226 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
Nutrient Carbohydrate Broth.— The addition of carbohydrates of 
various kinds to nutrient, sugar-free broth creates media of great 
importance for certain types of bacterial investigation. Usually a 
concentration of 1 per cent of the desired carbohydrate is a conven- 
ient amount. Glucose, lactose, saccharose, mannitol and other 
sugars, as well as alcohols, starches of various kinds, and glucosides, 
have been utilized to advantage in creating diagnostic media. 
It is absolutely necessary to sterilize these carbohydrates separately 
as aqueous solutions, usually in concentrations of 20 per cent.^ The 
requisite amount to make a final concentration of 1 per cent of the 
sterile sugar solution is added to the sterile nutrient medium. By 
so doing, the carbohydrate is left practically intact. 
Calcium Carbonate Nutrient Sugar Media.— BucteuR grown in sugar 
media frequently form acid products from the fermentation of the 
sugars— the amount of acid products may be sufficient to inhibit 
the development of the organisms even after one or two days' growth. 
The addition of insoluble carbonates— as calcium carbonate— neu- 
tralizes the acid as it is formed and thus maintains automatically a 
favorable reaction for prolonged development. Bolduan- has shown 
that pieces of marble about 0.5 cm. cube in 100 cc. of broth not only 
restrain the development of free acid— the marble appears to create a 
somewhat more favorable medium, especially for the pneumococcus 
and streptococcus as well. The bits of marble should be sterilized in 
the hot-air sterilizer before they are introduced into the broth. 
Nutrient Glycerin Broth.— To 1 liter of sugar-free broth add 3 to 5 
per cent pure, redistilled glycerin immediately before filtering. Sterilize 
in the autoclave fifteen minutes at 15-pounds pressure. Glycerin 
broth is extensively used for the cultivation of the human type of the 
tubercle bacillus-^ and it is frequently employed in the culture of 
bacteria which are susceptible to desiccation— the glycerin conserves 
the moisture and retards evaporation. 
The various sugar-broths may be prepared with meat extract as 
a basis; pathogenic bacteria develop less luxuriantly as a rule in 
extract media than in meat infusion media, however. 
Dunham's Solution.— Five grams of common salt and 10 gm. of 
Witte or similar peptone containing tryptophan are added to 1 liter 
of water and heated to boiling until the peptone is completely dissolved. 
Pass through filter paper until perfectly clear, tube, using 10 cc. to 
each test tube, and sterilize in the autoclave. The reaction does not 
require adjustment. 
This medium is frequently used to test the ability of bactei-ia to 
form indol. Indol is formed in the absence of utilizable sugars by 
Bacillus coli; members of the cholera group and other bacteria form 
1 Some carbohydrates are not soluble to this extent. An alternative procedure is to 
add the carbohydrate to fluid media and sterilize by passage through a Berkefeld filter. 
2 New York Med. .Jour., May 1.3, 1905. 
■' The reaction of glycerin broth designed for the cultivation of tubercle bacilli should 
be + 1 acid. The organism does not develop well in media neutral to phenolphthalein. 
