246 Development of a Standardised Agar Medium 



It will be seen that Cunningham's method of washing and filtering 

 has produced undesirable changes in the agar. The alteration in reaction 

 during autoclaving has been increased, while the number of colonies 

 which develop is distinctly lowered and spreading colonies are en- 

 couraged. 



Taking the media made up with agar washed in the shred con- 

 dition it will be seen that in the filtered medium, the colony develop- 

 ment is unaffected by the washing in acid but that when the medium is 

 not filtered the unwashed agar permits a rather higher number of 

 colonies to grow. In making up the medium for routine work, however, 

 filtering is nepessary, so that the washing will not produce a harmful 

 effect on the development of colonies. As a result 'of many trials it has 

 been found that agar washed in acid, while in the shred condition, gives 

 more regular results than unwashed agar. 



The process of washing in acid has the further advantage, also 

 observed by Cunningham (8), when using his method, that it renders 

 filtration of the medium easier and more rapid. For example, the medium 

 used in the last experiment was made up with unwashed agar and with 

 shred agar washed in 0-1 per cent. acid. The time taken to filter 200 c.c. 

 of medium through filter paper in a warm filter funnel was recorded. 

 It was found that washed agar medium passed through the filter paper in 

 55 minutes while the unwashed agar medium took 2 hours to pass 

 through. 



Experiments with various strengths of acid for use in the washing 

 have resulted in the adoption of a routine technique in which the agar 

 shred is soaked in 0-05 per cent. 112864^ for 15 minutes at room tem- 

 perature, washed in water till acid free, and then dried. 



Although the difference in composition between samples of agar may 

 be lessened by washing in acid, yet the removal of impurities is not com- 

 plete. The effect of these varying impurities must therefore be neutraHsed. 

 Small quantities of organic impurities, such as occur in the washed agar, 

 are unlikely to influence bacterial growth in a medium already richly 

 supplied with organic and nitrogenous food substances. In a similar 

 manner, the influence of traces of Ca, Mg, S, etc., may be masked by the 

 addition to the medium of quantities of these substances in excess of the 

 bacterial requirement. The necessity of this addition in a medium from 

 which standard results are expected, is sometimes overlooked. This is 

 the case, for example, in some of the "simple" media that have been 



^ For some time 0*5 per cent, acid was used, but it was sometimes found that this 

 affected the gel formation of the agar. 



