Technique. 



The various rules that apply to the utilisation of haemo- 

 globin and its derivates in the cultivation of Pfeiffer's bacillus, 

 have already been discussed. In this chapter therefore merely 

 the purely practical side of the technique of cultivation will 

 be dealt with. My own experiences will be related and then 

 I will pass on to a review of the literature on the different 

 media for Pfeiffer's bacillus. 



For the primary isolation of the haemoglobinophilic bac- 

 teria I have only used agar media in Petri plates. The agar 

 has practically always been the ordinary peptone broth agar: 

 25 kg. beef is minced; 25 litres water are added; the mixture 

 is allowed to stand in the cold room till the next day, when 

 it is boiled for a short time, and then the broth is pressed 

 out of the meat. About 16 litres of water is added to this, 

 it is again healed to boiling and the solid remnant of the 

 meat is compressed once more. The two portions of broth, 

 — about 50 litres in all, — are mixed. l<y peptone is added 

 (Witte's or a similar preparation; at first li/ 2 % was taken 

 but the difference seemed to be unimportant), and 1/2 °/o salt. 

 It is heated to boiling. 10 c.c. of the broth are titrated till the 

 red colour of phenol-phthalein begins to appear. The calcu- 

 lated amount of n. NaOH is added, the broth is boiled for 5—10 

 minutes and then the reaction tested again. After filtration 

 and cooling the broth should not give a colour with phenol- 

 phthalein even when the indicator is present in excess, but 



it should become tinted on the addition of 0.2 to 0.3 c.c. j^ 

 NaOH to 10 c.c. The P H . value will then be about 7.8 to 7.9. 



15 



