THE ACTION OF SOME DENITIIIFVING BAGTEItlA. 145 



II. Potassium Mai.ate Agar. 



Potassium malate (C.2H3(OH)< p. .,,,.] 



Sodium phosphate (NaoHP()^,12H.-.0) 

 Potassium nitrate (KNOg) 

 Sea-Avater . ... 



Agar Agar . ... 



The medium was only filtered through glass wool, so that a very 

 slight floccular precipitate of Calcium phosphate was retained. 



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

 employed (2 grammes to 1000 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, Laevulose, Mannite, Lac- 

 tose, etc., with sufficient Neutral IJed solution to colour them. 



In the case of Gelatin media, it was necessary to keep them artifi- 

 cially cooled to a temperature of between 20° and 25° C, as in the 

 Tortugas the Laboratory temperature occasionally rose as high as 

 37° C, at which temperature Gelatin media will not remain solid. 



When comparing various samples of water as to their power of 

 causing denitrification in culture media, the following uniform method 

 was employed : — 



10 c.c. of each sample was added to 1000 c.c. of a modification of 

 of Gran's medium (see Studien ilbcr Mcereshacterien, by H. H. Gran, 

 Bergens Museums Aarbog, No. 3, 1901). This culture fluid was 

 sterilized in glass flasks and has the following composition : — 



Potassium nitrate (KNO3) . . . 0*5 grammes. 

 Sodium phosphate (NaoHPO^, I2H2O) . 0*25 grammes. 



Calcium malate (C.,H3 (OH) < r^r\p. > Ca) . about 5'0 grammes. 



Sea-water 1000-0 c.c. 



Calcium malate is only slightly soluble in water (about Tr^th per 

 cent), and so can be added in excess. 



The reduction of the nitrate to a nitrite was tested for by the 

 addition of 5 c.c. of 10 per cent Sulphuric acid and 2 c.c. of a 1 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 Bismark brown) is an indication of the presence of a nitrite, and is 

 an extremely delicate reaction. 



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

 a 10 per cent solution of Potassium hydrate, and 5 c.c. of Nessler's re- 

 agent : the white precipitate formed on the addition of the Potassium 



