172 CLINICAL BACTERIOLOGY AND HEMATOLOGY 



9. Xylol, two lots (to render the section permeable to 

 balsam). 



10. Balsam and a cover-glass. 



The last three steps are practically the same as the first three, 

 but in the reversed order, and similar phenomena are seen. The 

 section is opaque whilst wetted with the alcohol, 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 back- 

 ground, an additional dose of alcohol must be used, and the 

 xylol applied again. 



11. Gram's method as applied to sections, suitable for sections 

 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. 



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, and 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. 



