STAINING TECHNIQUES 5-2 



siderable amounts of reducing substances, the dye is decolorized 

 if a large excess of yeast is used and the dead cells cannot be de- 

 tected. For this reason only a small loop of relatively dilute yeast 

 cells is placed in a large drop of the stain. The dead cells can be 

 detected without the stain by their characteristic disintegretation, 

 their coagulated cytoplasm, and the fact that the vacuole is irregu- 

 larly wrinkled rather than round or lobed if visible. The phase - 

 difference microscope can be used to advantage in observing these 

 cells. In addition to revealing the number of dead cells, the stain 

 usually gives slightly more color to the cytoplasm of the living cell 

 and helps to show the shape and condition of the vacuole. 



LUGOL'S SOLUTION 



Lugol's stain is valuable in studying yeast for it reveals the 

 presence of glycogen in the cells. The standard formula for Lugol's 

 solution is: 



5 gm. I 



10 gm. KI 



100 cc. H2O 



This is generally too strong and causes considerable shrinkage of 

 the cells. Lugol's can be diluted 1 to 100 and yeast cells can be sus- 

 pended in it, centrifuged to concentrate them and mounted in the 

 same liquid. The concentration of the stain may be varied by the 

 operator depending on conditions and on what he is looking for. The 

 preparations must be observed with the standard light microscope 

 since the stain makes the cells too opaque to show details under the 

 phase -difference microscope. Either a blue 45 or a green 58 Wrat- 

 ten filter, or both together, are very helpful. Gylcogen stains red- 

 dish brown with iodine and appears black with these filters. 



The centrosome in old cells is made visible by Lugol's if the 

 C3rtoplasm is not too dense; it stains yellow brown and has the ap- 

 pearance of being surrounded by a membrane, fig. 5-2. The vacu- 

 ole may be either a single round body or a lobed structure with nu- 

 merous interconnections. 



Lugol's makes the vacuole visible in cells in which it is sur- 

 rounded by glycogen, and therefore concealed, before applying the 

 stain. Cells without glycogen stain golden yellow. 



The nucleolus often appears in cells stained by Lugol's. 



In preparations fixed with formalin, the chromosomes often 

 stain well With iodine. 



ACETO-ORCEIN AND ACETO-CARMINE 



These dyes are only effective when the cytoplasm is not too 

 dense, (fig. 5-3). If much protein is present in the cytoplasm it 



