THE PRECIPITATION OF CALCIUM CARBONATE IN THE SEA. 519 



BACTEEIAL INVESTIGATION OF THE CHALKY MUD- 

 FLATS WHICH AKE BEING DEPOSITED TO THE WEST 

 OF ANDEOS ISLAND. 



Samples of the mud were taken from the western entrance of South 

 Bight, and from points two and three miles out from the shore : 

 practically identical results were obtained from all these localities. 

 The sample at the mouth of the Bight was taken in about 4 feet of 

 water, that two miles out in 7 feet, and that three miles out in 8 feet. 

 The samples were necessarily taken from the surface of the mud. 



For bacterial examination, one part of this mud was shaken up with 

 three parts of sterilized sea-water; this was allowed to settle for 

 15 minutes, and then the clearer surface layer was diluted 1 in 

 1,000,000 with sterilized sea-water. The diluted fluid was plated in 

 Peptone Agar, 1 c.c. being used for each plate. The count of a 

 number of plates after 48 hours gave 40 colonies as an average, and 

 thus the surface mud itself must contain about 40 x 4 X 1,000,000 

 = 160,000,000 bacteria per 1 c.c. The actual number in the mud 

 may exceed this figure, since a large proportion of the bacteria would 

 possibly settle with the larger particles after the first dilution. 



The bacteria found in these cultures were nearly all the B. calcis, 

 only occasionally were a few colonies of the non-denitrifying species 

 seen. 



A sample of the water taken from the surface at a spot three miles 

 out from the western entrance of South Bight gave a count of 35,000 

 colonies per 1 c.c, the great majority of these being B. calcis. 



Subcultures of B. calcis were made in Gran's medium, and in the 

 Calcium succinate, Calcium acetate, and Peptone Calcium acetate 

 media, whose composition has already been given. Denitrification in 

 all these media was rapid and eventually complete, and was accom- 

 panied by the precipitation of Calcium carbonate. In the last three 

 media, which contained no solid matter and were quite clear and 

 transparent before inoculation, this precipitation was manifested after 

 twelve hours by the formation of a thick white cloud in the fluid, 

 readily distinguishable from the cloudiness produced merely by bac- 

 terial growth. The development of this precipitate continued rapidly 

 during the first forty-eight hours, but in many cases it was composed 

 of such fine particles that they showed little tendency to settle to the 

 bottom of the flask ; in other cases larger particles were formed, and a 

 sediment similar in appearance to the chalky mud of the mud-flats 

 was produced. The exact conditions determining the size of tire 



