20 Papers from the Marine Biological Laboratory at Tortugas. 



This medium was boiled and filtered before sterilization to remove the 

 slight precipitate of calcium phosphate. It was found that this medium 

 with the addition of the phosphate gave a more vigorous growth than if it 

 was omitted. 



///. Calcium acetate medium: Calcium acetate (Ca(CH3COO)2), 5.0 grams; sodium 

 phosphate (Na2HP04, 12H2O), 0.25 gram; potassium nitrate (KNOj), 

 0.5 gram; sea-water, 1,000.0 c.c. 



Boiled and filtered before sterilization to remove precipitate of phos- 

 phate. 



IV. Peptone calcium acetate medium: Calcium acetate (CaCCHaCOO)^), 5.0 grams; 

 peptone (Witte's), 0.2 gram; potassium nitrate (KNO3), 0.5 gram; sea- 

 water, 1,000.0 c.c. 



Media II, III, and IV were also made up with the addition of 0.2 gm. 

 of magnesium tartrate per 1,000 c.c. 



The fluid media were made up in 1,500 c.c. resistance glass flasks, and 

 1,000 c.c. of medium were used for each culture. 



For other purposes a simple solution of peptone in sea-water was em- 

 ployed (2 gm. to 1,000 c.c), and media were also used consisting of this 

 peptone solution with the addition of 0.5 per cent of various carbohydrates, 

 such as cane sugar, dextrose, Isevulose, mannite, lactose, etc., with sufficient 

 neutral red solution to color them, in order to test the acid-forming proper- 

 ties of the bacteria in the presence of carbohydrates. 



The ordinary "Koch" steam sterilizer and iron oven for dry -heat 

 sterilization were used, and gasoline cooking stoves were found to be the 

 most satisfactory source of heat. It was found an advantage to use Petri 

 dishes with porous earthenware covers which enabled the water of con- 

 densation to evaporate partially; the evaporation could be checked at any 

 time by covering the dishes with a bell jar lined with wet filter paper. It 

 was usually found necessary to keep all cultures on tables with their feet 

 standing in dishes of kerosene, in order to prevent the attacks of ants and 

 other insects. In all other respects ordinary bacteriological routine was 

 followed, and the methods need not be further particularized here. 



The reduction of the nitrate to a nitrite in fluid culture media was 

 tested for by the addition of 5 c.c. of 10 per cent sulphuric acid and 2 c.c. 

 of a I per cent solution of metaphenylene diamine hydrochloride to 25 c.c. 

 of the culture. The production of a brown coloration (due to the formation 

 of Bismarck brown) is an indication of the presence of a nitrite, and is an 

 extremely delicate reaction. 



The diphenylamine and brucine sulphate reactions were also used when 

 testing for the presence of nitrates. 



The formation of ammonia was tested for by the addition of 5 c.c. of 

 10 per cent potassium hydrate and 5 c.c. of Nessler's reagent; the white 

 precipitate formed on the addition of the potassium hydrate does not 

 appreciably interfere with the test, though it renders it somewhat less 

 delicate. 



