Hi again fellow wormers --
I posted my 'crusty food' problem yesterday. As always, a whole bunch
of you
wrote back with many suggestions, which I've summarized in case this
info may
help someone else in the future. I've also included some of the longer
replies with more details. I'm sure some of these suggestions will
solve our
problem. Thank you all so much!!
Liz Ryder
Most of the suggestions had to do with saltiness in the plates, rather
than
problems with the bacteria. Here are some common suggestions:
Possible solutions:to crusy food problem:
1) Concentrations, pH of solutions for plates; in particular, too much
calcium was mentioned by several people
2) Try using 'NGM lite' (from Eric Lambie's WBG article. In
particular, no
CaCl2 or MgSO4 is added). This solution worked for Andrew Spencer, who
had
emailed the newsgroup concerning this problem awhile back.
3) Water source could cause problem, especially if too acidic
4) Quality of media components may vary, especially agar (may be too
salty)
5) Plates may be getting too dry (e.g. from wintertime low humidity)
6) Agar being poured at the wrong temp; may get precipitation during
cooling
of media (one person reported less crustiness when precipitation does
occur)
7) Broth in which bacteria are grown may matter (recommendation for TY
medium)
8) Bacterial strain may have accumulated mutations over time; get new
OP50
from CGC
Here are more details:
Erik Jorgensen
..here are also notes from Eric Lambie's recipe for NGM lite:
Calcium: Calcium or magnesium causes the bacterial lawn to be thick
and granular. Addition of 1M CaCl2 to an NGM-lite plate caused
bacteria to become crusty. For this reason calcium was eliminated in
the NGM-Lite recipe. Because autoclaving of CaCl2 and MgSO4 causes the
salts to precipitate these solutions had to be added after autoclaving
in the NGM recipe. Because they have been eliminated from the NGM-lite
recipe, no solutions need to be added after autoclaving. The absence
of calcium and magnesium does not alter the viability of sick strains
(E. Lambie, personal communication).
Creg Darby --
Witchcraft it ain't, I've seen this too, though not recently. Sometimes
OP50 seems to change phenotypes with prolonged passage, and I know from
work with a different bacterium that certain mutants make a crusty
lawn. Since you said you went back to frozen stocks that shouldn't be
the problem, but you might try ordering a different OP50 stock to be
sure. I've seen rather large differences between OP50 from different
labs.
I've found that the texture of the lawn varies with the broth the OP50
is grown in before it's spotted on plates. LB seems to make it
crustier, so my lab uses TY broth, which is 5 g tryptone, 2.5 g yeast
extract per L and nothing else (i.e. no NaCl).
Laura Berkowitz--
..the solution was to switch to
Eric Lambie's NGM Lite (to be found in WBG) or reduce the amount of Ca+2
in NGM to 1/10. The person that had this problem with their OP-50 was
Andrew Spencer at U of Wisconsin
Robert Reis --
I think many of us have had similar problems at one time or another
(and the
rest will, if they keep at it long enough!). I know you can
intentionally
simulate the problem by growing worms on rich medium (high-tryptone)
plates,
so maybe even when there is no cause evident, you could get rid of the
crustiness by making the agar "poorer" with respect to nutrients.
Josh Mcelwee --
.. it usually has something to do with the salt in the plates. I'd
check to
make sure that the calcium and magnesium stocks you're using are the
correct molarity. Also, we used to find that if we didn't find salt
precipitates in the plates, that this problem was more common (dunno
exactly what makes the salt crystals form in the plates, however).
Morris Maduro --
1. Dryish lawns. The lawns chip like you described. Solution seemed
to be
make new seeding stock from frozen stock; cause may be related to
dryness of
plates or age of seeding stock (i.e. multiple causes?)
2. The impenetrable lawn. The lawns don't chip; rather, worms crawl
'around' the bacteria. A worm pick won't even chip the bacteria. We
concluded the E. coli were somehow lysing; the problem could be
resolved by
using water from a different source. Our building distilled water in
Santa
Barbara flowed through 'scrubbers' that seemed to have different
effects on
E. coli growth depending on when they were last changed. Making
enriched
plates, with extra uracil, also seemed to alleviate the problem. (We
never
resolved if it was the media or the uracil that solved the problem here
though.)
Since starting my lab at UC Riverside we have used our own water
filtration
system (Barnstead, but Millipore is probably fine -- the resistance of
the
water is about 18 megohm) with good results.
It also probably wouldn't hurt to get OP50 from the CGC.
Peter Barrett:
1) Overdry plates (this is the major one), especially if the lab
humidity is very low (as is often the case with New England winters)--
if the plates are too dry, the worms will also mostly sit on the edges
of the lawn and will be too thin
2) Plates which have been allowed to sit too long (ditto)
3) Problems with the media; specifically, something wrong with or a
change in supplier of any of the agar, Bacto-tryptone/peptone, etc.
You didn't mention in your post if this was NGM, or MYOB (I switched
our lab over to MYOB when I arrived there-- in general, I like this
media better, it's much easier to make, and grows far fewer
contaminants than NGM) ...
Since you've been doing stuff for years without problems, then in your
position I would begin to suspect things like the media reagents. If I
remember right, Eric Lambie, in his original description of the MYOB
media, suggests switching from peptone to tryptone for making the MYOB,
as this makes the lawns "smoother" and less "crusty" or "bumpy". In my
lab, we've also had the experience of the media reagents being
different from different sources (e.g., agar more or less granular,
peptone and tryptone more or less finely milled and/or of a different
color... suggesting that the middleman has switched suppliers and/or
quality control isn't what it used to be...) At least, that's a good
place of where to start looking for ideas.
Another possibility (that just occurred to me) is your autoclave.
We've had the experience that it is not possible to re-autoclave the
media (if you wait too long before pouring, such that it cools down)--
theoretically there should not be any problem with re-autoclaving, but
we've recently had the experience with our current agar that if you do
that, you can't then use the plates because the worms burrow too much,
implying that the agar is changing chemically as a result of the
autoclaving. (This is a new problem, so I think it's because the agar
source has changed over time.) So the autoclaving can affect the
media... which could in turn affect the bacteria...
Hope this helps!
Ryder, Elizabeth F wrote:
> Hi --
>> We are having trouble with our OP50 lawns being 'crusty' -- that is,
> they
> form a hard surface that the worms have a hard time moving around on.
> The
> worms often get under the crust, which is a pain, and it 'chips' off
> like
> paint chips, which makes worms difficult to manipulate. We have been
> pouring
> and spotting plates for years with no problem, and have gone back to
> frozen
> strains of OP50 with no big improvement seen. We have grown the
> bacteria in
> LB and Hershey broth with similar results. We did move to new lab
> space
> recently, but the problem started a couple months later.
>> Has anyone ever encountered this problem? If you found a solution to
> it, we
> would be eternally grateful if you would share it with us. We are
> starting
> to suspect witchcraft... Thanks!
>> Liz Ryder
> ---
>>>>
---
