102 MICROSCOPIC METHODS. 



violet is mixed with lo parts of 5 per cent solution of carbolic acid. It is 

 used as No. 4. 



6. Carbol-fuchsin (see p. 104) . — This is a very powerful stain, and, when 

 used in the undiluted condition, \-\ minute's staining is usually sufficient. It 

 is better, however, to dilute with five to ten times its volume of water and 

 stain for a few minutes. In this form it has a very wide application. 

 Methylated spirit with or without a few drops of acetic acid is the most con- 

 venient decolorising agent. Then dehydrate thoroughly, clear, and mount. 



Various other staining combinations might be given, but the 

 above are the best and most widely used. If the reader has 

 thoroughly grasped the remarks made above on the general 

 principles which underlie the staining of bacteria, he will be able 

 to use any combination to which his attention may be directed. 

 We may only add here that different organisms take up and hold 

 different stains with different degrees of intensity, and thus 

 duration of staining and degree of decolorisation must be varied. 

 It may be laid down as a general rule that, so long as organisms 

 retain the stain, the greater the decolorisation of the tissues in 

 which they lie, the clearer will be the results. 



Gram's Method and its Modifications. — In the methods 

 already described the tissues, and more especially the nuclei, 

 retain some stain when decolorisation has reached the point to 

 which it can safely go without the bacteria themselves being 

 affected. In the method of Gram, now to be detailed, this does 

 not occur, for the stain can here be removed completely from the 

 ordinary tissues, and left only in the bacteria. All kinds of 

 bacteria, however, do not retain the stain in this method, and 

 therefore in the systematic description of any species it is 

 customary to state whether it is, or is not, stained by Gram's 

 method — by this is meant, as will be understood from what has 

 been said, whether the particular organism retains the colour after 

 the latter has been completely removed from the tissues. It must, 

 however, be remarked that some tissue elements may retain the 

 stain as firmly as any bacteria, e.g. keratinised epithelium, calci- 

 fied particles, the granules of mast cells, and sometimes altered 

 fed blood corpuscles, etc. 



In Gram's method the essential feature is the treating of the 

 tissue, after staining, with a solution of iodine. This solution is 

 spoken of as Gram's solution (sometimes as Lugol's), and has 

 the following composition : — 



