I've been struggling to grow an anaerobe (P. gingivalis), I take every precaution to keep the culture conditions free of oxygen, I was wondering if the conditions can be so reducing that they inhibit microbial growth of an obligate anaerobe.
To make the liquid media I do the following:
- TSB supplemented with yeast extract
- plus 0.5g/L L-cysteine HCl as a reducing agent
- plus resazurin as a redox indicator
- heat-sparge with nitrogen until the resazurin goes completely colourless
- add hemin (5 mg/L) and vitamin K1 (1 mg/L) from stock solutions (1mL/L hemin in water, 200uL/L vit K1 in ethanol)
- filter-inject this media into glass tubes which have had the gas in them removed (this takes place in an anaerobic hood)
- all materials (syringes, needles, filters, tubes) had been left in the anaerobic hood before use so that no residual oxygen is left in/on them
- swap the headspace for a gas mix of 80:10:10 N2:CO2:H2
I don't get any growth after over 72 hours @ 37oC.
Other protocols I have found are much more vague/lax about removing the oxygen, many just say that the cysteine should be added, and the media should be incubated in an anaerobic hood for 24 hours with the lid loose before being used. This got me thinking that maybe I'm removing too much of the oxygen.
What do you think?