48 AIDS TO BACTERIOLOGY 



particularly in cultures more than a day old, have a rest- 

 ing stage, when no motility is visible. 



Intra-Vitam Staining. A drop of a fluid culture is 

 placed on a sterile slide and covered with a sterile cover- 

 glass. A drop of stain is placed in contact with the edge 

 of the cover-glass. On application of a piece of filter- 

 paper to the edge diametrically opposite, the preparation 

 is irrigated with the stain. Non-toxic stains such as 

 neutral red or methylene green are used in 0'5 per cent, 

 aqueous solution. Other reagents may be similarly applied. 



Negative Staining. Burri's Indian-Ink Method. Liquid 

 Indian ink (Chin-chin, Giinther and Wagner, etc.) is 

 sterilised either by heating in an autoclave or allowing 

 it to stand for twenty-four hours mixed with one twenty- 

 fifth its volume of tincture of iodine and then centrifuging. 

 A drop of this is placed on a microscope slide, and by the 

 side of it a drop of the culture to be examined. (Dilution 

 of the ink with from one to six times its volume of water 

 is sometimes necessary.) Then the edge of another micro- 

 scope slide is allowed to rest on the two drops, which mix 

 and run along the line of junction of the slides. The second 

 slide is drawn across the lower one to make a smear as for 

 a blood-film (p. 46). Organisms show up white on a black 

 background. 



The Staining of Micro-Organisms. Bacteria take up 

 the basic anilin dyes with great avidity, and in some 

 cases peculiarity in respect of a certain staining method 

 serves for identification. 



Concentrated alcoholic solutions of stains are prepared 

 by allowing a large excess of the dye to digest for some 

 time in strong alcohol, shaking the solution from time to 

 time. The concentrated solutions are then filtered and 

 preserved in stoppered bottles. To increase the staining 

 properties, certain reagents (phenol, anilin, and alkalies) 

 are employed as mordants. 



Stains should be filtered before use, otherwise granules 

 of colouring matter may be deposited upon the preparation. 



Ehrlictis Anilin-Gentian Violet : 



Saturated alcoholic solution of gentian violet 11 c.c. 

 Saturated aqueous solution of anilin . . 100 c.c. 



The anilin solution is prepared by shaking about 5 c.c. of 

 colourless anilin with 100 c.c. of distilled water for some 



