MICROSCOPIC STUDY AND STAINING 113 



(1) Smearing. Bacteria from a fluid medium are transferred in 

 a small drop of the fluid, with a platinum loop, to a cover-slip and 

 carefully spread over the surface in a thin film. If taken from a 

 solid medium, a small drop of sterile water is first placed upon the 

 cover-slip and the bacteria are then in very small quantity carefully 

 emulsified in this drop with the platinum needle or loop and spread 

 in an extremely thin film. 



(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, inasmuch 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 bi- 

 chloride of mercury, Zenker's fluid, or acetic acid. If chemical fixa- 

 tives are used, they must be removed by washing in water before 

 the stain is applied. If a preparation 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, gen- 

 tian-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: 3 



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. 



3 After Wood, ' ' Chemical and Microscopical Diagnosis, ' ' Appendix. N. Y., 1909. 



