LIFE CYCLE AND NUCLEAR BEHAVIOR OF A SPECIES OFTHE YEAST GENUS SCHWANNIOMYCES

J. D. FERREIRA' AND H. J. PHAFF

Department of Food Technology, University of California, Davis, California

Received for publication February 16, 1959

The genus Schwanniomyces was established byKloecker (1909) who isolated the yeast from soilin the Antilles. The single species Schwanniomycesoccidentalis forms one or rarely two spores perascus. The spores have the shape of a walnut,are warty, have a ledge around the equator, andcontain pronounced lipid globules in the center.Conjugation tubes are formed before sporulationbut these do not complete conjugation with tubesof other cells. During germination only one halfof the spore swells and budding occurs on thatside. The other half remains intact and forms asort of cap on the thin-walled half which isgerminating. Guilliermond (1928) essentially con-firmed Kloecker's description. Stelling-Dekker(1931) also confirmed previous observations, butfound only one spore per ascus. She stated thatthe asci formed parthenogenetically. Phaff andMrak (1948), who reviewed the knowledge ofyeast life cycles, pointed out that the life cycleof Schwanniomyces and its nuclear behaviorremained to be worked out. Hjort (1956) re-ported a single instance where conjugation be-tween two cells of Schwanniomyces was observed.He postulated that alternation of generations oc-curs in this genus either by conjugation of twocells or by intracellular fusion of nuclei, but hedid not offer proof of this hypothesis. Recently,Capriotti (1957) isolated and described a secondspecies in the genus, which he named Schwannio-myces castellii. No details of the nuclear behaviorwere reported however. The purpose of this com-munication is to describe in detail the nuclearbehavior during sporulation of a new species ofSchwanniomyces. In addition, information willbe presented regarding its ploidy, mitotic divisionof the nucleus, and germination of the ascospores.

MATERIALS AND METHODS

Yeast culture. The yeast used in these investi-gations was a strain of a new species of the genus

1 Fellow of the Gulbenkian Foundation, Lisbon,Portugal. Present address: Estagao AgronomicaNacional, Oeiras, Portugal.

Schwanniomyces (no. 54-83) isolated from soilof the bank of Lytle Creek, Clinton County,Ohio, by Dr. W. B. Cooke. It will be named anddescribed in a separate publication. (Antonie vanLeeuwenhoek. J. Microbiol. Serol.)X-ray survival. The method of determining the

degree of ploidy of the vegetative cells wasessentially that of Beam et al. (1954). Thesource of X-rays was a low voltage berylliumwindow X-ray tube operated at 50 kv and 25ma. The dose rate was 246 roentgen/sec with thematerial 15.65 cm from the tube target.

Preparation of cells for X-ray studies. Cells weregrown for 24 hr on slants of yeast autolysateagar + 2 per cent glucose. Starvation beforeX-radiation is desirable (Beam et al., 1954), butin the case of Schwanniomyces, starvation resultsin the formation of one or more buds whichremain attached to the mother cell. Buds areeven formed if starvation is done in the absenceof nitrogen. Cells from a young slant cultureoccur mostly singly or in pairs, whereas liquidcultures produce many clusters. To eliminate asmany pairs of cells as possible, the suspensionfrom a slant culture was filtered asepticallythrough a glass tube containing cotton and drawnout to a capillary at one end. Filtration tookplace by gravity. This increased the percentageof single cells from about 50 to 70 per cent.Suspensions of cells in 0.05 M KH2PO4 werespread in known concentrations on plates con-taining yeast autolysate glucose agar. The cellnumbers varied with the dosage of X-rays re-ceived. All platings and irradiations were done insextuplicate. The colonies were counted after 48hr growth at room temperature. Uninoculatedplates were irradiated 5 min (maximal period ofirradiation used) and then inoculated and com-pared with nonirradiated plates. No toxic prod-ucts were formed as a result of irradiation.

Staining procedure. Nuclear staining was doneas follows. A very small droplet of Meyers ad-hesive (Johansen, 1939) was placed on a cleanslide with the aid of a loop, followed by a drop-

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let of cell suspension in distilled water. The twodroplets were mixed well and spread evenly overthe slide with a loop and allowed to dry at roomtemperature. Of a number of fixatives used,Schaudinn's fluid gave the best results. Fixationwas done by immersing the slide for 20 min inSchaudinn's solution, followed by washing withdistilled water. Digestion was tried with per-chloric acid at 5 C (Cassel, 1950) and withhydrochloric acid at various elevated tempera-tures and for various times (Jones, 1950). Bestresults were obtained with hydrochloric acid at60 C for 4 min when 18-hr cultures were stained.-Sporulating cultures (4 days old) were digestedfor 6 min. The slides were washed with distilledwater and stained for 24 hr with Azure A (certi-fication no. NAz 15, Allied Chemical and DyeCorporation, New York) plus S02-water. Weused 0.25 per cent of Azure A (DeLamater, 1951)and added 8 drops of S02-water to 30 ml ofAzure solution. The SO2 solution was made byadding 5 ml of 10 per cent solution of anhydrousNaHSO3 and 5 ml of 1 N HCl to 100 ml of dis-tilled water (Winge and Roberts, 1954). Lesssatisfactory results were obtained by the Feulgentechnique. The slides were washed with water,followed by S02-water to remove excess stain(clearer background) and finally with distilledwater, and dried in air.

Sporulation. Best sporulation was obtained onthe following medium: distilled water, 90 ml;yeast autolysate, 10 ml; glucose, 2 g; and agar,2 g. The yeast autolysate was prepared frombaker's yeast according to the procedure ofBouthilet et al. (1949). Sporulation began after3 days at room temperature, and, after 5 days,about 15 per cent of the cells transformed into

asci. Nearly all asci contained one spore. Asmall percentage of asci had two spores.

Single asci or single spores were isolated withthe aid of a de Fonbrune micromanipulator.Single asci were removed from a suspension of asporulating culture in a small droplet on a coverslip in a moist chamber and deposited on a verythin layer of yeast autolysate-glucose agar onthe same cover slip. The cover slip was thenplaced on a closed ring of another moist chamberand observed for germination.

EXPERIMENTAL RESULTS

Mitosis in Schwanniomyces. The mitotic pic-ture was obtained by nuclear staining of an18-hr culture. Cells without buds were uninucle-ate. Our observations have shown that the mi-totic division occurs nearly always in the mothercell rather than in the connection between mothercell and bud. After the daughter nuclei haveseparated, one nucleus moves into the bud andthe other remains in the mother cell. The lattermay immediately start a second mitotic divi-sion and its daughter nucleus then moves into asecond bud formed on the mother cell. Themitotic division takes place by an interesting andapparently unusual process. A schematic outlineof the series of events leading to the duplicationof the nucleus is given in figure 1. Photomicro-graphs are shown in figure 2 (18-hr culture).Since some of the mitotic figures stain moreclearly in 4-day-old cultures, a few of the stagesshown in figure 1 are illustrated in figure 3 inwhich cell material of this age was used. Theprobable reason for a clearer mitotic picture in4-day-old cells is that 18-hr cells appear toshrink during the staining process whereas older

. :A*F

A B C D E F G

H I J K L M NFigure 1. Schematic drawing of the various stages of mitosis in Schwanniomyces. The dotted lines

in E, F, and G indicate that a bud may or may not have formed at these stages. See text for full details.

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Figure 2. Various stages during mitosis in 18-hr cultures. A, cells indicated by arrows show rings ofwhich certain sections are not in focus; B, semicircle of chromatin material; a swelling has formed at one

end; C, the strand has become shorter, and a swelling occurs at both ends; D, E, and F, various examplesof continued shortening of the chromatin strand, the swollen terminals approach each other more andmore; G and H, the darkly stained bodies are very close and the connecting strand breaks; I, J, and K,the nuclei separate and the daughter nucleus moves into the preformed bud.

cells retain their natural size, more or less (figures2 and 3, which have the same magnification).The first stage consists of the formation of a

ring-like structure (figures iB; 2A; 3B, and C).Presumably during this stage the chromatin ma-

terial duplicates itself. In young cultures this ringis difficult to photograph, since all parts of thering are rarely in focus at the same time. Therings usually contain more or less pronouncedbead-like bodies. 'T'hey are most striking in 4-day-old cell material. Often these bodies seem tooccur in pairs and their total number approacheseight, but never exceeds this number. The dis-tribution of these bodies on the ring (four on

each half) suggests that the original number may

have been four and after ring formation andduplication the number becomes eight. Inthe second stage the ring breaks and the resultingstrand may assume a number of characteristicshapes (figure 1C, D, E, F, and G). Some ofthese are illustrated in the photomicrographs offigure 3. In figure 3D, the ring is broken and a

strong differentiation into bead-like structuresoccurs which are connected by a strand, whichis not in focus in its entirety in figure 3D. Thesame applies to figure 3E in which the strandassumes the shape of the Greek letter R. Infigure 3F the strand has shortened and hasassumed an S-shape. During the third stage thestrand becomes progressively shorter. This is

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Figure 3. Various stages during mitosis in cell material 4 days old. A, resting nucleus; B, ring-stage;C, ring containing several bead-like bodies. This ring represents a supernumerary mitosis, since meiosisis underway in the bud; D, ring is broken and it clearly shows granules. The two top granules form thetwo ends of the broken strand; E, the strongly beaded strand in the shape of the Greek letter Q; F, thestrand in the form of the letter S.

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S shown schematically in figure IH, I, J, K, and73.8 in the photomicrographs 2C, D (top), and E

(lower right). Finally the strand becomes veryshort and the two terminal bodies, which areto become the two daughter nuclei, approacheach other very closely. This is shlown in figures1L and 2D (bottom), 2E (top), F, G, and H.In the fourth stage the strand breaks, the twodaughter nuclei separate, and one moves intothe bud. This is shown in figures i1M, N, and in21, J, and K.

Ploidy of the vegetatime cells. The results ofX-radiation on 24-hr cells spread on yeast autol-ysate-glucose-agar plates are shown in figure 4.The survival curve is very typical of a single-hit curve, which is indicative of a haploid culture.

250 300 When the linear portion of the curve is extra-polated to zero X-ray dose it intercepts exactly

of Schwannio- at 100. The LDso of the culture of Schwannio-myces was about 4000 roentgens. This compares

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Figure 5. Diploidization and meiosis in Schwanniomyces in 4-day-old cell material. A, result of mi-totic division of the haploid nucleus; B, C, the two haploid nuclei move towards the "bud". Karyogamyhas occurred in D and the diploid nucleus moves into the "bud" (E). F and G show supernumerary mi-totic divisions in the mother cell; G also has a diploid nucleus in the "bud". The first meiotic division isshown in H, I, J and K. A good example of the mitotic ring-stage in the mother cell is shown in J.The second meiotic division is underway in L, resulting in a tetrad shown in M. M also has two extranuclei in the mother cell. N shows a tetrad in one "bud" and a single (diploid?) nucleus in a second"bud."

well with a figure of 3100 r for a haploid cul-ture of Saccharomyces cerevisiae (Beam et al.,1954) and 3500 r for another haploid strain of S.cerevisiae as reported by Lucke and Sarachek(1953).Nuclear behavior during sporulation. Since

Schwanniomyces was shown to be a haploid yeast,diploidization must occur prior to meiosis. Thiswas observed in cultures grown for 4 days onsporulation agar. The process starts with amitotic division of the haploid nucleus in themother cell as described above, whereupon thetwo daughter nuclei usually move to oppositesides of the cell (figure 5A). At this stage aspecial type of bud has formed which remains at-tached to the mother cell by a wide neck. Thesebuds (sometimes there are two or three) are theso-called "conjugation tubes" referred to in the

older literature. Our observations have con-sistently shown them to be bud-like structuresrather than tubes. The two nuclei then movecloser to each other and in the direction of thebud (figure 5B and C). Presumably, karyogamytakes place at about the time when the twonuclei have nearly reached the opening of thebud, or possibly after having entered the bud.Thus, figure 5D probably represents a diploidnucleus entering the bud, and figure 5E showssuch a nucleus in the bud.

Reduction division was found to take place inthe bud. This, for the first time, explains thefunction of the "conjugation tubes" or specialbuds which are found on nearly all asci. We sug-gest the name meiosis buds for these structures,since the name indicates their function. The firstmeiotic division is illustrated in figure 5H, I, J,

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Figure 6. Various stages in the formation of a spore primordium. A, one nucleus is just entering thespore primordium, whereas the other three nuclei from the tetrad are still in the meiosis bud. B, all fournuclei of the tetrad have moved back into the mother cell. C, all nuclei appear to be enclosed in the sporeprimordium. D, the spore primordium contains at least one nucleus, whereas three nuclei can be seenoutside the spore primordium.

and K. We interpret the photomicrographsshown in figure 5H, I, J, and K as the firstmeiotic division. A very early stage is shown infigure 5H where the "ring-stage" can be ob-served. The wall of the bud is not in focus in thispicture but it can be seen as a faint outline.Very often one or more supernumerary mitoses

occur prior to diploidization, resulting in threeor more nuclei. While two nuclei undergo kary-ogamy the extra nuclei may continue to divide,resulting in several haploid nuclei in the mothercell (figure 5F, G, I, J, and K).

Figure 5J in particular shows the abovementioned ring (early mitotic stage) veryclearly. It is assumed that the purpose of thesesupernumerary mitoses in the mother cell is toprovide nuclei for subsequent diploidization andmeiosis in a second or third bud attached to themother cell. Figure 5N shows a mother cell withtwo buds. In this case, meiosis in the top bud hasresulted in four haploid nuclei, whereas the sidebud contains' only a single, presumably diploid,nucleus. Figure 5M shows a quartet of fournuclei in the bud and two nuclei in the mothercell. Meiotic division appears to occur by asimilar process as is illustrated schematically infigure 1. The second meiotic division is shown infigure 5L. Two strands are visible. One is repre-sented by a heavily stained semicircle withswollen ends. The other is a more faintly out-lined loop for the most part inside the semicircle,except for the bend (middle portion) which iscovered by the heavier strand. Various granulescan be seen clearly in the faintly outlined strand.

After meiosis is complete, the nuclei migrateback into the mother cell (figure 6A and B)

and sporulation sets in. Only the very earlystages of spore development can be shown instained preparations, since spores of Schwan-niomyces, in contrast to those of Saccharomyces,are covered with a very dense outer envelope(possibly related to the warty surface) whichstains heavily and does not allow any light topass through. Figure 6A, B, C, and D showsvarious stages in the formation of a spore pri-mordium. In some cases, several nuclei can beseen outside the spore primordium, but in othercases the spore appears to contain all four of thenuclei resulting from meiosis (figure 60). It maywell be that the nuclei resulting from super-numerary mitoses remain outside the spore(figure 6B) or they may become incorporated ina second spore in the case of two-spored asci. Itmust be assumed that a mature spore contains avariable number of haploid nuclei.Abnormal diploidization and reduction division.

In rare cases we found that diploidization oc-curred as a result of conjugation between twocells. The various stages are shown in figure 7.Figure 7A shows two uninucleate cells in anearly stage of conjugation, followed by a secondstage (7B) in which the nuclei appear to havefused or possibly have begun meiosis. Figure 7Cshows a quartet of four nuclei in the conjugationtube. In this zygote, supernumerary nuclei areclearly visible.

Sporulation. An illustration of asci containingone or two mature ascospores is given in figure 8.The spores contain a distinct lipid globule, anequatorial ledge, and have a warty surface.

Single spore cultures. Single spores were isolatedfrom one-spored asci and from two-spored asci.

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4VA .B C.

Figure 7. Conjugation of two cells (A) followed by karyogamy (B) and meiosis (C). Figure 7C alsoshows supernumerary nuclei.

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oFo>=(W/e (4,Figure 8. A wet mount containing several single-spored asci and one two-spored ascus. In several

cases the ledge around the spores is distinct. All asci have an empty meiosis bud.

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HFigure 9. Germination of a one-spored ascus. A, intact ascus; B, 12 hr after isolation; C, after 13 hr;

D, after 134 hr; E, after 14'2 hr; F, after 16 hr; G, after 17 hr; H, after 18 hr. The spore formed fourseparate buds on its surface (B, C, E, G). The original meiosis bud is still visible in the first 6 pictures.

About half of the isolated spores germinated. sporic cultures sporulated as well as the parentWith two-spored asci sometimes only one of the strain. This indicates the homothallic nature oftwo spores germinated, whereas in other isola- the yeast. Always, three or four buds formedtions both spores grew. In all instances, mono- successively on the same spore.

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Figuire 10. I)rawing of a germinating two-spored ascus in which both spores germiniiated. Temperature22 to 24 C. A, ascus when it wN-as isolated; B, after 21 hr. Both spores are swollen; C, after 24 hir. Theascus has ruptured and one ol the spores is producing a bud; D, after 27 hr, when both spores are budding.After 31 hr a microcolony had formed consisting of 16 cells.

A B

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D EFigure 11. Drawing of a germinating two-spored ascus as observed microscopically. A, whern iso-

lated; B, after 26 hr. One spore is markedly swollen. C, after 34 hr. Beginning of germination of theswollen spore; D, after 43 hr a secondary bud has formed, which in turn is budding; E, after 54 hr. Twomore buds have been formed on the original spore and budding continues towards the right. The spore

adjacent to the meiosis bud was apparently unable to germinate. The germination of the other spore was

rather slow. Temperature, 22 to 24 C.

Figure 9 illustrates the germination of a single-spored ascus. It will be noted that in accordancewith Kloecker's (1909) original observation onehalf of the spore wall remains thick and highlyrefractive. This was called the cap in earlierpublications. After producing the original germi-nation bud, the spore produced successivelythree more buds. The fourth bud is not visible in

the photographs.In two-spored asci in which both spores

germinated, each spore produced germinationbuds which in turn produced secondary buds.No evidence was foun(d for conjugation betweentwo germinating spores. A drawing of the germi-nation of a two-spored ascus, based on micro-scopic observations, is given in figure 10. Figure

II shoxvs five stages in the germination of atwo-spored ascus, in whiich only onie spore grew.

D)ISCUSsION

The literature contalins no prev'ious stuiliesregarding the nuclear behavior during alterna-tion of generations in Schwanniomyces. M1ostinvestigators refer to the presence of abortiveor primitive conjugation tubes dluring sporulation.and assume that these have lost the ability toconjugate with those of other cells. Conjugationbetween two haploid cells is characteristic inZygosaceharomyces and in comparable haploidy-easts. Our observations have shown that thesetubes have a swollen tip (as is also evident fromKloecker's original drawing in 1909) and they

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should be considered as specialized buds with adefinite function, a chamber in which meiosisoccurs. These meiosis buds are empty exceptduring meiosis, after which the haploid nucleimove back into the mother cell. Yet, thereseems to be a relation between Schwanniomycesand haploid species of Saccharomyces (Zygosac-charomyces), since in rare instances Schwannio-myces follows the nuclear behavior as it occursin Zygosaccharomyces. This observation con-firms the single instance in which Hjort (1956)observed conjugating cells in Schwanniomycesoccidentalis. Whether this type of nuclear be-havior during sporulation also occurs in thegenera Debaryomyces and Torulaspora, in which"mother-daughter cell conjugation" or abortiveconjugation tubes have been described, remainsto be worked out.Supernumerary mitoses in young ascus mother

cells (in 4-day-old cell material on a sporulationmedium) are fairly common and two of theseextra nuclei, after karyogamy, may move into asecond meiosis bud on the mother cell. Althoughthe nuclear picture of a mature spore cannot beobserved easily in stained preparations due tothe extremely dense stainable material it con-tains, it appears from the cell contents outsidethe spore that most or in some cases all thenuclei are incorporated in the spore. The nucleinot incorporated into the spore presumably de-generate. Since during spore germination threeto four buds appear on the germinating spore, itis possible that each of these buds receives oneof the nuclei present in the spore. No spore con-jugation was observed during germination oftwo-spored asci or between buds arising fromsingle spores and the haploid nature of thevegetative cells (as shown by an X-ray survivalcurve) is in agreement with this.The behavior of the nucleus during mitosis as

revealed in stained preparations appears dif-ferent from the nuclear behavior described byvarious investigators working with differentspecies of yeast. We have described the variousstages observed in stained preparations, ratherthan applying the classical terminology used todenote the various phases of mitosis in higherplants. and animals. The formation of a ring ofchromatin material, followed by contraction ofthe strand after breaking of the ring and theformation of two distinct bodies at each endwhen the strand shortens are the most character-istic features of the process. Final separation

into two daughter nuclei, when the strand is veryshort, completes mitosis. These structures aremore distinct in cells which are about 4 daysold than in very young cells. Presumably, dupli-cation of the chromosomes occurs during forma-tion of the ring. Two equal halves are formed,which face each other in mirror image. Whetherthe bead-like structures visible on many of therings or strands shown in the various photo-graphs are chromosome-like bodies is a matterof conjecture. It is striking, however, that inwell stained material the number of these- bodiesappears to be constant, i. e. a maximal numberof eight can often be observed, although in somecells not all eight bodies are clearly visible. As aresult, each haploid nucleus after completion ofmitosis, presumably receives four of these bodies.Especially when the ring or strand is large thebead-like bodies can be observed in pairs, spacedat certain intervals from the next pair. A carefulanalysis of the photographs of nuclear behaviorin Saccharomyces by other investigators (e. g.,Winge, 1951; Winge and Roberts, 1954; De-Lamater, 1950) shows structures quite similar tothe ones observed here. It is possible that thetype of nuclear division described here forSchwanniomyces occurs also in other yeasts.One wonders whether there might be any

relation between the rings and chains describedhere and the chromosome catenations observedin Oenothera (Gates and Ford, 1938; Cleland,1936) and the chromosomal behavior in Ascaris(Wilson, 1925).

ACKNOWLEDGMENT

The authors are greatly indebted to Dr. R. K.Mortimer, University of California at Berkeley,for valuable advice and use of X-ray equipmentand to Dr. A. G. Marr, University of Califoriaat Davis, for the use of photographic equipment.

SUMMARY

The life cycle and nuclear behavior werestudied of a species of the yeast genus Schwan-nsiomyces. When young vegetative cells were ex-posed to X-radiation, the death rate curve wasfound to be typical of a haploid culture. Vegeta-tive cells are uninucleate, except for a- shortperiod following mitosis before the daughternucleus moves into the newly formed bud. Thesexual cycle is as follows. After a mitotic divisionof the nucleus, the two haploid daughter nucleipresumably fuse in the mother cell and the re-

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sulting diploid nucleus moves into a special bud,where it undergoes meiosis and forms a tetrad.Supernumerary mitoses prior to karyogamy arecommon, and more than one diploid nucleus maybe formed. In this case, extra buds in whichmeiosis takes place are formed on the mother cell.After meiosis the nuclei move back into themother cell. It appears that as a rule several orall nuclei become incorporated in the developingspore. Usually one, rarely two spores per ascusare formed. Upon germination, spores budindividually without any evidence of conjuga-tion and they produce haploid vegetative cells.In exceptional cases diploidization occurs as aresult of two conjugating haploid cells. Thenuclear division appears to occur by a not previ-ously described mechanism, which is illustratedin detail.

REFERENCES

BEAM, C. A., MORTIMER, R. K., WOLFE, R. G.,AND TOBIAS, C. A. 1954 The relation ofradioresistance to budding in Saccharomycescerevisiae. Arch. Biochem. Biophys., 49,110-122.

BOUTHILET, R. J., NEILSON, N. E., MRAK, E. M.,AND PHAFF, H. J. 1949 The fermentationof trehalose by yeasts and its taxonomic impli-cations. J. Gen. Microbiol., 3, 282-289.

CAPRIOTTI, A. 1957 New Blastomycetes iso-lated from soils of Spain I: Schwanniomycescastellii nov. spec. Arch. Mikrobiol., 26,434-438.

CASSEL, W. A. 1950 The use of perchloric acidin bacterial cytology. J. Bacteriol., 59, 185-187.

CLELAND, R. E. 1936 Some aspects of the cyto-genetics of Oenothera. Botan. Rev., 2, 316-348.

DELAMATER, E. 1950 The nuclear cytology ofthe vegetative diplophase of Saccharomycescerevisiae. J. Bacteriol., 60, 321-332.

DELAMATER, E. 1951 A staining and dehydrat-ing procedure for the handling of microor-ganisms. Stain Technol., 26, 199-204.

GATES, R. R. AND FORD, C. E. 1938 Chromo-some catenations in Oenothera. TabulaeBiologicae, 15, 122-153.

GUILLIERMOND, A. 1928 Clef dichotomique pourla determination des levures, 124 pp. Librairiele Frangois, Paris.

HJORT, A. 1956 Notes on Saccharomyces rouxiiBoutroux and other yeasts with special regardto their life cycles. Compt. rend. trav. lab.Carlsberg. S6r. physiol., 26, 161-181.

JOHANSEN, D. A. 1939 Plant microtechnique,523 pp. McGraw-Hill, Inc., New York.

JONES, R. McC. 1950 Handbook of microscopicaltechnique, 3rd ed. 790 pp. Paul B. Hoeber,Inc., New York.

KLOECKER, A. 1909 Deux nouveaux genres dela famille des Saccharomycbtes. Compt.rend. trav. lab. Carlsberg, 7, 273-278.

LUCKE, W. H. AND SARACHEK, A. 1953 X-ray in-activation of polyploid Saccharomyces. Na-ture, 171, 1014.

PHAFF, H. J. AND MRAK, E. M. 1948 Sporula-tion in yeasts, Part I. Wallerstein Lab.Communs. 11, 261-279.

STELLING-DEKKER, N. M. 1931 Die sporogenenHefen. Verhandel. Koninkl. Akad. Weten-schap. Amsterdam Afdeel. Natuurk., Sect. II,28, 1-547.

WILSON, E. B. 1925 The cell in development andheredity, 3rd ed. The Macmillan Co., NewYork.

WINGE, 0. 1951 The relation between yeastcytology and genetics. A critique. Compt.rend. trav. lab. Carlsberg. S6r. physiol., 25,85-100.

WINGE, 0. AND ROBERTS, C. 1954 Causes ofdeviations from 2:2 segregations in the tetradsof monohybrid yeasts. Compt. rend. trav.lab. Carlsberg. S6r. physiol., 25, 285-329.

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