52 METHODS OF CULTIVATION OF BACTERIA 



10 o.c, are prepared. The brilliant green (Bayer's Extra Oryst.) is used 

 as a 1 : 10,000 solution in distilled water. To a number of tubes of 

 peptone water, say half a dozen, varying amounts of the brilliant green 

 solution — from 0'1 c.c. to 0v e.e. — are added in series. Each tube is 

 then inoculated with a loopful of an emulsion of faeces in distilled water 

 and the tubes are incubated at 37° C. for twenty-four hours. At the end 

 of this time a loopful is taken from each tube and strokes are made on 

 plates of MacConkey's medium — three strokes with each loopful. Two 

 plates will be sufficient for the strokes from all the dilutions. After 

 incubation for another twenty-four hours the plates are examined for 

 typhoid colonies ; often a pure culture is obtained from one of the 

 dilutions. When the bacilli are scanty the results yielded by the method 

 are remarkable. The method is not suitable for the isolation of dysentery 

 bacilli. 



Whilst b. coli generally is inhibited by the brilliant green, Browning 

 and his co-workers have found that some strains, especially the inosite- 

 fermenters, e.g., b. lactis aerogenes, are equally resistant with b. typhosus, 

 but, on the other hand, are much less resistant to telluric acid. They 

 therefore recommend that 0'33 c.c. of a 1 : 1000 solution of telluric acid 

 be added to the tubes of peptone water along with the varying amounts 

 of brilliant green. 



Whilst a number of tubes, as above described, are essential for the best 

 results, a one-tube method may often be used with success. In this case 

 0'5 c.c. of the 1 : 10,000 solution of brilliant green is the optimunyruantity. 

 This is specially successful with paratyphoid B. 



Methods have also been devised, on the same principle as 

 the above, for inhibiting the growth of various organisms 

 present along with b. diphtherise, and thus aiding the isolation 

 of the latter. We give the following : — 



Conradi and Troch's Method for isolating the B. Diphtherias. —This 

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

 chloride, 20 grms. Witte's peptone, and 6 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 telluricum is added. The finished medium is dis- 

 tributed in Petri capsules and coagulated by a quarter of an hour's 

 exposure to 85° C. A tube of ordinary Lbffler'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. 



Smith's Method. — The following medium, containing telluric acid, 

 has been devised by J. F. Smith ; it gives excellent results. It has the 

 composition : — 



Peptone-water agar (neutral to litmus) . . . 100 c.c. 

 Sheep's serum (sterilised at 57° C. ) . . . 5 „ 

 1 per cent, telluric acid solution in distilled water - 9 ,, 

 The serum is added to the melted agar at a temperature of 50° C. On this 



