Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Automated Colony Picking Machine

Martin J. Pollard, Donald C. Uber, Jack S. Zelver
Human Genome Center and Engineering Division, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720

The LBL automated colony picking machine uses a room temperature CCD camera to image 100 x 100 mm colony plates. The Optimas software automatically selects colonies using criteria such as size, circularity, aspect ratio, nearest neighbor distance, etc. The colony image coordinates are then translated into mechanical colony picking coordinates.

The mechanical colony picker positions the source colony plate and the destination microtiter plate under a carousel of needles which pick and place the colonies. Each needle dips into the source plate onto a colony and then rotates until it is over the appropriate well in the 96-well destination microtiter plate. It then dips into the growth medium in that well to transfer picked cells. The needles move on to a semicircular cam that dips them into an ultrasonic cleaning bath. Picking, placing, and cleaning are performed simultaneously.

Usage of the colony picker has shifted from occasional picking of large libraries to more frequent picking of smaller libraries used as part of LBL's directed sequencing strategy. This mode requires picking 2000 colonies, five times every two weeks. To facilitate this, a new Windows-based graphical user interface was developed for the control software. The software now operates in two modes depending on how you start the application. The first picks a continuous stream of colonies into microtiter plates to generate large colony libraries. The second picks colonies into a single microtiter plate. Each microtiter plate constitutes a separate "experiment". Typically colonies are picked into 20 microtiter plates during a session. The new software makes it easier to use and more intuitive even for relatively inexperienced users.

The original programs were written in DOS-based QuickBasic and have been converted to Visual Basic. The code now takes advantage of the services provided by the object-oriented features of Visual Basic. Most modules were re-designed to be event-driven and parallel rather than in-line, and much of the code was replaced by object methods and property manipulations. Ease of maintenance was preserved because the two BASIC languages are mostly either identical or similar. An exception handling scheme was created to allow recovery from a variety of mishaps. This allows the user to restart picking into the middle of a destination plate, and allows a different sequence of input files to be chosen.

We have also upgraded the procedure which calibrates the imager and picker to each other. The picker pierces a sheet of aluminum foil with a predetermined array of holes, using the same needle. This array is imaged, and a nonlinear fit is performed between the locations of the holes in the image and their nominal locations when punched. This computation yields a set of polynomial coefficients that are used to transform colony centroids into picker coordinates. This transformation simultaneously handles the necessary scaling, translation, and rotation between imager and picker coordinates, and corrects for lens distortion and camera positioning errors.

To account for variations in straightness among the needles, the foil is punched with another grid of holes using all the needles. Using the coefficients described above, the hole for each needle is mapped from its image location back into picker coordinates, and compared with its nominal punched location. The difference, or offset, is applied to the needle when it picks a colony. The new calibration procedure has proven to be extremely convenient and accurate.