458 APPENDIX 



if the colonies of the plate appear to be distributed uniformly, we count 

 those in one of the divisions. They reach, we will suppose, the figure 

 of 60 ; 60 x 8 = 480 micro-organisms in the amount taken from the sus- 

 pected water and added to the melted gelatine from which the plate was 

 made. Let us suppose this amount was -25 c.c. Then the number of micro- 

 organisms in the suspected water is 60 x 8 = 480 x 4 = 1920 m.o. per c.c. 



Double Staining 1 Methods. These are various, and are used when 

 it is desired to stain the bacteria one colour, and the matrix or ground 

 substance in which they are situated another colour. Two of the 

 common methods are those of Ziehl-Neelsen and Gram. They are as 

 follows : 



Gram's Method. The primary stain in this method is a solution of 

 aniline gentian- violet (saturated alcoholic solution of gentian-violet 30 

 c.c., aniline water 100 c.c.), or Nicolle's carbol-gentian-violet, which stains 

 both ground substance and bacteria in purple. The preparation is next 

 immersed in the following solution for thirty or forty seconds : 



Iodine ..... 1 part 



Potassium iodide . . . 2 parts 



Distilled water . . . .300 parts 



In this short space of lime the iodine solution acts as a mordant by 

 chemical combination, fixing the purple colour in the bacteria, but not in 

 the ground substance. Hence, if the preparation be now (when it has 

 assumed a brown colour) washed in alcohol (methylated spirit), the 

 ground substance slowly loses its colour and becomes clear. But the 

 bacteria retain their colour, and thus stand out in a well-defined manner. 

 Cover-glass preparations decolorise more quickly than sections of 

 hardened tissue, and they should only be left in the methylated spirit 

 until no more colour comes away. The preparation may now be washed 

 in water, dried, and mounted for microscopic examination, or it may be 

 double-stained, that is, immersed in a contrast stain which will lightly 

 colour the ground substance. Eosin or Bismarck brown are commonly 

 used for this purpose. The former is applied for a minute or two, the 

 latter for five minutes, after which the specimen is passed through 

 methylated spirit (and preferably xylol also) and mounted. The result 

 is that the bacteria appear in a dark purple colour on a background of 

 faint pink or brown. Carbol thionine blue, picro-carmine, and other 

 stains, are occasionally used in place of the aniline gentian-violet, and 

 there are other slight modifications of the method. The application of 

 the method of Gram to coverslips and ordinary slide specimens for the 

 microscope may be shortly stated thus : 



1. Allow two or three drops of the gentian-violet stain to fall upon 

 slide and remain in contact with the film for t five seconds. 2. Wash off 

 the stain with the iodine solution applied from a drop bottle for five or six 

 seconds. The film should then be black or dark brown. 3. Wash off 

 the iodine solution with a mixture of 1 part acetone and 2 parts alcohol 

 absolute, but allow to remain in contact for two or three seconds only. 

 4. Wash off with absolute alcohol, applied until no more stain comes 



