THE KOCH-EPIRLICH METHOD OF STAINING 73 



reagents. These are known as decolorising agents, and 

 include amongst them water, alcohol, acetic, hydrochloric, 

 sulphuric, and nitric acids, iodine, &c. 



Upon decolorising with one or other of the above-named 

 fluids depend various methods which have acquired an 

 extraordinary significance for the diagnosis of micro-organ- 

 isms, and for the practical work of staining them in sec- 

 tions. 



Koch and Ehrlich method of staining, — The Koch- 

 Ehrlich method of staining tubercle-bacilli stands in the 

 first rank of these, and brings into action both the mordant 

 and bleaching processes. Aniline water is prepared as 

 described above, and alcoholic solution of fuchsine, gen- 

 tian violet, or methyl violet is added to it until a fairly 

 saturated solution in dilute alcohol is obtained. Small 

 masses of sputum, or of the wall of a pulmonary cavity, are 

 then conveyed on to a cover-glass and spread out by rub- 

 bing with a second, so that both glasses become coated 

 with a fine film of the mass under examination. The 

 cover-glasses, having been dried in air, are passed three 

 times through the flame with the prepared side up by 

 means of a forceps, and then deposited in the staining fluid 

 and either left for twenty-four hours at the temperature 

 of an ordinary room, or heated for fifteen minutes until 

 bubbles rise. The cover-glass is next lifted from the dye 

 with a forceps and plunged for a few seconds into a solu- 

 tion of about 33 per cent, of nitric acid until the prepara- 

 tion, previously of a red colour, becomes yellowish green, and 

 is then washed in 70 per cent, alcohol. If fuchsine has been 

 used for the staining, the after-staining may be done with 

 methyl blue, malachite green, or picric acid; but if the 

 first staining has been done with gentian or methyl violet, 

 Bismarck brown must be used for the second. The secondary 

 staining lasts from one to five minutes, until the particular 



