PREPARATION OF MEDIA 55 



the medium, whereas fermentation of only glucose will produce acid in 

 the butt but not in the slant. The addition of 0.2 g of ferrous sulfate 

 and 0.3 g of sodium thiosulfate to the above formula allows the determina- 

 tion of the production of hydrogen sulfide in the same medium {trij)le 

 sugar iron agar); cultures producing H2S show an extensive blackening of 

 the agar due to iron sulfide precipitation (Sulkin and Willett, 1940). 



Starch agar. Add 0.2 per cent of soluble starch to a suitable nutrient 

 agar basal medium, sterilize, and pour plates. After inoculation and 

 growth, test for starch hydrolysis by flooding the plate, which has been 

 streaked across center line, with dilute (Lugol's) iodine. Absence of the 

 bluish-purple color characteristic of the starch-iodine complex indicates 

 hydrolysis. 



Media to demonstrate gelatin liquefaction. Plain gelatin may be made 

 by adding 12 per cent of bacteriological grade gelatin to distilled water, 

 adjusting the pH to 7.0 before sterilization, sterilizing 12-15 min at 

 120°C, and cooling the tubes immediately. Nutrient gelatin is prepared 

 by adding the above amount of gelatin to a sugar-free nutrient broth. 

 For most obhgate anaerobes, it is necessary to add 0.25 per cent of 

 glucose and to tube the medium in deep columns. If growth does not 

 occur, sodium thioglycollate (0.1 per cent final concentration) is added to 

 serve as a reducing agent. Utilization of gelatin may be determined in an 

 agar medium by adding gelatin (0.4 per cent final concentration) to a 

 nutrient agar. A sufficient amount of the sterile medium is poured into 

 a petri dish to avoid a thin spot if center of dish is sUghtly raised. A 

 single streak of the culture is made across the surface of the hardened 

 medium, and following incubation, the plate is flooded with a gelatin 

 precipitant: acid HgCU (mercuric chloride 15.0 g; concentrated HCl, 

 20.0 ml; distilled water, 100 ml) ; or saturated ammonium sulfate solution. 

 A white precipitate indicates presence of nonhydrolyzed gelatin ; absence 

 of the precipitate in the region of growth indicates gelatin hydrolysis. 



Proteolysis. Gelatin hydrolysis (liquefaction) represents enzymatic 

 action upon an incomplete protein, and positive action is not necessarily 

 an indication that the organism can hydrolyze complex proteins; this is a 

 characteristic of particular value in the study of certain obhgate anae- 

 robes. The beef -heart infusion with particles of tissue represents one of 

 the media in which muscle protein hydrolysis may be observed. Coagu- 

 lated serum (as slants) represents another type of protein to be tested; 

 organisms with abihty to hydrolyze serum proteins will cause partial or 

 complete hquef action. For action on coagulated egg albumin one should 

 include a small cube of hard-boiled egg white in a tube of a suitable base 

 medium ; disintegration of coagulated egg white during growth is evidence 

 for proteolytic activity. Another medium for the indication of proteo- 

 lytic activity is alkaline egg medium which is prepared as follows: Mix the 



