Seed sterilization protocol

caspart at esvax.dnet.dupont.com caspart at esvax.dnet.dupont.com
Fri Jul 17 10:07:54 EST 1992


	Wayne Alphin, Florence Garlick and myself have developed 
a procedure for sterilizing small numbers of seed (up to 
about 200) from large numbers of independent lines.  We have 
used it for setting up plate screens of about 4000 lines from 
the DuPont T-DNA collection.  It may also be useful for 
others who are interested in plating large numbers of lines 
or families.  In brief, seeds are transferred to 96-well 
ELISA plates, sterilized using bleach, rinsed with sterile 
water, and resuspended in 0.15 % agar for plating.  All 
pipetting steps are done with a Beckman Biomek 1000 Automated 
Laboratory Workstation.  (Alternatively, this process should 
be adaptable for use with an ELISA plate washer or a hand-
held octapette and a support to hold the washer or pipette at 
the appropriate height above the plate.)  Using this 
procedure, it is possible to transfer 96 lines of seeds from 
tubes and sterilize them in about 15 minutes of hand work and 
about 25 minutes of robot work.  Seed sterility using this 
procedure is at least as good as when done by hand.  We have 
observed a low level (~1%) of cross-contamination of seeds.  
Minor changes in the protocol could probably reduce this 
cross-contamination, but we have not attempted them since the 
contamination has been so low that it does not affect our 
work.   The procedure is outlined below - if you would like 
more details contact me.  If you would like a copy of the 
Biomek program code, send me a PC-formatted 3.5 inch floppy 
disk.

Tim Caspar
Dupont Central Research and Development
P.O. Box 80402
Wilmington, Delaware  19880-0402


Overview of Protocol:
Seeds are removed from vials using dampened toothpicks and 
are washed into the wells of a 96-well ELISA plate in 50% 
isopropanol.  Water is added manually to dilute the 
isopropanol.  The Biomek robot then draws off the dilute 
isopropanol, adds a bleach/tween solution, then draws this 
off and washes the seeds 4 times in sterile water.  Finally 
the seeds are resuspended in dilute, sterile agar for manual 
plating.   A small amount of cross-contamination of the wells 
should be expected.  Some of the seeds (10-40%) are lost 
during the procedure (the number is roughly proportional to 
the initial number of seeds in each well).

Safety Precaution:
The Biomek robot arm is extremely powerful and has no sensors 
for objects which may block its path.  Never place your hands 
or other objects in the path of the robot arm while the 
indicator light on the top of the robot arm is illuminated.  
For an emergency stop of the robot, press the red emergency 
stop button at the front, middle of the robot unit.  For 
routine stopping, type a "/S" on the computer keyboard then a 
"/A" to abort the run or a "/C" to continue it. 

Seed Transfer to 96 Well Plates:
Place 150 ul of 50% isopropanol (see note 1) into each well 
of a 96-well flat bottom ELISA plate (we use Nunc-Micro Well 
plates, VWR #62409-068).  Wet a toothpick in a dish of 50% 
isopropanol, dip into a seed vial and transfer the seeds 
which stick to the toothpick to a well by dipping into the 
150 ul of isopropanol in the well (notes 2 and 3).  After 
seeds are distributed to all wells and within 20 minutes, add 
100 ul water to each well to dilute the isopropanol.  As soon 
as possible after the seeds are distributed, begin the 
sterilization treatment in order to minimize their exposure 
to the isopropanol.

Biomek Sterilization Protocol:  (note 4)
1.  Remove 190 ul from each well and transfer to a waste 
reservoir (note 5)
2.  Add 200 ul bleach/Tween solution (50% bleach, 0.02% 
Tween)
3.  Add 50 ul bleach/tween solution (note 6)
4.  Remove 200 ul to waste 
5.  Add 150 ul sterile water 
6.  Remove 200 ul to waste
7.  Wash subroutine, repeated 3 times:
	1.  add 200 ul sterile water  
	2.  remove 200 ul to waste 
8.  Rinse pipette tips in water, then agar
9.  Add 75 ul sterile 0.15% agar (note 7)


Notes:
1.  The isopropanol in the 96 well plate is critically 
important.  It wets the seeds so they sink in the sterilizing 
and rinse solutions and also reduces the swelling of the seed 
mucilage so they clump together less.  The seeds are not 
injured by exposure to isopropanol for the indicated times.
2.  During seed transfer to the plates, the seeds will stick 
to the moistened area of the toothpick.   By varying the 
depth the toothpicks are moistened or the type of toothpick 
used, the number of seeds transferred can be adjusted.
3.  To reduce the chances of cross-contamination during 
transfer of seeds to the 96 well plates, a plexiglass mask is 
used which covers the plate and has a single hole in it which 
is lined up over the target well.  In addition, the plate and 
the mask are placed on an anti-static mat (available from 
office supply stores) to reduce the static which causes the 
seeds to fly around.
4.  The entire Biomek unit is placed inside a laminar flow 
hood.  Reservoirs for solutions are sterilized by immersion 
in 70% ethanol.
5.  One set of tips is used for the pipetting series for an 
entire plate.  To minimize cross-contamination between wells 
caused by seeds sticking to the tips, the pipette tips are 
rinsed with 100% ethanol after each removal of solution from 
the plate in steps 1, 4, 6, and 7.2.   For applications which 
require less cross-contamination, the tips can be washed more 
vigorously or changed more frequently.
6.  Pipetting steps are determined by the capacity of the 
octapette (200 ul), the total volume of the wells in the 
plate, the height above the bottom of the well that the 
pipette must maintain to minimize loss of the seeds, and the 
volumes of the reservoirs on the Biomek.
7.  Resuspension of the seeds in 0.15% agar aids in plating 
since they remain suspended by the high viscosity of this 
liquid.  For some applications this is not useful and step 9 
can be omitted.




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