152 BACTERIOLOGY. 



that suggested by Van Erraengem. It is somewhat 

 more complicated than either of the preceding methods. 

 The steps in the process are as follows : 



In the centre of a perfectly cleaned cover- slip place a 

 drop of a very dilute suspension, in physiological salt 

 solution, of a 10- to 18-hour old agar-agar culture of the 

 organism to be studied. The suspension of the organ- 

 isms in the salt solution should be very dilute in order 

 to favor the isolation of single cells on the slip and also 

 to obviate the occurrence of excessive precipitation. The 

 slips are then to be dried in the air and in the gas-flame 

 in the usual manner. 



The mordant used consists of: 



Osmic acid (2 per cent, solution) 1 part. 



Tannin (10-25 per cent, solution) ...... 2 parts. 



To this 4 or 5 drops of glacial acetic acid may be 

 added, but experience has shown this to be hardly 

 necessary. 



Place a drop or two of this mordant on the cover-slip 

 to be stained, and allow it to act for one-half hour at 

 room temperature, or for five minutes at 50 to 60 C. 



Wash carefully in water and alcohol, and then im- 

 merse for a few seconds in the " sensitizing bath," viz., 

 a 0.25-0.5 per cent, solution of silver nitrate. Without 

 washing, bring the slip into a watch-crystalful of the 

 "reducing and reinforcing bath," viz. : 



Gallic acid . . . . . . . 5 grains, 



Tannin 3 " 



Fused pot. acetate 10 " 



Dist. water 350 " 



After a few seconds pass the slip back into a watch 

 crystal containing the dilute silver bath (0.25-0.5 per 

 cent, solution of silver nitrate in water) and keep it in 



