picking of isolated viral plaques or bacterial colonies has traditionally
been one of the most tedious and time consuming tasks associated with shotgun
sequencing. We have developed a picker capable of processing 1 input dish
in ~3 minutes. Furthermore the picker is equipped with plate handling servers
which allow the instrument to process up to 80 input
dishes without any human intervention. Assuming that the input dishes
have an average density of 200 plaques/dish, this should provide adequate
plaques/colonies to innoculate the 168 output microtiter
plates that are contained in 6 cassettes on a server
carousel. Including loading times, this instrument should be capable
of handling 10,000 daily samples in roughly 5 hours.
The picker features a rotating
turret of tungsten needles. These needles are cleaned
in a water jet station and heat sterilized
in a small furnace. High throughput is achieved by performing the various
steps in parallel. These steps are:
The input dishes are imaged
with a CCD camera and the images are
processed using a PC. The instrument operates in two distinct modes: plaque
picking mode and colony picking mode. When picking m13 plaques, the input
dishes are backlit using a collimated light source. Plaques are revealed
as bright points against a dim background of scattered light. A red filter
is placed over the camera so that blue plaques from ß-galactosidase
selection show up as dark objects which are easily rejected. When colonies
are being picked, the dishes are lit from above against a dark background.
Again, a red filter is used to distinguish between blue and white colonies.
needle in high pressure water jet.
Sterilize needle in furnace.
needle into plaque/colony.
needle several times into well containing sterile growth medium.
The optimization of the
dish lighting reduces the challenge of automatically finding the isolated
plaque/colony positions. Images are
background subtracted to remove any lighting or agar non-uniformities and
then features are selected based on brightness, roundness, blueness, and
separation from other features. All of these analysis parameters are under
operator control through a graphical user
interface, allowing efficient recovery of uncontaminated plaques/colonies.
Under typical operating conditions, approximately 90% of good plaques/colonies
are recovered while > 95% of blue plaques/colonies are rejected and <
3% of the selected innoculations may be of mixed population or sterile.
While commercial plaque/colony
pickers have recently become available, the principal advantages of our
system include increased throughput, and, more importantly, greater volume
of plates handled without user intervention. This instrument is currently
in production operation.
This instrument is now available
through Genemachines. For information
call them at 650-508-1634 or e-mail them.
The plaque picker
drawings are now available!
Indented Parts List (RTF) or Indented
Parts List (ASCII text).
Download the Drawings
as a zipped set of .DXF files.
Contact our Office
of Technology Licensing for access to the software.