5-7 THE YEAST CELL 



idly in 30 per cent acetic acid while holding the slide with a forceps. 

 The slide in not rinsed in water before destaining, and this prevents 

 the formation of the precipitate that usually occurs if it is transferred 

 directly to water from carbol fuchsin. Two or three rapid swishes 

 through the acetic acid should immediately be followed by a rinse 

 in water. If the slide is held too long in the acid, or not rinsed quickly 

 enough in water, it may be completely destained. The success of 

 this method depends on critical control of destaining. After the 

 water rinse, methylene blue is used as a counterstain. This pro- 

 cedure stains the spores red and the vegetative cells blue because 

 the waxes or lipoids in the spore retain the carbol fuchsin which is 

 washed out of the vegetative cells since they contain no wax. The 

 blue counterstain is incapable of staining the waxy spores so it does 

 not cover the red stain. 



DELAFIELD SPORE STAIN 



A tiny drop of cell suspension containing spores is placed on a 

 slide, a small drop of Delafield's haematoxylin added and covered 

 with a cover glass. This makes a preparation which dries out very 

 slowly, because the Delafield's contains glycerin (Frontispiece, S. 

 cerevisiae). The cell walls of the vegetative cells and spores may 

 be clearly defined. Some internal structures can be seen if 7 parts 

 of Delafield's is mixed with 3 parts of acetic acid, using the same 

 procedure. 



WEAK KILLERS 



The conventional cytological procedure of killing the cell with 

 concentrated solutions containing formaldehyde, acetic acid, osmic 

 acid, corrosive sublimate, picric acid, or chromic acid causes the 

 yeast cell to shrink to about one -fifth its normal size. It also causes 

 the precipitation of the cytoplasm and thus introduces an artifact of 

 some importance in addition to reducing the size of structures which 

 are already near the limit of visibility of the microscope. The ne- 

 cessity for high concentration of a toxic substance to kill cells 

 quickly to prevent necrotic changes from occurring is not so serious 

 in yeast cells as it is with animal tissues lor the distance which 

 killers must penetrate is only from 2 to 5 microns. I have used 

 various dilute cytoplasmic or enzyme killers such as sodium fluor- 

 ide or sodium azide with considerable success. Pretreatment with 

 these substances in concentrations of about 0.5 per cent renders the 

 cell permeable to dyes which are otherwise unable to enter the cell. 

 Without pretreatment concentrated toluidine blue is imable to enter 

 the cell but after pretreatment it enters the cell rapidly. The dye 

 is diluted to prevent over staining. Aqueous saturated toluidine blue 

 is diluted from 1 to 200 up to 1 to 800 and the cells are immedi- 



