396 A MANUAL OF BACTERIOLOGY 



end of incubation, the mixture is neutralised to litmus with 5 per 

 cent, hydrochloric acid, centrifuged and the deposit used for 

 inoculating the tubes. 



If the tubercle bacillus cannot be detected microscopically 

 after repeated examinations, and a certain diagnosis is important, 

 the inoculation method may be employed. A couple of guinea- 

 pigs are inoculated subcutaneously in the thigh or abdomen with 

 0-5 to 1 c.c. of the sputum. If tubercle bacilli are present the 

 animals will show signs of tuberculosis in three to six weeks (see 

 below, " Urine "). 



(b) Other methods have been devised for staining the tubercle 

 bacillus, but do not seem to be better than the Ziehl-Neelsen or 

 the Spengler. The following may be useful for those who are 

 colour-blind to red : 



a. Much's method. Prepare the following solution : 10 c.c. of 

 a saturated alcoholic solution of methyl violet B.N. in 100 c.c. of 

 2 per cent, aqueous carbolic ; (1) stain the film with this, warm- 

 ing over the flame, or for twenty-four to forty-eight hours at 

 37 C. ; (2) treat with Gram's iodine solution, one to five minutes ; 

 (3) treat with 5 per cent, nitric acid for one minute'; (4) treat 

 with 3 per cent, hydrochloric acid for ten seconds ; (5) treat with 

 a mixture of equal parts of acetone and absolute alcohol. 



j3. Herman's method. Prepare shortly before use the following 

 solution : 3 parts of a 1 per cent, aqueous solution of ammonium 

 carbonate, 1 part of a 3 per cent, solution of krystal violet in 95 per 

 cent, methyl alcohol. (1) Flood the film with this, warm until 

 it steams, and stain for one minute ; (2) decolorise with 10 per 

 cent, nitric acid for a few seconds, and then with 95 per cent, 

 alcohol until the film assumes a pale blue colour, then rinse in 

 tap- water followed by distilled water ; (3) counter-stain with 1 

 per cent, aqueous eosin. 



By both these methods the tubercle bacilli appear blue-black. 



2. Tissues. The histological appearance of the tubercle is 

 usually sufficient for diagnostic purposes without the demonstra- 

 tion of the tubercle bacilli, which in many instances may be diffi- 

 cult in human material, as they may be very scanty, or practically 

 impossible to find, e.g. in lupus. Sections should be prepared 

 either by the freezing or the paraffin method, stained with hsema- 

 toxylin, and counter- stained with eosin, or orange-rubin, or with 

 the Ehrlich-Biondi mixture. 



