52 AIDS TO BACTERIOLOGY 



over a colony (which should not exceed 2 millimetres in 

 diameter), in a slanting position, with one edge resting on 

 the nutrient medium, then allowed to sink gradually 

 down over the colony, and very gently pressed. The 

 cover-glass is carefully lifted with a needle, and allowed 

 to dry spontaneously. The preparation is ' fixed ' and 

 stained. This method shows the manner of growth and 

 the arrangement of the organisms. 



Staining of Spores. In ordinary cover-glass prepara- 

 tions spores resist the stain. All unstained spots are not 

 spores: they may arise from faulty staining due to air- 

 bubbles, the use of old staining solutions, or, in the case 

 of old cultures, the organisms may have become de- 

 generate tnd vacuolated. 



Heat Method. A cover-glass preparation is passed 

 through the flame twelve times in ' fixing,' stained for a 

 few minutes with warm Ehrlich's gentian- violet or Ziehl's 

 fuchsin solution, and then well washed in water. The 

 heating destroys the power of the organisms to take up 

 the stain, leaving only spores stained. 



Neisser's Method. The cover-glass preparation, made 

 in the usual way, is stained with warm carbol-fuchsin 

 solution for about ten to twenty minutes. It is best to 

 float the cover-glass on the surface of the stain contained 

 in a small dish on a sand-bath or piece of asbestos card- 

 board warmed with the Bunsen. The cover-glass is 

 removed, washed in water, decolorised for a few seconds 

 in a 3 per cent, alcoholic solution of hydrochloric acid, 

 well washed in water, counter-stained with Ldffler's 

 methylene blue for three minutes, washed in water, 

 blotted, dried, and mounted. The bacilli will be stained 

 blue and the spores red. 



Moeller's Method. The films are prepared and fixed, 

 and then treated with (a) absolute alcohol, two minutes; 

 (6) chloroform, two minutes; washed thoroughly; (c) 5 per 

 cent, chromic acid, one minute. They are stained in 

 warm Ziehl's fuchsin for ten minutes, decolorised in 1 per 

 cent, sulphuric acid for a few seconds (this has to be done 

 with care), washed, counter- stained with Loffler's blue for 

 two or three minutes, again washed, dried, and mounted. 

 The spores are stained red and the bacilli blue. 



Flagellum Staining. Flagella possess no affinity for 

 stains unless previously mordanted. The cover-glasses 



