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.
