GENERAL PRINCIPLES 163 



and cover with Lugol's (or Gram's) solution of iodine 

 (iodine i, KI 2, aq. dest. 300) for 30 seconds to 2 minutes. 

 Wash with 97 per cent alcohol (or methylated spirit) until 

 washings are no longer coloured (takes about 30 sec. to 

 2 min.). Examine in water, or dry and mount in balsam. 

 To counterstain : remove alcohol with water and cover 

 with dilute carbol-fuchsin for a few seconds (or saturated 

 watery solution of Bismarck brown for longer). Wash in 

 water, dry, and mount. Result : Bacteria blue-black, or 

 colourless, or red ; tissues red. Those bacteria which are 

 blue-black are said to be Gram-staining or Gram-positive. 

 The others are said to be Gram-negative. (See Table on 

 previous page.) 



Acid-fast Bacteria. Some bacilli stain with difficulty 

 with ordinary dyes, requiring the aid of heat or a mordant 

 (as carbolic). Such bacilli usually retain the stain even 

 when treated with dilute acids and alcohol, and hence are 

 called " acid-proof " or " acid-fast." This resistance is 

 believed to be due to the presence in the cell-body of a 

 waxy substance (an alcohol). The members of this group 

 are : Bacilli of human, bovine, avian, and fish tubercu- 

 losis; Moeller's Timothy-grass bacilli (i) and (2) ; Mist- 

 bacillus; Rabinowitch's butter bacillus; Korn's butter 

 bacilli (2) and others ; Johne's bacillus (of chronic bovine 

 pseudo-tuberculous enteritis) ; Bacillus smegmatis (smegma 

 bacillus) ; Bacillus leprae (leprosy bacillus). 



METHOD. Flood slide or cover-glass with carbol-fuchsin 

 and heat for 3 minutes. Wash and decolorize by dipping 

 into 5 per cent sulphuric acid and 60 per cent alcohol alter- 

 nately until film looks colourless. Wash in water. Counter- 

 stain with aqueous methylene-blue for a half to one minute. 

 W T ash and examine. The acid-fast bacteria are stained red, 

 while the others and the matrix are stained blue. 



Alcohol-fast Bacteria. In specimens of urine being 

 examined for tubercle bacilli, acid-fast smegma bacilli may 

 also be present. To distinguish: counterstain film in a 

 saturated solution of methylene-blue in absolute alcohol 

 for 5 minutes. Tubercle bacilli remain red, while smegma 

 bacilli become blue. 



Capsule Staining. Many bacteria possess a mucoid 

 or gelatinous envelope, though it is only in a few species 



