252 DIFGO MANUAL 



BACTO 



YEAST NITROGEN BASE (B392) 



DEHYDRATED 



Media for carbon assimilation tests may be prepared by adding various sources 

 of carbon to Bacto-Yeast Nitrogen Base. Wickerham^'-'^**'^ reported that some 

 strains of yeast require the presence of certain vitamins for the assimilation of 

 carbon. Bacto-Yeast Nitrogen Base is a suitable medium for such studies. 



For these demonstrations the tubes of media are inoculated very lightly, then 

 placed at 25 °C. After 6 to 7 days of incubation, and again at 20 to 24 days, the 

 tubes are shaken to suspend growth, then they are placed against a white card 

 bearing lines approximately ^ of a millimeter wide. The lines are drawn with 

 India ink. If the lines cannot be seen through the culture, or if the lines appear 

 as diffuse, broad bands, the test is positive. If the lines are distinguishable as such, 

 the test is negative. The two observations indicate which reactions occur rapidly 

 and which occur latently. 



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

 accomplished by suspending 6.7 grams of Bacto-Yeast Nitrogen Base and 5 grams 

 of dextrose or an equivalent amount of other carbohydrate in 100 ml. of distilled 

 water. It may be necessary to warm the distilled water slightly to effect complete 

 solution of some of the carbohydrates. 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, under aseptic conditions, 

 0.5 ml. into 4.5 ml. of sterile distilled w^ater in 16 mm. cotton stoppered tubes. 

 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. 



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

 hydrate solution separately. To accomplish this, dissolve 6.7 grams of Bacto- 

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

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

 carbon compound under test for 100 ml. (0.5 gram dextrose) in 90 ml. of dis- 

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

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

 aseptic conditions and mixing thoroughly by shaking. Final reaction of the basal 

 medium will be pH 4.5. 



BACTO 



YEAST CARBON BASE (B391) 



DEHYDRATED 



The ability of yeasts to assimilate nitrogeni»2,3,4,5 \^ determined by adding 

 various nitrogen sources to Bacto-Yeast Carbon Base and inoculating with the 

 test organisms. The inclusion of vitamins in this base was found necessary by 

 Wickerham as an aid for the utilization of nitrogen-containing compounds by 

 certain yeasts w^hich cannot assimilate these compounds in the absence of vita- 

 mins. Bacto-Yeast Carbon Base is a suitable medium for such studies. 



Yeasts which have grown on a rich medium may carry a reserve of nitrogen in 

 the form of protein. Possible errors due to this reserve are eliminated by making 

 two serial transfers in the complete medium. When the first transfer is seven days 

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

 complete medium containing the same source of nitrogen. If a positive test is 



