Denitrifier Pseudomonas aureofaciens care

It is constantly a guessing game. Here were my best bets from over 18 attempts. (all conclusions are hunches and cannot act as scientific conclusions without proper testing)

• I followed the Isolab procedure for a couple of runs and was mildly unsuccessful
• Andy made new ‘starters’ which seemed to work for about 4 runs
• Until they didn’t?? They were not metabolizing standard NO3 up to yield.
• Decided to use 3 starters in one media bottle instead of 1
• Concentrating the heck out of it to ‘add more machinery’
• This worked well!!
• I liked this run’s bacteria so well that I kept a ‘pellet’ from it and inoculated my next run’s media bottle with it. This involved what I like to call the “Happy Bacteria” Method:
• Running current run’s media through centrifuge like normal
• Determine ‘most pink’ pellet (or pellet with least amount of white)
• After preparing the ‘next run’ media bottle (i.e. adding the nutrient buffer to it)...
• Dump the current run’s media from the ‘most pink’ pellet’s tube into the old media’s container
• Re-suspend the ‘most pink’ pellet with the freshly prepared ‘next run’ media (use the vortex)
• Dump this into the ‘next run’ media to inoculate it
• Allow ‘next run’ media to grow on the shaker table for at least 24 hours.
• I tried less time than this and it was not very successful
• I tried 36 hours and it seemed successful
• Intuition: ~36 hours optimal
• Place bottle in fridge for at least 5 days
• I have used bottle before 5 days and was not successful
• This worked well for about 8 runs in a row :) until i noted ‘black specks’ on the pink pellet
• It worried me so instead of innoculting another bottle with it I used 3 new starters again
• Ended up that the ‘black specks’ had no harm in the ability of the bacteria to work so could have used it to innoculate :(
• Bacteria did not work the next time I tried the 3x concentration technique.
• Streak plated and noted different morphologies in colonies
• Staph infection?
• Would have been useful to have a contrast microscope
• Inoculated next run with a freeze dried vial Andy had prepared back in 2017.
• This run worked very well but the resources are more expensive and are limited.
• Inoculated a ‘pink pellet’ from this batch into another media bottle back in December so it may be too late to salvage this…
• If I were to move onto Run 17…
• I would try this December media bottle first
• If this bacteria is too old and lazy then I would inoculate with freeze dried bacteria
• Then hopefully continue forth by the “Happy Bacteria” method
Author: Emily Giedraitis - Last updated: 2022-02-01 10:50:25