250 DIFGO MANUAL 



MEDIA FOR 

 THE CLASSIFICATION OF YEASTS 



These media have been prepared according to the formulae of Wick- 

 erham^'2,3,4,5 ^^^^ ^^.q recommended for use in classifying yeast according to the 

 following criteria suggested by him. 



1. Colonial characteristics and cell morphology using Bacto-Yeast Morphology 

 Agar. 



2. Carbon assimilation using Bacto-Yeast Nitrogen Base. 



3. Nitrogen assimilation using Bacto-Yeast Carbon Base. 



4. Vitamin requirements using Bacto-Vitamin Free Yeast Base. 



These media will be considered separately, and the composition of each 

 medium is given on the following page. 



BACTO 



YEAST MORPHOLOGY AGAR (B393) 



DEHYDRATED 



Colonial characteristics and cell morphology are determined by a modified 

 Dalmau technique using Bacto-Yeast Morphology Agar. This medium is com- 

 posed of ingredients of known composition prepared according to the formula 

 suggested by Wickerham. 



The following technique has been suggested by Wickerham for the determina- 

 tion of typical colonial characteristics and cell morphology. 



Following sterilization, pour the sterile medium into plates to a depth of about 

 1.5 mm. Allow the plates to stand at room temperature for one or two days 

 before using to be assured of a dry surface. Inoculation is made using the Dalmau 

 plate technique, as described by Wickerham and Rettger^. Briefly, this method 

 consists of making a single streak inoculation near one side of the plate (as from 

 the relative positions 10 o'clock to 2 o'clock). The inoculum should be light and 

 taken from a slant culture. In addition to the single streak inoculation, two point 

 inoculations are made near the other sides of the plate (as at positions 4 o'clock 

 and 8 o'clock). A central section of the streak inoculation and one of the point 

 inoculations are covered with cover glasses. With forceps, remove cover glasses 

 from absolute alcohol, drain momentarily, and burn off the excess alcohol by 

 passing over a low flame. When the cover glass has cooled, place one edge on 

 the agar and allow the cover glass to fall across the central portion of the 

 inoculated streak and a second cover glass over one point inoculation. The plate 

 is incubated six or seven days at 25 °C. and then observed, using the high-dry 

 objective. This is an excellent method for studying the hyphae of filamentous 

 yeasts. 



To rehydrate the medium, suspend 35 grams of Bacto-Yeast Morphology Agar 

 in 1000 ml. of cold, distilled water. Heat to boiling to dissolve the medium 

 completely. Distribute in tubes or flasks and sterilize in the autoclave for 15 

 minutes at 15 pounds pressure (121°C.). The final reaction of the medium will 

 be pH 4.5. 



