480 BACTEBIOLOGIC TECHNIC. 



3. Wash with acid alcohol ^ (p. 476) until the preparation no 

 longer appears- red. 



4. Counter-stain with methylene-blue (a few seconds). 

 The spores remain red, the bacilli appear blue. 



6. Staining of Tubercle Bacilli. — ^This is accomplished under the 

 same principles as spore staining. The preparation is treated with a 

 hot, actively staining solution, and afterward with some acid solution. 

 Everything except the tubercle bacilli is decolorized. 



(a) The preparation is stained (according to Ziehl-Neelsen) ex- 

 actly the same as spores, except that it is passed only three times 

 through the flame. We employ this method exclusively. 



Another favorite method is the one recommended by A. Frankel 

 and Gabbet, in which decolorization and counter-staining are accom- 

 plished at the same time. Then the preparation, after being stained with 

 hot carbol-fuchsin and washed with water, is placed in the following 

 solution : Sulphuric acid, 1 ; distilled water, 3 ; pulverized methylene- 

 blue, enough to produce a most intense blue color. It is then again 

 carefully washed in water, dried, and mounted in Canada balsam. 



Although this method is convenient, it is better for those without 

 much experience to carry out the staining, differentiation with acid, 

 and counter-staining separately, since in this way success is more cer- 

 tain. 



(6) Ehrlich-Koch's method is also often employed. The prepara- 

 tion, after being dried and fixed in the flame, is treated with hot anilin 

 gentian-violet solution for one to two minutes, and then with acid 

 (usually 30% nitric) for one to four seconds, and for a few seconds 

 with 60 % alcohol. It is then placed in an aqueous solution of Bis- 

 marck brown for a few minutes and washed in water. The tubercle 

 bacilli now appear violet upon a brown background. 



These methods are suitable for cover-glass preparations made from 

 pure cultures and from tuberculous sputum containing many tubercle 

 bacilli. If very few or no tubercle bacilli are found in the first prepa- 

 rations, then some method must be employed for concentrating them. 

 We give two of the innumerable ones which are recommended : 



(a) According to Strolischein : 5 to 10 c.c. of sputum are mixed with 

 three times the quantity of Wendriner's borax-boracic acid solution,^ 

 and, after vigorous shaking, the mixture is set aside for four or five 

 days to settle. The mixture becomes fluid and the bacilli settle to 

 the bottom. Such sputum may be used for examination after years. 



(6) According to Dahmen, modified by Heim : The entire sputum is 

 boiled in a steam chamber for fifteen to twenty minutes and allowed 

 to cool. The opalescent fluid is poured oS and the crumbly sediment 

 \ised for smear preparations. 



^ In place of acid alcohol, 30% nitric acid or 5% to 25% sulphuric 

 acid may be employed, but they must be allowed to act for a shorter 

 time. 



' Eight gm. of borax are dissolved in hot water, 12 gm. of boracic 

 acid are added, and then again 4 gm. of borax ; after crystallization it 

 is filtered. 



