1 82 Methods of Observing Micro-organisms 



Loffler's Method. Certain bacteria that do not permit 

 ready penetration by the dye require some more intense 

 stain. One of the best of these is Loffler's alkaline methy- 

 lene-blue : 



Saturated alcoholic solution of methylene-blue . . 30 

 1 : 10,000 aqueous solution of caustic potash . . 100 



The cut sections of tissue are stained for a few minutes 

 and then differentiated in a i per cent, solution of hydro- 

 chloric acid for a few seconds, after which they are dehy- 

 drated in alcohol, cleared in xylol, and mounted in balsam. 



Bacteria, such as the typhoid fever bacillus, which de- 

 colorize rapidly, do not require the use of acid for the 

 differentiation, washing in water or alcohol being sufficient. 



Gram's Method of Staining Bacteria in Tissue. Gram 

 was the fortunate discoverer of a method of impregnating 

 bacteria with an insoluble color. It will be seen at a 

 glance that this is a marked improvement on the methods 

 given above, as the stained tissue can be washed thoroughly 

 in either water or alcohol until its cells are colorless, without 

 fear that the bacteria will be decolorized. The details of 

 the method are as follows: The section is stained from five 

 to ten minutes in a solution of a basic anilin dye, pure anilin 

 (anilin oil) and water. This solution, first devised by 

 Bhrlich, is known as Bhrlich's solution. The ordinary 

 method of preparing it is to mix the following: 



Pure anilin : 4 



Saturated alcoholic solution of gentian violet. . 11 

 Water 100 



Instead of gentian violet, methyl violet, Victoria blue, or 

 any pararosanilin dye will answer. The rosanilin dyes, 

 such as fuchsin, methylene-blue, vesuvin, etc., will not react 

 with iodin, and so cannot be used for the purpose. The 

 anilin-oil solutions do not keep well; in fact, seldom longer 

 than six to eight weeks, sometimes not more than two or 

 three; therefore it is best to prepare but a small quantity 

 by pouring about i c.c. of pure anilin into a test-tube, 

 filling the tube about one-half with distilled water, shaking 

 well, then filtering as much as is desired into a small dish. 

 To this the saturated alcoholic solution of the dye is added 

 until the surface becomes distinctly metallic in appear- 

 ance. 



