90 APPLIED BACTERIOLOGY 



may be broken off. Small drops of water are placed on a 

 number of perfectly clean cover-glasses, and each of these 

 inoculated with a trace of the dilution, which is gently 

 spread over the cover-glasses. Better results are obtained 

 by diluting a broth culture about twenty-four hours old. 

 Tap- water should always be used for diluting, as the micro- 

 organisms are so extraordinarily sensitive that they often 

 cast off their flagella if placed in distilled water. 



The prepared cover-glasses are now allowed to dry in the 

 air spontaneously. The ' fixing ' must then be done with 

 great care by passing through the flame three times, by 

 holding the cover-glass in the fingers instead of the forceps, 

 as the temperature which is endured by the fingers does 

 not injure, but is quite sufficient for the purpose. The 

 : preparations are now treated by one of the following 

 methods : 



Lqffler's Method.- — It is to Dr. Loffler that we are indebted 

 for the following method, which he devised as the result of 

 a long and tedious investigation. This process, when care- 

 fully carried out, gives very fine results. Loffler found that 

 some organisms require an alkaline, and others an acid, 

 mordant ; and, again, the exact amount of alkalinity or 

 acidity varies according to the particular organism under 

 investigation. To render the mordant alkaline, he recom- 

 mends the use of a 1 per cent, solution of caustic soda, 

 while for the acidification of the mordant he employs a 

 dilute solution of sulphuric acid of such a strength that a 

 given volume is exactly neutralised by the 1 per cent, soda 

 solution. 



The following is the composition of the mordant : Solu- 

 tion of tannin (20 parts tannin + 80 parts water) ; to 

 10 CO. of this tannin solution add 5 c.c. of a cold aqueous 

 solution of ferrous sulphate and 1 c.c. of a concentrated 

 solution, either aqueous or alcoholic, of fuchsine. 



