PROBLEMS OF METHOD 57 



It is possible that metabolites other than acids may accumulate in 

 inhibitory concentrations; for example, the accumulation of ethanol 

 has been suggested (181) as the explanation of thiamine toxicity to 

 Rhizopus suinus. 



Another set of problems is associated with contamination of sup- 

 posedly pure chemicals with growth-active substances. There is evi- 

 dent in the literature an unjustified confidence in the label placed on 

 a chemical by the manufacturer. Even compounds rated as "reagent" 

 quality or the equivalent may be contaminated with biologically sig- 

 nificant amounts of other substances, particularly inorganic ions and 

 organic growth factors. Such contamination is particularly likely in a 

 compound derived from natural sources, and most of the important 

 carbon and organic nitrogen sources are so derived. In a highly puri- 

 fied medium one source of carbon may appear superior to another 

 merely because it is contaminated with a required inorganic element 

 in which the basal medium is partially deficient. More commonly, 

 the fungus has an unrecognized vitamin requirement which is met by 

 the less pure of a series of carbon compounds; thus, the early and 

 anomalous finding that Diplodia macrospora grows with starch, mal- 

 tose, or sucrose but not with glucose or fructose is now known to re- 

 flect contamination of the first group of compounds with biotin, re- 

 quired by the fungus as a growth factor (199, 241). Growth factors 

 for fungi have been found in many carbon compounds, e.g., soluble 

 starch (172), glucose (121), galactose (44), and saponin (19). 



Growth inhibition by contaminants is less commonly encountered 

 but has been reported from the ions present in crude natural materials 

 (153) and from an unidentified alcohol-soluble contaminant of glu- 

 cose and galactose (44). 



It should not be necessary to point out that such materials as cellu- 

 lose and chitin as they occur in nature may be contaminated with or- 

 ganic materials in sufficient amount to provide the carbon source for 

 visible growth of fungi. It is not, for example, justified to claim that 

 an organism can utilize chitin merely on the evidence that it grows 

 on the exoskeleton of an insect, particularly since insect cuticle is less 

 than 50 per cent chitin (239). 



Breakdown of sugars during autoclaving constitutes another serious 

 and often unrecognized source of error in studies of carbon nutrition. 

 Growth inhibition from sugar breakdown products occurs in both 

 bacteria and fungi (9, 116, 193). Breakdown is more extensive if the 

 sugar is in contact during autoclaving with phosphate (59) or with 

 amino acids (86, 125). Ketohexoses, e.g., fructose and sorbose, break 

 down (to 5-hydroxymethylfurfural) more rapidly than do the aldo- 



