Colony Plating Instrument
An automated bacterial colony plating instrument has been developed for use in the high-throughput DNA sequencing production line at the U.S. Department of Energy Joint Genome Institute (JGI). This instrument processes approximately 150 bioassay trays to be picked on Genetix (Genetix Ltd.) colony pickers. The colony pickers produce 500 384-well microtiter plates per day of bacteria containing sequencing inserts. The use of this instrument reduces ergonomic risk factors associated with manual processing and standardizes the bacterial colony plating process.
Agar-filled Corning low-profile bioassay plates are purchased from Teknova Inc. (Hollister, CA). A transformation stock of bacterial colonies is supplied by the JGI Libraries Group with instructions prescribing the volume of stock to be added to each bioassay to achieve a colony count of 1000 - 1200 colonies per plate. The bioassay trays are dried in an incubator prior to plating. Two milliliters of transformation stock and SOC broth (Growcells Inc.) are added to the bioassay agar surface. A total of 15 to 20 5-mm-diameter sterile glass beads are added to the agar surface. Operators tilt and rock the bioassay plate to roll the beads through the liquid transformation stock to achieve a uniform spread of stock solution over the entire agar surface. The bioassay plate is incubated at 37 degrees C for 18 hours. The individual bacteria will grow into visible colonies. A successful plating event will result in approximately 1000-1200 well separated, uniformly spread visible bacteria colonies. These bioassay plates will be put into Genetix QPix II and QPix XT automated colony-picking machines. The quality of the bacterial spread on the bioassay trays is essential for efficient digital imaging and automated picking of the colonies into the destination 384-well microtiter plates.
The plating task, in a production-line environment, was resulting in fatigue in the operators. The fatigue was due to the repetitive nature of the task, the weight of plating up to five bioassays at a time, the grip strength required, awkward hand positions, and the total number of bioassays plated per person per day. An ergonomic assessment of the manual task of plating resulted in a “Hazardous” rating using the Moore-Garg Strain Index. In addition, the plating process resulted in variable quality between operators and a significant training time to train new operators.
The Joint Genome Institute has a very active ergonomics program directed primarily at the high-throughput DNA sequencing production line. In addition, the Lawrence Berkeley National Laboratory practices Integrated Safety Management in all of its facilities. Operator feedback and the ergonomic assessment identified the bacterial colony plating task as a priority area for engineering controls. An additional benefit to engineering controls was the potential to standardize the plating process and improve the operator-to-operator variability and training requirements.
The initial administrative control was to limit the number of bioassays plated at one time and to limit the total number of bioassays per person per day. This resulted in lower throughput and a need to train more operators. The initial engineering control was to build a manual fixture that would greatly reduce the weight component of the task. The initial design was contributed by the operators, and the collaboration between the production staff and the Instrumentation Group resulted in the fixture in Figure 1. The collaboration and device won the “Ergo Cup Award” at the 10th Applied Ergonomics Conference, Dallas, TX, March 2007. A fully automated version of the device standardized the plating process.
Figure 1
Automated Plating Instrument
The automated Plating Instrument is designed to produce bacterial colony spreads in bioassay plates with quality and efficiency comparable to manual plating. The challenge to the design is that the tilt and rocking action cannot be a regular repeated pattern. Rolling glass beads through 2 ml of transformation stock fluid on a dried agar surface is complicated by the surface tension effects of the liquid coating the beads and the absorption of the liquid into the agar during the one-to-two-minute process. The beads tend to clump and roll irregularly on the surface. It is also the case that the beads are not uniformly round nor is the agar surface uniformly flat. The agar surface tackiness varies as the liquid is absorbed. Previous experience with a commercial device that executed a regular repeated pattern resulted in inferior and unacceptable colony spreads.
Figure 2
The range of motion and accelerations were measured by using a goniometer attached to the top of a stack of bioassay trays that were plated manually. Data was collected from several operators. This data was used to define the mechanical design and select the motors.
Figure 3
The device is a custom-designed gimbal mechanism with the capacity of holding one to five bioassay trays. (See Figures 2 and 3.) The device is driven by two pairs of servo motors (Hitec HS-805BB), which tilt the trays around the two horizontal axes simultaneously. The pattern of motion is controlled by the operator using a joystick control attached to the computer. With practice, the operators can duplicate, using the joystick control, the sequence of motions that they know will yield an acceptable colony spread. While the operators control the plating motions, the joystick commands are recorded for later playback. The complete system is shown in Figure 4.
Figure 4
Video: automated plating instrument in action
Plating Software Description
The software for the Plating Instrument is written in VB.NET 2005 and does not rely on other commercial software packages or DLLs. The operator interface allows the operators to start and stop the instrument. There is a record feature which receives input from a Logitech USB joystick and then writes the joystick input to a file that can be replayed later. Files can be named and saved. Operators select a file and run the instrument.
The Configuration tab allows for selection of the COM port. The COM port value is stored in a configuration (.config) file that is generated by the VB.NET 2005 IDE. It can be created by creating settings for the project in the properties tab.
Input from the joystick is received when each timer tick event is triggered. The data is received via a declaration of the getJoyPos function in the winmm.dll file. The position is retrieved and adjusted if necessary, and then the information is sent to the motor controller via the instantiated COM port. The x and y positions are then written to a comma-separated file.
When a file is replayed, the program will read each individual line when the timer tick event is triggered. Data is sent to each servo motor via the instantiated COM port. Joystick control is locked out during replay.
Programming and Calibration
The device is programmed via the record mode while operators manually plate real samples using the manual joystick control. Once operators become used to the input and response of the instrument, using the joystick, they can then execute the plating process on the instrument and achieve results equivalent to their manual process. The instrument is calibrated by having several operators plate half of their bioassay plates manually and half using their previously recorded program using the automated instrument. The plating quality is assessed for each operator’s program. Reproducibility of results is also assessed. One program is then selected for production use by the operators.
There are several tasks associated with the plating process, including adding the transformation stock and the glass beads. The use of the plating instrument eliminates the most hazardous task. Use of the instrument doubles the number of bioassay plates that each operator is permitted to process each day from 40 to 80.
