STAINING METHODS. 33 



added to sixteen cubic centimetres of No. 1. For the bacillus of 

 typhoid fever one cubic centimetre of No. 2 is added to sixteen cubic 

 centimetres of No. 1. Bacillus subtilis requires twenty-eight to 

 thirty drops of No. 2 ; the bacillus of malignant oedema thirty-six to 

 thirty-seven drops, etc. 



By carefully conducted experiments Loffler has found that suc- 

 cess in staining the flagella depends upon adding just the right quan- 

 tity of acid or alkali, a very slight variation from the proper quantity 

 being sufficient to give an imperfect or negative result. In general, 

 those bacilli which produce an acid reaction in a neutral culture 

 medium require the addition of the alkaline solution, and those 

 which cause an alkaline reaction require the addition of an acid 

 acetic or sulphuric. 



Loffler gives the following detailed account of his method : 



A small quantity of a pure culture is suspended in a few drops of 

 distilled water. Upon perfectly clean glass covers small drops of 

 water are distributed by means of a platinum wire loop ; these are 

 sowed with bacilli from the first drop. These little drops are spread 

 out upon the cover with a platinum wire and allowed to dry in the 

 air, after which they are passed through the flame in the usual way 

 to fix the bacteria to the cover glass. Great care must be taken not 

 to heat the preparation too much, as this prevents the flagella from 

 taking the stain. Loffler recommends that the cover glass be held 

 between the thumb and forefinger in passing it through the flame, 

 instead of in forceps, as this insures it from being overheated. 



The mordant (solution No. 1) is now placed upon the cover glass 

 so as to completely cover it as an arched drop. The cover glass is 

 then carefully heated over a flame until steam commences to be given 

 off ; too much heat causes a precipitate which cannot be washed 

 away. The mordant is left upon the cover glass for from half a 

 minute to a minute, and during this time it is gently moved to and 

 fro. The cover glass is then washed by means of a stream of dis- 

 tilled water. All remnants of the mordant attached to the margins 

 of the cover glass should then be washed away with absolute alco- 

 hol. The staining solution is now dropped upon the surface of the 

 glass cover so as to completely cover it, and heat is applied as before 

 for about a minute until steam commences to be given off. The 

 staining solution recommended is a neutral, saturated aniline- 

 water-fuchsin solution. 



METHODS OF STAINING BACTERIA IN TISSUES. The solutions re- 

 commended for staining cover-glass preparations are also used in 

 staining bacteria in thin sections of the various organs, in which 

 they are found in certain infectious diseases ; but, in general, a 

 longer time is required to stain sections, and it is best not to hasten 

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