1 68 Methods of Observing Bacteria 



though somewhat cumbersome, and hence rarely employed 

 at the present time. Three solutions are used : 

 (A)- 



Twenty per cent, aqueous solution of tannic 

 acid 10 



Cold saturated aqueous solution of ferrous sul- 

 phate 5 



Alcoholic solution of fuchsin or methyl violet . 1 



(B) One per cent, aqueous solution of caustic soda. 



(C) An aqueous solution of sulphuric acid of such strength 

 that i c.c. will exactly neutralize an equal quantity of 

 solution B. 



Some of the culture to be stained is mixed upon a cover- 

 glass with a drop of distilled water making a first dilution, 

 which is still too rich in bacteria to permit the flagella to 

 show well, so that it is recommended to prepare a second 

 by placing a small drop of distilled water, upon a cover 

 and taking a loopful from the first dilution to make the 

 second, and spreading it over the entire surface without 

 much rubbing or stirring. The film is allowed to dry, and 

 is then fixed by passing it three times through the flame. 

 When this is done with forceps there is some danger of the 

 preparation becoming too hot, so Loftier recommends that 

 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, Loftier 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. 



