THE FIXING PROCESS 259 



of the protoplast Flemming's fixative has given on the whole 

 the best results. The formula for this is: 



One per cent, chromic acid 16 parts, 



Two per cent, osmic acid 3 parts, 



Glacial acetic acid i part. 



Make the i per cent, chromic acid solution by dissolving 

 i gram of chromic acid crystals in 99 c.c. of distilled water, and 

 dissolve i gram of osmic acid in 49 c.c. of distilled water to make 

 the 2 per cent, solution. Then mix together 16 c.c. of the chromic 

 acid solution, 3 c.c. of the osmic acid solution, and i c.c. of 

 glacial acetic acid. Of course more of the fixative can be made, 

 so only the ingredients are kept in this proportion. 



The osmic acid solution must be made with extreme care to 

 avoid all contamination with organic substances, which are 

 sure to spoil it, as shown by the formation after a time of a 

 black precipitate. For this solution procure a glass-stoppered 

 bottle, wash it thoroughly with soap and water, rinse it many 

 times, pour into it a saturated solution of bichromate of pot- 

 ash in strong sulphuric acid, stopper the bottle and shake it 

 vigorously, let it stand for a while and shake again, then pour 

 out the solution and rinse the bottle again and again with dis- 

 tilled water. Clean the stopper as thoroughly as the bottle. 

 The osmic acid comes sealed in glass tubes and it is best to 

 obtain it with one gram to the tube. Clean the outside of such 

 a tube in the manner described for the bottle, stirring it about 

 with a glass rod in the bichromate of potash solution and sub- 

 sequent rinsings, and keeping the fingers off of it; then guide 

 it with the rod into the clean bottle; pour into the bottle 10 c.c. 

 of distilled water, stopper the bottle and strike it against the 

 palm of the hand until the tube of osmic acid is broken; then 

 pour in the remaining 39 c.c. of distilled water necessary to 

 make the 2 per cent, solution. If the whole 49 c.c. of water 

 were poured in at first it would have been more difficult to break 

 the tube. Of course the distilled water must have been kept 

 in receptacles free from organic matter. The fumes of osmic 



