Abstract
Considerable difficulty has been experienced in estimating the abundance of marine anaërobic bacteria although they have been demonstrated 1 in nearly all samples of water or mud examined. Most of the conventional procedures 2 such as the incubation of plates in anaërobic jars have failed to yield reproducible results and moreover, the use of such complicated, space- and time-consuming apparatus is entirely impracticable aboard a rolling boat at sea. The application of oval tubes as described by Anderson 3 for the enumeration of anaërobes has exceeded expectations.
Ordinary round glass tubes with sealed ends as used by Roux 4 and Burri 5 are satisfactory for the cultivation of anaërobes but the curvature of the glass makes it virtually impossible to count the colonies which develop, particularly when it is necessary to use a hand lens. The Kimball Glass Company fabricated special oval tubes for us with flat, parallel sides, thereby obviating this difficulty. The oval tubes are 6×14 mm. in cross-section and 380 mm. long with one end permanently sealed and the other end flared to facilitate the introduction of the medium. The tubes are sterilized in a pipet can. A test-tube containing 10 cc. of nutrient agar recently heated to nearly 100°C. to expel oxygen and cooled to 45° is inoculated with the proper dilution of the sample to be analyzed for anaërobes. Without undue agitation (shaking has been found to be unnecessary to insure an even distribution of bacteria) the inoculated medium is poured into the oval tube. As soon as the medium has solidified, it is covered with a deep layer of reduced methylene-blue agar. The seal excludes oxygen and indicates the degree of anaërobiosis, Hall 6 having shown that conditions are suitable for the growth of anaërobes in an environment in which methylene blue remains colorless.
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