MICROSCOPIC STUDY AND STAINING 95 



(2) The film is allowed to dry in the air. 



(3) When thoroughly dried, fixation is carried out by passing the 

 preparation, film side up, three times through a Bunsen flame, at about 

 the rate of a pendulum swing. Fixation by heat in this manner is most 

 convenient for routine work, but is not the most delicate method, in- 

 asmuch as the degree of, heat applied can not be accurately controlled. 

 The other methods which, may be employed are immersion in methyl 

 alcohol, formalin, saturated aqueous bichloride of mercury, Zenker's 

 fluid, or acetic acid. If chemical fixatives are used, they must be re- 

 moved by washing in water before the stain is applied. If a prepara- 

 tion is made upon a slide instead of a cover-slip, passage through the 

 flame should be repeated eight or nine times. 



(4) Staining. The dyes used for the staining of bacteria are, for 

 the greater part, basic anilin dyes, such as methylene-blue, gentian- 

 violet, and fuchsin. These may be applied for simple staining in 5 

 per cent aqueous solutions made up from filtered saturated alcoholic 

 solutions, or directly by weight. They are conveniently kept in the 

 laboratory as saturated alcoholic solutions. The strengths of some 

 saturated solutions are as follows: 



Saturated Solutions 1 (Stains Gruebler or Merck). 



Fuchsin (aqueous), 1.5 per cent. 



Fuchsin (alcohol 96 per cent), 3 per cent. 



Gentian-violet (aqueous), 1.5 per cent. 



Gentian-violet (alcohol 96 per cent) , 4.8 per cent. 



Methylene-blue (aqueous), 6.7 per cent. 



Methylene-blue (alcohol 96 per cent), 7 per cent. 



The staining solution, in simple routine staining, is left upon the fixed 

 bacterial film for from one-half to one and one-half minutes according to 

 the efficiency of the stain used. Methylene-blue is the weakest of the 

 three stains mentioned; gentian-violet the strongest. 



(5) The excess stain is removed by washing with water. 



(6) The preparation is thoroughly dried by a blotter or between 

 layers of absorbent paper. 



(7) A small drop of Canada balsam is placed upon the film side of 

 the dry cover-slip, which is then inverted upon a slide. The prepara- 

 tion is now ready for microscopical examination. 



1 After Wood, " Chemical and Microscopical Diagnosis," Appendix. N. Y., 1909. 



8 



