Tim Burland (Last Update May 4, 1993)
Streak Escherichia coli HB101 over an LB plate and incubate 37o overnight or 26o for 2-4 days.
Flood plate with 10ml distilled water, then scrape off bacteria with a sterile glass spreader. Transfer suspension to test tube, vortex to remove clumps. Use 0.1ml suspension per plate. Use same or next day.
Inoculate 6 litres superbroth (1000ml per 2-litre flask) with 1ml overnight culture of Escherichia coli HB101. Shake overnight at 37o.
Add 15ml 37-40% formaldehyde solution per litre and shake for 60min.
Divide the culture equally among six 1000-ml Sorvall centrifuge tubes, cap firmly and spin at 5000rpm for 15min in the H6000A rotor at room temp.
Pour off supernatants and resuspend each pellet in a small volume of 0.5M glycine. Pool the pellets and rinse out the centrifuge tubes with more 0.5M glycine. Pool washings and make volume to 300ml with 0.5M glycine. Ensure that bacteria are resuspended and not clumped. Shake for 60min at room temp.
Divide bacterial suspension equally among six 50-ml tubes, then spin down at 5,000rpm for 10min in the H6000A rotor at room temp.
Decant supernatant, then wash each pellet once in 50ml 0.5M glycine, once in 50ml BSS, then resuspend each pellet in 20ml BSS. Store in 10-ml aliquots at 4o.
Check sterility of "killed" bacteria. Streak out a loopful of suspension from each aliquot on an LB plate and incubate 3 days at 30o. Nothing should grow.
Rinse out rotors with distilled water.
Inoculate 200ml superbroth with Escherichia coli HB101 and shake overnight 37o.
Divide culture among four 50-ml tubes and spin down in H6000A rotor for 10min at 5,000rpm at 4o.
Decant supernatants, resuspend each pellet in 1ml BSS, then make up to about 45ml each with more BSS and re-centrifuge as above.
Decant supernatants, resuspend each pellet in 1ml BSS, pool resuspended bacteria and make total volume to 10ml (i.e. 1/20th vol) with BSS.
USE SAME DAY.