Methods for Testing Stains 315 



For certification, samples of this stain should contain not less than 88% anhy- 

 drous dye. 



Biological Tests: Basic fuchsin is tested for several purposes: (1) For use as a 

 stain for the tubercle organism; (2) for use in the Endo medium for distinguishing 

 between the coli and aerogenes types of bacteria; (3) for its bacteriostatic action; 

 (4) for use in the Feulgen stain. 



(1) In testing as a stain for the tubercle organism, smears are covered with carbol 

 fuchsin (1 part of 3% alcoholic fuchsin to 9 parts of 5% aqueous phenol) and heated 

 on a water bath for 3-5 minutes. They are then rinsed in tap water and differ- 

 entiated in 70% alcohol containing 3% hydrochloric acid until practically no red 

 color remains visible to the naked eye. They are again rinsed in tap water; rinsed 

 once more and counterstained with dilute (i. e., about 0.1%) aqueous methylene 

 blue for 1 minute. The slides are then rinsed in tap water and dried and examined 

 under the microscope. The tubercle organisms should be distinctly red, while other 

 bacteria, leucocytes and debris appear blue. 



(2) In testing for use in the Endo medium, three separate solutions of the sample 

 to be tested are prepared: saturated alcoholic; 1% alcoholic; and 3% alcoholic. 

 Of these solutions, 0.5 ml. of the first and 1 ml. of each of the others are mixed 

 separately with 0.125 g. of anhydrous sodium sulfite dissolved in 5 ml. of hot dis- 

 tilled water. The tests are carried out separately with each of these three solutions; 

 good results must be obtained with at least one of them. The solution should be a 

 faint pink or straw color, or sometimes light brown, but without noticeable pre- 

 cipitate. This decolorized solution is added to 100 ml. of melted lactose agar of 

 the standard formula. The color of this medium should then be a light pink, which 

 fades almost entirely upon cooling. Before cooling it is poured into a Petri dish 

 and allowed to harden. The surf ace is then streaked in parallel lines with Escherichia 

 coli and Aerobacter aerogenes and allowed to incubate for 24 hours. A good sample 

 should show red growth with a strong metallic sheen for the former organism, pink 

 growth without metallic sheen for the latter, and no reddening of the medium 

 except where growth has occurred. 



(3) In testing for its bacteriostatic action, the same methods are followed as in 

 the case of brilliant green (p. 292) except that the dilutions for the spore-former 

 are from 1:100,000 to 1:1,000,000 and for the others 1:2,500 to 1:40,000. 



(4) In testing for its behavior in the Feulgen stain, the procedure followed is to 

 test the stain on properly prepared sections from which the paraffin has been re- 

 moved in the usual manner; these sections are rinsed in cold N HCl, placed 4-5 

 minutes in N HCl at 60° C, and then rinsed in cold N HCl, and finally in distilled 

 water. If the sections are of animal tissue they are then stained 2 hours, or if plant 

 tissue 3-5 hours, in the following solution: 0.5 g. of the sample to be tested is dis- 

 solved by pouring over it 100 ml. boiling distilled water, then shaken thoroughly 

 and cooled to 50° C, filtered, 0.5 g. K2S2O6 and 10 ml. A^ HCl are added; the solu- 

 tion is allowed to stand in the dark 24-48 hours; a good sample should then be almost 

 completely decolorized, at the most showing a very faint straw color. The sections, 

 after remaining for the specified time in this decolorized solution, are drained and 

 passed for 10 minutes each into three successive baths containing N HCl, 10% 

 K2S2OS, and distilled water in the proportion of 5:5:100 by volume (carrying out 



