Simple tissue-culture vessel permitting gas exchange with low moisture loss.

For the past 5 years, cultures and subcultures of excised lemon fruit tissue have been maintained in Kimble screw-cap vials (60957, no. 2-2k4), the vials being hermetically sealed with rubber-lined plastic screw caps, size 24 (Kordan, Nature 198: 867, 1963). Under these conditions, the only oxygen available to the excised tissue was that present within the vials at the time of tissue implantation just prior to being sealed with the rubber-lined caps. The culture vessels were hermetically sealed to prevent evaporation of water from the small volumes of nutrient solution employed for growing freshly excised tissue (0.15 ml/vial) and established cultures and subcultures (0.5 ml/vial). A relatively simple tissue-culture vessel is described which possesses the following characteristics: it allows for gas exchange between the interior of the vessel and the external atmosphere; it has an exceedingly low rate of water loss; and it forms a barrier against the entrance of microorganisms. The basic units comprising the modified tissueculture vessel are a Kimble (60957, no. 2-234) screw-cap vial, a size 24 plastic screw cap, a rubber liner, and a polyethylene disc. Size 24 plastic screw caps with paper liners are soaked in water until the liners can be easily removed from the caps. Any adhesive left on the caps by the liners is removed and a hole, l 6 in. (0.8 cm) in diameter, is made in the center of each cap. A Yl6-in. hole is also made in the center of a rubber liner (Aloe no. 17324, white rubber, or Aloe no. 17322, pink rubber). Discs 13f6 in. (2.06 cm) in diameter are punched out from Du Pont no. 1000 natural polyethylene film, 10 mils (0.254 mm) thick, with an arch punch. The plastic caps and rubber liners with holes in their centers are sterilized by autoclaving in petri dishes and are dried before use. The polyethylene discs are surface-sterilized by immersion in full-strength commercial bleach (4.25% NaClO) for 1 hr with frequent agitation. The sterilized discs are rinsed thoroughly with sterile distilled water, and are dried between layers of sterile filter paper. Rubber-lined solid-top plastic screw caps (Aloe no. 17260 or Arthur H. Thomas G.M.C.I. no. 24) normally employed for making the vials air-tight serve as temporary covers during autoclaving of the vials, after the addition of sterile-filtered nutrient solution, and after inoculation of the tissue onto the nutrient solution. The final step consists of replacing the solid-top plastic screw caps with the open-top polyethylenelined caps as described below.