Dictyostelium HELP

micprf at lure.latrobe.edu.au micprf at lure.latrobe.edu.au
Tue Mar 15 03:41:22 EST 1994


In article <199403091732.JAA24694 at net.bio.net>, MUNGO at UVVM.UVIC.CA writes:
> We are using Dictyostelium discoideum in a developmental biology teaching
> lab.  Our problem is that we can't get it to grow predictably.  We are
> using a culture from Carolina and their instructions say that we should
> streak bacteria then sporocarps on a lactose agar plate.  When we do we
> get a hell of  a lot of bacteria, and the mature sporocarps arise straight
> out of the thick bacterial colonies.  We need the migrating slugs and they
> are not apparent on the plates.  Can anyone help us here PLEASE, any method
> that will allow us to get the slugs will help.  We aren't sure if we are
> supposed to be making a lawn of bacteria from liquid culture (we aren't) then
> innoculating with sporocarps.  Any help appreciated, if we are being just
> plain ignorant (as oppossed to stupid...) tell us
> Thanks
> Mungo

Hi. Here is what you need to do:
1.
Medium: a. SM agar
           MgSO4.7H2O 1g
           KH2PO4 2.2g
           K2HPO4 1g
           Agar 10g
           Peptone 10g
           Yeast extract 10g
           dist. water 500ml and autoclave 121 deg/20min

           glucose 10g
           dist. water 500ml and autoclave 121 degC/20min

After autoclaving, before pouring the plates mix the glucose solution
and the other solution together aseptically. Allow the medium to cool
sufficiently that you can hold the flask in your hand without discomfort.
If the plates are poured too hot plasticizers will leach from the plastic
and inhibit growth. Pour thick plates - about 30 ml per plate.

        b: Water agar
           Agar 10g
           dist. water 1 litre
           Autoclave 121 deg C/20 min
           When the agar is cool enough to hold, pour thick plates
           as above.

        c: Bonner's slat solution (SS)
           NaCl 0.59g
           KCl 0.75g
           CaCl2.2H2O 0.4g
           Distilled H2O to 1 litre. Autoclave 121 degC/20 min.

2: The SM agar is used to grow the Dicty on a bacterial host, normally
a lawn of Klebsiella aerogenes or E. coli B/r. It is also used to
maintain a stock culture of the bacterial host. With a sterile loop
scrape several mm of growth from a stock plate of the bacteria
and suspend it in 1 ml of the sterile saline (in a sterile test tube).
Spread 0.1 ml per plate of this suspension onto each SM agar plate
you wish to use, and allow the plate to dry in a laminar flow hood.
Take spores from several sorocarps of Dictyostelium (preferably from
a single colony) or amoebae from the growing edge of a colony and
streak them onto the bacterial lawn exactly as you would a bacterial
culture. This streak dilution should give you single colonies (plaques)
in the lawn after several days. Incubate the plates at 21 degC or up to
24 deg C (Maximum would be 27 deg C for a strain that has no temperature
sensitivity mutations in it).

3. The water agar is used to make slugs. The simplest way is to
use a sterile toothpick and transfer ca. 3 mm growth from the edge of
a Dicty colony on the bacterial lawn. Transfer as a small patch
(2-3 mm diameter) to a water agar plate. Wrap the plate in alfoil
and incubate in the dark, or in a lighted room with a hole scratched
in the side of the alfoil covering (to let light in for phototaxis).
Incubation temperature should be 21-24 deg C and time of incubation
24-48 hours.

4. We always use 21 deg C in our experiments.

5. Dicty can also be grown axenically (no bacteria) in broth ...
but this applies only to certain strains and not to wild strains.
Axenically grown cells can be harvested by centrifugation and also used
to make slugs. You don't say why you want the slugs and on what scale
so I am assuming your needs are simple. Let me know if you need more advice.

                                  Have fun,
                                  Paul Fisher





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