5-5 THE YEAST CELL 



becomes overstained and the centrosome is concealed. If protein is 

 not too abundant, the centrosome stains well with aceto -orcein or 

 aceto -carmine, and these stains also reveal its structure. To see 

 this structure the ordinary light microscope should be used. 



METAPHOSPHATE (VOLUTIN) STAIN 



I have devised a specific stain for volutin which contains 4 cc. 

 saturated aqueous toluidine blue, 20 cc. commercial formalin adjusted 

 to pH of 2.5 with about 0.6 cc. of glacial acetic acid. Volutin (meta- 

 phosphate) stains red with this stain while basophilic proteins stain 

 light blue . A large loop of the sediment from a centrifuge tube 

 is introduced into 10 drops of the dye in a 4 x 1/2 inch test tube and 

 allowed to stand about 20 minutes. It may stand over-night without 

 harm. The suspension is centrifuged and the cells mounted in the 

 same fluid imder an 18 nrmi. cover slip, blotted, and sealed with para- 

 ffin. The glacial acetic acid in the stain prevents complete overstain- 

 ing of the cytoplasmic ribose nucleoprotein. The preparations must 

 be observed with an ordinary light microscope, since the light blue 

 C5rtoplasm makes the cell opaque in phase -difference microscopy. 



ALKALI -TOLUIDINE BLUE STAIN 



Cells are centrifuged and allowed to stand for ten minutes in two 

 per cent sodium hydroxide, which probably dissolves out much of the 

 ribose nucleoprotein but leaves most of the desoxy ribose nucleo- 

 protein. The cells are centrifuged and washed with water and then 

 brought down again. They are moimted in a 1 to 100 dilution of satu- 

 rated aqueous toluidine blue. No color appears in the cjrtoplasm, 

 but the heterochromatin stands out clearly differentiated from the 

 colorless cytoplasm. The fine desoxyribose nucleoprotein filaments 

 which form the chromosomes and which are clearly revealed by 

 Rafalko's modified Feulgen stain are also visible in some prepara- 

 tions, although probably they usually disintegrate due to the sodium 

 hydroxide treatment. This procedure works very well with non- 

 dividing yeast cells. When yeast cells are dividing rapidly they con- 

 tain very large amounts of ribose nucleoprotein and overstain very 

 readily; it may be helpful to follow the alkaline washing with a 10- 

 minute wash of 1 per cent sulfuric acid before applying the stain. 

 When this is done the the heterochromatin will stain with dilute tolu- 

 idine blue. 



ACID FAST STAIN FOR YEAST SPORES 



The asci are smeared on the slide from the gypsum slope. The 

 smear is fixed by heat and placed in a coplin jar of carbol fuchsin for 

 20 minutes or longer. Destaining is accomplished by washing rap- 



