176 BACTERIOLOGY 



nection with this method that require to be emphasized in 

 order to insure success: the culture to be employed should 

 be young, not over 18-20 hours old; it should have developed 

 for this time on fresh agar-agar at 37° to 38° C. ; the mordant 

 should not be perfectly fresh, as the best results are obtained 

 from the use of old solutions that have stood exposed to the 

 air and that have been filtered just before using; when 

 placed on the cover-slip and held -over the flame never heat 

 the mordant to the boiling-point; indeed, the best results are 

 obtained when the preparation is held high above the flame 

 and removed from it at the first evidence of vaporization, or, 

 better still, a little before this point is reached.^ 



Duckwall's Method^ is a modification of the Lofiler method, 

 and the results obtained thereby are very satisfactory. 



Preparation of the Staining Agents. — The fixing agent is 

 mordant, and the stain is carbol-gentian-violet or, prefer- 

 ably, carbol-fuchsin. 



The Mordant. 



Desiccated tannic acid 2 grams 



Cold saturated solution ferrous sulphate (aqueous) 5 grams 



Distilled water 12 c.c. 



Saturated alcoholic solution of fuchsin 1 c.c. 



The tannic acid is dissolved in the water first by the 

 application of gentle heat, then the ferrous sulphate, and 

 then the alcoholic solution of fuchsin are added. To these 

 ingredients it is advisable to add from 0.5 to 1 c.c. of a 

 1 per cent, sodium hydroxide solution. The best grade of 

 filter-paper is used for filtering the mordant, and there 

 should be left a heavy precipitate. After filtering, the color 



' I am indebted to Dr. James Homer Wright, Thomas Scott Fellow in 

 Hygiene, 1892-1893, University of Pennsylvania, for some of the suggestions 

 in connection with the modification of this method. 



2 The Canner, vol. xx, p. 23. 



