422 PHYSIOLOGY OF THE FUNGI 



times with distilled water. Wash the agar twice with 95 per cent alcohol, 

 allowing the alcohol to stand on agar overnight before draining. Dry 

 the agar in thin layers between cheesecloth. This procedure takes about 

 10 days (see Robbins, 1939). In some instances the agar and other con- 

 stituents of the medium may be autoclaved separately and the two solu- 

 tions mixed, using antiseptic precautions. This should be done when it 

 is required to have a very acidic agar medium. A known amount of 

 sterile acid may be added to the agar medium after sterilization. 



pH or reaction of the medium. See Chap. 8 for a discussion of pH. 

 An approximate method of determining pH is sufficiently accurate for 

 many purposes. On a white porcelain spot plate place one drop of 

 Hellige (or other) wide-range indicator in each depression. Have the 

 drops of indicator of equal size. Then add one drop of the medium to 

 a drop of indicator in one of the depressions of the spot plate. The color 

 of the mixed drops indicates the pH of the medium. Thus, red indicates 

 a pH in the neighborhood of 4, light green 7, purple 10. Standard buffers 

 (solutions of known pH) may be provided so that the student may have 

 standards with which to make comparisons. 



The buffers used in testing pH are most conveniently made from buffer 

 tablets (Coleman). Dissolve one tablet in 100 ml. of distilled water. 

 Add a crystal of thymol as a preservative. Thymol aids in preventing 

 contamination and does not appreciably affect the pH of the buffer. It 

 is convenient to store the buffers in brown-glass dropping bottles fitted 

 with pipettes. 



Unless otherwise specified, media used in the laboratory should be 

 adjusted to pH 6 before autoclaving. This may be done by the use of 

 the spot plate, adding a drop of pH 6 buffer to a drop of indicator. This 

 is the standard color to which the media should be adjusted. Add either 

 QN NaOH or QN HCl to the medium until the color produced by one drop 

 of medium matches the color produced by the standard buffer. Always 

 agitate the medium after each addition of acid or base and then test the 

 pH. The use of concentrated acid and alkali is recommended so that 

 dilution of the medium may be avoided. More precise methods of 

 measuring pH may be used if desired. 



Autoclaving usually lowers the pH of a medium. In general, this 

 change will not be great, but the student should never assume that the 

 pH will remain unchanged in autoclaving. 



Sterilization of media and glassware. Except in special instances, the 

 autoclave may be used to sterilize both media and glassware. Fifteen 

 minutes at 15 lb. steam pressure is adequate for test tubes and flasks 

 which do not contain over 150 ml. of medium. Larger lots of media 

 should be autoclaved 20 min. at 15 lb. steam pressure. Petri dishes may 

 be sterilized 20 min. in the autoclave. It is convenient to wrap two Petri 



