224 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
permanganate solution and the acid is finally removed by repeated 
washings with water. If the filter becomes so clogged with organic 
matter that it can no longer deliver a reasonable amount of filtrate, 
the filter may be placed in a muffle-furnace, gradually heated to about 
250° C, and as gradually cooled. It is then cleaned as before with 
permanganate solution, to remove the last traces of organic matter. 
Storage of Media. — If media are not to be used at once it is necessary 
to protect them from evaporation and contamination. Flasks of 
media are preserved best by tying paper caps over the cotton plugs 
if the period of storage does not exceed a few days, or by pouring 
melted paraffin over the plugs if longer periods of storage are con- 
templated. It is necessary to burn the surface of the plug to destroy 
surface contamination, then to push the plug into the neck of the 
flask for a distance of 1 cm. to make room for the paraffin. Flasks 
hermetically sealed in this manner may remain visibly unchanged for 
weeks or even months. It is good practice to place a lead foil cap 
over the paraffin plug, and lead foil caps are better than paper caps 
as coverings for cotton plugs Media in storage should be maintained 
at a temperature not exceeding 45° C, in a dry ice-box. 
The Preparation of Nutrient Bouillon (Broth).— Meat Infusion Broth.— 
To 1000 cc. of meat infusion (see page 210) for preparation), in a 
tared, agate-ware boiler, add 5 gm. of common salt (NaCl) and heat 
to boiling. Dust 10 gm. of Witte or other peptone over the surface 
and stir until it is thoroughly dissolved. Restore the loss by evapora- 
tion and adjust the reaction to the desired degree of acidity. Boil for 
five minutes, verify the reaction and filter through filter paper until 
the filtrate is perfectly clear. Sterilize in the autoclave. 
Meat E.rtraet Broth. ^— To 1000 cc. of meat extract (see page 210 
for preparation) in a tared agate-ware boiler, add 10 gm. of Witte or 
other peptone, dusting the peptone on the surface. Heat to boiling, 
restore loss by evaporation and adjust the reaction. Continue the 
boiling for five minutes, verify the reaction and cool to room temperature.^ 
Filter when cold through filter paper until perfectly clear and sterilize. 
Trypsin Broth and Trypsin Media.—'Douglas^ and others have found 
that an excellent substitute for peptone broth may be made by digesting 
lean beef or beef heart with trypsin for a few hours then making the 
medium directly into nutrient broth, which may be converted into an 
agar medium or reinforced with various carbohydrates. 
Five hundred grams finely groimd lean beef, or beef hearts, and 1000 
cc. tap water are made just alkaline to litmus with XaOH solution (20 
per cent) . The mixture is heated to 80° C. for fi\'e minutes then cooled 
to body temperature, and digested for several hours with 0.5 gm. 
• It is unnecessary to add salt to meat extract. 
2 A precipitate containing phosphates, soluble in the hot medium, settles out upon 
cooling. It must be removed before the medium is used. 
3 Lancet, 1914, ii, 891. 
