152 Methods of Observing Bacteria 



the glass be held in the fingers while the passes through the 

 flame are made. 



The cover-glass is now held in forceps, and the mordant, 

 solution A, dropped upon it until it is well covered, when 

 it is warmed until it begins to steam. The mordant must 

 be replaced as it evaporates. It must not be heated too 

 strongly; above all things, must not boil. This solution 

 is allowed to act from one-half to one minute, is then washed 

 off with distilled water, and then with absolute alcohol 

 until all traces of the solution have been removed. The 

 real stain, Loffler recommends an anilin-water fuchsin 

 (Ehrlich's solution), which should have a neutral reaction, 

 is next dropped on so as to cover the film, and heated for 

 a minute until vapor begins to arise, after which it is washed 

 off carefully, dried, and mounted in Canada balsam. To 

 obtain the neutral reaction of the stain, enough of the i 

 per cent, sodium hydrate solution is added to an amount 

 of the anilin-water-fuchsin solution having a thickness of 

 several centimeters to begin to change the transparent 

 into an opaque solution. 



A specimen thus treated may or may not show the flagella. 

 If not, before proceeding further it is necessary to study 

 the chemic products of the micro-organism in culture 

 media. If by its growth the organism elaborates alkalies, 

 from i drop to i c.c. of solution C in 16 c.c. must be added 

 to the mordant A, and the staining repeated. It may be 

 necessary to stain again and again until the proper amount 

 is determined by the successful demonstration of the fla- 

 gella. On the other hand, if the organism by its growth 

 produces acid, solution B must be added, drop by drop, 

 and numerous stained specimens examined to see with 

 what addition of alkali the flagella will appear. Loffler 

 fortunately worked out the amounts required for some 

 species, and of the more important ones the following 

 solutions of B and C must be added to 16 c.c. of solution 

 A to attain the desired effect: 



Cholera spirillum |-1 drop of solution C 



Typhoid fever 1 c.c. of solution B 



Bacillus subtilis 28-30 drops of solution B 



Bacillus of malignant edema .36 or 37 drops of solution B 



Part of the success of the staining depends upon having 

 the bacteria thinly spread upon the glass, and as free from 



