H. Gr. Thornton 271 



spreading of B. dendroides occurred on the plates. Mannitol was conse- 

 quently adopted as the energy source, in addition to the asparagine, in 

 the count medium, and subsequent work has shown its advantage, both 

 on account of the slight change in reaction produced by it on autoclaving, 

 and of the good development of colonies on the medium. 



4. Preparation of the Medium. 



On account of the changes and interactions which take place in a 

 nutrient medium in the course of its preparation, it is necessary, in order 

 to obtain uniform results with it, that the method of preparation should 

 be carefully standardised. The need for this precaution is well shown by 

 the effect of variations in the method of filtration, discussed below. 



The medium here developed has the following composition : 



In making up this medium, the following technique was finally 

 adopted. The phosphate, nitrate and asparagine are dissolved in the 

 distilled water and the MgS04, CaCla, NaCl and FeClg added from 

 standard solutions, in the order named. The agar is then added and dis- 

 solved at 100° C. The medium is then filtered at this temperature, by 

 being passed twice through a layer of absorbent cotton-wool half an inch 

 thick. The mannitol is then dissolved in the filtrate. It is then allowed 

 to cool to 60° C. and its reaction adjusted against Brom Thymol Blue 

 to pH 7'4. The medium is then poured into tubes and sterilised at 15 lbs. 

 pressure for 15 minutes. 



In the earhest work done with this medium, some differences were 

 found between different batches of medium. These differences were 

 eventually traced to variations in the temperature at which filtration 

 was carried out. The effect of the temperature of filtration is shown in 

 the following experiment. Three Htres of the medium were made up and 

 divided into two portions, one of which (A) was filtered through cotton- 

 wool at 100° C. and the other (B) at 50° C. A single diluted suspension 

 of Rothamsted soil was plated on the two media and the mean number 

 of colonies on ten parallel platings on each medium was taken. The 

 results are shown in Table VII, and indicate a perceptible fall in the 

 nutritive value of the medium when filtered at the lower temperature. 

 (See also Fisher, Thornton and Mackenzie(2).) 



