104 A MANUAL OF BACTERIOLOGY 



from the alcohol when no more colour dissolves out, or 

 the stain may be entirely removed ; usually twenty to 

 forty seconds in the alcohol suffices, thick preparations 

 taking longer than thin ones. After decolorising, films 

 are washed in water, dried, and mounted, or, after washing, 

 the ground substance may be counter-stained, if required, 

 with eosin for a few seconds, or Bismarck brown for two 

 or three minutes, washed again in water, dried, and 

 mounted. With films it is important to remember on 

 which side of the glass the film is, for it may be very 

 difficult to ascertain this after decolorisation. Sections 

 after decolorising are passed through absolute alcohol and 

 xylol before mounting, or, if required to be counter- 

 stained, are immersed in eosin for fifteen to thirty seconds, 

 or Bismarck brown for three to five minutes, and then 

 passed through alcohol, absolute alcohol, and xylol. 



Sections frequently are somewhat difficult to decolorise 

 with alcohol alone, in which case it is well to treat them 

 with a slightly acid alcohol (3 per cent, of hydrochloric 

 acid) for a few seconds, and then return to the alcohol 

 (Giinther's method). 



The iodine in Gram's method seems to act as a mordant, 

 precipitating the stain in a relatively insoluble form in 

 certain species of bacteria. The staining of organisms 

 by Gram is relative ; some forms do not stain at all, are 

 Gram-negative i.e. the colour is removed by the alcohol 

 with the greatest facility ; others stain intensely, are 

 Gram-positive, but even these may become decolorised 

 by prolonged treatment with alcohol. In order to ascer- 

 tain whether an organism is or is not stained by Gram's 

 method, it is sometimes useful to mix with it in making 

 the preparation some undoubted Gram-staining organism 

 e.g. if a bacillus, the Micrococcus pyogenes ; if a coccus, 

 B. anthracis or B. subtilis. The admixed organism then 

 serves as an index. 



