42 Papers from the Marine Biological Laboratory at Tortiigas. 



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 i in i ,000,000 with sterilized 

 sea-water. The diluted fluid was plated in peptone agar, i c.c. being used 

 for each of the plates. The count of a number of plates after 48 hours gave 

 40 colonies as an average, and thus the mud itself must contain 40 X 4 X 

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

 mud possibly 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; only 

 occasionally were a few colonies of the non-denitrifying species seen. 



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

 the western entrance of South Bight gave a count of 35,000 colonies per 

 I 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 accompanied by the precipi- 

 tation 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 12 hours by the formation of a thick 

 white cloud in the fluid, readily distinguishable from the cloudiness pro- 

 duced merely by bacterial growth. The development of this precipitate 

 continued rapidly during the first 48 hours, but in many cases it was com- 

 posed 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 the particles 

 precipitated could not be ascertained, 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 precipi- 

 tation 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 rhombohedral crystals of calcium carbonate. Occa- 

 sionally 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 gm. of magnesium tartrate per liter had been added. The deposition 

 of calcium carbonate in a distinctly crystalline form was only noted in old 

 ■cultures, and then it was in an amount relatively extremely small when 



