METHODS OF PURE CULTUKE STUDY 525 



good growth in one or both media, proceed as follows before 

 concluding that the organisms do not reduce nitrate: 

 Inoculate into: 



A. Nitrate-peptone agar or broth. 



B. Peptone agar or broth without nitrate. 



C. Synthetic nitrate medium (formula recommended: Ni- 

 trate 1 g, K2HPO4 0.5 g, CaCU 0.5 g, glucose 10 g, distilled water 

 1000 cc, with or without agar according to the organism under 

 investigation). 



D. Peptone agar with 2 p. p.m. potassium nitrite. 

 Prepare the following reagents for the Thomas test for ammonia: 



1. A 5 per cent solution of phenol, 



2. A solution of sodium hypochlorite containing one per cent 

 available chlorine. To obtain this amount of available chlorine, 

 the solution should be so adjusted that 1 cc. of it should neutral- 

 ize 2.86 cc. of a N/10 solution of sodium thiosulphate (i.e., 

 24.8 grams to the hter), titrating with starch as an indicator 

 in the presence of acetic acid and potassium iodide. 



In making the Thomas test 1 cc. of each of the above reagents 

 should be added to the broth or agar slant' on which the culture 

 has been growing and allowed to stand half an hour. A blue 

 color indicates the presence of ammonia. 



After incubation, test A and B for ammonia by the Thomas 

 method; test C for nitrite as usual, and for ammonia by the 

 Thomas method; test D for nitrite. The results give the follow- 

 ing indications: 



On A: Presence of ammonia indicates nitrate reduction if no am- 

 monia is present in B. Absence of ammonia on both media indicates 

 non-reduction provided this conclusion is confirmed by the results on 

 C and D. Otherwise inconclusive. 



On C: Presence of cither nitrite or ammonia indicates nitrate reduc- 

 tion. Absence of both, if the growth is good, strongly indicates non- 

 reduction. 



On D: Presence of nitrite indicates non-reduction in case this is 

 confirmed by the two above tests, for if the organism cannot destroy 



' This test can be made for this purpose to great advantage with agar slant cul- 

 ture as well as with liquid media, as recentlj- shown by Hucker and Wall (1922). 



