Fungal contamination of Arabidopsis seeds

charles logsdon clogsdon at indiana.edu
Mon Jun 18 10:29:04 EST 2001


Here's the fast technique that I use for filter sterilization:

Materials:

sterile ddH2O
Bleach
Detergent (Triton, whatever)
95% EtOH
"Vortex"-type mixer or equivalent
Buchner(sp?) funnel
Vacuum pump
Whatman filter paper

This procedure should be performed in a STERILE work environment - a
laminar flow hood or equivalent.

1.  Place seeds into a 1.5ml eppendorf tube and add 1ml of a 30% bleach,
1% detergent  solution.

2.  Vigorously shake the tube for 20 minutes.  I attach the tubes to the
top of a vortex and run it all nearly full speed.  I don't know if a
regular shaker would be sufficiently fast - it is important that all of
the seeds are fully suspended in solution.

3.  Place a whatman filter paper into a Buchner funnel, place the funnel
into the top of a vacuum flask and attach to a vacuum pump - a standard
filter setup.

4.  Fill the funnel halfway with sterile ddH2O and add the bleach-treated
seeds - you might want to use a squirt-bottle to get all of the seeds out
of the tube.  Make sure the seeds are evenly distributed in the water.

5.  Apply vacuum.  The seeds should be evenly distributed on the filter
paper.  Rinse the seeds 2X with sterile ddH2O.  Do a final rinse with a
few ml of 95% EtOH.

6.  When the filter is dry (1-2 min), turn off the vacuum and transfer the
filter paper to a sterile petri dish.  Place the open dish at the back of
the laminar flow hood (or other suitable sterile location) and allow the
seeds to dry.  In a laminar flow hood, this takes about 30-40 minutes.
The seeds are now sterile and ready for plating - I get excellent
germination rates and rarely have problems with any sort of conatmination.

Hope this helps

Charles Logsdon
Indiana University

vijay wrote:

>  Hi :
>          We are one of the Arabidopsis research  group in Hong Kong.
>
>  We got a very serious fungal contamination of Arabidopsis seedlings
>  grown on the petridishes and paper medium.In addition, on the adult
>  plant itself during the silique maturity stage a lot of fungal colonies
>  formed on the matured siliques.
>
>  We are looking for  some effective seed treatment methods from
>  Arabidopsis researchers to avoid this fungal contamination of
>  Arabidopsis seeds.
>
>  Vijay
>  ----------------------------------------------------
>
>  ---




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