AUTOTROPHIC BACTERIA 69 



The dishes are incubated in an inverted position at 25° to 30°C. The 

 development of an organism on the plate is first detected by chemical 

 tests for the formation of nitrous acid and disappearance of ammonia. 

 The tests are made by cutting a piece of the gel with a sterile loop 

 and placing them in dishes containing the proper reagent. The culture 

 can then be "fed" with 1 or 2 drops of a sterile 10 per cent ammonium 

 sulfate solution to bring about further development of the organism. 



The minute microscopic colonies are better studied on the clear 

 (sodium carbonate) medium and soon after the addition of a fresh por- 

 tion of ammonium sulfate. The colonies are at first colorless, then they 

 become yellowish to brownish and finally dark brown; after a certain 

 time (10 to 14 days), the dark colonies clear up, beginning from the 

 edge toward the center, till finally the dark colony becomes colorless. 

 A pure colony will show uniform fine granulation up to the edge, while 

 contaminated colonies have an hyaline rim. A clear zone is formed 

 around each colony on the MgC0 3 medium due to the fact that the 

 latter is gradually dissolved by the nitrous acid; it is often difficult, 

 however, to demonstrate this zone. The plate is carefully examined 

 with a magnification of 50 to 100, and several clear surface colonies are 

 selected for transfer. Winogradsky recommended the use of finely 

 drawn sterile glass rods which are first dipped into the colony, then 

 transferred into the flasks with sterile liquid medium and, by striking 

 the bottom of the flask, the tip of the rod with the inoculum is broken 

 off and left in the flask. A number of transfers are made, since, in many 

 cases, the organism fails to develop. It is much more difficult to obtain 

 a culture from the dark colonies (zooglea) than from the colorless 

 colonies. To prove the purity of the culture, a few drops (about 0.5 

 cc.) of the liquid are inoculated into bouillon and meat peptone agar. 

 No growth should take place after two weeks incubation; this combined 

 with microscopic examinations indicates the absence of contaminations. 



The silica gel can also be prepared by the method of Stevens and Temple, 22 

 as modified by Doryland: 23 24 grams of K 2 Si0 3 and 8.4 grams of Na 2 Si0 3 

 are dissolved in 500 cc. of water to give a concentration of 34.2732 grams of 

 H 2 Si0 3 per liter. One-half this concentration of H 2 Si0 3 per liter gives a 

 medium, which will solidify in approximately five minutes, thus making it 

 suitable for plating. The mixture of the two salts lessens the danger of too 



22 Stevens, F. L., and Temple, J. C. A convenient mode of preparing silicate 

 jelly. Centrbl. Bakt. II, 21: 84 -87. 1908. 



33 Doryland, C. J. T. Preliminary report on synthetic media. Jour. Bact. 1: 

 135-152. 1916. 



