28 MATERIALS AND PROCESSES OF SLIDE-MAKING 



Johansen's Quadruple Stain: 

 First staining solution 



Johansen's safranin (see page 22) 

 Second staining solution 



1% Methyl violet 2B 

 First differentiating solution 



95% Alcohol 30 ml. 



Methyl cellosolve 30 ml. 



Tertiary-butyl alcohol 30 ml. 

 Third staining solution 



Mix 6 ml. of methyl cellosolve with 6 ml. clove oil and saturate thiS' 



mixture with fast green FCF. Filter the saturated solution and add to it 



35 ml. of 95 per cent alcohol, 35 ml. of tertiary-butyl alcohol, and 12 ml. 



of 1 per cent acetic acid. 

 Second differentiating solution 



95% Alcohol 50 ml. 



Tertiary-butyl alcohol 50 ml. 



Glacial acetic acid 0.5 ml. 



Fourth staining solution 



Prepare separately saturated solutions of orange G in methyl cellosolve 



and 95 per cent alcohol. Mix fifty ml. of each solution. 

 Third differentiating solution 



Clove oil 30 ml. 



Methyl cellosolve 30 ml. 



95% Alcohol 30 ml. 



Special dehydrating solution 



Clove oil 30 ml. 



Absolute alcohol 30 ml. 



Xylene 30 ml. 



STAIN IS USED AS FOLLOWS: 



1. Accumulate sections mounted on slides in 70 per cent alcohol and place 

 them in the first staining solution for 1 to 3 days. 



2. Wash slides in running tap water until no more color comes away. 



3. Transfer them to the second staining solution for 10 to 15 minutes. 



4. Rinse slides in running tap water. 



5. Differentiate them for 10 to 15 seconds in the first differentiating solution. 



6. Stain sections in the third staining solution from 10 to 20 minutes or 

 until sufficient green dye has been absorbed. 



7. Rinse them for 5 to 10 seconds in the third differentiating solution. 



8. Place sections in the fourth staining solution from 3 to 5 minutes or 

 until the cytoplasm of the cells has become bright orange. 



9. Dip them up and down 3 or 4 times in the special dehydrating solution. 

 10. Transfer slides to xylene and mount in the ordinary manner. 



