156 Methods of Observing Micro-organisms 



its original color^ If it is simply desired to find the bacteria, the 

 section can be dehydrated in absolute alcohol for a- moment, cleared 

 in xylol, and mounted in Canada balsam. If it is necessary to 

 study the relation of the bacteria to the tissue elements, a nuclear 

 stain, such as alum-carmin or Bismarck brown, may be previously 

 or subsequently used. Should a nuclear stain requiring acid for 

 its differentiation be desirable, the process of staining must precede 

 the Gram stain, so that the acid shall not act upon the stained 

 bacteria. 



Gram's method rests upon the fact that the combination of bac- 

 terial substance, anilin dye, and the iodids forms a compound insoluble 

 in alcohol. 



The process described may be summed up as follows: 



Stain in Ehrlich's anilin-water gentian violet five to thirty minutes; 



Wash in water; 



Immerse two to three minutes in Gram's solution; 



Wash in 95 per cent, alcohol until no more color comes out; 



Dehydrate in absolute alcohol; 



Clear in xylol; 



Mount in Canada balsam. 



No matter how carefully the method is performed, an unsightly 

 precipitate is sometimes deposited upon the tissue, obscuring both 

 its cells and contained bacteria. Muir and Ritchie obviate this 

 (i) by making the staining solution with i : 20 aqueous solution of 

 carbohc acid instead of the saturated aniUn solution, and (2) by 

 clearing the tissue with oil of cloves after dehydration with alcohol. 

 The oil of cloves, however, is itseK a powerful decolorant and must 

 be washed out in xylol before the section is mounted in Canada 

 balsam. 



The Gram-Weigert Stain can be employed with beautiful results 

 for staining many micro-organisms in tissue. It differs from the 

 Gram method in that anilin oil instead of alcohol is used for decolor- 

 izing. To secure the most brilliant results it is best first to stain 

 the tissue with alum, borax, or lithium carmin, and then — 



1. Stain in Ehrlich's anilin-oil- water gentian violet, five to twenty minutes; 



2. Wash o£f excess with normal salt solution; 



3. Immerse in dilute iodin solution (iodine 1, iodide of'potassium 2, water loo) 



for one minute ; 



4. Drain off the fluid and blot the section spread out upon the slide, with 



absorbent paper; 



5. Decolorize with a mixture of equal parts of anilin and xylol; ' 



6. Wash out the anihn with pure xylol. 



7. Mount in xylol balsam. 



Gram's Method as an Aid in the Identification of Species.— 



Gram's method does not stain all bacteria, hence can be used to 

 aid in the differentiation of species. The following lists show the 

 reaction of the well-known species to the stain. 



