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 Loffler, 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. Bacillus subtilis requires twenty-eight to 

 thirty drops of No. 2 ; the bacillus of malignant oedema 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 

 fingera, 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 he de- 

 scribes as follows : 



"The method consists in the use of but a single solution, which is at once 

 mordant and stain. The solution should be made in two parts, which are 

 filtered and mixed. 

 3 



