agar 2.0 per cent, pH unad-justed), and incubating at 28 to 30 C. Cellswere collected at appropriate time intervals forstudy by electron microscopy. Sporulation wasessentially complete within 48 to 72 hr.Specimen preparation. Cells were collected

from agar surfaces by washing off with 0.06M phosphate buffer at pH 6.6. Cells were thensedimented by centrifugation and resuspendedin a 1.5 per cent aqueous solution of KMnO4 for40 min at 4 C. All subsequent steps, except for thepolymerization were also carried out at 4 C.Specimens were dehydrated by serial passage (1hr each) into 70, 95, and 100 per cent ethanol,transferred to a 50 per cent solution of butylmethaerylate in absolute alcohol, and left for 1to 2 hr. Cells were then passed twice (fi hr each)through cold butyl methaerylate, and trans-ferred to partially polymerized butyl methacry-late. Specimens were left in the latter solutionsfor 6 to 8 hr, transferred to gelatin capsules, andallowed to settle for 4 hr at room temperature.Polymerization was aecomplished by placing thecapsules in an oven at 56 to 60 C for 8 to 12 hr.Ultrathin sections were obtained by means of aPorter-Blum ultramicrotome equipped with aglass knife. Difficulty in sectioning due to thesoftening of the block could be overcome by

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leaving the blocks at 4 C before sectioning. Sec-tioning could also be facilitated by exposing thetip of the block to a stream of liquid nitrogen for20 to 30 see, approximately 20 min before sec-tioning. Condensed water was removed by care-fully blotting with filter paper. The technique ofSatir and Peachey (1958) was employed to de-crease decompression artifacts. Sections of lessthan 0.1 ,u were picked up on 200 mesh coppergrids on which a collodion, Formvar, or carbonfilm had been mounted. Sections were examinedin an RCA-EMU 2B electron microscopeequipped with a 50 or 100 ,u objective aperture.

Cytochemical techniques and light microscopy.Several cytochemical methods were used to cor-relate structures observed in electron micro-graphs with structures observable by light micros-copy. Neotetrazolium salts an(l dilute Lugol'ssolution were used, respectively, for the demon-stration of mitochondria and glycogen. Lipoidal

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inclusions were identified by their stainabilitywith Sudan black 1B. Copulation was also fol-lowed by means of phase microscopy to confirmthe general appearance of the cells during thesequence of events leading to ascus formation.The shape of the cells and previous studies byphase and electron microscopy (Conti andNaylor, 1959a) of dividing cells of S. octosporusmade it possible to clearly distinguish betweendividing and conjugating cells. Since vacuolesare absent from actively dividing cells of S.octosporus (Ganesan and Swaminathan, 1958;Conti and Naylor, 1959b) and present in con-jugating cells, the development and fate of thisstructure was of particular interest.

RESULTS

Light microscopy. Observations by light micros-copy confirmed many of the previous observa-tions of the conjugation process (Guilliermond,

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Figure 1. An oblique section of two cells during an initial stage of conjugation. Structures labeledM are double membrane chambers containing cristae. Note particularly the prominent cytoplasmic mem-brane, and the partial fusion of the cell walls. Small, unidentified, electron-dense incltusions (G) canalso be observed. Few lipoidlal inclusionis are observable, and vactuoles appear to be absent.

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Figures 2 and 2a. Figure 2 is a micrograph of two cells immediately prior to cytoplasmic fusion. Thecytoplasmic rnembranes (at site of arrow) are thin, distinct, and still intact. Figture 2a illustrates thatdissolution of the central portion of the connecting cell wall occurs, thereby leading to cytoplasmicftusion (arrows point to area of cytoplasmic intermixing).

Figutre 3. This micrograph illustrates the increase in length and width of the conjugation canal causingthe ascus to appear halter-shaped. Note that the nuclei appear to be migrating towards the canal.

1920; Widra and DeLamater, 1955). The con-jugation process appears to be initiated by theattachment of the cells in pairs by a portion ofthe cell wall. The area of contact then increases,and the cells appear to be firmly joined by a com-mon cell wall. The central portion of the con-necting cell wall then disintegrates, thereby form-ing a short, narrow conjugation canal. This canalsubsequently increases both in length and width,giving the ascus a dumbbell appearance. Furtherincrease in the width of the conjugation canalgives the ascus a rectangular shape. Ascosporeformation appears to occur after the ascus hasassumed a rectangular appearance. It appeared,contrary to the observations of Guilliermond(1920), that the copulation canal is not formedby the fusion of short projections from each cell.

Examination of actively dividing vegetativecells of S. octosporus by means of phase micros-copy and staining with dilute Lugol's solutionand Sudan black B revealed that a vacuole wasnot present, and lipoidal inclusions lacking or fewin number (Conti and Naylor, 1959a). Similarstudies of conjugating cells revealed that vacuolescould be clearly observed only in cells joined bya well developed conjugation canal. There wasno increase in the number of inclusions stainablewith Sudan black B3. Tetrazolium salts stainingof actively dividing vegetative cells, and of copu-lating cells revealed a noticeable increase in thenumber of stainable bodies (mitochondria) duringthe initial stages of copulation.

Electron microscopy. Figures 1 to 10 are electronmicrographs of S. octosporus illustrating suc-

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Figure 4. Migration of the two ticlei towards one another, immediately prior to fusionFigure 5. Nuclear fusion. Note that the ntuclear membrane remains intact except at the site of fu-

sion. The presence of an internial membrane system is also apparent.

cessive stages of copulation and nuclear division.The sequence of events is basedl on the prelimi-nary studies by phase microscopy, and previousreports of other investigators (Guilliermond,1920; Widra and DeLamater, 1955). The shapeand size of the growing ascus was useful in re-constructing the successive stages from electronmicrographs. Widra and DeLamater (1955) alsoused the shape and size of the ascus as an in-dependent indicator of timing and reported thatthere was a correlation between nuclear develop-ment and the appearance of the ascus.

Nuclei are regularly observed in sections ofconjugating cells during both initial and laterstages of the conjugation process. The structurelabeled N3 in the micrographs is considered to be

I CC = conjugation canal; CM = cytoplasmicmembrane; CW = cell wall; IM = internal mem-branes; L = lipoidal inclusion; Al = mitochon-drion; N = nucleus; and, V = vacuiole.

the nucleus on the basis of its behavior duringcell division (Conti and Naylor, 1959a) and theoccurrence and behavior of a similar structure individing (Hashimoto et al., 1959), sporulating(Hashimoto and Gerhardt, 1959), and germinat-ing cells (Hashimoto et al., 1958) of Saccharomycescerevisiae. The nuclei in conjugating cells appearto be similar in structure to the nuclei of dividingcells (Conti and Naylor, 1959a). Nuclei are sur-rounded by a double membrane, are ovoid tospherical in shape, and the nucleoplasm appearsto be granular and homogeneous in texture.The ascus wall, like that of the vegetative cell

wall, was relatively wide, and appeared to be oflow electron density in electron micrographs. Thecytoplasmic membrane appeared to be double,and closely adherent to the ascus wall. Sphericalor elliptical inclusions, which appear to be emptyin the micrographs, are similar in size, shape,

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and number to the structures stainable witiSudan black B.

Figure 1 illustrates the appearance of twovegetative cells during the initial stages of copu-lation. It can be seen that the cell walls are atleast partially fused, with no indications of theprior formation of projection tubes. Nuclei can-

6

not be observed due to the plane of sectioning.The absence of nuclei facilitates observations ofother cytostructures within these cells. In agree-ment with the results from the light microscopystudies, vacuoles were not present, and lipoidalinclusions absent or few in number. Structureshaving circular or ellipitical profiles, and pos-

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Figure 6. This figure illustrates the dumbbell appearance of the ascus, and the size of the nucleussubsequent to nuclear fusion. Note that the fusion nucleus (approximately twice the size of the vege-tative nucleus) spans almost one-half of the length of the ascus. A conspictuous vacuole can be readilyobserved. Note also the low-electron dense, and continuous ascus wall.

Figure 7. A cross section of a mature ascus, i.e., an ascus in which the conjugation canal is no longerevident. At this stage, immediately prior to nuclear division, the nucleus appears to be contracted, andassumes an irregular shape. Note again the conspicuous cytoplasmic membrane.

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Fiqgure 8. This particularly informative micrograph illustrates the behavior of the nucleus duringthe first division. The nucleus appears to elongate, and divide by a process of constriction. Note thatthe vacuole is separated from the dividing nucleus, and appears to contain granutlar or amorphousmaterial. It is evident that the nuclear membrane remains intact.

sessing an internal membrane system, can be ob-served in the cytoplasm of the conjugating cells.These structures are considered to be mito-chondria on the basis of their ultra structure inelectron micrographs, and the number and sizeof similar structures in conjugating cells stainedwith tetrazolium salts and observed with thelight microscope. These mitochondria are ran-domly distributed throughout the cell, and ap-pear to be similar in structure to the mitochondriaof other yeast cells (Agar and Douglas 1957;Hashimoto et al., 1958, 1959). Previous studies(Conti and Naylor, 1959a) revealed that themitochondrialike structures were few in numberin dividing vegetative cells, and cristae difficultto observe. It thus appears that during the initialstages of copulation, cellular reorganization in-

eludes an increase in number of mitochondria, andan alteration of mitochondrial structure.

Subsequent to cell wall fusion, the centralportion of the connecting cell wall appears to dis-integrate (figures 2 and 2A), resulting in a cyto-plasmic intermixing. The copulation canal thenappears to increase in both length and width,giving the ascus a characteristic appearance (fig-ure 3). At this stage of the process the nuclei thenbegin to migrate towards the copulation canal(figure 4). Nuclear fusion then occurs (figure 5).It is interesting to note that the limiting mem-branes of the fusing nuclei remain intact, exceptin the region where fusion occurs. Nuclear fusionwas always found to occur within the conjuga-tion canal. It is pertinent to mention at this pointthat vacuoles do not appear to be present in the

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ascus until nuclear fusion has occurred (figures1-6). The preliminary studies employing iodinestaining, and observations by phase microscopy,also indicated that vacuoles were formed in themajority of cells during, or immediately after,fusion nucleus formation. The vacuole, whenpresent, usually appeared to contain granular oramorphous material.

Figure 6 illustrates the appearance of theascus after the fusion of the nuclei. Note thecharacteristic dumbbell appearance of the ascusand the conspicuous vacuole. The fusion nucleus(presumably diploid) appears to be elongate inshape, and spans approximately half the lengthof the ascus. Calculations from electron micro-graphs of the volume of the nucleus at this stagereveal that the fusion nucleus is approximatelytwice that of the vegetative cell nucleus.

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The fusion nucleus, soon after its formation,often appears to assume an irregular shape (figure7). Presumably, this stage precedes division of thenucleus by the process of constrictioni illustratedin figure 8. This figure is particularly informative.It clearly demonstrates that the vacuole is sepa-rated from the dividing nucleus, and that thenuclear membrane remains intact during thefirst division of the nucleus. The process of nu-clear division can best be described as a constric-tion within the central region of the nucleus,yielding two nuclei of equal size, and presumablyalso equal in content.The two nuclei then migrate to opposite ends

of the ascus (figures 9A and 9B). At this stagethe nuclei again appear to begin to constrict andseparate, thereby resulting in the formation offour nuclei. Note particularly the size of the

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Figure 9 and 9a. These micrographs illustrate that after the first division, the nuclei migrate to op-posite ends of the ascus. Note that the nuclear membrane is still intact, and that these nuclei appearto be approximately the same size as vegetative cell nuclei.

Figure 10. The size of the nuclei, shape of the ascus, and the observable constriction (NC) of one ofthe nuclei indicates that the second nuclear division is occurring. Note again that the nuclear membranesare still intact. The presence of the internal membrane system and low-electron dense ascuis wall arestill apparent.

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nuclei in figure 10, and the invagination of thenuclear membrane of one of the nuclei, indicatingthat constriction of the nucleus is occurring. Un-fortunately, electron micrographs which couldclearly illustrate the subsequent stages of nu-clear division were not obtained. Some electronmicrographs indicated that nuclear division con-tinued in the manner described, resulting in theformation of eight nuclei, each being surroundedby a limiting membrane. The interpretation ofthese micrographs is, however, difficult due to avariety of reasons, and therefore subject to criti-cism. Attempts are now being made to obtaincomplete serial sections of a single cell in order toovercome difficulties in interpretation.

DISCUSSION

The observations made by means of electronmicroscopy are generally in agreement with theprevious observations by light microscopy(Guilliermond, 1920; Widra and DeLamater,1955; Yoneyama, 1958). Although there areseveral points of disagreement, the previous ob-servations by light microscopy and the presentstudies by electron microscopy clearly supple-ment each other.Widra and DeLamater (1955) utilizing re-

fined nuclear staining techniques were able toobserve the behavior of the nucleus during con-jugation and sporogenesis of S. octosporus. Theseinvestigators, however, found it difficult to dis-cern the presence or absence of a nuclear mem-brane due to the resolution limit of their opticalsystem. The present studies by electron micros-copy, however, illustrate that the nuclear mem-brane persists throughout the first two stages ofnuclear division, and probably also persiststhroughout the third division. The observationsof Widra and DeLamater on the size, locationand behavior of the nucleus, particularly duringearly stages of conjugation, are in close agree-ment with the results of the present study. It isclear that the structure stained and observed byWidra and DeLamater corresponds to the struc-ture considered to be the nucleus in the electronmicrographs. These authors, however, also ob-served chromosomes within the nucleus, andreported the occurrence of a classical meioticprocess during nuclear division. Chromosomescould not be observed in the nucleus utilizing thetechniques employed in the present study. Thedifficulties involved in observing intranuclearstructure particularly chromosomes, by means

of ultrathin sectioning and electron microscopyare briefly discussed by Cosslett (1958) and Contiand Naylor (1959a). It is apparent, however,that despite the limitations of both light andelectron microscopy, observations by both tech-niques can be closely correlated. It is also ap-parent that investigation of structure and be-havior of chromatin material within the yeastnucleus by electron microscopy must await fur-ther development and refinement of techniques.Application of methods in this laboratory, similarto those employed by Moses (1956), to a studyof the yeast nucleus have not been successful todate. Until further techniques are developed thedescription of structure and behavior of the intra-nuclear material of the yeast cell remains pri-marily in the realm of light microscopy. Presentstudies in this laboratory utilizing various nuclearstaining techniques indicate the presence ofchromosomelike structures within the membranesof ascus nuclei of S. octosporus, however classicalmeiotic figures have not been observed. Cutter(1951), and Ganesan and Swaminathan (1958)among others, are aware of the deficiencies ofthe techniques of light microscopy that are em-ployed to study the fungal and yeast nucleus andcite the need for the development of more re-fined procedures. It is hoped that newer tech-niques for the study of yeast and fungal nucleiwill become available in the near future.

Guilliermond (1920) indicates that conjugationis initiated by the formation and fusion of pro-jections from each cell. Although the appearanceof conjugating cells under the light microscopemay give this impression, this apparently is notthe case. The evidence obtained in the presentstudy by employing both light and electron mi-croscopy strongly indicates that such projectionsare not formed. The views of Guilliermond areprobably due to the difficulty in distinguishingbetween cell wall material and cytoplasm-in un-stained yeast cells examined with the light mi-croscope. The conflicting observations also maybe attributed to differences in strains andmethods of inducing copulation and sporulation.Widra and DeLamater (1955) state that

t two vegetative haploid cells unite bymeans of fusion tybes to form an ascus." Evalua-tion of their evidence for this statement is difficultsince their micrographs are poorly reproduced,and the figures referred to are of cells which havebeen subjected to nuclear staining. The use of theterm "fusion tubes" is also unfortunate since

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these authors may be referring to the copulationcanal. It is recommended that the term "fusiontube" not be used in the description of the earlystages of copulation in yeast.The process of copulation in the yeast cell has

been described by Lindegren (1951). The be-havior and identification of various structures,and particularly the involvement of the vacuolein the conjugation process, are not confirmed.The evidence from the present study supportsthe view of Widra and DeLamater (1955) andGuilliermond (1920) that the vacuolar contentsare ergastic in nature and that the vacuole is notan integral part of the yeast nucleus. The struc-ture and behavior of the organelle consideredto be the centrosome by Lindegren, seems tocorrespond to the structure considered to be thenucleus in this study.

These studies shed no light on the question ofthe presence of an intranuclear nucleolus inyeast (Caspersson and Brandt, 1941; Lietz, 1951;Widra and DeLamater, 1955). A structure cor-responding to the centriole also was not observedin any electron micrographs. The failure to ob-serve these structures in electron micrographsdoes not establish or indicate that these struc-tures are not present. The inability to observesuch structures may be ascribed to a variety offactors (Moses, 1956; Cosslett, 1958; Conti andNaylor, 1959a).The cytological changes accompanying asco-

spore formation ("free cell formation") andgermination of ascospores are currently underinvestigation.

SUMMARY

Electron micrographs illustrate the cytologicalchanges occurring during plasmogamy, kary-ogamy, and subsequent nuclear division inSchizosaccharomyces octosporus. The conjugationprocess appears to be initiated by the attach-ment of the cells in pairs by a portion of the cellwall. Fusion of cells by means of projection tubeswas not observed. The central portion of theconnecting cell wall then disintegrates, therebyforming a short, narrow conjugation canal whichsubsequently increases both in length and width.At this stage, the nuclei migrate toward oneanother, and fuse within the canal. The limitingmembranes remain intact except in the region ofnuclear fusion. Subsequent nuclear division ap-pears to occur by a process of constriction. Intra-nuclear structures were not observed.

The structure and behavior of other com-ponents of the yeast cell during the conjugationprocess are also described. Observations on thevacuole lend further support to the view that thevacuole is not an integral part of the nucleus.

REFERENCES

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CASPERSSON, T. AND BRANDT, K. M. 1941 Nu-cleotidumsatz und Wachstum bei Presshefe.Protoplasma, 35, 507-526.

CONTI, S. F. AND NAYLOR, H. B. 1959a Electronmicroscopy of ultrathin sections of Schizo-saccharomyces octosporus. I. Cell division. J.Bacteriol., 78, 868-877.

CONTI, S. F. AND NAYLOR, H. B. 1959b Conju-gation, sporulation, and ascospore structureof Schizosaccharomyces octosporus. Bacteriol.Proc. 1959, 50-51.

COSSLETT, V. E. 1958 Recent developments inelectron microscopy. Brit. J. Appl. Physics,9, 253-256 and 273-275.

CUTTER, V. M., JR. 1951 The cytology of thefungi. Ann. Rev. Microbiol., 5, 17-34.

GANESAN, A. T. AND SWAMINATHAN, M. S. 1958Staining the nucleus in yeasts. Stain Tech-nol., 33, 115-121.

GUILLIERMOND, A. 1901 Recherches histolog-iques sur la sporulation des Schizosaccharo-myc&tes. Compt. rend., 133, 242-244.

GUILLIERMOND, A. 1917 Sur la divisionnucl6aire de levures. Ann. inst. Pasteur, 31,107-113.

GUILLIERMOND, A. 1920 The yeasts. TranslatedF. W. Tanner. John Wiley & Sons, Inc.,New York.

HASHIMOTO, T. AND GERHARDT, P. 1959Structural development during yeast sporo-

genesis. Bacteriol. Proc. 1959, 49.HASHIMOTO, T., CONTI, S. F., AND NAYLOR, H. B.

1958 Fine structure of microorganisms. III.Electron microscopy of resting and germi-nating ascospores of Saccaromyces cerevisiae.J. Bacteriol., 76, 406-416.

HASHIMOTO, T., CONTI, S. F. AND NAYLOR, H. B.1959 Studies of the fine structure of micro-organisms. IV. Observations on buddingSaccharomyces cerevisiae by light and electronmicroscopy. J. Bacteriol., 77, 344-354.

HOFFMEISTER, C. 1900 Zum Nachweise desZellkerns bei Saccharomyces. Lotos, 20, 250-262.

LIETZ, K. 1951 Beitrag zur Hefecytologie.Arch. Mikrobiol., 16, 275-302.

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LINDEGREN, C. C. 1951 The mechanics ofbudding and copulation in Saccharomyces.Exptl. Cell Research, 2, 305-311.

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SATIR, P. G. AND PEACHEY, L. D. 1958 Thinsections. II. A simple method for reducingcompression artifacts. J. Biophys. Biochem.Cytol., 4, 345-348.

SCHIONNING, H. 1895 Eine neue und eigen-tutmliche Ascusbildung bei einem Hefepilz(Abstract). Botan. Jahresber., 1, 172.

WIDRA, A. AND DELAMATER, E. D. 1955 Thecytology of meiosis in Schizosaccharomycesoctosporus. Am. J. Botany, 42, 423-435.

YONEYAMA, M. 1958 Some observations on themultiplication and sporulation of Schizo-saccharomyces versatilis. Seibutsugakkaishi8, 9-13 (Biol. Soc. of Hiroshima Univ., Hiro-shima, Japan).

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