EXPERIMENTAL EQUIPMENT AND PROCEDURES 21 



d. Heldenhaln' a Iron haematozylin - still the most reliatle and satisfactory 

 nuclear stain. The alum must be in form of violet crystals when the mordant 

 is made up. Mordant in k'jL for 12 hours, stain in 0.5^ haematoxylin for 

 3-12 hours (shorter time if 0.1^ Turgitol is used) and deatain in 2']t alum 

 under hinocular magnification. The slide should be rinsed in water when the 

 tissue has become grey and the nuclear constituents first become visible. 

 Elnse 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 instead 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^ 



HCl _ 0.2'f, 



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

 stain about 20 minutes; destaln in a weak ferric chloride (O.l^) under 

 binocular magnification. 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 modifications recommended are: 



1. Hydrolysis 10-12 minutes at 6o°C. in N-HCl 



(use 82.5 cc. cone. HCl to 1000 cc. water) 



2. Blnae In cold N-HCl. 



5. Elnse in d,lstllled water. 



k. Stain in acld-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 solution becomes colorless It la ready for use. 

 Keep in the dark, and do not use if it becomes discolored. 



5. Pass sections throi;igh 5 baths of dilute sulfuroua acid 



Distilled water 200 cc. 



10^ aq. solution of anhydrous 



sodium bisulphite 10 cc. 



Dilute (N) HCl 10 cc. 



6. Binse In distilled water. 



7. Counterstaln (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 - 1^ in emillne water, wash in tap water. 



d. Orange G - sat. solution in clove oil, use only 3O-60 seconds. 



e. Masson stain - differential stain; excellent for pituitary cell types. 



