212 PHYSIOLOGY OF THE FUNGI 



to certain amino acids, especially tryptophane. Alkaline hydrolysis of 

 proteins has been recommended for this amino acid (Greene and Black, 

 1944). 



The concentrations of the standard compound and of the sample for 

 assay should be so chosen that the response of the test organism is roughly 

 linear. Every concentration should be run in duplicate. Control flasks 

 to which neither the standard compound nor the assay sample have been 

 added should form a part of every assay. This provides a means of 

 evaluating the basal medium and should never be omitted. 



The type of culture vessel and the volume of the basal medium used 

 will depend upon the test organism. Bacteria are frequently cultured 

 in test tubes. These are also useful for yeasts. Uniform test tubes 

 which can be used in a photoelectric colorimeter allow measurement of 

 turbidity without transfer (Lindegren and Raut, 1947). The filamentous 

 fungi are usually cultured in Erlenmeyer flasks. The volume of medium 

 should be so chosen that the liquid is less than 1 cm. deep. All glassware 

 must be clean. Accuracy in measuring the basal medium and the known 

 and unknown solutions is essential. 



Inoculation and incubation. The medium upon which the inoculum 

 is grown should be complete and contain an adequate but not excessive 

 amount of the factor under investigation. Certain fungi, especially the 

 yeasts, cease to be deficient for certain vitamins when continuously cul- 

 tured upon media free from these factors. 



Spore inoculum may be used with advantage with many filamentous 

 fungi. Frequently it is desirable to use germinated spores for inoculum. 

 Phycomyces blakesleeanus spores require the Z factors for rapid germina- 

 tion (Robbins, 1940). If the test sample contains these factors and the 

 basal medium does not, early growth will be more rapid in the sample 

 series. It is convenient to germinate the spores of this fungus and others 

 by preparing a spore suspension in dilute peptone solution a few hours 

 before inoculation. These germinated spores grow essentially without 

 interruption and shorten the time of incubation. Fragmented mycelium 

 may also be used to advantage. A uniform amount of inoculum must be 

 used. This is easy to achieve when a suspension of spores or fragmented 

 mycelium is used. Inocula of these types provide a multitude of growing 

 points, which results in uniform growth. Disks of mycelium on agar 

 are, in general, unsatisfactory. 



An obvious advantage of using a large amount of inoculum is the 

 shorter time required for an assay. However, there is danger of intro- 

 ducing with a large inoculum enough of the substance under investigation 

 to give abnormally high blanks. Washing the inoculum with sterile 

 distilled water reduces this hazard but increases the work and multiplies 

 the chances of contamination. A very small inoculum results in a longer 

 lag period, and the time required for analysis may be prolonged. 



