CYTOCHEMICAL TESTS ON EMBRYOS 329 



2. Tissue Smear Method : 



a. Making smears: Tease small piece of tissue on slide. Moisten tissue with 

 physiological saline, distribute in thin film. Do not allow film of cells to dry. 



b. Fixation: Place slide face down carefully in shallow dish of absolute methyl 

 alcohol, supporting ends of slide with glass rods. Immediately transfer to 

 methyl alcohol in Coplin jar with screw top. Fix for several 24 hr. periods. 

 Slide can remain in methyl alcohol indefinitely. 



3. Ascites Fluid Smear Method : 



a. Shake bottle of fluid thoroughly so that sediment is well distributed throughout. 



b. Centrifuge samples of fluid at 3000 + rpm for 5 minutes. 



c. Decant supernatant fluid from centrifuge tube. 



d. With straight 6" tube dropping pipette transfer sediment from centrifuge tube 

 to glass slide. Distribute in thin film. 



e. Fix fluid film as in tissue smear method. Do not allow to dry. 



4. Preparation of Methyl-Green-Py ronin Y Staining Solution, 

 a! Acetate Buffer Solution (0. 2 M, pH 4. 2) 



Using only analytical reagents: 



Glacial acetic acid M. W. 60. 05 



Sodium acetate M. W.165. 09 



For 0. 2M Acetic Acid, add 1. 15 cc Acetic to 98. 85 cc distilled water 

 For 0. 2 M Sodium Acetate, add 2. 72 gr. Sodium Acetate to 100 cc distilled 

 water. 



Mix 75 cc of 0. 2 M Acetic Acid + 25 cc of 0. 2 M sodium Acetate for pH 4. 16. 

 Adjust pH with either sodium Hydroxide or Acetic Acid. 



b. Purification of Methyl Green in Acetate Buffer: 



Wash 0. 5% Methyl Green in Acetate Buffer with Chloroform in a separatory 

 funnel (abont 5 liters may be needed) until all violet color has been removed 

 from the stain-buffer solution. Let solution stand in open flask until residual 

 chloroform has evaporated (may be overnight). 



Use Commercial Certified Methyl Green. Different batches of this dye appear 

 to present varying shades of blue-green in the final reaction result with D N A. 



c. Purified Methyl-Green Pyronin Y Staining Solution. 

 100. cc Purified Methyl Green in pH 4. 2 Acetate Buffer 



0. 1 gr. Pyronin Y (Commerc. Cert.) 

 Keep refrigerated in glass-stoppered bottle. 



Solution may be reused for months, but always bring to room temperature 

 before using. 



5. Methyl Green-Pyronin Y Staining Procedure: 



a. (1) Sections cut at SfJ.. 



Decerate and hydrate from absolute ethyl alcohol in fresh alcohols to 2 

 quick rinses in distilled water. Let stand in 50% alcohol if necessary, 

 not distilled water. 

 (2) Tissue and Ascites fluid smears placed directly into absolute alcohol and 

 proceed as in (1) above. 



b. Stain: Sections 5 min. 



Smears 1 hour 



c. Handle each slide individually. 



d. Quick plunge into distilled water. Use fresh water frequently. 



e. Quick blot with No. 1 filter paper (7 cm circles good). 



f. Immediately place in Tertiary Butyl-alcohol mixture - 30 sec. with continu- 

 ous agitation. 



Alcohol mixture: 



Tertiary Butyl Alcohol 3 parts 



Absolute Ethyl Alcohol 1 part 



g. Quick rinse in xylene 



