472 APPENDIX 



Search for B. Ooli 



Method. — The committee recommend either — 



(a) The glucose-formate broth method of Pakes. 

 (6) The bile-salt broth method of M'Conkey. 



Incubation anaerobically at 42° C. increases the chances of success with either 

 medium, and is strongly recommended. 



It has also been suggested that the neutral-red (Griibler's) glucose broth medium 

 may be employed. 



The committee do not regard with favour the Parietti method, or the use of 

 carbohc acid media. 



Quantity of Water to be Examined. — As a routine 50 c.c. should be the minimal 

 quantity examined for the presence of the B. coli, quantities from a minimum of 

 0"1 c.c. to a maximum of 25 c.c. being added to the tubes of culture media. 



The committee are of opinion that it is preferable to add the water directly to the 

 tubes of culture medium, even with the larger amounts, rather than first to concen- 

 trate by filtration through a porcelain filter (the filter-brushing method). The 

 culture media recommended may be diluted with at least an equal volume of the 

 water without interfering with their cultural properties, and large tubes or small 

 flasks may be used for the larger amounts. 



In the case of the bile-salt-lactose-peptone water, the medium may for the larger 

 amounts be prepared of double strength. 



Isolation of B. coli, if Present. — If indications of the presence of the B. coli be 

 obtained in the preliminary cultivations, the organism must be isolated and identified. 



This may be done by making surface cultures on plates of either (o) litmus-lactose 

 agar, reaction + 10 ; (ft) bile-salt agar ; (c) nutrose agar of Conradi and Drigalski ; 

 or {d ) ordinary nutrient gelatin. 



The best medium of all is, probably, the nutrose agar of Conradi and Drigalski. 

 Agar media have the advantage of saving time. 



Identification of, and Tests for, the B. coli. — Having obtained coli-like colonies on 

 the plates made from the preliminary cultivations of the water, sub-cultures must be 

 made in order to identify the organism. The following, at least, should be made : 



(a) Surface agar at 37° C. The abundant growth so obtained enables many sub- 

 cultures and preparations to be made if required. 



(6) Stab and surface cultures in gelatine. This may be done in the same tube. 



(c) Litmus milk incubated at 37° C. 



(d) Glucose litmus medium. 



(e) Lactose litmus medium. 



(/) Peptone water for indol reaction. 



Characters of the B. coli. — The B. coli is a small motile, non-sporing bacillus, 

 growing at 37° C. as well as at room temperature. The motility is well observed in 

 a young culture in a fluid glucose medium. It is decolorised by Gram's method of 

 staining. It never liquefies gelatin, and the gelatin cultures should be kept for at 

 least ten days in order to exclude a liquefying bacillus. It forms smooth, thin 

 surface growths and colonies on gelatin, not corrugated, growing well to the bottom 

 of the stab (facultative anaerobe). 



It produces permanent acidity in milk, which is curdled within seven days at 

 37° C. It ferments glucose and lactose, with the production both of acid and of gas. 



The typical B. coli must conform to the above description and tests. 



It generally also forms indol (best obtained in peptone-water cultures), gives a 

 thick yellowish-brown growth on potato (greatiy dependent on the character of the 

 potato), sometimes (about 50 per cent.) ferments saccharose, changes neutral-red 

 (Griibler's), and reduces nitrates, and half the gas produced by it from glucose is 

 absorbable by KOH ; and these tests, if time and opportunity permit, may be per- 

 formed in addition to the foregoing. 



The committee recognise that atypical B. cvli are met with, but in the present 

 state of our knowledge hesitate to make any suggestion with regard to their 

 significance. 



