APPENDIX 467 



growth in one to three days should be plated out on ordinary or pheno- 

 lated gelatine and colonies of B. coli examined for. Some authorities 

 recommend incubating Parietti's tubes at 42° C, a temperature favour- 

 able to B. coli but unfavourable to ordinary water organisms ; 



Or, tubes of glucose-formate bouillon (meat infusion, 1 per cent, 

 peptone, 0-5 per cent, salt, 2 per cent, glucose, and 0-4 sodium formate) 

 may be inoculated with 0-1 to 0-5 c.c. of the water, and incubated in 

 Buchner's tube at 42° C, and the tubes which show turbidity in 24 hours 

 may be plated out on gelatine or glucose-litmus agar and B. coli, if 

 present, thus isolated (Pakes' method) ; 



Or, inoculate tubes of M'Conkey's medium, bile-salt-glucose peptone, 

 with 1-5, or 10 c.c. of the water. " When this test yields negative results, 

 the absence of B. coli and of glucose fermenting coli-like microbes may 

 be accepted without reserve " (Houston) (see p. 484) ; 



Or, place 1-20 c.c. of the water, or -Ol-'S of the suspension, into tubes 

 of bile-salt solution, and incubate at 37° C. aerobically for twenty-four 

 hours, or at 42° C. anaerobically (in Buchner's tubes) for twenty-four hours; 

 and if, after this period, there is (a) presence of growth, (6) formation 

 of acid, or (c) formation of gas, plate out on gelatine agar or bile-salt- 

 lactose-peptone agar, and sub-culture coli-like colonies on suitable media ; 



Or, incubate at 38° C. 1 c.c. of the water in a Smith's fermentation 

 tube with glucose broth. If after twelve hours' growth gas collects, it 

 may be B. coli, and the species must be further tested. If there is no 

 gas there is probably no B. coli. 



In the examination for B. coli, the important media for sub-culturing 

 are as follows : gelatine shake cultures * (for gas production and lique- 

 faction), glucose gelatine or glucose agar (for gas production), milk or 

 litmus milk (for acidity and coagulation), peptone water (for indol),t and 

 potato. Eisner medium, | neutral-red agar, lactose and maltose media may 

 also be used. 



*" Shake Cultures." — To 10 c.c. of melted gelatine, a small quantity of the 

 suspected organism is added. The test-tube is then shaken and incubated at 22° C. 

 In this medium the B. coli have opportunity for gas production. 



t The Indol Reaction. — Indol and skatol are amongst the final products of 

 digestion in the lower intestine. They are formed by the growth, or fermentation 

 set up by the growth, of certain organisms. Indol may be recognised on account of 

 the fact that with nitrous acid it produces a dull red colour. The method of testing 

 Is as follows. The suspected organism is grown in pure culture in broth or peptone 

 water (or Dunham's solution), and incubated for forty-eight hours at 37° C. Two c.c. 

 of a •01 per cent, solution of potassium nitrite is added to the test-tube of broth 

 culture. Now 1 c.c. of concentrated sidphuric acid (unless quite pure, hydrochloric 

 should be used) is run down the side of the tube. A pale pink to dull red colour 

 appears almost at once, or in a few minutes, and may be accentuated by placing the 

 culture in the blood-heat incubator for half an hour. The presence of much dextrose 

 (derived from the meat of the broth) inhibits the reaction. B. typhosus does not 

 produce indol, and therefore does not react to the test ; B. coli and the bacillus of 

 Asiatic cholera do produce indol, and react accordingly, 



t Blsner's Medium. — This special potassium-iodide-potato gelatine medium is used 

 for the examination of typhoid excreta. It is made as follows : 500 grams of potato 

 gratings are added to 1000 c.c. of water ; stand in ice-chest for twelve hours, and 

 filter through muslin ; add 1,50 grams of gelatine ; sterilise and add enough deci- 



