28 CLINICAL BACTERIOLOGY AND 



4. Formalin (a 10 per cent, aqueous solution) fixes in 

 two or three minutes. 



As we shall see (page 33), where Jenner's or Leishman's 

 stains are used no fixing is required, as these stains are 

 already supplied with fixing agents. 



Stains. 



Where one is examining pathological fluids, blood, etc., 

 for bacteria, their presence is more readily detected when 

 they are stained. Unfortunately, however, other elements 

 in the field take up the staining material, thus rendering 

 the presence of bacteria less effective. We possess a 

 method of staining which has the distinct virtue of 

 staining only bacteria, leaving pus cells, debris, etc., intact 

 — i.e., Gram's method. Moreover, this method of staining 

 has another advantage, for by it some bacteria are stained 

 and some are not. It is therefore used as a diagnostic 

 stain, as we have certain bacteria which stain by Gram's 

 method, and called " Gram-positive," and other bacteria 

 which do not stain — " Gram-negative." 



Gram's Method of Staining. 



Method. — Spread the fluid on a slide, dry, and fix as 

 already described. Stain for two or three minutes with 

 carbol gentian violet or aniline gentian violet; wash in 

 water to remove excess of stain. Flood with " iodine solu- 

 tion " (iodine, 1 part ; pot. iodide, 2 parts ; water, 300 parts) 

 for about one minute ; wash off in alcohol until no more stain 

 comes away. If the slide is left very pale, one may usually 

 take it for granted there are no Gram-positive bacteria 

 present, or at least very few. The film should now be sub- 

 jected to a contrast stain, carbol fuchsin (diluted) or neutral 

 red ; wash in tap-water, dry with filter-paper, mount, and 

 examine. If the bacteria are Gram-positive they will be 

 dark violet, almost black, in colour; those Gram-negative 

 will follow the contrast stain, as will blood-corpuscles, 

 pus, cells, debris, etc. 



