METHYL^ VIOLET NUTRIENT MEDIA 315 



has been removed from everything but the tubercle bacilli. 

 The double staining is accomplished by now pouring over the 

 preparation a mixture of 3 parts of water with i part of a concen- 

 trated alcoholic solution of methylene-blue. After a few minutes 

 the methylene-blue is washed off with water, and the prepara- 

 tion is allowed to dry; when dry, it may be mounted in Canada 

 balsam. Other bacteria than the tubercle bacilli are decolor- 

 ized by the acid-bath, and are subsequently stained blue by 

 the methylene-blue. 



Methyl-violet. Starch grains may be stained by treatment 

 with a dark aqueous solution of methyl-violet. If the starch 

 grains after staining are treated with a very dilute solution of 

 calcium nitrate, the stain becomes deposited particularly in the 

 less dense layers of the grains. Useful as a stain for elaioplasts. 

 See under this head in the next chapter. See also in this chapter 

 under Staining Intra Vitam. 



Millon's Reagent. This should be prepared fresh, as 

 needed, by dissolving mercury in an equal weight of nitric 

 acid and then diluting this solution with an equal weight of 

 distilled water. Proteids are colored a brick-red with this 

 reagent. Sections to be tested are to be mounted in a drop 

 of the reagent on a glass slip. Warming the slip hastens the 

 reaction. 



Nutrient Media. Nutrient media must be sterilized by 

 heat to keep them from spoiling and to make it possible to grow 

 in them pure cultures that is, cultures of organisms of any 

 desired single species. Sterilization may be accomplished 

 by steaming the medium for about twenty minutes each day 

 on three days in succession, after having poured it into test- 

 tubes or flasks which have previously been tightly plugged with 

 cotton rolled into the form of a stopper of the proper size and 

 baked in an oven until the cotton is slightly scorched. The 

 tubes and cotton plugs should be baked together. Or, if an 

 autoclav is available in which steam can be generated under 

 pressure, and accordingly at a higher temperature than that 

 of boiling water at ordinary atmospheric pressure, the cotton 



