144 REPORTS ON INVESTIGATIONS AND PROJECTS. 



40 X 4 X 1,000,000 = 160,000,000 bacteria per cubic centimeter. The actual 

 number in the mud possibly far exceeds this figure, since a large proportion 

 of the bacteria would probably settle with the larger particles after the first 

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

 previously described ; only occasionally were a few colonies of the non-deni- 

 trifying species seen. 



A sample of the water taken from the surface of the sea at a spot 3 miles 

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

 nies per cubic centimeter, 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 com- 

 position has already been given. Denitrification in all these media was rapid 

 and eventually complete, and was accompanied by the precipitation of cal- 

 cium carbonate. In the last three media, which contained no solid matter 

 and were quite clear and transparent before inoculation, this precipitation 

 was manifested after 12 hours by the formation of a thick white cloud in 

 the fluid, readily distinguishable from the cloudiness produced merely by 

 bacterial growth. The development of this precipitate continued rapidly 

 during the first 48 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 ap- 

 pearance to the chalky mud of the mud-flats was produced. The exact con- 

 ditions determining the size of the particles precipitated could not be ascer- 

 tained, as the size varied largely in cultures made at the same time, in the 

 same media, and kept apparently under the same conditions. The addition 

 of magnesium tartrate in small quantities (0.2 gram per 1,000 c. c.) to the 

 culture media seemed to induce the precipitation of larger particles, but it 

 did not appreciably affect the rate of growth of the bacteria. In some of 

 the older cultures, that had been kept for a week or more, the sides of the 

 flasks were coated with a thin layer consisting of extremely minute rhom- 

 bohedral crystals of calcium carbonate. Occasionally these crystals formed 

 around small bubbles that had remained near the surface of the fluid, the 

 weight of the crystals eventually caused the bubbles to sink, and then the 

 contained gas became dissolved. In this way a number of small hollow 

 spheres were formed, their walls consisting of minute crystals of calcium 

 carbonate. The formation of these curious bodies occurred especially readily 

 in the calcium succinate medium to which 0.2 gram of magnesium tartrate 

 per liter had been added. The deposition of calcium carbonate in the crys- 

 talline form was only noted in old cultures, and then it was in an amount 

 relatively extremely small when compared to the precipitate of unorganized 

 and amorphous calcium carbonate. 



A consideration of these observations shows firstly that B. calcis is found 

 in enormous numbers in the chalky mud-flats of the Great Bahama Bank, 

 and secondly that this bacterium is capable of precipitating calcium car- 

 bonate from fluid media containing soluble calcium salts. It would seem a 

 fair deduction that these mud-flats have been precipitated by the action of 

 B. calcis on the soluble calcium salts carried into the sea by drainage from 

 the land, where extensive and rapid weathering of the limestone rock is in 

 progress. 



