158 Methods of Observing Micro-organisms 



Method of Staining Flagella. This is more difficult than the 

 staining of the bacteria or the spores. 



Loffler's Method.* This is the original and best method, 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 sulphate 5 



Alcoholic solution of fuchsin or methyl violet i 



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



(C) An aqueous solution of sulphuric acid of such strength that i cc. 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 plac- 

 ing 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 Lofifler 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 

 Lofifler 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 rise, 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 cc. of solution C in 16 cc. 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 flagella. 

 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 fol- 

 lowing solutions of B and C must be added to 16 cc. of solution A to attain the 

 desired effect: 



Cholera spirillum J^-i drop of solution C 



Typhoid fever i cc. 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 using a very young 

 culture and having the bacteria thinly spread upon the glass, so 

 as to be as free from albuminous and gelatinous materials as possible. 

 The cover-glass must be cleaned most painstakingly; too much 

 heating in fixing must be avoided. After using and washing off 

 the mordant, the preparation should be dried before the applica- 

 tion of the anilin-water-fuchsin solution. 



* Ibid., 1890, Bd. vii, p. 625. 



