KILLING AND FIXING 193 



Material is generally left in chrom-acetic acid for twelve hours or more, 

 but very delicate structures require only an hour or two. Some algge with 

 soft cell walls, such as Polysiphonia, will go all to pieces if left in the weak 

 formula more than five or ten minutes. Solutions employed upon the marine 

 algae must be made up in salt water instead of distilled water, and the fixed 

 material must also be washed in salt water. Tissues with hard or very firm 

 cell walls are improved by being left for longer periods, perhaps several 

 days, in the fluid, for the chromic acid acts on the cell walls, softening them 

 somewhat. 



Material fixed in chrom-acetic acid must be washed thoroughly before 

 being carried up into alcohol for final preservation (Sec. 178). This may be 

 done most satisfactorily with firm tissues in a gentle stream of tap water 

 circulating through the vessel (a wide-mouthed bottle with a piece of gauze 

 tied over the mouth to hold the material within is convenient). Washing 

 may occupy several hours or, with firm tissues, a much longer time without 

 danger. It is necessary to get all of the chromic acid out of the material, 

 otherwise a precipitate will be formed by the alcohol and the protoplasm 

 will not stain well. 



It is not generally known that the chromic acid can be washed out by 

 running the material through the grades of alcohol to 70% (Sec. 178), provided 

 the bottle of material is kept in the dark. The precipitate referred to in the 

 paragraph above is only formed by alcohol in the presence of light. The 

 70% alcohol must of course be changed until there is no trace of chromic 

 acid. This method works especially well with small objects and saves 

 much time and the somewhat difficult operation of washing small objects 

 in water. 



173. Chrom-osmo-acetic acid (Flemming's fluid). The most successful of the 

 formulae containing chromic, osmic, and acetic acids were developed and 

 perfected by Flemming and are accordingly called Flemming's fluids. The 

 addition of osmic acid to the chrom-acetic basis gives somewhat better 

 fixation of material than chrom-acetic acid alone. This better fixation 

 appears in the cytological details of nuclear division and in a more brilliant 

 reaction of material to the stains safranin and gentian violet, which with 

 orange G form a group often used together as a triple stain (Sec. 199, D) after 

 this fixing agent. Flemming's fluids penetrate slowly, and material should 

 be cut up into small pieces or slices, perhaps an eighth of an inch in thick- 

 ness, to obtain the best results. The expense of the osmic acid rather pre- 

 cludes the use of these fluids for general morphological and histological 

 studies where fortunately the cheap chrom-acetic formula^ are in the main 

 quite satisfactory. Flemming's fluids do not keep in the light, and it is best 

 to make them up fresh just before fixation. Solutions of osmic acid must be 

 kept in the dark. 



