896 PROTOZOOLOGY 



Fixation for 5-30 minutes; wash with 70% alcohol until picric acid 

 is completely washed away from the smears. 



Sublimate-acetic 



Saturated sublimate solution 100 cc. 

 Glacial acetic acid 2 cc. 



This is the original fixative for Feulgen's nucleal reaction (p. 897). 

 Fixation and after-treatment similar to Schaudinn's fluid. 



Fixation for 5-30 minutes; wash in 95% alcohol. 

 Osmium tetroxide 



The vapor from or the solution itself of 1% Osmium tetroxide may 

 be used. Fixation in 2-5 minutes; wash in running water. 



Flemming's fluid 



1% chromic acid 30 cc. 



2% osmium tetroxide 8 cc. 



Glacial acetic acid 2 cc. 



Fixation for 10-50 minutes; wash for one hour or longer in running 

 water. 



The most commonly used stain is Heidenhain's iron haematoxy- 

 lin, as it is dependable and gives a clear nuclear picture, although it 

 is unsatisfactory for voluminous organisms or smears of uneven 

 thickness. It requires a mordant, ammonio-ferric sulphate (iron 

 alum) and a dye, haematoxylin. Crystals of iron alum become yellow 

 and opaque very easily. Select clear violet crystals and prepare 2% 

 aqueous solution. Haejnatoxylin solution must be well "ripe." The 

 most convenient way of preparing it is to make 10% absolute alcohol 

 solution as it does not require ripening. By diluting this stock solu- 

 tion with distilled water, prepare 0.5 or 1% slightly alcoholic solution 

 which will be ready for immediate and repeated use. Smears are left 

 in the mordant in a jar for 1-3 hours or longer. Wash them with run- 

 ning water for 5 minutes and rinse in distilled water. Place the smears 

 now in haematoxylin for 1-3 hours or longer. After brief washing in 

 water, the smears are decolorized in Petri dish in a diluted iron alum, 

 0.5% HC1 in water or 50% alcohol, or saturated aqueous solution of 

 picric acid under the microscope. Upon completion, the smears are 



