METHODS OF OBSERVING BACTERIA. 159 



The cover is warmed until it begins to steam, and the 

 stain 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 in distilled water, then in 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 now 

 dropped on so as to cover the specimen, and heated for a 

 minute until vapor begins to arise; it is then washed off 

 carefully, dried, and mounted in Canada balsam. To 

 obtain this neutral reaction enough of the 1 per cent, 

 sodium-hydrate solution is added to an amount of the 

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

 eral centimeters to begin to change the transparent into 

 an opaque solution. Such a specimen may or may not 

 show the flagella. If not, before proceeding farther it is 

 necessary to study the products of the bacterium in cul- 

 ture-media. If by its growth the organism elaborates 

 alkalies, Solution C, in proportion from 1 drop to 1 c.cm. 

 in 16 c.cm. of the mordant A, must be added, and the 

 process repeated again and again until the proper amount 

 is determined. On the other hand, if the organism by 

 its growth produces acid, Solution B must be added, 

 drop by drop, until 1 in 16 cm. have been attained, and 

 numerous experiments made to see when the flagella 

 will appear. LofBer has fortunately worked out the 

 amounts required for some of the species, and of the 

 more important ones the following amounts of Solutions 

 B and C must be added to 16 c.cm. of Solution A to 

 attain the desired effect : 



Cholera spirillum, %-i drop of Solution C ; 



Typhoid fever, 1 c.cm. of Solution B ; 



Bacillus subtilis, 28-30 drops of Solution B ; 



Bacillus of malignant edema, 36-37 drops of Solution B. 



Part of the success of the staining depends upon 



