M9 precipitate
Leon Avery
Does anyone know how to solve this stupid, annoying problem? When we
make up M9 according to the recipe in the 1988 worm book, a white
precipitate forms after autoclaving. I have in past lives seen that
this precipitate didn't always form, or that, when it did, it would
redissolve if the bottles were allowed to sit on the shelf for a
couple of weeks before use. However, for the last year or more, the
precipitate has been 100% reliable, and has shown no sign of
redissolving even after several months. It does cause problems, so I
have taken to sterile-filtering my M9, which is expensive.
What is this precipitate, and how can one get rid of it?
--
Leon Avery (214) 648-2420 (office)
Department of Biochemistry -2768 (lab)
University of Texas Southwestern Medical Center -8856 (fax)
5323 Harry Hines Blvd le...@eatworms.swmed.edu
Dallas, TX 75235-9038 http://eatworms.swmed.edu/~leon/
Norio Suzuki
Do you add MgSO4 before autoclaving?
I think this precipitate is Mg salt. So, adding steriled 1M MgSO4
AFTER autoclaving, like making NGM agar plates, will never make
any precipitate.
In my experience, the precipitate always disappears within a
month. And I wonder why the precipitate doesn't disappear for over
monthes. How about pH?
Does anyone have any problemes by using the M9 that the precipitate
apeared but now it have diappeared?
Norio Suzuki
The Graduate University for Advanced Studies
School of Life Science, Department of Genetics
(National Institute of Genetics)
E-mail: nosu...@wormhole.lab.nig.ac.jp
Home page: http://wormhole.lab.nig.ac.jp/
Carlos Eduardo Winter
Dear Leon,
The precipitate you are seeing is probably calcium (or magnesium)
phosphate. I assume you add MgSO4 after autoclaving, thus this must be a
Ca2+ contaminant from either NaCl or phosphate stock salts. To confirm
that, take a sample of the precipitate and look at the microscope;
calcium phosphate cristals look like micro sea-urchins or short needles.
Sometimes I have seen this kind of thing occuring with concentrated
phosphate buffers that have been autoclaved. To correct that you could add
a little EGTA to the buffer, but take care not to chelate all Mg2+ out of
the M9. I have never tried that. To be sure of you can quantitate Ca in
all salt stocks by flame spectrophotometry or through analytical methods.
Please, tell us what was the final results.
Good luck,
Carlos
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
CARLOS EDUARDO WINTER - Department of Parasitology - ICB2 - USP
Av. Prof. Lineu Prestes, 1374 - 05508-900 Sao Paulo - BRAZIL
FAX: (55)(11)818-7417 - PHONE: (55)(11)818-7269
http://www.icb2.usp.br/~cewinter/
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Patrick Phillips
I had this problem for several years, but was too embarrassed to ask
about it since I figured all the "in" worm people would know what was
wrong (sounds like the makings of a good TV commercial). Anyway, the
culprit seems to be the magnesium sulfate solution. It must react with
another salt when hot (I never figured out which one). This is
presumably why you need to add the salts after autoclaving in NGM too.
We have therefore slightly modified the book recipe by adding the MgSO4
after autoclaving. Works great now. It is a bit of a pain since we
aliquot to 50ml bottles before autoclaving. (Thanks to Phil Anderson's
old recipe cards for helping me figure this one out).
On a related note, what exactly is the history of these solutions? I
believe that M9 is originally from bacterial culture (some recipes I've
seen call for glucose, for instance). Was M9 ever optimized for worms,
or just taken over from the microbiology literature? The reason I ask
is that it seems very salty (judged by taste), and it is not clear that
it is isotonic with the worms (never tasted the worms :-). The worms
don't always seem happy after long periods in M9 (say longer than an
hour). Of course many people just use S-basal instead. Is the choice
between these two all just individual preference and history?
---------------------------------------------------------------------
Patrick C. Phillips pphi...@uta.edu
Biology Department, Box 19498 http://www.uta.edu/biology/phillips
University of Texas at Arlington Tel. (817) 272-2409
Arlington, TX 76019 USA FAX (817) 272-2855
Genes in theory, Worms in practice.
Carlos Eduardo Winter
Dear worm people,
I don't know if this will interest many of you. I have noticed that the
worm I work with (Oscheius n.sp., a Rhabditid related to C. elegans) "does
not like" M9. When you leave the worms in M9 for some time they do not
float anymore in 30% sucrose (density variation = water loss). I have
noticed that same behavior with C. elegans also if they are left for
longer times in M9. In our lab we use only S medium, or MilliQ-water, to wash
worms. I think M9 was originally developed for E. coli, that lives inside
the human intestine. Solutions with much less salt are probably better
suited for C. elegans and other free-living nematodes, taking into
consideration what happens to Oscheius.
I hope to hear more about water balance in nematodes.
Sincerely,
Carlos Winter
I.Johnstone
Check the hydration of the Na2HPO4. The recipe is for anhydrous. If you
are using the dodecahydrate form it should be 15g/L (I think) instead of
6g. ie your pH would be wrong. The recipe I use for M9 is also slightly
different from the worm book one. It contains no Mg, and instead has 1g/L
Ammonium chloride. I have never been sure if this would cause worms
problems, however it seems to be fine for our purposes. We have a media
kitchen that makes the stuff. We get it made as a 10X stock and even at
that concentration there is no problem with ppt formation.
--
*****************************************************************
This message is from:
Iain Johnstone
Wellcome Unit of Molecular Parasitology
University of Glasgow
Anderson College
56 Dumbarton Road
Glasgow G11 6NU
Scotland
Tel: 44 141 339 8855 Ext 2000
Fax: 44 141 330 5422
email: i.joh...@udcf.gla.ac.uk
********************************************************************
I.Johnstone
Check the hydration of the Na2HPO4. The recipe is for anhydrous. If you
your p
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