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METHODS Preparation of protoplasts. Haploid strains of Kluyveromyces lactis (laboratory strain ERY%~) and Saccharomyces cerevisiae (A~Cc26500) were used. Actively growing cultures (about 3 x 10' cells d-l) in GYP medium [0.5 % (w/v) yeast extract, 1.0 % (w/v) peptone, 2 % (w/v) glucose] at 30 "C were washed twice with water. Washed cells were resuspended to a final density of about 10" cells ml-1 in citrate/phosphate buffer (pH 5.4; 0.2 M-N~,HPO,. I 2H,O, 0.1 M-citric acid.H,O) containing 0.6 M-KCl as osmotic stabilizer. Driselase (Kyowa Hakko Kogyo Co., Tokyo, Japan) and snail gut juice (Industrie Biologique Francaise, 921 15, Clichy, France) were added to final concentrations of 2 % (w/v) and 0.5 % (v/v) respectively to the washed cells, which were allowed to stand at 30 "C for about 30 min in the case of K. lactis and 60 min for S. cerevisiae. This procedure for producing protoplasts was carried out directly in 10 rnl graduated centrifuge tubes. Fixation and staining. Protoplasts were harvested by centrifuging at 150 g for 10 min and the pellet was fixed immediately (without any washing) in methanol/acetic acid (3 : I, v/v) as described previously (Brody & Williams, 1974) except that the centrifugation was extended to 10 min at each step. The protoplasts were air-dried on to clean slides and stained with 10 % (v/v) Gurr's Giemsa stain (Improved R66) in 0.067 MSorensen's phosphate buffer (pH 6.8) for 40 min. Usually slides were treated with 0.25 % (w/v) trypsin (Difco, I : 250) in 0.85 % (w/v) NaCl for 5 min immediately before Giemsa staining. Microscopy and photographic techniques. Slides were examined with a Carl Zeiss photomicroscope using bright field optics (plan apo 1oo/1.3 objective) and a green filter. Some photographs were taken with Kodak Panatomic X film (Robson & Williams, 1977) but Agfa Copex Pan rapid film was found to be more suitable and was used in most experiments. The Agfa film was developed with Agfa Rodinal I : 50 (v/v) for 7 min at 20 "C.

Giemsa Staining of Mitotic Chromosomes in Kluyveromyces lactis and Saccharomyces cerevisiae