STAINING TECHNIQUES 5-8 



ately centrifuged, mounted In the dye and sealed. There Is no 

 shrinkage of the cell and the chromosomes are readily stained. 



Sodium fluoride or azide can be mixed with toluidine blue with- 

 out precipitating the dye and mixtures of various concentrations 

 have been used successfully. This makes staining of the chromo- 

 somes possible In a single operation. This method does not stain 

 the centrosome. The use of delicate cytoplasmic poisons which In- 

 activate the metabolic enzymes Instead of grossly precipitating the 

 cytoplasm may be effective In the cytologlcal examination of larger 

 organisms. 



CONVENTIONAL CYTOLOGICAL TECHNIQXJES 



Dr. J. S. Rafalko (1945) devised a modified Feulgen stain with 

 which the yeast chromosomes were successfully stained for the 

 first time. The procedure Is as follows: 



The leuco basic fuchsln Is prepared by gentle bubbling of sul- 

 phur dioxide gas from a small aperture In glass tubing Into 1/2% 

 solution of basic fuchsln until decolorlzatlon occurs (In about 10-30 

 minutes). The solution Is then ready for use. Distilled water Is 

 similarly saturated for the sulphurous acid rinsing bath and may 

 be stored for many weeks In a tightly -corked flask. The sulphur 

 dioxide can be produced easily by a simple flask -and -funnel gen- 

 erator using sodium bisulphite and dilute sulphuric acid. 



The following schedule gave satisfactory results for smear pre- 

 parations of yeasts. 



1. Fixation, usually 2 to 20 minutes. 



2. Washing In tap water, minimum time according to fixative 

 used (not over 20 minutes). 



3. Distilled water, 2 minutes. 



4. Normal HCl at room temperature, 2 minutes. 



5. Normal HCl at 60^0., 8 to 10 minutes. 



6. Normal HCl at room temperature, rinse. 



7. Distilled water, rinse. 



8. Sulphurous acid bath, 2 minutes. 



9. Leuco basic fuchsln, 1^ to 2 hours. 



10. Sulphurous acid bath, for sufficient time to remove the free 

 unreacted leuco basic fuchsln (usually two or three 1 -minute 

 changes). 



11. Tap water, 10 to 15 minutes. 



12. Dehydration through alcohol, and counterstalnlng In 95% al- 

 cohol tinted with fast green. 



In collaboration with Mrs. Margaret Rafalko (Lindegren and Raf- 

 alko, 1949), the use of the standard killers such as PFA, Navashin, 

 corrosive sublimate, Flemmlng's chrome -aceto-formalln and others 



