52 METHODS OF CULTIVATION OP BACTERIA 



Conradi and Troch's Method for isolating the B. Diphtheriae, This 

 medium is made by mixing 1000 c.c. water, 10 grms. Lemco, 5 grms. sodium 

 chloride, 20 grms. Witte's peptone and (j grms. calcium bimalicum, steaming 

 for half an hour and filtering. To this slightly acid fluid 1 per cent, 

 of glucose is added and one part is mixed with three parts fresh ox serum. 

 To each 100 c.c. of the bouillon-serum medium 2 c.c. of a 1 per cent, 

 solution of potassium tellurosum is added. The finished medium is dis- 

 tributed in Petri capsules and coagulated by a quarter of an hours 

 exposure to 85 C. A tube of ordinary Loffler's serum is inoculated with 

 the material to be examined for the diphtheria bacillus and incubated for 

 three hours. The surface is then scraped and two plates of the special 

 medium are inoculated, and incubated for twenty hours. Any diphtheria 

 colonies present are a deep black from a reduction of the dioxide of 

 tellurium ; pseudo-diphtheria colonies show yellow-grey or greyish-black. 



Media for growing Trichophyta, Moulds, etc. 



1. Beer Wort Agar. Take beer wort as obtainable from the brewery 

 and dilute it till it has an s.g. of 1100. Add l - 5 per cent, of powdered 

 agar, and heat in the Koch till it is dissolved (usually about two hours 

 are necessary). Filter rapidly' and fill into tubes. Sterilise in the Koch 

 for twenty minutes on three successive days. If the medium is heated too 

 long it loses the capacity of solidifying. 



2. Sabourauds Media. Sabouraud recommends the following 

 media, the first being that most frequently used : 



(1) Pure tap water 1000 c.c. 



Maltose (" brute de Chanut ") . . . 40 grms. 

 Peptone ("granulee de Chassaing") . . 10 ,, 

 Agar 18 ,, 



(2) Pure tap water 1000 c.c. 



Glucose ("massee de Chanut ") . . . 40 grms. 

 Peptone (" granulee de Chassaing") . . 10 ,, 

 Agar 18 



In order to secure uniformity of results over as long a series of observa- 

 tions as possible, it is advisable to make up these media in large 

 quantities, say three litres at a time in a five-litre flask. The agar is 

 put to soak in the water for an hour, the other ingredients are added and 

 dissolved by gradually heating to 120 C. in an autoclave. The medium 

 is then thoroughly mixed by stirring and rapidly filtered th rough papier 

 Chardin (Cogit, 36 Boulevard Saint Michel, Paris). For this purpose, 

 Sabonraud recommends that ten 500 c.c. flasks should be fitted with 

 funnels and filtration simultaneously carried on in the whole series ; 

 whenever in any one of the flasks the filtrate begins to pass only in 

 drops, a new filter paper is substituted. In this way the three litres of 

 medium can be filtered in a few minutes. We have found that the pro- 

 cedure can be simplified without apparently affecting the efficiency of the 

 medium, by dissolving the agar and sugar in one flask, and the peptone in 

 another. The contents of each are filtered and the two filtrates are then 

 mixed ; in this procedure only two or three filter papers are required for 

 the rapid filtration of a large quantity of the agar and sugar moiety. If 

 filtration in a number of flasks is practised, the contents of all are 

 mixed and then distributed in 6 x |th inch test-tubes (plugged with non- 

 absorbent cotton) and sterilised by one exposure in the autoclave at 



