MUCH'S METHOD 327 



of the ether and water ; this is pipetted off and films are made with 

 it and stained. Antiformin (a mixture of sodium hypochlorite and 

 sodium hydrate) has also been recommended. Into a boiling- tube 

 or small flask of 50 c.c. capacity, 5 c.c. of the sputum are introduced. 

 To this are added 25 c.c. of antiformin solution (10-20 per cent, 

 aqueous solution) diluted with 10-20 c.c. of water according to the 

 density of the sputum. The mixture is well shaken until homo- 

 geneous (about 15 minutes), then centrifuged, the deposit is washed 

 three times with salt solution by centrifuging, and films are made 

 with the washed deposit and stained by the Ziehl-Neelsen or 

 Spengler method. 



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 "). 



(6) 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. Muck'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, warming 

 over the flame, or for 24-48 hours at 37 C. ; (2) treat with Gram's 

 iodine solution, 1-5 minutes ; (3) treat with 5 per cent, nitric acid 

 for 1 minute ; (4) treat with 3 per cent, hydrochloric acid for 

 10 seconds ; (5) treat with a mixture of equal parts of acetone and 

 absolute alcohol. 



/3. 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 1 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 demonstration of the 

 tubercle bacilli, which in many instances may be difficult in human 



