SECTION TWO 



Technique: 



1. Fresh semen is allowed to stand for about one half to one 

 hour until it liquifies. 



2. Prepare thin even smears of the liquified semen on scrupul- 

 ously clean and dry slides or coverslips. 



3. Fix for 3 minutes in a mixture consisting of equal volumes 

 of ether and absolute alcohol ; then allow to dry in the air. 



4. Flood smears with solution B and warm over a steam bath 

 or hot plate while the stain is allowed to act for 5-7 minutes at 

 40-60° C. 



5. Pour off excess stain. 



6. Wash the preparation thoroughly with distilled water. 



7. Drain and allow to dry thoroughly in air. 



8. Mount in a neutral synthetic resin such as D.P.X., Clear- 

 mount or Cristallite. 



Results : 



Sperm structure Stain reaction 



Galea capitis Pale bluish-grey; sharply outlined 



Cell membrane Bluish-grey; sharply outlined 



Nuclear membrane (shell) Bluish-grey ; sharply outlined 



Acrosome Slate blue 



Nucleus Pink 



Neck Sharply outlined (dark blue), inside 



colourless 



End knobs Dark blue 



Middle piece Sheath, dark blue ; centre dark pink 



Axial filament Dark blue 



Tail Dark blue 



Notes: 



It is suggested that the technique which has been tried out 

 only on dog and human semen, might be applied to other species. 



The technique gives good differentiation and preservation of 

 cytological structure, reliable fixation and staining and undistorted 

 and easily recognizable detail, upon which assays of semen for 

 fertility depend. 



Casarett states that there is a tendency for abnormal forms of 

 spermatozoa to stain more intensely than the formal forms. 



Reference: Casarett, George W. (1953). 



