PEPTONE SOLUTION 21 



To make, simply dissolve i or 2% of the sugar in sugar-free bouillon or that 

 made from meat extract. Tube in Durham's or the ordinary fermentation tubes and 

 sterilize in the autoclave at only about 5 pounds' pressure for 15 minutes, or in the 

 Arnold. Ordinary peptone solution is a good substitute for sugar-free bouillon. 



Too high a degree of heat may turn the sugar bouillon brownish. The nature 

 of the sugar itself ma} 7 further be affected by too high a temperature. 



CALCIUM CARBONATE BOUILLON. 



Where we wish to cultivate such organisms as streptococci and pneumococci in 

 massive cultures we may add small fragments of marble (calcium carbonate) so 

 that any inimical excess of acid may be neutralized. North used a glucose bouillon 

 containing calcium carbonate in the production of massive cultures of B. bulgaricus. 



GLYCERINE BOUILLON. 



Add 6% of glycerine to ordinary bouillon. It is chiefly used in the cultivation 

 of tubercle bacilli. 



PEPTONE SOLUTION (DUNHAM'S). 



Dissolve i% of Witte's peptone and 1/2% of sodium chloride in distilled water. 

 Filter, tube, and sterilize. Peptone solution may be used as a base for sugar media 

 instead of bouillon. It is the medium used in testing for indol production. This 

 test is made by adding from 6 to 8 drops of concentrated H 2 SO 4 to a twenty-four- to 

 forty-eight-hour-old peptone culture of the organism to be tested. If the organism 

 produces both indol and a nitroso body, we obtain a violet-pink coloration, "cholera 

 red. " If no pink color is produced on the addition of the sulphuric acid, add about 

 i c.c. of an exceedingly dilute solution (i : 10,000) of sodium nitrite. 



It is very important in determining the "cholera red" reaction to know that the 

 peptone used will give the reaction as it is not given by true cholera strains with 

 certain samples of peptone. 



For the Voges-Proskauer Reaction. Fill fermentation tubes with a 2% glucose 

 Dunham's peptone solution and sterilize. After inoculation with the organism to 

 be tested incubate for three days. Then add 2 to 3 c.c. of strong caustic potash 

 solution. The development of a pink color on exposure to the air is a positive 

 reaction (the color of a weak eosin solution). 



Hiss' SERUM WATER MEDIUM. 



Take one part of clear beef serum and add to it about 3 times it's bulk of water. 

 Heat the mixture in the Arnold for 15 minutes to destroy any diastatic ferment which 

 might be present. Color to a deep transparent blue with litmus solution and then 

 add i% of any of the various sugars used in fermentation tests. Sterilize in 

 the Arnold by the fractional method. 



NUTRIENT AGAR. 



In making agar medium it is preferable to use powdered agar, as this goes into 

 solution more readily than the shredded agar. The reaction of agar is slightly 



