26 A MANUAL OF BACTERIOLOGY 



are diluted to 100 c.c. with distilled water at the time of 

 making the test, and 1 c.c. of this dilution is added to 

 every 10 c.c. of the culture. The addition of the acid 

 liberates free nitrous acid, which reacts with any indole 

 present, and yields a pink colour. Sometimes when the 

 reaction is apparently absent or feeble, it may be obtained 

 or intensified by placing the tube in the blood-heat 

 incubator for half an hour. The sulphuric acid should be 

 pure and free from oxides of nitrogen, hence hydrochloric 

 acid is often preferable. 



A more delicate method of testing is to run a little 

 hydrochloric acid down the side of the tube, so that a layer 

 forms at the bottom, the nitrite having been previously 

 added to the culture if required. A pink ring at the 

 juncture of the hydrochloric acid and culture indicates 

 the presence of indole. The pink pigment, the product 

 of the reaction, may be extracted by shaking with a little 

 amylic alcohol. 



Other delicate and more certain reagents for the detection of 

 indole are para-dimethylamidobenzaldehyde (14 grm., dissolved in 

 absolute alcohol 380 c.c., hydrochloric acid 80 c.c.). To about 

 10 c.c. of culture 5 c.c. of this solution are added, and then 5 c.c. 

 of a saturated aqueous solution of potassium persulphate ; indole 

 gives a pink or red colour. Another test is /3-naphthaquinone- 

 sodium-mono-sulphonate (2 per cent, aqueous solution), which gives, 

 when the mixture is rendered alkaline with caustic potash, a blue 

 or blue -green colour or precipitate. The coloured compound may 

 be extracted with chloroform, in which it yields a red solution. 



Peptone water is by no means a good culture medium, 

 and broth may therefore be employed, but it should be 

 free from dextrose. Peptone water with the addition of 

 a little rabbit's serum is perhaps the best culture medium 

 for the production of indole. 



The presence of dextrose, saccharose, glycerin, or lactose 

 in quantity exceeding about 0-25 per cent, prevents the 

 formation of indole in broth by bacteria. Broth prepared 



