710 GENERAL TECHNIQUES FOR CLASSES OF PLANTS 



cent, solution of crystal violet. Allow the drop to evaporate to 

 dryness at 22° to 24° C. ; this takes eighteen to twenty-four hours. 

 In humid climates the last stages must be carried out in a desic- 

 cator, allowing twenty-four to thirty-six hours over concentrated 

 sulphuric acid. Now add clove oil with the least possible delay 

 and differentiate under the microscope. Remove the oil with 

 xylol and mount in balsam. 



1400. Yeasts. Wager {Ann. BoL, xii, 1898, p. 449) first 

 demonstrated the nucleus of Saccharomyces by the following 

 method. Fix in a saturated aqueous solution of corrosive subli- 

 mate for at least twelve hours. Wash successively in water, 

 30 per cent, alcohol, 70 per cent, alcohol and methyl alcohol. 

 Place a drop of the alcoholic suspension on a slide and, when 

 nearly dry, add a drop of water. When the cells have settled, 

 drain off the water and allow the preparation to dry. Place the 

 slide in water for a few seconds and then stain with a mixture of 

 fuchsin and methyl green, or fuchsin and methylen blue. Mount 

 in glycerin or balsam. See also Wager and Peniston, Ann. Bot., 

 xxiv, 1910, p. 45. 



Fixation of a smear in Flemming and staining with Heidenhain's 

 hsematoxylin gives good results. 



Kater {Biol. Bull, lii, 1927, p. 438) prepares films on slides 

 previously smeared with albumen and fixes them immediately 

 with corrosive sublimate-acetic-alcohol or preferably with Bouin's 

 fluid. Stain with iron-alum haematoxylin with or without light 

 green as a counterstain. 



Maneval {Stain Tech., iv, 1929, p. 21) states that in some kinds 

 the nucleus may be demonstrated by fixing smears with heat, 

 staining one minute with aqueous acid fuchsin, followed by 

 5 per cent, tannic acid for twenty seconds and a final washing 

 with acidulated water. A mixture of equal parts of acid fuchsin 

 and methyl green in water also is satisfactory. He also describes 

 some of the most useful of Gutstein's methods {Centrhl. f. Bakt., 

 xciii, 1924, pp. 233 and 393 ; xciv, 1924, p. 145 ; xcv, 1925, p. 1 ; 

 c, 1926, p. 1) of staining yeasts. 



Gutstein's general procedure is : Fix smears by means of heat 

 (three times over a flame) and stain two to three minutes with a 

 1 per cent, solution of a basic dye. Then wash with water and 

 treat two minutes with a 5 per cent, aqueous solution of tannin, 

 wash in water, counterstain, wash and examine. 



The most useful combinations are : For vegetative cells of yeast: 



(1) Tannin followed by safranin ; (2) carbol methylen blue, tannin, 

 safranin ; (3) methylen blue, tannin, safranin. For spores of 

 yeast : (1) Carbol fuchsin, 5 per cent, acetic acid, tannin, safranin ; 



(2) carbol methylen blue, 5 per cent, acetic acid, tannin, safranin. 

 Maneval prefers to use 2 to 3 per cent, sulphuric acid in place of 

 the acetic acid. 



