396 THE DOUBLE STAINING OF SPORES AND BACILLI, 



glass is used to hold the fluid. There are spores, as, for example, 

 Bar,, lactis XII. (Fliigge), which stain quite readily with carbol- 

 fuchsin in the usual manner after fixation by heat, while others, 

 as a Bac. leptosporus sp., stain but faintly when Klein's method is 

 employed. The refractory spores are stained a deep red by the 

 following method : — Four drops of normal saline are pipetted 

 into a small test-tube, and the spore-bearing material is rubbed 

 up with this until a homogeneous suspension is obtained. Four 

 drops of fresh carbol-fuchsin are pipetted into the tube and the 

 mixture shaken. A plug of cotton wool is inserted and the tube 

 placed into a beaker of boiling water. The water is boiled for a 

 quarter of an hour, when the tube is taken out and shaken. A 

 loopful of the bacterial suspension is withdrawn and spread 

 uniformly over a cover-glass which is dried either in the air or 

 high over a bunsen-flame. The film is next fixed by passing the 

 cover-glass three times through the flame in the usual manner. 

 The bacilli are decolorised in methylated spirit containing 1*5% 

 (by volume) of concentrated hydrochloric acid. When the film 

 appears colourless, the cover-glass is withdrawn and moved about 

 in water to remove the alcohol, after which the film is stained 

 with carbol-methylene-blue in the ordinary manner; it is then 

 washed, dried and mounted. 



The acidified alcohol appears to give a cleaner film than when 

 sulphuric acid is employed. Klein apparently means 1% sulphuric 

 acid by volume — that is, 1 c.c. of strong sulphuric acid to 99 c.c. 

 of water; 1% by weight of sulphuric acid does not decolorise the 

 film sufficiently. The spores of some of the water-bacilli are so 

 readily decolorised by acidified alcohol that it is advisable to 

 extract the excess of colour with methylated spirit, then to dip 

 the cover-glass for a moment only into the acidified alcohol and 

 quickly place in water. 



Generally speaking, spores can be recognised by the fact that 

 they are not coloured by the usual stains, and that the remains 

 of the mother-cell stain sufficiently to enable one to make out the 

 regular shape of the unstained spore. There occur cases, however, 



