316 Biological Stains 



this step in closed jars); they are then rinsed in distilled water and counterstained 

 in 1% aqueous orange G (for animal tissue) or 0.5% alcoholic fast green FCF or light 

 green (for plant tissue), dehydrated, cleared and mounted in balsam. A good basic 

 fuchsin should show almost complete decolorizatiou of the solution after standing 

 24 or at the most 48 hours, and on staining should give a reddish violet color to the 

 chromosomes. A sample which does not thus decolorize, however, need not be re- 

 jected if a clear solution can be obtained by the following procedure: To 85 ml. 

 distilled water, add 1 g. of sample, 1.9 g. Na2S205, 15 ml. N HCl. Place in a bottle 

 with 50-60 ml. free air space above the fluid. Shake for 2 hours at intervals or on a 

 mechanical shaker. Add 500 mg. fresh activated charcoal, and shake for 2 minutes. 

 Filter on a plaited hard filter. Solution should be colorless. 



"special" basic fuchsin for flagella staining 



Samples sold under this designation are presumed to be mixtures of pararo- 

 sanilin acetate and chloride in the proportion of 3 : 1 by weight, as recommended 

 by Leifson (1951). 



Biological Tests: They are tested by Leifson's method: Treat slides 1 week in 

 KjCrO* in cone. H2SO4, wash thoroughly with final rinsing in distilled water, drain 

 dry, hold in colorless flame for few seconds, cool, make heavy wax line across 

 middle and along margin to distal half. Place a loopful of sediment from a centri- 

 fuged 24-hour broth culture of a motile species of bacteria at end of slide; tilt, 

 causing liquid to flow to opposite wax line; air dry. Add quickly 1 ml. of stain 

 mixture (equal proportions of the following 3 solutions mixed together: 1.5% NaCI 

 in distilled water; 3% tannic acid in distilled water; 1.2% of sample in 95% alcohol). 

 Leave at room temperature 10 min., rinse slide with tap water under faucet, but 

 do not pour stain off before rinsing and drain dry. Satisfactory sample should 

 show the flagella bright red. 



Gentian Violet 



Dyes furnished under this label are frequently poorly defined mixtures of methy- 

 ated rosanilins, of the type usually designated as methyl violet. In some cases 

 crystal violet is marketed under the name gentian violet, and at times products of 

 this nature have both names on the label. Since crystal violet is more satisfactory 

 for bacteriological work and for those histological purposes where a deep blue- 

 violet is required, and methyl violet 2B more desirable in histological procedures 

 where a reddish-violet is called for, users should specify which of the two is desired 

 and should avoid entirely the term gentian violet. It is hoped that when users have 

 been suflSciently educated, distributors of stains can be induced to label these 

 violets methyl or crystal, as the case may be, and to abandon the meaningless name 

 gentian violet. 



When a dye is submitted under the name gentian violet, the nature of the dye 

 which predominates is determined spectrophotometrically, and the analysis and 

 biological tests are made as described under that dye. (See methyl violet, C. I. No. 

 680, and crystal violet, C. I. No. 681.) 



