16 BULLETIN 227, U. S. DEPARTMENT OF AGRICULTURE. 
A culture medium made according to the following formula was 
used: 
Extract of 1 pound lean beef in distilled water 1,000 c.c. 
Lofflund's malt extract 25 grams. 
Agar-agar 20 grams. 
(Carefully filtered, but reaction not adjusted; slightly acid.) 
This is the formula largely used by German investigators. It is a 
good medium for the development of fungi, but, like all other media 
of organic and often unknown composition, offers the objection 
of possible chemical reaction with certain preservatives. How- 
ever, such synthetic media as were experimented with proved very 
poor substrata for the development of the organisms. 
The above medium after melting was measured * into 50-c. c. glass 
bottles with carefully ground glass stoppers, usually 17 c. c. to a 
bottle, using a standardized 17-c. c. pipette or a 25-c. c. graduate. 
One check was usually prepared for each series of concentrations 
and to this was added sufficient distilled water to make 20 c. c. 
The stoppers were then sealed in with a rubber-glycerin burette-cock 
grease and capped with a small piece of muslin. The bottles were 
clamped in specially constructed frames (PI. I, fig. 1) and given a 
sterilization of 25, 20, and 20 minutes, respectively, at 100° C. on 
successive days. 
The handling of the preservatives involved slight modifications 
for individual cases, but hi all instances concentrations are based 
on the actual weight of the preservatives in grams in 20 c. c. agar- 
preservative mixture. 
With inorganic salts soluble in water solutions were prepared 
varying from 3 to 10 per cent concentration (grams in 100-c. c. solu- 
tion), and these were used by measuring into 50-c. c. bottles, similar 
to those used for agar, the desired amount of solution, using either a 
10-c. c. or a 25-c. c. standardized burette graduated in twentieths 
or tenths of a cubic centimeter, respectively. To each bottle was 
then added sufficient distilled water to make 3 c. c. In all cases 
concentrations were based on the weight of dry salt present. 
All other preservatives were weighed into the 50-c. c. bottles on an 
analytical balance, and enough distilled water was added to make 
3 c. c. In the case of a few viscous oils, namely, coal-tar creosote, 
coal-tar creosote Fraction V, wood tar, and wood creosote, which do 
not readily emulsify with water, 5 to 33 J per cent stock emulsions 2 
were prepared, using equal amounts of gum arabic and preservative 
and diluting with distilled water to the desired concentration. These 
emulsions were then used in place of the crude preservatives. 
1 In all measurements of agar one-half c. c. excess was allowed to cover the amount adhering to the 
glass containers. 
2 This method usually produced a quite permanent emulsion. 
