EXAMINATION OF BACTERIA WITH THE MICROSCOPE. 29 



(&) Allow the stain to act for about thirty seconds. 



(c) Wash in water. 



{d) Examine with the microscope in water directly or after 

 drying and mounting in Canada balsam. 



The rapidity and intensity of staining may be increased by 

 wanning the solution slightly. The bacteria will usually ap- 

 pear more distinct if, directly after pouring off the stain, the 

 preparation is rinsed for a few seconds in i per cent, solution 

 of acetic acid, and then thoroughly washed in water without 

 materially affecting the stain in the bacteria, which are thus 

 brought out more strongly. The acetic acid solution serves to 

 remove in a measure any color which has been imparted to the 

 background. 



Preparations that are mounted at first in water may be made 

 permanent by letting a drop of water fall at the edge of the 

 cover-glass so that it may easily be removed from the slide, then 

 drying and mounting in Canada balsam. Cover-glass prepara- 

 tions which have been stained are examined with oil-immersion 

 objective, employing the plane mirror, having the iris dia- 

 phragm open and the condenser close to the lower surface of 

 the glass slide. The purpose is to obtain the most intense 

 illumination possible over a small field. The watery solutions 

 of aniline dyes prepared as above described deteriorate in a short 

 time, and it is best to prepare them freshly each time they are 

 required. A very useful solution, which is permanent, is 

 Loffler's alkahne methylene-blue : 



Concentrated alcoholic solution of methylene blue 30 c.c. 



Potassium hydrate (caustic potash), 1—10,000 watery 



solution 100 c.c. 



Loffler's methylene-blue is a good stain for general purposes. 

 It is perhaps more in use than any other formula for coloring the 

 diphtheria bacillus. 



Aniline-water Staining Solutions. — The intensity with 



