104 STAINING METHODS 



with 8 c.c. distilled water, filtering and replacing the loss from 

 evaporation. 



Alternative Mordant (B tinge's Mordant). 



Tannic acid, 20 per cent, aqueous solution . . 10 c.c. 

 Ferrous sulphate, saturated aqueous solution . . 5 c.c. 

 Fuchsin, saturated alcoholic solution i c.c. 



(6) Stain 



Weigh out 



Methylene-blue | 



Or methylene-violet .... [ 4 grammes 



Or fuchsin J 



and dissolve in 



Aniline water, freshly saturated and filtered ... 100 c.c. 



METHOD. 



1. Prepare and fix films as above. 



2. Pour the mordant on to the film and warm cautiously over 

 the flame till steam rises; keep the mordant gently steaming for 

 one minute. 



3. Wash well in distilled water till no more colour is discharged; 

 if necessary, wash carefully with absolute alcohol. 



4. Filter a few drops of the stain on to the film, warm as 

 before, and allow the steaming stain to act for one minute. 



5. Wash well in distilled water. 



6. Dry and mount. 



NOTE. The flagella of some organisms can be demonstrated 

 better by means of an alkaline stain or an acid stain a point to 

 be determined for each. Speaking generally, those bacilli which 

 give rise to an acid reaction in the culture medium require an 

 alkali; those which form alkali in cultivation require an acid. 

 According to requirements, therefore, LoefHer recommends the 

 addition of sodium hydrate, i per cent, aqueous solution, i c.c.; 

 or an equal quantity of an exactly comparable solution of sul- 

 phuric acid. 



5. Van Ermengem's Method. This method, being merely a 

 precipitation of a silver salt on the micro-organisms and not a 

 true stain, creates a false impression as to the relative proportions 

 of bacteria and flagella. 



(a) Fixing Fluid. 



Osmic acid, 2 per cent, aqueous solution . 10 c.c. 

 Tannic acid, 20 per cent, aqueous solution . 20 c.c. 

 Acetic acid, glacial i c.c. 



