SECT. 2] BIOASSAY OF TRACE SUBSTANCES 225 



and organic materials, including glycerophosphate, betaine, adenylic acid, 

 glucose and acetate which, although not a problem of assays in the laboratory, 

 might constitute a limitation on the use of the assay at sea. This disadvantage 

 could be overcome by returning samples to the laboratory for assay. Addition 

 of volatile preservative (Hutner and Bjerknes, 1948) would maintain the 

 sterility of the sample. These authors have not as yet reported any data on the 

 application of this assay to sea-water samples. 



Burkholder (1959) has applied a rather different method to the assay of 

 biotin, thiamine and niacin. The preceding techniques are all carried out in 

 liquid culture, and the quantitation of the assays done by densitometry or 

 some method to estimate increase in cell mass. Burkholder uses an agar-plate 

 method whiqh should lend itself well to studies at sea, as well as in the labora- 

 tory. His assay organisms are bacteria isolated from the marine environment. 

 An agar plate containing a basal medium, but lacking the vitamin to be 

 assayed, is seeded with the assay bacterium. The sample to be tested is applied 

 to a small sterile filter pad, which is placed on the surface of the seeded plate. 

 The growth stimulation is estimated by measuring the diameter of the zone 

 surrounding the pad. The major disadvantage of this technique is its relative 

 insensitivity. The responses of the three assay organisms are : niacin, 15 m[xg- 

 1.0 ^g/ml; thiamine, 4 mfi.g-1.0 fj.g/ml; and biotin, 1 mfxg-0.5 [xg/ml. Although 

 no specific data are available, Burkholder asserts that physiologically sig- 

 nificant quantities of all three of these vitamins have been found in sea- water 

 samples and natural marine sediments. 



Vishniac and Riley (1959) have assayed thiamine using a non-filamentous 

 fungus which is as yet unidentified. The organism responds to concentrations 

 from 25 y.y.g to 200 (j.jj.g/ml of thiamine. They report that detectable concentra- 

 tions (i.e. greater than 20 [i[ig/m\) were rarely encountered in Long Island 

 Sound, although near-shore samples, subject to enrichment by land drainage, 

 showed relatively high concentrations (e.g. 63-65 [Xfj-g/ml at a river outfall 

 and 30-40 (j.|j.g/ml near Charles Island). 



Niacin and biotin in sea-water are also being assayed by Belser (1959) 

 utilizing organisms with artifically produced gene mutations. In all of the pre- 

 ceding cases, the assay organisms are naturally occurring auxotrophs ; that is, 

 they have evolved in such a way that they lack the synthetic capacity for the 

 compounds they are used to assay. Belser takes advantage of an observation 

 from microbial genetics that gene mutation in bacteria sometimes resulted in 

 alteration or loss of enzymes. When such a mutation occurs, and an enzyme is 

 no longer manufactured, the organism cannot synthesize the end-product of 

 the biosynthetic chain in which the enzyme occurs. 



Belser employs Serratia marinorubra, a red-pigmented coccobacillus (ZoBell 

 and Upham, 1944), which can synthesize every single compound it requires for 

 growth, given a mixture of inorganic salts and with glycerol as the sole source 

 of carbon. By inducing mutations with ultraviolet light, biochemically deficient 

 strains have been isolated, each of which is incapable of synthesizing one specific 

 end-product necessary for maintaining life. If the end-product in question is 



