MICROSCOPIC METHODS 75 



Staining Spores. We have already noted that during certain 

 stages in the growth of a number of bacteria spores are formed which 

 refuse to take up color when the bacteria are stained in the ordinary 

 manner. Special methods have been devised for causing the color to 

 penetrate through the resistant spore. In the simplest method a cover- 

 slip after having been prepared in the usual way is covered with Ziehl's 

 carbolic fuchsin solution and held over the Bunsen flame until the 

 fluid steams. This is continued for one or two minutes. It is then 

 washed and dipped in a decolorizing acid solution, such as a 2 per 

 cent, alcoholic solution of nitric acid, or a 1 per cent, solution of 

 sulphuric acid in water, until all visible color has disappeared, then 

 it is washed off and dipped for one-half minute in a saturated watery 

 solution of methylene blue. The bacilli will then be blue and the 

 spores red. Sometimes, however, the spores refuse to take the stain in 



FIG. 52 



* 



V 



. * *l - 



v:H $ 



-,'-> - '*!" 





Culture stain by Hiss' method. 



this manner. We then can adopt Moeller's method, which is designed 

 still further to favor the penetration of the coloring matter through the 

 spore membrane. The prepared cover-slip is held for two minutes in 

 chloroform, then washed off in water, then placed from one-half to 

 three minutes in a 5 per cent, solution of chromic acid, again washed 

 off in water, and now stained by adding to it carbolic fuchsin, which 

 is steamed for several minutes. The staining fluid is then washed off 

 and the preparation decolorized in a 3 per cent, solution of hydrochloric 

 acid or a 5 per cent, solution of sulphuric acid. The preparation is 

 finally stained for a minute in methylene-blue solution. The spores 

 will be red and the body of the cells blue. The different spores vary 

 greatly in the readiness with which they take up the dyes, and we have, 

 therefore, to experiment with each variety as to the length of time they 



