LABORATORY AND TEACHING METHODS 59 1 



3. Wash. 



4. Stain with Aq. Vesuvin five seconds. 



5. Wash. 



6. Mount. 



Diphtheria bacillus should show the polar granules stained blue and the body 

 brown. Pseudo-diphtheria show no polar granules. 



Auerbach's Stain. — Atjeebach, Leopold: Untersuchungen iiber die Spermato- 

 genese von Paludina vivipara. Jenaische Zeitschrift fur Naturwissenschaft, 30: 

 40S-SS4- 



B. Acid fuchsin and Methyl green 



Ba. Simultaneous. 



I part methyl green \ I. 

 1000 parts of water j 



I part acid fuchsin \ II. 

 1000 parts of water. J 



To so grams of the red solution add i drop of 10 per cent, glacial acetic acid. 



Solution I: 3 parts 1 ^^.^ 



Solution II: acid 2 parts J 



If necessary to filter, use a filter paper moistened with solution i, as the paper 

 absorbs the methyl green. Take slides from alcohol and stain slides five to fifteen 

 minutes, having dried the glass leaving only the sections moist before immersion. 

 20° to 25° is best temperature; more heat hastens the absorption of methyl green, 

 cold retards it. Place in absolute alcohol and destain five to fifteen minutes, or 

 even an hour. 



Polychrome Methylene Blue. — See McFakland, Joseph: Pathogenic Bacteria 

 and Protozoa, 1912, p. 197. 



To a o.s per cent, aqueous solution of sodium bicarbonate add methylene 

 blue (B X or "medicinally pure") in the proportion of i gram of the dye to 100 c.c. 

 of the solution. Heat the mixture in a steam sterilizer at ioo°C. for one full hour 

 counting the time after the sterilizer has become thoroughly heated. The mixture 

 is to be contained in a flask of such size and shape, that it forms a layer not more 

 than 6 cm. deep. After heating, the mixture is allowed to cool, placing the flask 

 in cold water, if desired, and is then filtered to remove the precipitate which has 

 formed in it. It should, when cold, have a deep purple-red color, when viewed in 

 either layer by transmitting a yellowish artificial light. It does not show this 

 color, while it is warm. To each 100 c.c. of the filtered mixture, add 500 c.c. of a 

 o.oi per cent, aqueous solution of yellowish water soluble eosin and mix thoroughly. 

 Collect the abundant precipitate which immediately appears on a filter. When the 

 precipitant is dry, dissolve it in methylic alcohol (Merck's reagent) in the proportion 

 of 0.1 grain to do c.c. of alcohol. In order to facilitate the solution, the precipitate 

 is to be rubbed up with methyl alcohol in a porcelain dish, or mortar with a metal 

 spatula, or pestle. 



This alcoholic solution of the precipitate is the staining fluid. It should be kept 



