microscopical examination of bacteria. 89 



Double Staining of Cover-glass Preparations. 



To double stain cover-glass preparations they can be treated hy 

 Ehrlicb's method for staining tubercular sputum, or by Neelsen's 

 modification, or by staining with eosin after treatment by the 

 method of Gram. 



Ehrlich.'s method is as follows : Five parts of aniline oil are 

 shaken up with one hundred parts of distilled water, and the 

 emulsion filtered through moistened filter-paper. A saturated 

 alcoholic solution of fuchsine, methyl-violet, or gentian-violet, is 

 added to the filtrate in a watch-glass, drop by drop, until precipitation 

 commences. Weigert recommended that exactly eleven parts of the 

 dye should be used to one hundred parts of the aniline solution. 



Cover-glass preparations are floated in this mixture for fifteen 

 minutes to haK an hour, then washed for a few seconds in dilute 

 nitric acid (one part nitric acid to two of water), and then rinsed 

 in distilled water. The stain is removed from everything except 

 the bacilli ; but the ground substance can be after-stained brown 

 if the bacilli are violet, or blue if they have been stained red. 



Hifeelsen's Solution and Methylene Blue. — Ziehl suggested the use of 

 carbolic acid as a substitute for anUine oil, and Neelsen recommended 

 a solution composed of 100 cc. of a 5 per cent, watery solution 

 of carbolic acid, 10 cc. of absokite alcohol, and 1 gramme of 

 fuchsine. This stain is commonly known as the Neelsen or Ziehl- 

 Neelsen solution. Cover-glass preparations are floated on the hot 

 dye for two minutes, they are then rinsed in dilute sulphuric acid 

 25 per cent., washed in water, immersed in watery solution of 

 methylene blue for three minutes, again washed in water, dried, 

 and mounted in balsam. 



Oram's Solution a7ul Eosin. — Double staining of cover-glasses can 

 be obtained by combining Gram's method with eosin. The method 

 is very useful for differentiating the sheath of Streptococcus 

 pyogenes and Bacillus anthracis, from the protoplasmic contents, 

 and for staining preparations of pneumonic sputum, or of micrococci 

 and other micro-organisms in pus. After decolorising the prepara- 

 tion in alcohol, the cover-glass is transferred to a weak solution 

 of eosin for two or three minutes, then washed again in alcohol, 

 immersed in clove-oil, dried between fllter-paper, and mounted in 

 balsam. 



Staining of Spores. 

 A slight modification of the ordinary process employed in making 

 cover-glass preparations has to be adopted to stain the spores of 



