Chapter 5 

 STAINING TECHNIQUES 



The phase -difference microscope has proved extremely valu- 

 able in yeast cjrtology. This instrument increases slight contrasts 

 immensely, making lightly stained substances appear deep black. 

 K the cytoplasm is lightly stained it will also appear opaque; this makes 

 the phase difference microscope useless when the cytoplasm is col- 

 ored even faintly. It is necessary to shift from the ordinary light 

 to the phase -difference microscope as the occasion requires. 



An essential part of the equipment for yeast cytology is a small 

 centrifuge. A dilute suspension of cells can be concentrated in thir- 

 ty seconds, the supernate discarded, and the sediment used for ob- 

 servation, thus insuring an adequate amount of material on the slide. 

 Wet mounts are preferred because there is so much shrinkage of 

 the cell in a dry mount that much detail is lost. One of the first es- 

 sentials in yeast cytology is that the cells must never be allowed 

 to dry, for this invariably produces artifacts. The wet mounts are 

 pressed out using blotting paper (small pads of "bibulous" paper 

 can be purchased) and then sealed with melted paraffin. Cover 

 glasses 18 mm. square make sealing easier because they leave a 

 wide margin at the edge of the slide. 



DEAD CELL STAIN 



Yeast should first be observed in the dead cell stain, methylene 

 blue, diluted 1-10.000, at pH 5.5. It stains the dead cells blue and 

 stains living cells only slightly. The number of dead cells in a cul- 

 ture is quite variable and judicious use of the dead cell stain makes 

 it possible to avoid staining dead cells with their abundant artifacts 

 on the assumption that they are alive. There is one important pre- 

 caution: When a yeast cell contains an abimdance of stored glyco- 

 gen the cytoplasm does not take the dead cell stain. In these cells 

 some cytoplasm and the nuclear vacuole are crowded to one side 

 or one end of the cell and when the cells are presumably dead these 

 stain deeply with the dead cell stain suggesting that the cell, which 

 is otherwise completely unstained, may actually be dead. The slight 

 coloration of the living cells by the dead cell stain is usually con- 

 centrated in the mitochondria which appear black with the phase - 

 difference lens (fig. 5-1) but are much more difficult to see under 

 the ordinary light microscope. 



In applying methylene blue it is important to remember that 

 this dye is decolorized when reduced. Since yeasts contain con- 



5-1 



