202 LABORATORY EXERCISES IN BACTERIOLOGY, 



(k) Acetic Acid Solution: 



Acetic acid (glacial), ... 2 



Water, 100 



Sometimes used as a mordant, the film preparation being flooded with the solution 

 for half a minute, then washed in water or acid blown off with fine blowpipe. A good 

 decolorizing agent for ordinary stains. 



STAINING METHODS. 



In the study of any unknown organism it should be a rule to subject film prepara- 

 tions first to the simple aqueous solution of gentian violet. or fuchsin, then to Loeffler's 

 blue, to Gram's method, and finally to the Ziehl's carbol-fuchsin and Gabbet's blue, 

 recording the result of each method. 



In staining a film with the ordinary aqueous solutions it is customary to 

 flood the film with a few drops of the staining fluid. Usually there is no need of heating ; 

 but should the stain be taken up but slowly, the slide or cover may be held over the 

 flame until vapor arises (not boiled). The staining is usually accomplished, when the 

 solution is warmed, in one-half to one minute ; when cold, usually from three to five 

 minutes are required. If preferred, the staining solution may be placed in a dish or 

 watch-crystal, and the cover on which the film is spread floated on the surface of the 

 fluid, film side downward. After sufficient exposure to the stain the preparation is 

 held under the tap in a gentle stream of water or waved gently through a dish of water 

 until color is no longer discharged. This is usually sufficient; but should the film 

 retain a deep tint, it is well to wash for a moment in alcohol, and again rinse in water. 

 The film is then dried carefully, being held at some height over the flame until quite 

 dry. It is then to be examined directly in oil, or a cover is added with Canada balsam, 

 as usual, and the preparation then examined with the microscope. 



Practically all bacteria may be stained by this method. The mycobacterium of 

 tuberculosis may be stained only with such difficulty, however, that in ordinary prepara- 

 tions containing this organism it is not found. So with tissues containing the Myco- 

 bacterium leprce; and the organisms of glanders, Asiatic cholera, and influenza also 

 stain very poorly, but may be demonstrated in deeply stained preparations made with 

 heat. 



Loeffler's alkaline blue is of general value, but many of the organisms take up 

 the stain but faintly. It is especially useful for staining the organisms of diphtheria 

 and gonorrhea. The stain is applied by flooding the film with several drops. It 

 acts best if somewhat warmed (not hot), and successful preparations are made in 

 from a few seconds to one or two minutes. The film is then washed well in water, 

 dried, and examined either after application of balsam and cover, or directly in oil 

 {oil-immersion lens). 



In the use of Gram's method probably the most important differential staining 

 process is met. For prosecution of this method the film is well stained, with use of 

 moderate warmth, with Ehrlich's aniline-water solution of gentian violet or fuchsin 

 for three or four minutes. The excess of the stain is washed in water, and the film 

 is flooded with Gram's iodine-iodide solution for one minute, the film now becoming 

 dark. It is next washed in ninety-five per cent, alcohol until the color is almost dis- 

 charged. vShould the original tint of the film return persistently, the preparation is 

 again treated with Gram's solution and again washed in alcohol. After almost total 



