Lyophilizing Cells
- Inoculate 200 ml L-broth supplemented with appropriate antibiotics with the bacteria to be lyophilized.
- Incubate the culture at 37°C with vigorous shaking (200-250 rpm) overnight.
- Add 2 ml (1/100 volume) of formaldehyde solution to culture. Incubate at 37°C for one hour on a rotary platform to kill the cells.
- Aliquot 100 ml of the culture to each of two 250-ml centrifuge bottles. Recover the cells by centrifugation at 7,000 rpm, 4°C, for 15 minutes.
- Wash the cells by resuspending each pellet in 100 ml (1 volume) sterile 1x PBS. Pellet the cells by centrifugation as before.
- Resuspend each pellet in 10 ml (1/10 volume) sterile 1x PBS, and recombine into a 50-ml conical bottom, polypropylene centrifuge tube.
- Freeze the contents of the tube at -70°C for approximately one hour, or until frozen solid. Freeze the tube in an inclined position to maximize surface area and facilitate lyophilization.
- Place the frozen tube, with the cap loosened, in the glass lyophilization vessel. Attach the vessel to the lyophilizer, and desiccate under vacuum until dry (may take several days).
- Store the tightly-capped tube at 4°C in a zip-lock plastic bag containing dessicant.
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