EXPERIMENTAL EQUIPMENT AND PROCEDURES 27 



c. Harris' acid haemalum - dilute to 25% with distilled water, stain as with haema- 

 toxylin and rinse in tap water. 



d. Heidenhain' s Iron haematoxylin - still the most reliable and satisfactory nuclear 

 stain. The alum must be in form of violet crystals when the mordant is made up. 

 Mordant in 4% for 12 hours, stain in 0. 5% haematoxylin for 3-12 hours (shorter 

 time if 0. 1% Turgitol is used) and destain in 2% alum under binocular magnifica- 

 tion. The slide should be rinsed in water when the tissue has become grey and 

 the nuclear constituents first become visible. Rinse thoroughly and dehydrate 

 quickly. 



There is a modification of the Heidenhain' s Iron Haematoxylin method which 

 shortens the staining time and makes the nuclei and chromosomes blue-black in- 

 stead of intense black, and the cytoplasm retains a slight stain which increases 

 the visibility of the spindle fibres. Two solutions are needed: 



1. Haematoxylin: 1% in absolute alcohol 

 Ferric chloride, C. P 1.2% 



2. HCl 0. 2% 



Prepare solutions separately. Before using, mix equal volumes of 1 and 2; stain 

 about 20 minutes; destain in a weak ferric chloride (0. 1%) under binocular magni- 

 fication. Intensity of stain relative to concentration of HCl. 



e. Feulgen stain - this is a chemical test for thymo-nucleic acid and if properly used 

 will give excellent chromosome stain without any trace of the cytoplasm. Polar 

 bodies of the frog's egg will stand out as red or violet in color. The modifica- 

 tions recommended are: 



(1) Hydrolysis 1 0-1 2 minutes at 60°C. in N-HCl 



(use 82. 5 cc. cone. HCl to 1000 cc. water) 



(2) Rinse in cold N-HCl. 



(3) Rinse in distilled water. 



(4) Stain in acid-fuchsin about 80 minutes. 



Basic fuchsin 1 gm. 



Distilled water 200 cc. 



This is made up as follows: Bring water to boil, add basic fuchsin and stir 

 thoroughly. Cool to 50°C. ; filter through coarse filter; add 20 cc. of dilute 

 HCl; cool to 25°C. ; add 1 gm. of anhydrous sodium bisulphite. When the so- 

 lution becomes colorless it is ready for use. Keep in the dark, and do not 

 use if it becomes discolored. 



(5) Pass sections through 3 baths of dilute fulfurous acid 



Distilled water 200 cc. 



10% aq. solution of anhydrous 



sodium bisulphite 10 cc. 



Dilute (N) HCl 1 cc. 



(6) Rinse in distilled water. 



(7) Counter stain (if desired) with 0. 5% Grubler's light green in 95% for 5 to 7 

 seconds only. 



(8) Mount in gum damar. 



f. Mayer's haemalum - very good for sections containing chromosome figures. 



Cytoplasmic stains : 



a. Light green - 0. 25% in 95% alcohol. Good for spindle fibres. Stain 1-2 minutes. 



b. Eosin - 0. 5% in 95% alcohol. Merely dip the slides into this quickly. Should not 

 be used when chromosomes are to be studied or photographed. 



c. Safranin O - 1% in aniline water, wash in tap water. 



