In article <35C19D5D.6B0DEAD7 at post.rwth-aachen.de>, Boris Braun
<Boris.Braun at post.rwth-aachen.de> writes
>I´m working on PAH degrading bacteria in liquid cultivation, but want to
>bring them on agar-plates. Who knows anything about cultivating bacteria
>on solid PAH-containing media, especially about cultivating on
You can try one of Carl Cerniglia's recipes for minimal salts.
see Cerniglia C and Hietkamp MA Methods in Enzymology 188, p.148 or one
of his many other papers.
Adding BaP can be fun. If youre very brave with lots of PPE you can try
spraying PAH dissolved in a flash solvent like ether onto the plate
surface after inoculation. This prodces a plate covered in crystals of
pure compound. Do not try this at home ! Also probably best not to
tell your safety rep about it. Alternatively you can dissolve the PAH
in solvent and add to the liquid agar just before you pour. Try to get
the agar fairly cool (50 C-ish or less) before you start. The PAH will
crash out of solution when it mixes in the agar but you should be able
to get fairly dispersed, fairly small crystals.
Beware : growth on BaP alone will likely be pretty sloooooooooowwww and
you may find your organisms get more interested in the agar itself. The
spray technique above is nice 'cos you can visualise quite small zones
of clearing to give confidence that you really have BaP degradation.
If you have a PAH degrader which you just want to keep going on PAH but
dont need the selection pressure of using a 3,4, or 5-ring compound then
life is easy. Spread organism on the minimal salts agar. Invert the
plate then put a few crystals of naphthalene in the lid. The vapour
pressure of naphthalene is sufficient to allow good growth this way,
especially if you incubate at say 30C.
best of luck
Dr Alwyn Hart...........................http://www.alwyn-h.demon.co.uk
Loughborough UK.............................Tel/Fax +44 (0)1509 269086
Email ajh at alwyn-h.demon.co.uk