360 METHODS IN PLANT HISTOLOGY 



Professor Gregoire and his students have made this their principal 

 fixing agent. 



In spite of the weight of authority, we beheve that the Chicago 

 chromo-acetic-osmic formula is better. In the Flemming's weaker for- 

 mula we believe the chromic acid is too weak; the acetic acid, with its 

 tendency to cause swelling, is too strong ; and the osmic acid, unneces- 

 sarily strong. 



The osmic acid undoubtedly accelerates the killing of the proto- 

 plasm. This is seen more readily in animals. If Cyclops be brought into 

 30 c.c. of the solution A, the animals will swim for a while; if 5 or 6 

 drops of 1 per cent osmic acid be added to the solution, the animals 

 cease their movements almost instantly. Doubtless the osmic acid has 

 the same effect upon plant protoplasm. Where fixing is slow, very few 

 mitotic figures are found with the chromosomes midway between the 

 equator and the poles. 



Farmer and Shove, in studying these mitoses .and also vegetative 

 mitoses in Tradescantia, claimed better results with a mixture of 2 

 parts of absolute alcohol and 1 part glacial acetic acid. They allowed 

 the fixing agent to act 15-20 minutes, then washed in absolute alcohol, 

 and imbedded by the usual methods. This proportion of acetic acid 

 seems entirely too large for any accurate work with chromatin, and we 

 doubt whether the structure of the cytoplasm is normal when so much 

 acetic acid is used. 



The entire pollen mother-cell may be stained and mounted without 

 sectioning. Two descriptions of technique appeared in 1912, one by 

 Mann and the other by Pickett. Mann removes the pollen mother- 

 cells before fixing and staining; Pickett fixes and stains the anther in 

 toto and teases out the pollen mother-cells just before mounting. 



In Mann's method, the anther is placed in a drop of water and the 

 tip is cut off; a gentle tapping with a needle will then cause the pollen 

 mother-cells to float out into the drop. Fix in Bouin's fluid, 4-8 hours, 

 wash in 50 per cent alcohol until no color remains, and then stain in 

 iron-haematoxylin. At this stage we should put the material into 10 

 per cent glycerin and follow the Venetian turpentine method. 



Pickett fixed entire anthers in chromo-acetic acid for 30 hours, 

 washed in water for 24 hours, and then passed up to 80 per cent alco- 

 hol. At this point, he stained in strong cochineal or Kleinenberg's 

 haematoxylin for 5 days, then completed the dehydration, cleared in 

 cedar oil, teased out the mother-cells, and mounted in balsam. 



