132 MICROBIOLOGY 



to ascertain the length of time they should be exposd to the 

 maceration of the chromic acid. Even under the best condi- 

 tions it is almost impossible to stain some spores. 



Staining capsules. Many methods of demonstrating the 

 capsules have been devised. The glacial acetic method, as 

 described by Welch, is as follows: 1. Cover the preparation 

 with glacial acetic acid for a few seconds. 2. Drain off and 

 replace with aniline gentian violet solution ; this is to be re- 

 peatedly added until all the acid is replaced. 3. Wash in 1 

 to 2 per cent solution of sodium chloride and mount in the 

 same. 



Hiss' copper sulphate method. The organisms are grown, 

 if possible, on ascitic fluid or serum media. Spread the or- 

 ganisms on the cover-glass by mixing them with a drop of 

 serum, or, better, a drop of one of the diluted serum media. 

 Dry in the air and fix by heat. 



The capsules are stained in a 5 to 10 per cent aqueous 

 solution of gentian violet or fuchsin (5 cc. saturated alcoholic 

 solution gentian violet or basic fuchsin to 95 cc. distilled 

 water). The stain is placed on the dried and fixed cover- 

 glass preparation and gently heated for a few seconds until 

 steam arises. The dye is washed off with a 20 per cent solu- 

 tion of copper sulphate (crystals). The preparation is then 

 placed between filter-paper and thoroughly dried. 



Staining flagella. 1 Johnson and Mack have modified 

 Loeffler's method with very satisfactory results. The process 

 which they recommend and which we have used with success 

 is as follows : 



Make a culture of the organism to be stained on slant 

 agar and incubate for from 18 to 24 hours. Prepare a tube 

 with 6 to 8 cc. of sterile water, and keep it in the incubator 

 until it is of the same temperature. With a sterile platinum 

 loop scrape away some of the growth from the agar surface, 

 using care not to remove any of the agar, and rinse it off 

 carefully in the tube of water previously prepared for this 

 1 Johnson and Mack. Am. Med., Vol. VII (1903) p. 754. 



