28 GENERAL BACTERIOLOGY 



a -piece of sheet iron or in hot air sterilizer for one hour at about 

 200° C. They are best kept in a clean Petri dish and handled 

 with forceps. (Novy). Old slides and covers having balsam on 

 them should first be dropped one by one into a cleaning solution 

 (potassium bichromate 60, sulphuric acid 60, water 1,000), and 

 boiled for one-half hour and then treated as above. 



Special Directions. Clean J4 oz. of cover-glasses and place 

 them in a clean Petri dish. 



EXERCISE 17. PREPARATION OF STAINING SOLUTIONS. 



General Directions. The dyes most useful for staining bac- 

 teria are the basic anilin dyes which come in powdered or crystal- 

 line form. (Gruebler's dyes are standard). Those in most com- 

 mon use are Fuchsin, Methylen blue, Gentian violet and- Bismarck 

 brown. They keep in powdered form, with perhaps the exception 

 of Methylen blue, but because of greater convenience and equally 

 good keeping qualities, saturated alcoholic solutions are kept in 

 stock. These are made by adding the dry dye to 95% alcohol to 

 saturation and filtering. This form can not be used for staining 

 bacteria. The following solutions are required to begin work with : 



1. Aqueous solution of Gentian violet. 



Saturated alcoholic solution of Gentian violet 2.5 cc. 



Distilled water 47.5 ec. 



2. Saturated aqueous solution of Bismarck brown. 



3. Zieil's earbol-fuchsin. 



Saturated alcoholic solution of Fuchsin 5 cc. 



Solution of carbolic acid (5%) 45 cc. 



4. Loeffler's Methylen blue. 



Saturated alcoholic solution of Methylen blue 15 ee. 



Potassium hydrate (1:10,000)' 50 cc. 



6. Ehrlich's Anilin Oil Gentian violet.' 



Saturated alcoholic solution of Gentian violet 6 cc. 



Absolute alcohol 5 cc. 



Anilin water 50 cc. 



'•This dilution can be readily made by taking 1 cc. of a 10% potassium 

 hydrate solution, making this up to 100 cc, then taking 5 cc. of this and 

 making it up to 50 cc. 



' Some prefer anilin oil made as follows : Solution A., 2 ec. of anilin oil 

 and 100 cc. of distilled water; Solution B., 25 cc. of filtered saturated alco- 

 holic solution of gentian violet and 75 cc. of Solution A. Mix and filter. 

 This stain remains good for a long time. 



