Denitrifier_Lab3_Response.txt Hi there, As I am using a different bacteria I cannot give specific instructions for P. aurofaciens but have used this growing method for P. Chlororaphis, a subspecies of P aurofaciens. 1) I keep all of original which has been rehydrated and centrifuged, in 1 ml eppendorfs in -80C freezer. They are kept in the original supernatant (but glycerine is apparently a good cryogenic agent). I hand thaw and inoculate 100 ul into 8 ml lots of unamended TSB and grow to mid log stage, then centrifuge and resuspend in 1 ml supernatant. I refreeze in a domestic freezer(-4 to -12C) and use these cultures. 2) For plates I thaw by hand, streak, (cool the loop in agar first) incubate 24h or longer if necessary at temps recommended by supplier. It would pay to grow at different temps around recommended ( is it 26C?) to get an idea of how the colonies appear, large mucoid colonies being probably too old. The aim is to get vegetative cells before the stationary phase. 3)For inoculum I lift a small colonies and inoculate into 8 ml lots of TSB, grow to mid log and use one loopful to inoculate into 400 ml broth, (100 ml headspace) with NO3 and antifoam. I then centrifuge and store the remaining inocula in the domestic freezer. I repeat (3)until the cells grow less well, then start at 1) again. 5) I grow the 400 ml culture to mid - late log phase. For all broth cultures I use a reciprocal shaker, it ensures all cells are exposed to all the nutrients. 4) Light/dark does not seem to be important but cultures are kept away from direct sunlight. 5) For a harvest I decant 10 ml lots (one 10 ml lot = one sample) and centrifuge and resuspend in one ml. 6) Autoclaving is 121 C for 15 mins, but the autoclave cycle takes about 1.5h from start to finish. I have focused on the suppliers recommendations for growth as I found the Casciotti/Sigman methods outlined too unspecific regarding temperatures and incubation times. If your survival rate has fallen off, most cells may be too old, try to revive by growing a culture in broth for a longer time to allow the younger cells to regenerate a population. -------- Second email from same individual -------------------- Hi Andrew, I read my last message and decided it may be a bit cryptic. My experience has been that growing bacteria for a certain period of time ? usually 24h at recommended temps gives the best growth ie vegetative cells. If they are grown for longer they can go to stationary death/decline phase, usually seen by sediment at the bottom of the bottle. Growing at room temp (? 18 ? 22C?) for 6 ? 10 days is very unspecific and could cause Slow growth or death and decline, depending on if the room is cold or warm. Usually 24 ? 48h should be fine for 200 ml and 400 ml respectively at the suppliers recommended temperature. It is probably not important if broth gets inoculated with a colony or a quantity of inocula, but making plates, 24h incubation, enables visual examination of colony type, ensures no contamination is present and selection of good colonies (not too large) for use. I am very wary of using antifoam as I have found it can contaminate even after washing . Bottles which have had broth and antifoam have caused problems if subsequently used for agars, by way of producing translucent, uncharacteristic colonies. Bottles need to be rinsed with alcohol after washing to clean properly. Regarding regenerating cells, probably do a few cycles of 24h cultures to enable the younger cells to regenerate the population.