STAINING METHODS. 33 
No. 2. 
A one-per-cent solution of caustic soda. 
No. 3. 
A solution of sulphuric acid of such strength that one cubic centimetre 
is exactly neutralized by one cubic centimetre of the soda solution. 
According to Léffler, solution No. 1 is just right for staining the 
flagellum of Spirillum concentricum, but for certain other bacteria it 
is necessary to add to this some of No. 2 or of No. 3. Thus, for the 
cholera spirillum from half a drop to a drop of the acid solution is 
added to sixteen cubic centimetres of No. 1. For the. bacillus of 
typhoid fever one cubic centimetre of No. 2 is added to sixteen cubic 
centimetres of No. 1. Bagillus subtilis requires twenty-eight to 
thirty drops of No. 2; the bacillus of malignant cedema thirty-six to 
thirty-seven drops, etc. 
This method has not been very successful in the hands of other 
bacteriologists, and improvements in the technique have been made 
since it was first published. Van Ermengem (1893) points out the 
fact that a principal condition of success is that the cover glasses shall 
be absolutely clean. He boils them in a mixture composed of potas- 
sium bichromate, sixty grammes; concentrated sulphuric acid, sixty 
grammes; water, one hundred grammes. After coming from this they 
are thoroughly washed in water, then in absolute alcohol, and then 
dried in an upright position under a bell-jar. Recent agar cultures 
(ten to eighteen hours) are preferred, and the suspension in water 
should be very much diluted so that in the cover-glass preparation 
the bacteria are well isolated. The cover glass, held between the 
fingers, is passed three times through a flame. <A drop of the follow- 
ing solution is then placed upon it: Osmic acid two-per-cent solution, 
one part; solution of tannin (ten to twenty-five per cent) two parts. 
This is allowed to act for about five minutes at a temperature of 50° 
to 60° C.—or half an hour at the room temperature. After careful 
washing with water and alcohol the cover glass is immersed for a 
few seconds in a bath containing one-quarter to one-half per cent of 
nitrate of silver. Then without washing it is placed for a short 
time in the following: Gallic acid, five grammes; tannin, three 
grammes; fused potassium acetate, ten grammes; distilled water, 
three hundred and fifty grammes. It is then returned to the silver 
bath and kept there, with constant movement of the bath, until this 
commences to turn black. It is then thoroughly washed in water, 
dried, and mounted in balsam. 
Pitfield (1895) has devised a much simpler method which, as 
modified by Muir, is as follows: 
‘Prepare the following solutions : 
3 
