288 BACTERIOLOGY. 



thumb and forefinger, with the specimen side down, and 

 then carefully dropped upon the surface of the stain. It is 

 allowed to float on the dye for 3-5 minutes. Sometimes, it 

 is necessary to stain for a longer period, or to warm the 

 dye on the radiator or on an iron plate (Pig. 22, p. 150), 

 in order to obtain a rapid and intense stain. The best 

 results are obtained when the specimen is treated with a 

 strong dye for a few minutes. The longer the dye acts, 

 the more difficult it will be to subsequently obtain a good 

 decoloration. 



The cover-glass is then picked up with the forceps, 

 thoroughly washed with water, and immersed in a solution 

 of iodine in potassium iodide. This is made by dissolving 2 

 g. of potassium iodide and 1 g. of iodine in 300 c.c. of dis- 

 tilled water. The specimen is allowed to remain in the 

 iodine solution for 3 to 5 minutes. Care must be taken not 

 to expose the specimen too long to the action of iodine, in- 

 asmuch as this tends to cause the protoplasm to contract 

 into granules. The cover-glass is then removed from the 

 iodine solution, washed with water, and placed in 95 per 

 cent, or in absolute alcohol. This can be kept in a watch- 

 glass or in an Esmarch dish, which should be tilted from 

 time to time to facilitate the decoloration. In the case of 

 greatly over-stained specimens, a drop of dilute acetic acid 

 may be added to the alcohol. 



From time to time, the cover-glass should be washed 

 with water and examined with the No. 7 objective in order 

 to ascertain the progress in decoloration. If the material 

 is spread over the cover-glass in a perfectly thin, even 

 layer the decoloration will be rapid and thorough. On the 

 other hand, if thick masses are present, it will not be pos- 

 sible to obtain complete decoloration without also decolor- 

 ing many of the bacteria. When, therefore, the greater 

 part of the back-ground has been decolored, the treatment 

 with alcohol should be discontinued. 



The cover-glass is then washed with water and stained 



