226 CLINICAL BACTERIOLOGY AND H^MATOLOGY 



and becomes transparent when the xylol is added, and this 

 transparency is the proof that the steps have been carried 

 out properly. If the section looks opaque when held against 

 a perfectly dark background, an additional dose of alcohol 

 must be used, and the xylol applied again. 



II. Gram's method as applied to sections, suitable for sec- 

 tions of diphtheritic membrane, organs containing anthrax 

 bacilli, streptococci, staphylococci, etc. : 



1. Xylol, two lots. 



2. Absolute alcohol, two lots. 



3. Water. 



These steps are always the same with paraffin sections, no 

 matter what stains are to be used subsequently. 



4. Aniline gentian violet five minutes. Rinse in water. 



5. Gram's iodine solution three minutes or more. 



6. .Absolute alcohol or methylated spirit until no more 

 colour comes out. This step is best carried out as follows : 

 Hold the slide by one end, keeping the fingers clean by using 

 a duster or pair of dissecting forceps, arid pour a little spirit 

 on the section; rock it gently from side to side and notice 

 the ,clouds of colour which it takes up. After a little time 

 pour off the spirit and add a fresh lot; repeat the rocking, 

 and pour off again. Do this until the spirit comes away quite 

 clean, and does not take up any colour from the section. 

 This may take a long or short time, and no definite rules can 

 be laid down. 



In some cases decolorization can be carried out best by 

 the use of clove oil. This is applied when the section is wet 

 with absolute alcohol (for it will not mix with water), and 

 must be entirely removed by the same fluid before the section 

 is mounted, or it will cause it to fade. Clove oil is a very 

 powerful decolorizing agent, and requires careful use, or the 

 colour may be removed from the bacteria. 



7. Eosin half a minute or more. This is a counterstain, 

 and is used to demonstrate the structural elements, which are 

 not coloured by the gentian violet. It may be omitted in 

 some cases. 



8. Absolute alcohol two lots (to remove the water). 



9. Xylol two lots, or until the section becomes trans- 

 parent. 



10. Balsam and a cover-glass. 



