148 Methods of Observing Micro-organisms 



the flame from apex to base three times. This is supposed to be 

 exactly the requisite amount of heating. The rule is a good one for 

 the inexperienced. 



Inequality in the size of various flames may make it desirable 

 to have a more accurate rule. Novy* suggests that as soon as it is 

 found that the glass is so hot that it can no longer be held against the 

 finger it is sufficiently heated for fixing. 



After fixing, the preparation is ready for the stain. Every labora- 

 tory should be provided with "stock solutions," which are saturated 

 solutions of the ordinary dyes. For preparing them Woodf gives 

 the following parts per 100 as being sufficiently accurate: 



Alcoholic solutions (96 per cent, alcohol) Aqueous solutions (distilled water) 



Fuchsin 3.0 grams. 



Gentian violet 4.8 " Gentian violet 1.5 grams. 



Methylene-blue 7.0 " Methylene-blue 6.7 " 



("O per cent, alcohol) 



Scharlach R 3.2 " 



Soudan III 0.2 " 



(50 per cent, alcohol) 

 Thionin 0.6 " Thionin 1.2 " 



Of these it is well to have fuchsin, gentian violet, and methylene- 

 blue always made up. The stock solutions will not stain, but form 

 the basis of the staining solutions. For ordinary staining an aqueous 

 solution is employed. A small bottle is nearly filled with distilled 

 water, and the stock solution added, drop by drop, until the color 

 becomes just sufficiently intense to prevent the ready recognition 

 of objects through it. For exact work it is probably best to give 

 these stains a standard composition, using 5 cc. of the saturated 

 alcoholic solution to 95 cc. of water. Such a watery solution pos- 

 sesses the power of readily penetrating the dried cytoplasm of the 

 bacterium. 



Cover-glasses are apt to slip from the fingers and spill the stain, 

 so when using them it is well to be provided with special forceps 

 which hold the glass in a firm grip and allow of all manipula- 

 tions without danger of soiling the fingers or clothes. The ordi- 

 nary sharp-pointed forceps are unfit for the purpose, as capillary 

 attraction draws the stain between the blades and makes certain 

 the soiling of the fingers. In using the special forceps the glass should 

 not be caught at the edge, but a short distance from it, as shown in 

 the cut. This altogether prevents capillary attraction between the 

 blades. When the material is spread upon the slide no forceps are 

 needed, and the method correspondingly simplified. Sufficient stain 

 is allowed to run from a pipet upon the smear to flood it, but not 

 overflow, and is allowed to remain for a moment or two, after which 

 it is thoroughly washed off with water. The smear upon a slide is 

 then dried and examined at once, a drop of oil of cedar being placed 



* "Laboratory Work in Bacteriology," 1899. 



f "Chemical and Microscopical Diagnosis," N. Y., 1905, D. Appleton & Co., 

 p. 683. 



