POSSIBLE APPLICATION OF A BACTERIAL BIOASSAY 

 IN PRODUCTIVITY STUDIES 



William Belser * 



An increasing number of reports in the litera- 

 ture, demonstrating requirements for growth 

 factors by various marine algae (Levin, 195^-, 

 Provasoli and Pintner, 19^3a, Sweeney, 195^) , 

 the effects of external nutrilites on feeding 

 responses (Loomis, 1953, Collier, 1950), and 

 the possible implication of organic micro- 

 nutrients in the discontinuous distribution of 

 marine plants and animals (Lucas, 1939, Prova- 

 soli, 1956, Margalef, 1956, Wilson, 1956) has 

 led to the formulation of this program. Many 

 of these organic materials are required in 

 extremely small amounts, and might be expect- 

 ed to be present in sea water in very low con- 

 centration. Previous attempts to isolate and 

 characterize some of them have been moderately 

 successful (Johnston, 1955, Provasoli and 

 Pintner, 1953^), although somewhat cumbersome. 

 Relatively high salt concentrations in sea 

 water preclude direct chromatography of the 

 organic materials, and require their preisols- 

 tion, either by absorption or desalting. Deal- 

 ing with materials present in micrograms-per- 

 liter quantity presents a formidable task. 



With these facts in mind, I have considered 

 the possibility of establishing a series of 

 biochemical mutants with a wide spectrum of 

 nutritional requirements, which might be 

 employed directly as bioassay organisms in 

 sea water. Attempts to train Escherichia 

 coli, in which many mutants are already in 

 culture, to grow in sea water- were time con- 

 suming and impractical. Therefore, a number 

 of marine bacteria were screened for desirable 

 characteristics, and Serratia marinorubrum 

 (ZoBell, 19^4) was selected as the most suit- 

 able of these organisms for this purpose. 

 S. mar i nor ub rum is an easily distinguishable 

 red pigmented organism, which will grow well 

 in a medium composed of inorganic salts, with 

 glycerol as the sole source of carbon. In 

 addition, it shows a wide range of salt 



* Public Health Service Research Fellow of 

 The National Cancer Institute. 



tolerance, growing in media with the salinity 

 of fresh water, as well as in threefold con- 

 centrated sea water. This heterosmotic 

 feature suggests the value of the organism 

 for bioassay of rivers and lakes, as well as 

 the ocean, in tide pools, estuaries and 

 seasonally landlocked sloughs . 



To date, several mutants have been obtained 

 by ultraviolet irradiation of S. marinorubrum . 

 One of these requires biotin, and will respond 

 to concentrations in the order of 1 to 5 

 mug/ml. (see Table l) . The second mutant has 

 a specific requirement for uracil, and responds 

 to concentrations between 10 and 100 mug/ml. 

 The third mutant thus far obtained has a non- 

 specific purine requirement, and will grow 

 when supplied with any of the purine bases or 

 their ribosides. The most sensitive respc: 

 obtained with this mutant is to hypoxanthi 

 in the range from 10 to 100 mug/ml. 



In a preliminary field trial designed to test 

 the bioassay system, some 38 sea water samples 

 were tested. These samples were taken for me 

 or. SCOPE in waters off the coast of Mexico 

 and Central America. The results of these 

 tests shoved quite definitely that the bio- 

 assay system has merit, since a fairly wide 

 distribution of biotin was observed, with 

 sporadic occurrence of uracil, a ' one 

 instance of purine (see Table 2). Controls 

 failed to show any evidence of either rever- 

 sion of any of the mutants or contamination 

 of the water samples 



MATERIALS AND TECHNIQUES 



The technique for mutant induction involves 

 the irradiation of cultures in the logarithmic 

 phase of growth and screening for mutants 

 after incubation. This has been done by 

 minimal enrichment and delayed enrichment 

 techniques. The mutants, so isolated, are 

 identified with regard to their specific re- 



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