16 BULLETTX 227, TJ. 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 grama. 



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 (PL 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 omulsion. 



