DECOLOUBISING AGENTS 85 



Saturated alcoholic solution of methylene blue 30 c.c. 

 Caustic potash solution (1-10,000) 100 c.c. 



This solution keeps well, and is very useful for those 

 organisms which are very apt to overstain with the two 

 preceding staining solutions, such as the Bacillus diphtherioB, 

 sarcina, yeasts, etc. 



All the above staining reagents should be preserved in 

 the dark when not in use. 



Decolourising Agents. — It is found that when bacteria are 

 stained by any of the above methods, they can be made to 

 give up their stain, partly or wholly, by the application of 

 various reagents, such as acids, iodine, alcohol, etc. The 

 great importance of this fact will be seen when the staining 

 of bacteria in the tissues is dealt with. 



Cover-glass Preparations. — A chemically clean cover-glass* 

 is taken up with a pair of forceps and drawn through the 

 flame of a Bunsen burner or spirit-lamp to remove any 

 faint trace of grease remaining on the cover-glass. A small 

 droplet of water is then placed in the centre of the cover by 

 means of a clean glass rod. A trace of the culture or other 

 material to be examined is taken on the end of a sterile 

 platinum wire and mixed with the drop of water, and the 

 mixture spread out as evenly as possible over the cover- 

 glass. Care should be taken not to convey particles of 

 nutrient medium with the bacterial material on to the cover- 

 glass, as in the subsequent treatment these are apt to become 



* Mere rubbing with a clotb is not sufficient to clean cover-glasses. 

 They are best cleaned as follows : The cover-glasses are first cleaned 

 by rinsiag in water, and wiped with a clean rag, after which they are 

 heated for ten minutes ia a mixture of strong sulphuric acid and 

 bichromate of potash. After rinsing them in distUled water, they are 

 immersed in dilute ammonia, and after that polished on a clean linen 

 rag which is quite free from grease. The clean cover-glasses are best 

 preserved in strong alcohol, or in a clean stoppered bottle, so as to be 

 kept free from dust. 



