ZoBell — 42 — Marine Microbiology 



After sterilization at 120° C. for 20 minutes, Medium 2216 has a reaction 

 of pK 7.5 to 7.6 when in equilibrium with atmospheric CO2. 



Medium 2216 differs very little in composition from the medium rec- 

 ommended by Fischer (1894a), which contained i.o per cent peptone 

 and 0.5 per cent fish extract in sea water. Fischer used either 10 per cent 

 gelatin or 2 per cent agar as the solidifying agent. Although in his 

 earlier work Fischer used Koch's nutritive gelatin prepared with fresh 

 water, he soon discovered that sea water was far superior for the cultiva- 

 tion of marine microorganisms. He relates that many bacteria found in 

 the sea failed to grow unless the media were prepared with sea water. 

 Neither mussel, shrimp, copepod, diatom, nor beef extract gave results as 

 good as fish extract. 



Although several workers have used fish extract media for the culti- 

 vation of marine bacteria, we have failed to note any increase in plate 

 counts by fortifying Medium 2216 with various fish extracts. Similarly, 

 after trying extracts of various marine organisms including fish, Lloyd 

 (1930) concluded that they are not essential for the growth of marine 

 bacteria in spite of the claims of many earlier workers. Of course, it 

 should be pointed out that the commercial peptones available today 

 are much more growth-promoting for most bacteria than are the peptones 

 which Fischer and others used during the last century. Not nearly as 

 many colonies or kinds of bacteria developed on a fish-extract peptone 

 agar prepared according to the directions of Fischer as on Medium 2216. 



After experimenting with various media, bacteriologists (Reuszer, 

 1933) at the Woods Hole Oceanographic Institution adopted the following 

 one which consistently gave them the highest counts of bacteria: 



Peptone i . o gram 



Glucose I . o gram 



K2HPO4 0.05 gram 



Agar 15.0 gram 



Sea water 1 000.0 ml. 



Except for the presence of glucose, this medium is similar to the one em- 

 ployed by Drew (1913) and in which Lipman (1929) found higher counts 

 than in any other medium with which it was compared. Reuszer's 

 formula differs from Medium 2216 in that it contains glucose and a lower 

 concentration of peptone. In many comparisons with marine materials 

 from the Atlantic Ocean as well as from the Pacific Ocean, ZoBell (1941a) 

 found only 53 to 78 per cent as many colonies on Reuszer's medium as on 

 Medium 2216. The addition of glucose to Medium 2216 failed to increase 

 either the numbers or kinds of bacteria which developed. 



The addition of o.oi per cent ferric phosphate to Reuszer's formula 

 resulted in counts which were nearly as high as those obtained on Medium 

 2216. It has been found that the addition of o.oi per cent of either ferric 

 citrate, ferric phosphate, or ferrous sulfate to nutrient sea- water agar 

 increased the plate count by 18 to 76 per cent, the iron being more bene- 

 ficial in slightly alkaline than in acidic media. 



Maximum plate counts were obtained on media having a reaction of 

 from ^H 7.5 to 7.8. The results were nearly comparable throughout the 

 range of ^H 7.0 to 8.0. Very few colonies develop on media more alkaline 

 than pH. 8.5 or more acid than pH. 5.5. The influence of the hydrogen-ion 

 concentration upon the development of colonies is illustrated by the data 

 summarized in Figure 5. 



The media employed by Berkeley (1919), Lipman (1926), Baven- 



m 



