DEHYDRATED CULTURE MEDIA 253 



obtained when the second culture is seven days old, the organism under test 

 assimilates this particular nitrogen source. 



The medium should be filter sterilized in lOX strength for best results. This is 

 accomplished by dissolving 11.7 grams of Bacto-Yeast Carbon Base and the 

 nitrogen sources as desired in 100 ml. of distilled water. It may be necessary to 

 warm the distilled water slightly to efTect complete solution. This lOX strength 

 solution is then filter sterilized. The lOX strength sterile medium is kept in the 

 refrigerator and used as needed. The final medium is prepared by pipetting 

 0.5 ml. into 4.5 ml. of sterile distilled water in a 16 mm. cotton stoppered tube. 

 The required number of such water blanks are prepared and autoclaved in 

 advance. After receiving the lOX strength solution, the tubes are shaken, and are 

 then ready for inoculation. 



At present, the most important nitrogen-containing compound being used for 

 nitrogen assimilation studies is potassium nitrate. This medium may be prepared 

 by dissolving 0.78 gram potassium nitrate in 100 ml. distilled water containing 

 11.7 grams of Bacto-Yeast Carbon Base. 



Some laboratories may prefer to filter sterilize the basal medium and nitrogen 

 source solution separately. To accomplish this, dissolve 11.7 grams of Bacto- 

 Yeast Carbon Base in 100 ml. distilled water and filter sterilize. Distribute in 

 0.5 ml. amounts in sterile 16 mm. cotton stoppered tubes. Dissolve sufficient 

 source of nitrogen for 100 ml. (0.078 gram potassium nitrate) in 90 ml. of dis- 

 tilled water. Filter sterilize. The final medium is prepared by adding 4.5 ml. of 

 the sterile nitrogen solution to each 0.5 ml. of the sterile basal medium under 

 aseptic conditions and mixing thoroughly by shaking. The final reaction of the 

 basal medium will be pH 4.5. 



BACTO 



VITAMIN FREE YEAST BASE (B394) 



DEHYDRATED 



Vitamin requirements of yeast may be determined by adding various vitamins 

 to Bacto-\'itamin Free Yeast Base. This base contains sufficient nitrogen and 

 carbon sources to permit growth of yeast after the addition of the required 

 vitamins, as described by Wickerham-^'^'^'-^'S 



Because the inoculum may carry a large supply of vitamin reserve, it is neces- 

 sary to make two serial transfers in a medium containing either no vitamins or 

 the particular combination to be tested. When the first transfer is seven days old, 

 the culture is shaken and one loopful is transferred to a second tube of medium 

 having the same combination of vitamins. This second tube is likewise incubated 

 for seven days. If growth occurs, the yeast does not require an exogenous source 

 of whatever vitamins are absent from the medium. 



The medium should be filter sterilized in lOX strength for best results. This is 

 accomplished by dissolving 16.7 grams Bacto-Vitamin Free Yeast Base in 100 ml. 

 of distilled water containing the desired vitamins. It may be necessary to warm 

 the distilled water slightly to effect complete solution. This lOX strength solu- 

 tion is then filter sterilized. The sterile lOX strength solution is kept in the 

 refrigerator and used as needed. The final medium is prepared by pipetting, 

 under aseptic conditions, 0.5 ml. into 4.5 ml. of sterile distilled water in 16 mm. 

 cotton stoppered tubes. The solutions are mixed thoroughly by shaking and are 

 then ready for inoculation. 



Some laboratories may prefer to filter sterilize the basal medium and the 

 desired vitamins separately. To accomplish this, dissolve 16.7 grams of Bacto- 

 Vitamin Free Yeast Base in 100 ml. distilled water and filter sterilize. Distribute 



