152 Methods of Observing Micro-organisms 



The cut sections of tissue are stained for a few minutes and 

 then differentiated in a i per cent, solution of hydrochloric acid for 

 a few seconds, after which they are dehydrated in alcohol, cleared in 

 xylol, and mounted in balsam. 



Some bacteria, such as the typhoid fever bacillus, decolorize 

 readily so that the use of acid should be avoided, 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 Ehrlich, 

 is known as Ehrlich's solution. The ordinary method of preparing 

 it is to mix the following: 



Pure anilin 4 



Saturated alcoholic solution of gentian violet n 



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 cc. 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 appearance. 



Friedlander recommends that the section remain from fifteen to 

 thirty minutes in warm stain, and in many cases the prolonged 

 process gives better results. 



From the stain the section is given a rather hasty washing in 

 water, and then immersed from two to three minutes in Gram's 

 solution (a dilute Lugol's solution): 



Iodin crystals i 



Potassium iodid 2 



Water 300 



The specimen while in the Gram solution turns a dark blackish- 

 brown color, but when removed and carefully washed in 95 per 

 cent, alcohol again becomes blue. The washing in 95 per cent, 

 alcohol is continued until no more color is given off and the tissue 

 assumes'its original color. If it is simply desired to find the bacteria, 

 the section can be dehydrated in absolute alcohol for a moment, 



