FUNGI AS TEST ORGANISMS 213 



Test organisms during an assay should be cultured under uniform 

 conditions with respect to light and temperature. In general, the fila- 

 mentous fungi should not be agitated during the period of incubation. 

 Yeasts are frequently grown with continuous or intermittent shaking. 



There are two schools of thought concerning the time of incubation 

 for assay. The first recommends a uniform short period of growth and 

 determination of the yield before the organism reaches its maximum 

 development. There is a saving in time in this method, but the influence 

 of accessory factors in the sample may make such results unreliable. A 

 comparison should always be made between the analytical data for a 

 short and a long period of incubation before choosing the length of incu- 

 bation period. In general, we feel that assays tend to be more reliable 

 when the period of incubation is long enough to allow maximum 

 development of the test organism. 



Measuring the response. The methods used for measuring the 

 response of test organisms vary. The growth response of bacteria may 

 be measured either by titrating the acid produced or by determining the 

 turbidity with a suitable photoelectric colorimeter. The growth response 

 of yeasts may be measured as turbidity, or the cells may be weighed. 

 The first procedure is by far the simpler. The growth of filamentous 

 fungi is commonly measured by collecting the mycelium and determining 

 the drj^ weight (see discussion in Chap. 3). 



Calculation of results. A growth curve (acidity, turbidity, or weight) 

 is plotted from the response of the test organism to the different concen- 

 trations of the standard substance. The concentration of the substance 

 in the sample is then calculated from the standard curve. It is necessary 

 to use a new standard curve for each series of assays. Unsuspected 

 variations in the basal medium and in technique from day to day make 

 this precaution necessary. In making the calculations, it is assumed that 

 equal amounts of the substance, whether as a pure compound or in the 

 sample, will cause the same amount of response by the test organism. 

 It is customary to report the concentrations of vitamins and micro essen- 

 tial elements in micrograms per gram of original sample. 



As an example of the type of calculation involved in an assay, the 

 standard curve (Fig. 43) and protocol of a biotin assay are given below. 

 The substance assayed was air-dry yeast cells. Biotin was liberated 

 from the sample by acid hydrolysis, and the cell extract was neutralized 

 and made up to such volume that 1 ml. of hydrolysate was equivalent 

 to 50 mg. of original yeast cells. The test organism, Saccharomyces 

 cerevisiae, Gebriide Mayer strain, was incubated for 72 hr at 25°C. 

 Twenty-five milliliters of glucose-casein hydrolysate medium was used 

 per 250-ml. flask. The cultures were agitated 10 min. every hour. The 

 data for the response of the test organism to varying amounts of yeast 

 hydrolysate are given in Table 35. 



