ELECTRON MICROSCOPY OF ULTRATHIN SECTIONS OFSCHIZOSACCHAROMYCES OCTOSPORUS

I. CELL DIVISION

S. F. CONTI' AND H. B. NAYLOR

Laboratory of Bacteriology, Department of Dairy Industry, Cornell University, Ithaca,New York2

Received for publication June 8, 1959

Detailed studies of the process of cell divisionin yeast, particularly of the genus Schizosac-charomyces, have been few (Guilliermond, 1920;Knaysi, 1941), and little is known of the detailsof this process. Duraiswami (1953) points outthat "It would be interesting to study therelationship between karyokinesis and cytokinesisin those yeasts where cell division is effected notby budding but by the formation of a septum inthe middle-as for instance in the Schizosac-charomycetes. It is very likely that a closecorrelation between these two processes exists inthese yeasts."

Since the Schizosaccharomycetes appear todivide in a manner which is analogous to thatdescribed for bacterial cells, a study by electronlmicroscopy of cytological changes occurring inSchizosaccharomyces octosporus during cytokinesisand karyokinesis was carried out not only toelucidate details of the process of cell division,but also to compare and relate these to the,process of cell division described for bacterialcells.

MATERIALS AND METHODS

The culture used in these studies was S.octosporus strain NRRL Y-854, obtained fromDr. L. J. Wickerham. Cells were routinely grownin a glucose-yeast extract medium (glucose,1.0 per cent; yeast extract, 2.0 per cent; peptonie,0.5 per cent; KH2P04, 0.1 per cent; and MgSO4,0.05 per cent). The pH of the medium wasadjusted to 7.0 prior to sterilization. When asolid medium was required, 1.5 per cent agar was

1 Present address: Department of Biology,Brookhaven National Laboratories, Upton, NewYork.

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

included. Cells were prepared for study bystreaking on agar plates and incubating at 30 Cfor 24 hr. The culture was then transferred at 12-hr intervals for 2 days. Cells were then washedfrom the surface of the agar and suspended in40 ml of liquid medium in 250-ml flasks. Theflasks were incubated with vigorous aeration at30 C and cells were collected at appropriateintervals for examination by light and electronmicroscopy.

Specimen preparation. The technique used forthe preparation of ultrathin sections was a modifi-cation of the method described by Hashimotoet al. (1958b). Cells were fixed in a 1.5 per centaqueous solution of KMnO4 for 40 min at 4 C.After prolonged treatment with partially poly-merized n-butyl methacrylate, cells were placedin gelatin capsules and subjected to centrifugationat 2000 X G for 2 min or by allowing them tosettle for 4 hr at room temperature. Polymeriza-tion was accomplished by placing the capsulesin an oven at 60 C for 8 to 12 hr. Specimens wereleft at room temperature for at least 3 days beforesectioning by means of a Porter-Blum microtomeequipped with a glass knife. The technique ofSatir and Peachey (1958) was employed todecrease compression artifacts. Sections of lessthan 0.1 ,u were picked up on 200-mesh coppergrids on which a thin collodion membrane hadbeen mounted and dried. All specimens wereexamined in an RCA-EMU 2B electron micro-scope equipped with a 50 or 100 ,u objectiveaperture.

Cytochemical techniques and light microscopy.The nuclear stain of Ganesan and Swaminathan(1958) and Lindegren's modification of theCarnoy-perchloric acid Giemsa staining tech-nique (Lindegren et al., 1956) were employed forthe preliminary investigation of nuclear behaviorduring vegetative cell division. Cell division wasalso followed by means of phase microscopy.

868

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ULTRATHIN SECTIONS OF S. OCTOSPORUS

Dilute Lugol's solution and Sudan black B wereused, respectively, for the demonstration ofglycogen and lipoidal inclusions.

RESULTS

Considerable difficulty was encountered in theearly attempts to obtdin adequately fixed cellswhich were resistant to explosion during poly-merization. These difficulties were overcome bystrict adherence to the procedure outlined above.The concentration of KMnO4 and temperaturecontrol during fixation and subsequent stepswere critical factors. Cells could be fixed for 20,30, or 40 min, the latter time yielding the mostconsistent and satisfactory results. Light micro-scopic observation and measurements revealedthat there was less than 10 per cent shrinkageof the cells as a result of the various treatments.It was found that the yeast cell wall was of suchlow electron density that its detection wasdifficult in electron micrographs. This difficultycould be partially overcome by treating thesections with lanthanum nitrate (Hashimotoet al., 1958b), or by not washing the cells com-pletely free from adhering medium prior tofixation, thereby delineating the outer border ofthe cell wall. Since many of the cell walls appearedto be refractory to the electron stain, the latterprocedure was regularly employed.

Staining of the cells with Giemsa according tothe procedure of Ganesan and Swaminathan(1958) revealed the presence and general behaviorof the nucleus. Application of Lindegren's modifi-cation of the Carnoy-perchloric acid Giemsastaining technique (Lindegren et al., 1956) yieldedsimilar results. The nucleus in most of the cellsappeared to be uniformly stained by eithertechnique. However, occasional cells containeddeeply stained areas within the nucleus whichhave been interpreted as being chromosomes byGanesan and Swaminathan (1958) and Lindegrenet al. (1956). Alternative interpretations of thesignificance and nature of these deeply stainingareas are discussed by Mundkur (1954), andHashimoto et al. (1959). Although the stainingprocedures made it possible to follow the generalbehavior of the nucleus during cell division,detailed observations of structural changes werenot possible. Observation of stained cells andphase microscope studies of living cells, indicatedthat during vegetative division the cells elongatedand formed a transverse partition, usually inthe medial portion of the cell. The transversepartition appeared to be continuous with thecell wall. Cells then either separated or remainedattached, but cell partitioning could proceedwithout cell separation. Individual cells weregenerally found to contain a single nucleus in

Figure 1. Actively dividing cells. Note the partitioned appearance of the cells, and the nucleus whichhas elongated and is in the process of constriction. Initiation of cross wall formation is not evident.

1959] 869

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CONTI AND NAYLOR

2

Figure 2. Actively dividing cells illustrating that the plane of division is not always perpendicularto the long axis of the cell. Note the low electron density of the cell wall and the conspicuous nuclei.

stained preparations. These observations are inagreement with the reports of other investigators(Guilliermond, 1920; Knaysi, 1941; Ganesan andSwaminathan, 1958; Yoneyama, 1958). Moredetailed observation was beyond the resolutionlimit of the light microscope. Vacuoles were

not seen in young cells observed under phasemicroscopy, or when stained with dilute Lugol'ssolution. Sudan black B staining revealed thatlipoidal inclusions were absent or few in number.

Figures 1 to 7 are electron micrographs ofS. octosporus in various stages of cell division.The micrographs illustrate successive stages ofcell division based on the preliminary studies bylight microscopy and reports in the literature(Guilliermond, 1920; Knaysi, 1941; Ganesan andSwaminathan, 1958; Yoneyama, 1958).The structure labeled N3 is considered to be

3 CM = cytoplasmic membrane; CW = cellwall; I = invagination of the cell wall; L =

lipoidal inclusion; N = nucleus; TM = transversecell wall membrane.

the nucleus on the basis of its behavior duringcell division, and the occurrence and behavior ofa similar structure in Saccharomyces cerevisiae(Hashimoto et al., 1958a, b; 1959). This structurewas found to be present in almost all of the cellssectioned and observed with the electron micro-scope. The absence of this structure from some ofthese cells can be ascribed to the plane of sec-tioning.

Nuclear structure and behavior during cell divi-sion. Figures 1, 2, and 3C illustrate that thenucleus appears to be internally undifferentiatedin electron micrographs, although details of itsstructure, not observable by light microscopy,are apparent. The nuclear membrane is quitedistinct, appears to be double, and persiststhroughout all stages of vegetative division.The nucleus appears to be ovoid to sphericalin shape, in contrast to its lobulate shape inactively dividing cells of S. cerevisiae (Hashimotoet al., 1959). The nucleoplasm appears granularand homogenous in texture, with few indications

870 [VOL. 78

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

...... _. u isw

I

If

3A

Figures SA, B, and C. Early and intermediate stages of transverse wall formation. Note in figure 3Athat the nuclei are separated and cross wall formation is being initiated. Figures 3B and 3C furtherillustrate that cross wall formation occurs by the annular centripetal growth of an inner portion of thelateral cell wall. The cytoplasmic membrane is observed to be closely associated with the advancingcross wall in figure 3C. Internal membranes can be observed in the area of the yeast cell labeled IM.

871

:.,:. 19-I.......- - M I

.#'L---l Q

.1.ik

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CONTI AND NAYLOR

of internal differentiation during any of the stagesof cell division. The pores observed in the nuclearmembrane of a variety of animal cells (Kautzand DeMarsh, 1955; Rhodin, 1954; Watson,

c-cw

4.

1955), Coccidioides immitis (O'Hern and Henry,1956), and in the vegetative cell of S. cerevisiae(Agar and Douglas, 1957), were 'not observed.At the onset of nuclear division the nucleus

W6 aM

TjM'"... ,

_iA_ P4.S .4AFigures 4 and 4A. Figure 4 illustrates the appearance of the newly formed transverse cross wall. Figure

4A shows a somewhat thicker section, which has been over-exposed in printing. Note the appearanceof membrane-like structures within the cross wall.

5 i -WM(fi+ t 0t sh E0a~~ ht $

*~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.1w..~*.. d.mu__;:_...... .S. 9Z ..

r

cw2r;:

Figures 6, 6, and 7. These electron micrographs illustrate the development of the membrane-likestructures within the connecting cross wall with eventual separation of the daughter cells. Note thatthese membrane-like structures appear to serve initially as the outer edge of the newly formed portionof the cell wall.

872 [VOL. 78

0Jdo ...::...

-1 :;:.:.

41: %"

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ULTRATHIN SECTIONS OF S. OCTOSPORUS

i..*.1

6,- E-------'-----

FIGS. 6-7

19591

6:.873

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CONTI AND NAYLOR

appears to increase in size and to elongate, takingon a more or less dumbbell appearance (figure 1).Constriction of the nucleus then occurs, pre-sumably in a manner similar to that describedin S. cerevisiae (Hashimoto et al., 1958a, b;1959).

Transverse cell wall formation. During the laterstages of nuclear division or after the nucleiare completely separated, transverse cell wallformation occurs by means of the annularcentripetal growth of a portion of the inner cellwall into the cytoplasm (figures 2, 3A, B, C).The newly formed cross wall then appears toincrease in thickness prior to separation of thedaughter cells. The transverse wall, as well asthe cell wall, is of low electron density. Thecell wall of S. cerevisiae (Hashimoto et al., 1959),Saccharomycodes ludwigii, Schizosaccharomycespombe, and Nadsonia fulvescens (Conti andNaylor, unpublished observations) also appear tobe nonelectron dense in electron micrographs ofsectioned cells.

There are no indications of the prior formationof a cell plate as described in bacteria (Knaysi,1941, 1949; Chance, 1953; Webb and Clark,1954; Bisset, 1955; Clark et al., 1957). The positionof various cytoplasmic inclusions within theregion of cross wall formation, and absence of anobservable cross plate supports this contention.The absence of an observable cell plate in dividingcells of S. octosporus is in agreement with observa-tions (Knaysi, 1941) on cell division in S. pombeby light- and dark-field microscopy. Inwardgrowth of the transverse cell wall is usually notperpendicular to the long axis of the cell, asshown in figures 1 and 2. This, combined withthe lag of cell separation after cell division,gives rise to the partitioned appearance of thecells described by various investigators (Guillier-mond, 1920; Lodder and Kreger-Van Rij, 1952).

After the cross wall has thickened (figure 4),but prior to cell separation, electron dense,membrane-like areas can be observed withinthe transverse cell wall (figure 5). The membranesappear to originate in the distal portions of thetransverse cell wall. These structures then elon-gate and thicken, eventually partitioning theconnecting cell wall (figures 6 and 7). The innerpartition finally disintegrates resulting in com-plete separation of the two daughter cells inthe majority of cases. However, some cells wereobserved where disintegration was not complete,

such cells being attached only by a portion ofthe cell wall. Attachment in this manner causesthe cells to appear in the shape of a "V" whichis characteristic of the Schizosaccharomycetes.

Figure 2 reveals that the internal membranesystem described in S. cerevisiae (Agar andDouglas, 1957) is also present in S. octosporus.This membrane system appears to be a normalcomponent of yeast cells, and may be analogousto the endoplasmic reticulum of animal cells(Palade and Porter, 1954; Palade, 1955).A central vacuole and lipoidal inclusions were

regularly observed in sections of vegetative cells ofS. cerevisiae (Agar and Douglas, 1957; Hashimotoet al., 1959). However, vacuoles appear to beabsent and lipoidal inclusions few in numberin the actively dividing S. octosporus. Vacuolesare regularly observed in cells of S. octosporusundergoing sporogenesis (Conti and Naylor,1959).

Mitochondria-like bodies were occasionally ob-served in the cytoplasm. In general, these struc-tures appeared to be randomly distributedthroughout the cells, in contrast to their mainlyperipheral location in S. cerevisiae (Agar andDouglas, 1957; Hashimoto et al., 1959). It wasnoted, however, that these bodies appeared toaccumulate in the vicinity of the transverse cellwall during its centripetal growth and remainedthere while complete separation of the daughtercells occurred. The internal membrane (cristaemitochondriales) system described by Palade(1952, 1953) and Sjostrand (1953) as beingcharacteristic of mitochondria was infrequentlyobserved. However, the mitochondria-like bodieswere found to be enclosed within a double mem-brane, and possessed circular or elliptical profilescharacteristic of yeast mitochondria (Agar andDouglas, 1957; Hashimoto et al., 1958b, 1959).

DISCUSSION

Knaysi (1941) observed cell division in S.pombe by both dark- and bright-field microscopy,and reported that a cell plate was not formed,which is in agreement with our results. Knaysistated further that "cell wall material is depositedat both bases of a cylindrical portion, in themiddle of the cell, where chromatin materialdisappeared," and that there is "no indicationof the formation of a single wall with subsequentsplitting...." However, later studies by Knaysi(personal communication) indicated that trans-

874 [VOL. 78

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ULTRATHIN SECTIONS OF S. OCTOSPORUS

verse cell wall formation occurs in S. pombe bythe centripetal growth of the cell wall. Thesestudies support the data obtained by electronmicroscopy, i. e., that cross-wall formation in theSchizosaccharomycetes occurs by the annularcentripetal growth of an inner lateral portionof the cell wall. Furthermore, the present studiesshow that the cytoplasmic membrane remainsin close contact with the cell wall and the develop-ing transverse cell wall during the various stagesof cytokinesis. Unfortunately, satisfactory pic-tures were not obtained to show the final stagesof the closing of the transverse cell wall.

Comparison of our results with studies byRobinow (1945), Chapman and Hillier (1953),and Chapman (1959) indicates that the processof cytokinesis in bacterial cells and the Schizosac-charomycetes is quite similar. Chapman andHillier (1953) also observed that after the trans-verse cell wall is formed it appears to thickenand to divide into two layers. Knaysi (1941)reported that in Bacillus cereus ". . . the portionof the mother-cell wall corresponding to theplace of division becomes less and less stain-able ... until it withers away liberating thetwo cells." On the basis of these observations itis tempting to propose that B. cereus, and perhapsother bacterial cells, form transverse cell wallmembranes, the cells then separating in a mannersimilar to that observed in S. octosporus. Thetransverse membranes appear to be analogousto the splitting plate observed in budding cellsof Histoplasma (Edwards et al., 1959).The identification of the structure labeled N as

the nucleus is in agreement with the views ofother workers (DeLamater, 1950; Mundkur,1954; Agar and Douglas, 1957; Hashimotoet al., 1958b, 1959). Examination of the electronmicrographs of dividing cells of S. octosporusreveal that the nucleus, like that of S. cerevisiae(Hashimoto et al., 1959), is surrounded by alimiting membrane which remains intact duringnuclear division. The nucleus appears to elon-gate and divide by constriction, further detailsof the process remaining beyond the scope of thetechniques employed. It is particularly significantthat a large central vacuole, such as that regularlyfound in S. cerevisiae, is absent from activelygrowing vegetative cells of S. octosporus. Asimilar observation has also been reported byGanesan and Swaminathan (1958).Although chromosomes have not been observed

in electron micrographs of yeast nuclei, this doesnot in any way establish or indicate that thechromosomes, described by other investigators,are absent. The studies of Moses (1956) andGibbons and Bradfield (1957) illustrate the dif-ficulties involved in observing chromosomes bymeans of the ultrathin sectioning technique. Theabsence of observable chromosomes in the yeastnucleus may be due to one or more of the follow-ing factors: (a) The components of the nucleushave similar electron scattering properties,thereby leading to low contrast in the final image.(b) Intranuclear structures are not delineated bymembranes. (c) The preparative procedures mayresult in changing the contents, form, or distribu-tion of intranuclear material. (d) Difficulties ininterpretation and observation of the dimensionsof structures not well delineated due to thethinness of the section. (e) Chromatin may bedistributed uniformly throughout the nucleusin structures beyond the resolution limit of theelectron microscope. It therefore should beemphasized that the granular and homogeneousappearance of yeast nuclei in electron micrographscan be reconciled with the reports that chromo-somes are present in the yeast nucleus. Studiesof alternate thick and thin sections, employingthe methods described by Moses (1956) havenot been rewarding. Further attempts are nowbeing made to determine the structure andnature of chromatin material in the yeast nucleus.Although it has been postulated (Mundkur,

1954; Hashimoto et al., 1958b, 1959) that chromo-somes may be dispersed uniformly throughoutthe nucleus in the form of submicroscopicchromatinic particles, evidence for this is in-direct. Many yeast cytologists consider that thenucleus contains chromosomes comparable instructure to those observed in other organisms,but even among these cytologists, disagreementas to number, size, location, and behavior isapparent. The views of Winge (1951) on thissituation are particularly illuminating. His state-ment that "the numerous technical difficultiesinvolved in yeast cytology have rcsulted in thisfield of study being in a most uncertain state"unfortunately still holds true. Progress in thefield has been made, however, and most yeastcytologists are now in agreement as to whichstructure in the yeast cell contains the geneticmaterial. It now seems clear that the yeastvacuole is not an integral part of the nucleus

19591 875

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

CONTI AND NAYLOR

(DeLamater, 1950; Mundkur, 1954; Agar andDouglas, 1957; Hashimoto et al., 1958a, b; 1959).

Recent studies of cell division in Blastomycesdermatitidis (Bakerspigel, 1957), Mucor hiemalisand Mucor fragilis (Robinow, 1957a), andPhycomyces blakesleeanus (Robinow, 1957b), areof particular interest since the mode of divisionof the nuclei in the vegetative parts of thesefungi appears tobe similar to the process observedin S. cerevisiae (Hashimoto et al., 1958a, 1959)and S. octosporus. The nuclei of these fungi appearto be lacking nuclear membranes in the lightmicroscope whereas the presence of a limitingmembrane is established in all the yeasts studiedto date by means of ultrathin sectioning andelectron microscopy. The comment of Baker-spigel (1957) that "the results during this studysuggest that B. dermatitidis is another instanceof a fungus in which vegetative nuclei do notseem to divide in the classical mitotic manner"is particularly intriguing. Robinow (1957a) tenta-tively proposes that the vegetative nuclei ofPhycomyces and Mucor divide by a form ofendomitosis. Although this proposal is subjectto criticism, Robinow clarifies his position bystating that "endomitosis may seem a ratherfar-fetched device with which to explain thebehavior of Mucor nuclei, but it can hardly besaid that more conventional concepts have beenmore successful in this. Whatever the truth aboutthese nuclei may turn out to be, the contempla-tion of some of the varieties of chromosomebehavior known in the protozoa will probablyhelp us to attain it sooner than Procrustes-likeloyalty to the canons of classical mitosis." Thevalidity and applicability of these views to thefield of yeast cytology are difficult to question.

SUMMARY

Cell division in Schizosaccharomyces octosporuswas studied by means of ultrathin sectioningand electron microscopy. It was established that(a) karyokinesis occurs well in advance ofcytokinesis; (b) transverse cell wall formationoccurs by the annular centripetal growth of aninner lateral portion of the cell wall; (c) thecytoplasmic membrane is closely associated withthe cell wall throughout all stages of cytokinesis;(d) after initial formation, the transverse cellwall increases in thickness eventually splittingand completely separating the daughter cells;(e) electron dense membranes are formed which

partition the transverse cell wall prior to itssplitting, these membranes serving initially asthe outer portion of the cell wall; (f) the nucleusis surrounded by a limiting membrane whichremains intact during all the observed stagesof nuclear and cytoplasmic division; (g) nucleardivision can only be described as a process ofelongation and constriction, further observa-tions of chromatin behavior being beyond thescope of the techniques used; (h) internal mem-branes and cytoplasmic inclusions similar instructure to those found in other yeast arepresent.

REFERENCESAGAR, H. D. AND DOUGLAS, H. C. 1957 Studies

on the cytological structure of yeast: Electronmicros3opy of thin sections. J. Bacteriol.,73, 365-375.

BAKERSPIGEL, A. 1957 The structure andmode of division of the nuclei in the yeastcells and mycelium of Blastomyces dermati-tidus. Can. J. Microbiol., 3, 923-936.

BISSET, K. A. 1955 The cytology and life historyof bacteria, 2nd ed. The Williams & WilkinsCo., Baltimore.

CHANCE, H. L. 1953 Cytokinesis in Gaffkyatetragena. J. Bacteriol., 65, 593-595.

CHAPMAN, G. B. 1959 Electron microscopy ofultrathin sections of bacteria. III. Cell wall,cytoplasmic membrane, and nuclear material.J. Bacteriol., 78, 96-104.

CHAPMAN, G. B. AND HILLIER, J. 1953 Electronmicroscopy of ultrathin sections of bacteria.I. Cellular division in Bacillus cereus. J.Bacteriol., 66, 362-373.

CLARK, J. B., WEBB, R. B., AND CHANCE, H. L.1957 The cell plate in bacterial cytokinesis.J. Bacteriol., 73, 72-76.

CONTI, S. F. AND NAYLOR, H. B. 1959 Conjuga-tion, sporulation and ascospore structure ofSchizosaccharomyces octosporus. Bacteriol.Proc., 1959, 50-51.

DELAMATER, E. D. 1950 The nuclear cytologyof the vegetative diplophase of Saccharomycescerevisiae. J. Bacteriol., 60, 321-332.

DURAISWAMI, S. 1953 Studies on the cytologyof yeast. VIII. Karyokinesis and cytokinesisin a diploid brewery yeast. La Cellule, 55,381-395.

EDWARDS, M. R., HAZEN, E. L., AND EDWARDS,G. A. 1959 The fine structure of yeast-likecells of Histoplasma in culture. J. Gen.Microbiol., 20, 496-503.

GANESAN, A. T. AND SWAMINATHAN, M. S. 1958

876 [VOL. 78

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

ULTRATHIN SECTIONS OF S. OCTOSPORUS

Staining the nucleus in yeasts. Stain Tech-nol., 33, 115-121.

GIBBONS, I. R. AND BRADFIELD, J. R. G. 1957The fixation of nuclei in locust testis. InElectron microscopy, pp 108-110. Proc. Stock-holm Conf., 1956.

GUILLIERMOND, A. 1920 The yeasts. Trans-lated by F. W. Tanner. John Wiley & Sons,New York.

HASHIMOTO, T., CONTI, S. F., AND NAYLOR, H. B.1958a Nuclear changes occurring during thebud-formation in Saccharomyces cerevisiae as

revealed by ultra-thin sectioning. Nature,181, 454.

HASHIMOTO, T., CONTI, S. F., AND NAYLOR, H. B.1958b Fine structure of microorganisms.III. Electron microscopy of resting and ger-

minating ascospores of Saccharomyces cere-

visiae. 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.

KAUTZ, J. AND DEMARSH, Q. B. 1955 Finestructure of the nuclear membrane in cellsfrom the chick embryo. On the nature of theso-called "pores" in the nuclear membrane.Exptl. Cell Research, 8, 394-396.

KNAYSI, G. 1941 Observations on the cell divi-sion of some yeasts and bacteria. J. Bacte-riol., 41, 141-153.

KNAYSI, G. 1949 Cytology of bacteria. II.Botan. Rev., 15, 106-151.

LINDEGREN, C. C., WILLIAMS, M. A., AND Mc-CLARY, D. 0. 1956 The distribution ofchromatin in budding yeast cells. Antonievan Leeuwenhoek. J. Microbiol. Serol., 22,1-20.

LODDER, J. AND KREGER-VAN RIJ, N. J. W. 1952The yeasts. A taxonomic study. IntersciencePublishers, Inc., New York.

MOSES, M. J. 1956 Studies on nuclei using cor-

related chemical, light and electron micro-scope techniques. J. Biophys. Biochem.Cytol. (Suppl.), 2, 397-407.

MUNDKUR, B. D. 1954 The nucleus of Saccharo-myces: A cytological study of a frozen-driedpolyploid series. J. Bacteriol., 68, 514-529.

O'HERN, E. M. AND HENRY, B. S. 1956 A cyto-logical study of Coccidioides immitis by elec-tron microscopy. J. Bacteriol., 72, 632-645.

PALADE, G. E. 1952 The fine structure of mito-chondria. Anat. Record, 114, 427-452.

PALADE, G. E. 1953 An electron microscopystudy of the mitochondrial structure. J.Histochem. Cytochem., 1, 188-211.

PALADE, G. E. 1955 Studies on the endoplasmicreticulum. II. Simple dispositions in cellsin situ. J. Biophys. Biochem. Cytol., 1, 567-582.

PALADE, G. E. AND PORTER, K. R. 1954 Studieson the endoplasmic reticulum. I. Its identifi-cation in cells in situ. J. Exptl. Med., 100,641-656.

RHODIN, J. 1954 Correlation of ultrastructuralorganization and function in the normal andexperimentally changed proximal convolutedtubule cells of the mouse kidney. KarolinskaInstitut, Stockholm, Sweden, AktiebolagetGodvil.

ROBINOW, C. F. 1945 Nuclear apparatus andcell-structure of rod-shaped bacteria, InDubos, The bacterial cell, p. 353-377. HarvardUniversity Press, Cambridge, Mass.

ROBINOW, C. F. 1957a The structure and be-havior of the nuclei in spores and growinghyphae of Mucorales. I. Mucor hiemalis andMucor fragilis. Can. J. Microbiol., 3, 771-789.

ROBINOW, C. F. 1957b The structure andbehavior of the nuclei in spores and growinghyphae of Mucorales. II. Phycomyces blakes-leeanus. Can. J. Microbiol., 3, 791-798.

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

SJOSTRAND, F. S. 1953 Electron microscopy ofmitochondria and cytoplasmic double mem-

branes. Ultra structure of rod-shaped mito-chondria. Nature, 171, 30-31.

WATSON, M. 1955 The nuclear envelope. Itsstructure and relation to cytoplasmic mem-

branes. J. Biophys. Biochem. Cytol., 1, 301-314.

WEBB, R. B. AND CLARK, J. B. 1954 Cell divi-sion in Micrococcus pyogenes var. aureus. J.Bacteriol., 67, 94-97.

WINGE, 0. 1951 The relation between yeastcytology and genetics. A critique. Compt.rend. trav. lab. Carlsberg. S6r. physiol., 25,85-99.

YONEYAMA, M. 1958 Some observations of themultiplication and sporulation of Schizosac-charomyces versatilis. Seibutsugakkaishi, 8,9-13. Biol. Soc. of Hiroshima Univ., Hirosh-ima, Japan.

1959] 877

on August 26, 2020 by guest

http://jb.asm.org/

Dow

nloaded from