( anaerobic) and B. subtilis 168 ...)A membrane-lid tube for bacteria cultivation and freeze-drying

anaerobic) and B. subtilis 168 (strictly aerobic) were inoculated at a concentration of 1% into the LB medium. 1 ml of this medium was then transferred into each 1.5 ml Lid_Bac tube and 5 ml of the medium was placed into each 50 ml Erlenmeyer flask. The vessels were then sealed (Lid_Bac with its membrane-lid, the Erlenmeyer flasks with cellulose stoppers) and incubated under the same conditions as for bacteria cultivation. After 3, 6,16, and 24 hours, samples were removed from both vessel types and examined under a microscope. To prevent any effect on the morphology of the bacteria caused by the increase in the oxygen supply as a result of the tubes being opened, a new tube was used for each measurement. Freeze-drying In five parallel preparations, a comparison was made between freeze-drying (Beta 2-16, Christ, Osterode, Germany) E. coli HB101 in membrane-lid tubes and in standard tubes.

The medium used for lyophilization was 5 % lactose (in distilled water).

1 ml of each bacteria culture, grown in both Lid_Bac and standard tubes, was pelleted in the early stationary phase (in the Eppendorf Centrifuge 5402 at 4 C and 14,000 x g), resuspended in 1 ml of 5 % lactose, centrifuged, and then diluted in 500 l /200 l medium.The Lid_Bac tubes were then sealed with their membrane-lid and placed into a custom-made aluminum block with the corresponding bores. The standard tubes were transferred, unsealed, to the same block. For a eutectic point of the cultures in 5 % lactose of approx. 13 C, a vacuum of 1.03 mbar was set. To dry the samples, the heating temperature of the sample area was raised gradually from 5C to +5
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