ANTI-.FORMIN METHOD 395 



cent, alcohol ; (4) treat with 15 per cent, nitric acid until yellow 

 (about thirty seconds) ; (5) rinse again with 60 per cent, alcohol ; 

 (6) counter- stain with the picric acid alcohol until yellow ; (7) 

 wash with distilled water. This is an excellent method, and thick 

 films may be used. In material which has been preserved a long 

 time, e.g. sputum with carbolic, or tissue in spirit, the bacilli may 

 be much less acid -fast than in fresh material. 



Various methods have been recommended for the solution of 

 the sputum and the examination of the sediment for the bacilli. 

 In one method 5 c.c. of sputum are mixed with 50 c.c. of normal 

 KOH solution ; the mixture is warmed in a water-bath to 60- 

 65 C. until the sputum is dissolved (about three hours) ; 50 c.c. of 

 cold water are next added, the whole is well shaken, and again 

 warmed for half an hour. Petroleum ether 2 c.c. is next added, 

 the whole is well shaken, and is then kept at 60 C. until the ether 

 has separated. The bacilli will be concentrated in the fluffy 

 layer at the junction 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 recom- 

 mended. 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 1020 c.c. of water according to the density of the sputum. 

 The mixture is well shaken until homogeneous (about fifteen 

 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 loopfuls of the antiformin deposit are inoculated on to suit- 

 able media, pure cultures of the tubercle bacillus can frequently 

 be obtained. 



For obtaining cultures of the tubercle bacillus directly from 

 sputum and post-mortem material Soparkar l recommends the 

 use of caustic soda. From 2 c.c. to 5-10 c.c. of the material 

 is used, according to its richness in bacilli. It is mixed with an 

 equal volume of normal sodium hydrate solution and the mixture 

 is kept at 37 C. for about half an hour until the material is 

 quite fluid ; with fluid sputum ten minutes may suffice. At the 



1 Indian Journ. Med. Research, vol. iv, 1916, p. 28. 



