STAINING OF FILM PREPARATIONS 729 



3. Stain with Ziehl's fuchsin as for flagella (p. 151). 



Reitmann's method. This is an application of Sclavo's method for staining 

 bacterial flagella. 



1. Fix thin films in absolute alcohol for 10 minutes. 



2. Wash in distilled water. 



3. Wash for 5 minutes in a 3 per cent, solution of phosphotungstic acid. 



4. Wash in water, then in 70 per cent, alcohol. 



5. Stain in Ziehl's fuchsin, warming until steam just begins to rise. 



6. Wash in 70 per cent, alcohol, then in distilled water. Dry. 

 Herxheimer's method. 1. Prepare films on slides and fix in absolute alcohol. 



2. Stain for a quarter-of-an-hour in an aqueous solution of gentian- violet saturated 

 in the warm. 



3. Wash. Dry. Mount in balsam. 



Herxheimer and Huber's method. 1. Fix for 10 minutes in absolute alcohol. 



2. Stain in the following solution, which must be filtered just before use, for 

 16-24 hours. 



Nile blue (or Capri's blue), - 010 gram. 



Distilled water, - 100 c.c. 



3. Wash in distilled water. Dry. 



Proca and Vasilescu's method. 1. Fix for 30 minutes in absolute alcohol. 



2. Immerse in the following mordanting solution for 10 minutes. 



Liquid carbolic acid, - - 50 grams. 



Tannin, - 40 



Distilled water, - - 100 



Basic fuchsin, - 2 '5 



Absolute alcohol, - 100 c.c. 



Mix the carbolic acid, tannin and water and then add the fuchsin after dissolving it in 



the alcohol. 



3. Wash in distilled water. 



4. Stain for 5 minutes in carbol-gentian- violet. 



5. Wash in water. Dry. 



Oppenheim and Sachs' method. No preliminary fixing of the film is required and 

 the treponemata are stained without having been dehydrated in alcohol, with the 

 result that the transverse diameter of the parasites is said to appear larger than 

 when they are treated by the classical methods. 



1. Dry the film. 



2. Without fixing, flood it with the following solution : 



Saturated alcoholic solution of gentian- violet, 10 c.c. 



5 per cent, carbolic acid in water, - - 100 ,, 



Heat the stain over a small flame until steam just begins to rise. 



3. Wash. Dry. Mount in balsam. 



Davidson's method. 1. Dry the film. Fix in absolute alcohol for 10 minutes. 



2. Stain for 110 hours in a saturated aqueous solution of Muhleimer's cresyl- 

 violet R extra. The stain must be freshly prepared and filtered before use. 



3. Wash in distilled water. Dry. Mount in balsam. 



Zabolotny's method. Dry. Fix. Mordant with a 5 per cent, solution of carbolic 

 acid. Stain in the warm for 15 minutes in a mixture containing 01 per cent, azur 

 solution and 0*2 per cent, eosin solution. 



Simonelli and Bandi's method. In this method, as in Marino's, preliminary 

 fixation is eliminated, the stain being dissolved in methyl alcohol. 



Method of preparing the stain. Dissolve 1 gram of water-soluble eosin in a litre 

 of water, and, in another vessel, 1 gram of methylene blue in a litre of water. Mix 

 the two solutions and let the mixture stand for a week. Filter through an unfolded 

 filter paper and wash the precipitate with distilled water. Dry the precipitate in 

 the air then dissolve it in sufficient methyl alcohol to yield a saturated solution. 



Method of staining. 1. Dry the film and, without fixing, stain in the above 

 solution for about 10 seconds. 



2. Wash very quickly in distilled water. 



3. Blot with filter paper. Examine without a cover-glass. 



Goldhorn's method is merely a complicated modification of the preceding. 

 Hoffmann and Halle's method. Fix the wet film with osmic acid. 



