J. Cell Sci. 29,77-84(1978) 7 7

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FLUORESCENT STAINING OF FUNGAL

NUCLEI WITH A BENZIMIDAZOL

DERIVATIVE

PAUL A.LEMKE, BERTHA KUGELMAN, HIDEO MORIMOTO,ERNEST C.JACOBS AND JOHN R.ELLISONMellon Institute and Department of Biological Sciences,Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213, U.S.A.

SUMMARYA direct staining procedure is described for fluorescence microscopy of fungal nuclei,

chromosomes and mitochondria. The fluorochrome is a benzimidazol derivative (33258Hoechst) known to bind selectively to deoxyribonucleic acid at neutral pH. The advantages°f 33258 Hoechst relative to Feulgen compounds used previously to stain these structuresinclude a greater intensity of fluorescence, the absence of fading or rapid quenching of thefluorescence, and the omission of acid hydrolysis from the procedure for removal of ribonucleicacid. 33258 Hoechst has been evaluated as a nuclear stain with a yeast (Saccharomyces cerevisiae)and a filamentous fungus (At>aricus bisporus) and appears to penetrate easily vegetative cellsand spores of both fungi.

INTRODUCTION

Improved methods for resolving structures containing deoxyribonucleic acid (DNA)in fungi by fluorescence microscopy have been developed recently (Laane & Lee,1975; Lemke et al. 1975; Slater, 1976; Williamson & Fennell, 1975). These methodsinvolve fluorescent dyes that are known either to act as Feulgen reagents (Laane &Lee, 1975; Lemke et al. 1975), to intercalate with double-helical DNA (Williamson &Fennell, 1975), or to bind to DNA without intercalation (Slater, 1976; Williamson &Fennell, 1975). The compound 2-[2-(4-hydroxyphenyl)-6-benzimidazol]-6-(i-methyl-4-piperazyl)-benzimidazol-trihydrochloride, or 33258 Hoechst, is recognized to bea dye of the third type and to bind most efficiently with certain classes of constitutiveheterochromatin (Hilwig & Gropp, 1972; Latt, 1976). We have examined 33258Hoechst as a nuclear and mitochondrial stain in 2 fungi, Saccharomyces cerevisiae andAgaricus bisporus.

MATERIALS AND METHODS

Strains and growth media

Fungal strains examined were a haploid culture of S. cerevisiae (XJB1-1B, a met-iade-2), a diploid culture of S. cerevisiae (E280, a his-4 trp-i can' x A 4840A, OL his-5ade-2),and a strain of A. bisporus (D-26, prototrophic). The yeast strains were grown on yeastextract-peptone medium (YEP) containing 2 % Bacto-peptone, 2 % dextrose, and 1 % yeast

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78 P. A. Lemke and others

extract. Yeast cells were grown overnight in YEP at 30 CC to an approximate titre of io8 cellsper ml. Petite strains were selected from the haploid yeast (ade-2, red colour) as slow-growingstrains of white colour on YEP medium containing 2 % agar, and petite phenotype was con-firmed by lack of growth on YEP medium containing a non-fermentable carbon source (3 %glycerol) in place of dextrose. The diploid yeast strain was brought to sporulation on biphthalate-acetate medium (Kiienzl, Tingle & Halvorson, 1974), and these cells were harvested 18-20 hfollowing inoculation. The A. bisporus strain was grown on 5 cm1 membranes (PD 150, DuPont,Wilmington, Delaware, U.S.A.) spread upon the surface of an agar-based growth medium(Saksena, Marino, Haller & Lemke, 1976). Spores of A. bisporus were obtained from mush-rooms grown commercially (Butler County Mushroom Farm, Inc., Worthington, Pa. 16262).

Staining procedure

Yeast cells, freshly harvested by centrifugation (iooog) from YEP, and mushroom tissue orspores were routinely fixed in absolute isopropanol for at least 30 min prior to staining. All cellswere washed at least once in distilled water, recovered by centrifugation (1000 g), and resus-pended in 10 ml of water containing 50//g/ml of 33258 Hoechst (Farbwerke Hoechst AG,Frankfurt, Germany). A stock solution of 33258 Hoechst in absolute ethanol (1 mg/ml) can bestored indefinitely at —20 °C and added dropwise to obtain the concentration desired for stain-ing. Staining time varied from 3 h (yeast vegetative cells and mushroom spores) to 16 h(meiotically dividing cells), and all cells were destained in 95 % ethanol for 30 min prior tomicroscopic examination.

Microscopic examination

Cells were mounted for examination in a 65 % solution of sucrose. A Leitz Orthoplan micro-scope equipped for incident-light fluorescence with a Ploem vertical illuminator was used. Thelight source was an Osram superpressure xenon lamp (XBO 75W/z) and the power supply wasan Optiquip 1020 unit. Conditions for microscopic examination were determined from theknown emission and excitation frequencies of 33258 Hoechst bound to DNA at neutral pH(Latt & Wohlleb, 1975); this spectrum indicates a maximum emission at 470 nm with abouta 45-fold increase in fluorescence over free 33258 Hoechst. The microscope was set at position 1with the following: one excitation filter (UG-i), one dichroic mirror (K 400), and a barrierfilter (K 420). A fluorite objective (95 x , 1.32 NA) was used. Photographs were taken on Adox135-KB14 film (Fotokemika, Zagreb, Yugoslavia) with a 20-s exposure time and processedwith Diafine developer (Acufine, Inc., Chicago, Illinois, U.S.A.).

RESULTS AND DISCUSSION

The effective use of 33258 Hoechst to stain DNA-containing structures in theyeast Saccharomyces cerevisiae is shown in Figs. 1 and 2. Cells depicted in these2 figures represent, respectively, those of a haploid yeast strain and a meioticallydividing yeast strain.

Dividing nuclei of haploid cells assume an elongated configuration during mitosis(Fig. 1 A), an atypical configuration for anaphase which has been described previouslyin yeast (Peterson & Ris, 1976) as well as in several filamentous fungi (for review seeFuller, 1976). Unfortunately, resolution with 33258 Hoechst is too poor and yeastchromosomes are too small to be counted in this configuration, but it appears thatchromosomes are slightly condensed and aligned with the axis of elongation. In thesecells mitochondrial DNA is evident as weakly fluorescent regions present mainly inthe peripheral cytoplasm. A small percentage of yeast cells, significantly less than 1 %of the population, contain mitochondria but lack a nucleus (Fig. IB). Cells

Fluorescent staining of fungal nuclei

Fig. i. Vegetative cells of S. cerevisiae stained with 33258 Hoechst. A, uninucleated cellscontaining numerous mitochondria (faint fluorescent spots) mainly in the peripheralcytoplasm; elongated nuclei (arrows) represent stages of mitosis. B, group of cells withmitochondria evident in the cytoplasm; one cell (arrow) lacks a nucleus. C, cells of apetite variant lacking mitochondria, D, dividing cell of petite variant showing totaldistribution of chromatin into bud. A, B, D, X 5000; c, x 4000.

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Fig. 2. Stages of meiosis and ascospore formation in cells of S. cerevisiae stained with33258 Hoechst. A, prophase I nuclei (arrows) with chromosomes evident as con-densed fluorescent structures, x 2500. B, elongated nucleus (small arrow) of cell duringanaphase I; meiotic tetrads (large arrows) in proasci. x 2500. c, nucleus during lateprophase I. x 3500. D, elongated nuclei during anaphase II. x 2500. E, stages of meiosisII; paired nuclei (large arrow) prior to elongation and elongated nuclei (small arrow)of anaphase II in parallel configuration, x 2500. F, elongated nuclei of anaphase II incrossed configuration, x 4000. G, mature ascus containing 4 uninucleated ascospores.X4000.

from a spontaneous segregant of petite or respiratory-deficient phenotype are shown inFig. ic . These cells lack evidence for mitochondrial-DNA when stained with 33258Hoechst. Dividing nuclei in this petite strain often assume an elongated configuration,and nuclei in other cells may be extruded totally into a bud without apparent division(Fig. ID). In some preparations this latter phenomenon was observed repeatedly,resulting in a higher than expected frequency of progeny lacking both mitochondria

Fluorescent staining of fungal nuclei 81

Fig. 3. Vegetative cells of A. bisporus stained with 33258 Hoechst. A, branched terminalcell containing numerous mitochondria (faint fluorescent spots) and 5 elongatednuclei, x 3500. B, mycelial network showing irregular distribution of nuclei, x 3000.C-E, cells containing, respectively, 2, 3 and several nuclei per cell, x 3000.

and chromosomal DNA. Another interpretation of this phenomenon is that it is anartifact of fixation.

Diploid yeast cells as well as asci are evident in Fig. 2. Also present in this figureare stages of meiosis with evidence for an elongated configuration of nuclei duringanaphase I (Fig. 2B). Chromosomes are seen in a condensed state during prophase I(Fig. 2 A, c) but appear to be more diffuse during subsequent stages of meiosis.Elongated configurations of dividing nuclei reappear during terminal stages of meiosisII (Fig. 2D-F). These configurations agree with models for chromosome movementsduring meiosis based mainly on ultrastructural analysis of non-chromosomal structures

P. A. Lemke and others

Fig. 4. Cells from mushrooms of A. bisporus stained with 33258 Hoechst. A, meiotictetrad in young basidium. x 6500. B, prophase I of meiosis with chromosomes evidentas condensed fluorescent structures, x 6000. c, basidium with nuclei and mitochondriamigrating into terminal sterigmata. x 4500. D, E, basidiospores containing, respectively,4 and 8 nuclei, x 3000. F, portion of a thin-walled cell from internal tissue of mushroomcap containing several nuclei and elongated mitochondria (arrows) in the peripheralcytoplasm, x 2000.

associated with meiosis and ascospore formation (for review see Heywood & Magee,1976). Young asci with meiotic tetrads are shown in Fig. 2B and E and a mature4-spored ascus is shown in Fig. 2G.

Vegetative cells of the mushroom Agaricus bisporus are shown in Fig. 3. Individualcells contain several nuclei, and mitotic divisions are again represented by elongatedconfigurations (Fig. 3 A). In these cells nuclei are irregularly distributed (Fig. 3B) andmitochondria are evident as faint fluorescent regions abundant in apical or tip cells

Fluorescent staining of fungal nuclei 83

(Fig. 3 A, c, D). In older cells nuclei may be clustered (Fig. 3E) and mitotic figures, asevidenced by elongation of nuclei, are less frequent.

Cells associated with mushrooms of A. bisporus are seen in Fig. 4. These cellsinclude basidial cells at different states of maturation (Fig. 4A-C), basidiospores(Fig. 4D, E) and a cell from the cap tissue of a mushroom (Fig. 4F). A basidium con-taining the meiotic tetrad is seen in Fig. 4A and a young basidium containing condensedchromosomes during prophase I is shown in Fig. 4B. In Fig. 4c a binucleated basidiumis evident and in this cell one nucleus and several mitochondria are seen entering theapical prongs or sterigmata.

Details of the life-cycle of A. bisporus have been published elsewhere (Saksena et al.1976) and indicate that spore number per basidium as well as the number of nucleiper spore can vary. The 2 basidiospores in Fig. 4D and E have, respectively, 4 and8 nuclei. The cap tissue of A. bisporus is composed mainly of inflated thin-walled cellswith a large central vacuole. Nuclei and mitochondria in this cell type are distributedin the peripheral cytoplasm just inside the cell wall (Fig. 4F). Mitochondria in thesecells are more filamentous in appearance than mitochondria in vegetative cells.

The advantages of 33258 Hoechst as a fluorescent dye for fungal structures con-taining DNA are basically three, and all are really refinements on methods employingother fluorescent dyes. First, 33258 Hoechst is broadly applicable as a stain for nucleiin both vegetative cells and spores following a single and simple procedure for fixation.Secondly, unlike Feulgen reagents, 33258 Hoechst binds selectively to DNA and canbe used without acid hydrolysis of cells to remove ribonucleic acid. Finally, 33258Hoechst is a fluorochrome of high intensity and good resolution, and this fluorescenceunder the experimental conditions described does not fade.

This work was supported in part by a fellowship to the Carnegie-Mellon Institute of Researchfrom the Butler County Mushroom Farm, Inc., of Worthington, Pennsylvania 16262, U.S.A.

REFERENCES

FULLER, M. S. (1976). Mitosis in fungi. Int. Rev. Cytol. 45, 113-153.HEYWOOD, P. & MAGEE, P. T. (1976). Meiosis in protists. Bad. Rev. 40, 190-240.HILWIG, I. & GROPP, A. (1972). Staining of constitutive heterochromatin in mammalian

chromosomes with a new fluorochrome. Expl Cell Res. 75, 122—126.KOENZL, M. T., TINGLE, M. A. & HALVORSON, H. O. (1974). Sporulation of Saccliarowyces

cerevisiae in the absence of a functional mitochondrial genome. J. Bad. 117, 80-88.LAANE, M. M. & LEE, T. (1975). Examination of fungal nuclei with the Feulgen-fluorescence

method. Mikroskopie 31, 85-90.LATT, S. A. (1976). Optical studies of metaphase chromosome organization. A. Rev. Biophys.

Bioeng. 5, 1-37.LATT, S. A. & WOHLLEB, J. C. (1975). Optical studies of the interaction of 33258 Hoechst with

DNA, chromatin, and metaphase chromosomes. Chromosoma 52, 297—316.LEMKE, P. A., ELLISON, J. R., MARINO, R., MORIMOTO, B., ARONS, E. & KOHMAN, P. (1975J-

Fluorescent Feulgen staining of fungal nuclei. Expl Cell. Res. 96, 367-373.PETERSON, J. R. & Ris, H. (1976). Electron-microscopic study of spindle and chromosome

movement in yeast Saccharcmryces cerevisiae. J. Cell Set. 22, 219-242.SAKSENA, K. N., MARINO, R., HALLER, M. N. & LEMKE, P. A. (1976). Study on development of

of Agaricus bisporus by fluorescent microscopy and scanning electron microscopy. J. Bad.126, 417-428.

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SLATER, M. L. (1976). Rapid nuclear staining method for Saccliaromyces cerevisiae. J. Bad.126, 1339-1340.

WILLIAMSON, D. H. & FENNELL, D. J. (1975). The use of fluorescent DNA-binding agent fordetecting and separating yeast mitochondrial DNA. In Methods in Cell Biology, vol. 12 (ed.D. M. Prescott), pp. 335-351. New York: Academic Press.

{Received 16 February 1977)