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Chapter IV 



METHODS OF ENUMERATING MARINE 

 BACTERIA 



Plating procedures, the extinction dilution method, and direct micro- 

 scopic counts are the principal methods employed for estimating the 

 abundance of bacteria and allied microorganisms in sea water, mud, or 

 other marine materials. The success of the first two methods depends 

 largely upon the capacity of the nutrient medium to satisfy the cultural 

 requirements of the microorganisms. The cultural requirements embrace 

 not only energy and food requirements but also the proper hydrogen-ion 

 concentration, osmotic pressure, surface tension, gas tension, oxidation- 

 reduction potential, physical consistency, temperature, and other environ- 

 mental conditions. 



Because different microorganisms differ widely in their cultural re- 

 quirements, no one nutrient medium or combination of environmental 

 conditions can be expected to provide for the growth of more than a small 

 percentage of the microorganisms in a sample of raw sea water or mud. 

 The most one can hope for is a medium in which many microorganisms 

 will grow and with which results may be duplicated. Such a general- 

 purpose medium must be supplemented by numerous selective or differ 

 ential media for estimating the abundance of any particular kind or phys- 

 iological type of microorganism. 



Though often invaluable for estimating the total number of micro- 

 organisms in certain materials, direct microscopic counts give very little 

 information concerning the nature of the microorganisms, often failing to 

 distinguish inactive or dead ceils from living ones. 



Media for plate counts: — As pointed out by Lepman (1929), the com- 

 position of the medium is an important determinant of the numbers and 

 kinds of microorganisms found in the sea. Apparently there are almost 

 as many kinds of nutrient media for estimating the bacterial population 

 of marine materials by platt-count procedures as there are workers in the 

 field, each medium yielding very different results qualitatively as well as 

 quantitatively. 



Over a period of several years bacteriologists at the Scripps Institution 

 of Oceanography have had occasion to compare the growth-promoting 

 properties of a large number of nutrient media which have been used or 

 recommended by various workers for plating marine bacteria. New 

 formulae have been developed as indicated by the results. These studies 

 indicate that Medium 2216 (ZoBell, 1941a) is one of the best for maxi- 

 mum and reproducible plate counts on marine materials and for the culti- 

 vation of most aerobic heterotrophs which occur in the sea. It has the 

 following composition: 



Sea water ("aged," see p. 58) 1000. o ml. 



Bacto-peptone 5.0 gram 



Ferric phosphate o . i gram 



Bacto-agar 15.0 gram 



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