seeds

STEPHEN A ROLFE S.Rolfe at sheffield.ac.uk
Mon May 23 06:38:35 EST 1994


Dear netters,

Thank you all for you overwhelming reply to my question about spreading Arabidopsis seeds on plates.  Here are the replies.  Thank you once again

Steve Rolfe
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Hi Steve: We resuspend the seeds in water and apply them to the petri dish 
using a P1000 w/ a cutoff tip (or simply using a 5 ml pipette). Inintially the seeds will be "floating" in the excess water; by rotating the dish you can get a
pretty good distribution. Remove the excess water after few minutes when the
seeds have settled. Good luck
Antonio Reinero
----------------------------------------------------
We put a round piece of sterile cheesecloth (2 layers)  on the surface of the 
plate for "traction". Take the sterile seeds up into an 11 cm paster 
pipet, put the plate on a bacteriological turntable and spin the 
plate.  Expel the seeds onto the spinning plate by gently dragging 
the pipet tip in a tight spiral from the center of the plate 
outwards and gentle pressure on the pipet bulbb.  With a
 little practice this gives a very uniform 
distribution and takes only a minute or so per plate.
Chris Somerville
Carnegie Institution
290 Panama Street
Stanford, CA 94305
fax 415-325-6857
----------------------------------------------------
0.1% agar and a transfer pipette is what I use, it works pretty well.

James Keddie
UC Berkeley
---------------------------------------------------------
This method works great for me.

      Place seeds in microfuge tube and add 70% ethanol containing
0.1% triton.  Soak for 4-5 minutes shaking several times.  Remove as
much of the ethanol/triton solution as possible.  Squirt 95% ethanol into
the tube (approx. 400ul), remove seeds with a pipette tip and spread on sterile
filter paper.  Allow the ethanol to evaporate and then you can sprinkle the
seed on the plate.  Good Luck
--------------------------------------------------------
In our lab we use the laborious but effective method of placing the seeds 
individually with forceps, using a grid for even spacing where helpful. 
If you get better suggestions please post them to the network.

Paul Talbert

University of Washington
-------------------------------------------------------
After sterilization and washings, we have plated the seed on the agar 
plates with a forceps. plated 50 seed per plate and it takes @5 min to 
plate 50 seeds. It worked well in our lab. Hope this helps!

Deena Errampalli, Ph.D. (derrampa at credit.erin.utoronto.ca)       
Department of Botany, University of Toronto   
Mississauga, Ontario, L5L 1C6, Canada  
--------------------------------------------------------
Hi, Steve!

        My solution to this oft encountered challenge is to wash bleach-treated
seeds over an 8.5cm Whatman #1 filter (custom cut; cat. # 1001-085) mounted in
an autoclaved Fisher scintered glass filtering unit (9cm dia.).  I pour seeds
onto pre-wetted filter with ~25 ml overlying sterile H2O -- vac off -- then pour
in ~100 ml sterile H2O to stir up seeds and rapidly turn on vacuum. Seeds
distribute pretty evenly across filter.  I subsequently wash with another ~100
ml H2O (taking care to deflect incoming H2O off of side of glass chimney so as
not to dislodge seeds -- though you can stir them up again if you're not
satisfied with initial distribution), then transfer filter w/ seeds to 10cm
petri dish containing desired medium.  I learned some years ago that it is
important to wash filter with sufficient H2O (150ml or so, seems to be adequate)
to minimize residual bleach in filter.  If you don't wash thoroughly enough, you
may find seeds germinate, begin to green, then bleach out and die after about
3-5d post-germination. (Etiolated seeds don't show this same sensitivity --
seems that residual bleach only hits seedlings when they try to proceed in
development beyond what's normally achievable in darkness).

I have used this for sowing anywhere from 100-2500 seeds per dish, either for
physiological expts or for genetic selections.

One other note: seedlings on filters act as though they are "seeing" a somewhat
lower concentration of drug than if directly on agar, so I somwtimes bump up
concentration of, say, hygromycin from 25 to 30 or 35 ug/ml. 

George Karlin-Neumann
--------------------------------------------
You can add the seeds with some water on to a sterile filter paper in a 
petridish.  Let it dry in the LFH with the lid open for a few hours. 
Later on you have to scrape the seeds off with Sterile forceps over the 
media to get an even spread.  It is time consuming but it works.  
Good luck !
hema
------------------------------------------------------
Steve,
0.15% agar in water. No problem. We plate individual seed (500 at
a time) and have yet to find a better solution.
Andrew.
s
--------------------------------------------------------
We make up bottles of 0.7% agar in water, autoclave them, and then microwave
them to melt before use. When the agar is cooled to 50-55 degrees we pipet
about 3 mls/90 mm Petri plate on which sterilized seeds in a small amount
of water have previously been placed. We swirl and tilt the plates rapidly
and manage to disperse the seeds quite effectively in the top agar. The
small amount of agar does not interfere with kan selections. It helps if the
plates are room temperature before the agar is spread on them so it doesn't
solidify too fast. Also, be sure the top agar has solidified before turning
the plates over.
Lawrence Hobbie (Estelle lab)
--------------------------------------------------------
Hi Steve,  We actually do resuspend seeds in 0.5 ml 0.12% agarose, and then
spread them on the plate using a sterile Blue Tip (attatched to pipetperson).
This method is fast, easy, and we rarely get contamination.  Also, you can do
multiple lines/plate by using less 0.12% agarose.  The samples won't spread
into eachother.
-------------------------------------------------------- 
 I've tried several methods of spreading seeds, including using 0.1%
 agarose, pouring them onto the plate in water-based "top agar" &
 spreading them around with a bent glass rod the way we do bacteria. 
 I haven't been satisfied with the evenness of the spread for any of these.
My tried & true method is to take up the seeds in the residual water left
 from the sterilization and, keeping a gentle, steady pressue on the pipetman,
let the seeds out one by one.  It's tedious, but its the only method that's really 
given me a good spread.
Best wishes!
                                Kathy_|_
                                      |
> 

-------------------------------------------------------

Me, too unfortunately:  I spread in tiny drops using the P1000, often getting
only 2-3 seeds per drop.  Then I thin them out using the P20.
		good luck
				--b0b kuhn
> Best wishes!

ditto
				--b0b kuhn
>                                 Kathy_|_
>                                       |
------------------------------------------------------
Robert Kuhn, PhD              "Twenty years of schoolin'
Sinsheimer Laboratories        and they put you on the day shift
University of California    
Santa Cruz, CA 95064  USA      (brown shoes don't make it)"
                                           -F. Zappa (RIP) 
email: rkuhn at orchid.ucsc.edu
Hi,

I routinely put seeds on Kan plates without prior sterilization.  I use MS
salts without sucrose, and by not adjusting the pH of the medium, mold growth
seems to be discouraged.  I put the seeds on the plates using a round pointed
toothpick.  I just touch the plate with the toothpick to moisten the end, and
simply touch a seed, then transfer it to the plate by touching it to the
plate.  I can easily put seeds out in a nice grid, and I would guess that
fewer than 4% of my plates  have any mold contamination.
Good Luck!  Leslie Sieburth
------------------------------------------------------
We routinely surface sterilise as you do, and then rinse
briefly in ethanol before tipping the seeds in ethanol
out onto a filterpaper on top of tissues in a flowhood.
The tissues soak up excess Etoh and the filter can 
then be placed in a sterile petri dish
(lid displaced or off) in the flowhood to evapourate dry.
The seeds can then be sprinkled or transferred aseptically
onto the agar as you wish.

OK..if you don't have a flow hood..or it is heavily
booked this could be impractical...but it works well,
without contamination.
Good luck
Sean.
--------------------------------------------------------
As far as I know, there is no easy answer.  I like Kathy's method, but I 
recently tried a new one that you may like.  Sterilize the seeds in an eppy
tube and pour them onto a sterile scintered glass vacuum filter aperatus
(with or without filter).  Vacuum the bleach soln through, and follow with 
several rinses.  After sucking the last rinse through, use a sterile 
disecting needle to pick the seeds up one by one and touch them to the agar.
Certainly no easier than Kathy's method, but no harder either.  depending 
on your taste, you make like this one better.  Good luck.
...
Lenny Bloksberg
--------------------------------------------------------
hi stephen,
	I have sterilized seeds using uv-c before planting them on agar 
plates.  I do about 10 minutes of light treatment.  good luck...

	stuart Baum
------------------------------------------------------
Dear Steve
I find the easiest way to spread Arabidopsis seeds on an agar plate
is to take them up in about 0.5 ml of sterile water (the same
that I use to wash away the bleach) and squirt them onto the plate.
Then this is runny enough to allow you to spread them evenly with
the Gilson tip, yet not too wet that they are waterlogged. 

However, I suspect that the afficionados will tell you that runny agar
is the most effective, but I agree with you its a bit inconvenient.
Cheers
Alison Smith
Dept of Plant Sciences
University of Cambridge
------------------------------------------------------
Dear Steve,
	In response to your question about seeds on agar plates, I usually
use a sterilised cocktail stick. I made little grids photocopied onto acetate
sheets which I stuck on the bottom surface of the plate, so that I could place seeds
in a 10x10 grid, which made them easy to score. We sterilise seeds in folded
whatman filterpaper sachets dipped in bleach and detergent. like your method.
We then wash them twice in sterilised dd water. When you fold open the filter
paper in a sterile petri dish, the seeds are in a wet clump. Taking a cocktail
stick which has also been bleached, you can scrape up a few seeds and, with a
wee bit of practise, you can set them down approximately one per square in the
grid. It takes a bit longer than smearing them out, but it makes them much
easier to score afterwards. Alternatively, you could use a sterilised spatula
to smear the seeds about on the agar.

Hope this helps

Steve Rutherford
York University
SMR7 at VAXA.YORK.AC.UK
Dear Steve,
------------------------------------------------------
I have found that as you are placing onto Kanamycin it is not
necessary to surface sterilise the seed. To test for Kanamycin
resistance we just gently scatter dry seed. We have not observed
any contamination problems.

Mary Anderson
NASC



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