[BERKELEY] MARINE BACTERIA 23 



It will be seen from what follows that the same thing has been found 

 to hold in my own experiments. 



The following media were used to follow the course of nitrate 

 reduction under the influence of the bacteria in the three samples. 



Medium B. 



Calcium Acetate 5*0 grams 



Potassium Nitrate -5 gram 



Sodium Phosphate (Na2HP04) -25 gram 



Sea-water 1000 cc. 



Medium C. 



The same as Medium B with addition of • 2 gram Peptone (Witte's) . 

 Both these media were boiled and filtered before sterilizing to remove 

 the precipitate which formed. The media, after sterilizing, were 

 alkaline to litmus and neutral (or very slightly alkaline) to phenol- 

 phthalein. 



A series of six, 120cc. Erlenmeyer flasks each containing 25cc.^ 

 of Medium B was inoculated from the three sea-water samples using 

 •3cc. of each sample and inoculating two flasks with each. One 

 series of three was kept at 28° (Series Bl), the other series at air 

 temperature (series B2) and blanks were kept under the same con- 

 ditions. 



A second series of six similar flasks containing the same amount 

 of Medium C was inoculated in the same way and kept under the 

 same conditions (series CI and C2 respectively) and blanks were kept 

 under the same conditions. 



The following reagents were used for detecting the products of 

 the reduction: — 



For nitrites. The Griess — Ilosvay reagent. 

 This is prepared as follows: — 



(i) • 5 gram sulphanilic acid is dissolved in ISOcc. dilute acetic 

 acid. 



(ii) • 2 gram of a naphthylamine is boiled with 20cc. of water, 

 the colourless solution is poured off from the bluish-violet residue 

 and 150cc. dilute acetic acid added. 



' The effect of using only a small volume of liquid in a comparatively large flask 

 was to have a thinly spread layer of medium thoroughly exposed to aeration. It is 

 possible that this condition inhibited the development of one class of denitrifying 

 bacteria. Organisms producing denitrification in similar media to those in question 

 here, under conditions of both very free and very limited aeration, can be isolated 

 from soil, but they are of different type to one another and their modus operandi is 

 probably also different. It is possible that this also holds of marine bacteria. A 

 second series of tests should be carried out to determine this point. 



