Ogg et al. (54) designed a tube with double wells so that agar with cultures can be introduced into the tube and form a thin film. Anaerobiosis is initially achieved by boiling and autoclaving of the medium. By the action of reducing agent in the medium and minimum reabsorption of O2 due to the design of the tube (commercially called the Lee tube), good anaerobiosis is maintained in the system. The glass cylinder forming the inner wall (the inverted smaller tube) makes it easy for enumeration and observation of colony morphologies. The Lee tube has the advantage of ease of operation and low operating cost (no gas or special equipment needed). The disadvantages include (1) difficulty in picking isolates from the agar; (2) difficulty in cleaning the tubes; and (3) fragility of the system.
Fung and Lee (41) developed a double-tube system for anaerobic cultivation of bacteria from foods. It consists of a glass tube (15 x 1.0 cm o.d.) placed into a larger glass tube (15 x 1.5 cm o.d.) with anaerobic agar (designed for specific organisms) and culture sandwiched between the two tubes. The screw cap of the larger tube when closed seals the system and makes it anaerobic. Anderson and Fung (29) were successful in cultivating strict anaerobic rumen microbes, such as Megasphaera elsdenii, Butyrivi-bro fibrisolvens, Clostridium perfringens, Eubacterium li-mosum, and anaerobes from beef and poultry. Ali et al. (31) successfully used this system to isolate C. perfringens from meat. Aramouni et al. (32) used the double-tube system to study C. sporogenes in home-style canned quick bread. The advantages of the system include (1) ease of operation; (2) inexpensive and ease for cleanup; (3) ease of enumeration and observation of cultures; (4) ease of obtaining colonies (by removing the inner tube and pick colonies); and (5) flexibility and ease of adaptation by other laboratories. Disadvantages include (1) operation of samples in the open environment and (2) occasional breakage of agar by gas-producing organisms.
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