154 Methods of Observing Micro-organisms 



Nicolle* suggests the following modification of the technic: 



(a) For Cover-glass Specimens: 



1. Stain for one to five minutes in a warm solution made as follows: lo cc. 



of saturated alcoholic solution of gentian violet, loo cc. of a i per cent, 

 aqueous solution of carbolic acid. 



2. Immerse from four to six seconds in the iodine-iodide of potassium solu- 



tion. 



3. Decolorize in a mixture of 3 parts of absolute alcohol and i part of acetone. 



4. Counterstain if desired. 



(&) For Sections: 



1. Stain the nuclear elements of the tissue with carmine. For this Nicolle 



prefers Orth's carmine solution (5 parts of Orth's carmine with r part 

 of 95 per cent, alcohol). 



2. Stain in the carbol-gentian violet, as indicated above. 



3. Immerse for four to six seconds in the iodine-iodide of potassium solu- 



tion. 



4. Differentiate with absolute alcohol containing 0.33 per cent, (by volume) 



of acetone. 



5. Treat with gs per cent, alcohol containing some picric acid until the 



tissue is greenish yellow (one to five seconds). 



6. Dehydrate with absolute alcohol. 



7. Clear with xylol or other appropriate reagent. 



8. Mount in balsam. 



The Gram-Weigert Stain can be employed with beautiful results 

 for staining many micro-organisms. It differs from the Gram 

 method in that anilin oil instead of alcohol is used for decolorizing. 

 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 off excess with normal salt solution; 



3. Immerse in dilute iodin solution (iodin i, iodid of potassium 2, water 100) 



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 anilin with pure xylol. ^ 



7. Mount in xylol balsam. 



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

 aid in the differentiation of species: 



Gram-negative 

 Bacillus anthracis symptomatici; 

 Bacillus coli (whole group) ; 

 Bacillus ducreyi; 

 Bacillus dysenterise 

 Bacillus icteroides; 

 Bacillus influenzae; 

 Bacillus mallei; 

 Bacillus oedematis maligni; 

 Bacillus pestis bubonica; 

 Bacillus pneumoniEe (Friedlander); 



Gram-positive 

 Bacillus aerogenes capsulatus; 

 Bacillus anthracis; 

 Bacillus botulinus; 

 Bacillus diphtheriae; 

 Bacillus subtilis (whole group); 

 Bacillus tetani; 



Bacillus tuberculosis (whole acid- 

 fast group); 

 Diplococcus pneumoniae; 

 Micrococcus tetragenus; 



'■ "Ann. de ITnst. Pasteur," 1895, ix. 



