Stains and Staining 95 



4. Place in second staining solution for from 5 to 20 min or until sec- 

 tions assume bluish tinge. 



5. Differentiate in differentiating solution until nuclei are dark blue on 

 pink background. 



6. Rinse in 95% alcohol. 



7. Dehydrate in absolute alcohol and clear in two changes of xylol. 



Complex Botanical Stains. Most of the combinations used for zoological 

 staining, such as hematoxylin-eosin, can also be used in plant structures, 

 where it is desired to differentiate between the nucleus and the cytoplasm. 

 It is only a matter of convention that the safranin and light green com- 

 bination, so little used in zoological techniques, is preferred by most 

 botanists for their material. 



The complex stains are an altogether different matter, since the chemical 

 nature of plant structures is naturally different from that of animals. The 

 best of these complex botanical stains is: 



Johansen's Quadruple Stain: 



First staining solution 



Johansen's safranin 

 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 mix- 

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



35 ml of 95% 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% alcohol. Mix 50 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 



