44 C'l'LTURE METHODS AND TECHNIQUE 



Chrom-osmo-acetic acid. This mixture, commonly known as 

 Flemming's solution, is very satisfactory for cytological work with 

 plant tissues. It is particularly desirable as a fixing agent to pre- 

 cede the triple stain, also iron haematoxylin ; and these are two of 

 the best cytological stains. As commonly employed, the Flemming 

 solution varies greatly in strength. The weaker solution is ordina- 

 rily to be recommended. This should include as an aqueous 

 solution chromic acid from \ to 1- per cent, osmic acid ^ per cent, 

 acetic acid ^ per cent. It is possible, however, to employ the 

 solution at least twice as strong as the formula given, and it is 

 convenient to make stock solutions of each substance rather than 

 to make up at one time a large quantity of the fixing fluid. More- 

 over, the stock solutions mentioned may be so prepared as to serve 

 for any strength of the Flemming which may be required. Stock 

 solutions should be as follows : 



Chromic acid i per cent 



Osmic acid i per cent 



Acetic acid i per cent 



In order to prepare the weaker fluid, the following quantities 

 will be required : 



i per cent chromic acid . . . 25 cc. 



i per cent osmic acid . . . 10 cc. 



i per cent acetic acid . . . 10 cc. 



Water 55 cc. 



Stock solutions are very desirable on account of the fact that the 

 Flemming does not keep well, especially when constantly opened 

 for use. Any solution containing osmic acid turns black promptly 

 upon contact with certain organic material or dust. This effect is 

 facilitated by light, although light alone is noninjurious. It is 

 advisable, however, to store the osmic acid in a brown bottle,' or to 

 keep it from the direct sunlight. 



Ordinarily, material should be left in this mixture from twenty- 

 four to forty-eight hours, and it should then be washed in running 

 water two to four hours and finally passed through the different 

 grades of alcohol beginning at i 5 or 30 per cent until the desired 

 storage grade is reached, or until the material is thoroughly dehy- 

 drated, if it is to be immediately imbedded. After the use of the 

 Flemming mixture, however, it is often necessary to decolorize the 



