MEDIA FOR SEPARATING BACTERIAL GROUPS 49 



incubated for twenty-four hours. At the end of such a period b. coli 

 colonies are 2 to 6 mm. in diameter, stained distinctly red, and are non- 

 transparent. Colonies of the b. typhosus are seldom larger than 2 mm., 

 they are blue or bluish-violet in colour, are glassy and dew-like in 

 character, and have a single contour. Sometimes in the plates b. subtilis 

 and its congeners appear, and colonies of these organisms have a blue 

 colour. Their growth is, however, more exuberant than that of the 

 typhoid bacillus, being often heaped up in the centre, and the contour 

 of the colony is often double. 



Conradi's Picric Acid Brilliant Green Method. Applying his principle 

 of seeking for anilin bodies which while inhibiting the action of ordinary 

 intestinal bacteria rather favour the growth of b. typhosus and b. para- 

 typhosus, Conradi in 1908 used for this purpose crystalline brilliant 

 green (Hoechst, extra pure), acting along with picric acid (Grubler). The 

 medium is made as follows : 900 c.c. water, 20 grms. Liebig's meat extract 

 and 100 c.c. of a 10 per cent, watery solution of Witte's peptone are 

 mixed and filtered ; 30 grms. agar in threads are dissolved in the fluid, 

 and the whole filtered. The reaction is then adjusted with normal sodium 

 hydrate or normal phosphoric acid to an acid content of 3 per cent, 

 (phenol-phthalein being the indicator), i.e., the finished medium is such 

 that to make it neutral would require the addition to each 100 c.c. of 

 3 c.c. of normal sodium hydrate. The acid medium is then sterilised, 

 and kept in bulk in this form. For use the remaining substances are 

 added in the proportions of 10 c.c. of 1-1000 watery solution of the 

 brilliant green and 10 c.c. of 1 per cent, watery picric acid to 1^ litres of 

 the peptone-agar, and the finished medium is poured in large Petris and 

 allowed to stand at 37 C. till the surface is firm. The capsules are 

 inoculated in the way already described. Typhoid colonies appear sharp- 

 edged, round, flat-surfaced but slightly thicker in the middle, transparent, 

 and of light green colour. Colonies of the paratyphoid bacillus are 

 similar, but tend at the same age to be slightly larger and have a some- 

 what yellowish green tint. 



Fawcus's Picric Acid and Brilliant Green Medium. This is a 

 modification of Conradi's medium which has been used with great 

 success at the Royal Army Medical College in the investigation of 

 typhoid carriers. It is made as follows : To 900 c.c. tap water add 

 5 grms. sodium taurocholate (which is commercially prepared from ox 

 bile), 30 grms. powdered agar, 30 grms. Witte's peptone, 5 grms. sodium 

 chloride ; steam for three hours, clear with white of egg, filter through 

 cotton wool, and bring to a reaction of +15 with normal lactic acid or 

 caustic soda, and sterilise. Dissolve 10 grms. lactose in 100 c.c. sterile 

 distilled water, and add to melted agar. Mix and filter through Chardin 

 paper, sterilise carefully, and store in 100 c.c. flasks. For use, add to 

 each 100 c.c. flask 2 c.c. of a 1-1000 watery solution of brilliant green 

 and 2 c.c. of a 1 per cent, watery solution of picric acid. Pour into 

 large Petri dishes, and leave these to stand inverted at 37 C. till the 

 surface hardens. Inoculate as usual. Colonies of b. typhosus of twenty- 

 four hours' growth are of about 1 mm. in diameter, transparent and re- 

 fracting ; those of b. coli, on the other hand, have a deep green centre, 

 though later typhoid colonies may also present a pale green centre. 



In the case of several of the special media used for the isolation of 

 typhoid bacilli under circumstances where other bacteria are present, a 

 difficulty arises from the fact that the agglutinability of the strains 

 isolated appears to be affected by substances present in the media. 

 The application of this important confirmatory diagnostic method is thus 

 4 



