PhenoBooth+ and Stinger Picking Accuracy Report


MATERIALS AND METHODS
S. cerevisiae culture
S. cerevisiae were cultured in YPD medium with 2% (w/v) glucose at 30°C. S. cerevisiae was spreaded onto YPD agar using sterile glass beads or ROTOR with RePads at 96-array or 384-array. The plates were incubated for 12-60 hours at 30°C. The plates were imaged and analysed with PhenoBooth+. The images and Stinger coordinates were exported. This was performed with an uncalibrated PhenoBooth+, and then the same PhenoBooth+ was calibrated.


TRANSFER EFFICIENCY ASSAYS
To measure the transfer efficiency of S. cerevisiae by Stinger, 1 pick from each colony was made to a fresh YPD agar plate. Following incubation for 24 hours at 30°C, the number of successful transfers were counted. The transfer efficiency was calculated using the formula:


NUMBER OF SUCCESSFUL TRANSFERS AND TOTAL NUMBER OF PICKS
Using Image J, the size or area of each colony was determined and the colony diameters were calculated. Transfer efficiency at a range of known diameters was also determined.


PICKING ACCURACY ASSAYS
Using the same plate that the single pick was made to determine the transfer efficiency, the Stinger was used to make 20-30 more picks to a fresh agar plate.
This will produce hole in the centre of the colonies in the source plate. Phenobooth+ was used to image the source plates following the picks. The images were processed using Image J. The centroids of both the internal pick and the whole colony were measured using image J and Sypder.


STATISTICAL ANALYSES
Unpaired t-tests were used to determine significant differences between uncalibrated and calibrated diameters and errors.


RESULTS
the stinger is able to transfer s. cerevisiae cells with extremely high efficiency. The diameters of the colonies used in the study range between 0.33 and 5.66 mm.
The average colony size is 2.50mm. A total number of 1138 picks were performed by Stinger using coordinates exported from an uncalibrated PhenoBooth+. The 1135 picks were successful. The transfer efficiency across the range of colony diameters tested was 99.7%. The transfer efficiency by Stinger using coordinates exported from a calibrated PhenoBooth+ is 99.9%. . Although calibration did not significantly affect the already excellent transfer efficiency (see Table 1), a correctly calibrated PhenoBooth+ can pick with greater accuracy.


Table 1: Summary of diameter sizes and transfer efficiencies of calibrated and
uncalibrated PhenoBooth+’s.


Figure 1: Histogram representing the range of colony diameters that were transferred for both calibrated and uncalibrated PhenoBooth+s.


 

CALIBRATION INCREASED THE PICKING ACCURACY OF STINGER
To determine the picking accuracy, Stinger was used to make 20-30 more picks to produce holes in colonies. The distance (mm) between the centre of each colony and the centre of the pick, which is the error, is measured. ~140 colonies from the transfer efficiency study were used to investigate the accuracy of Stinger. The diameters of colonies between uncalibrated and calibrated data sets were not found to be significant (p = 0.217), and therefore the picking accuracy of the two groups could be directly compared. As shown in the summary table (Table 2), the uncalibrated error was 0.59±0.3 mm over a range of diameter sizes between 2.94 and 5.49 mm. Following calibration the error was significantly reduced to 0.45±0.25 mm (p = 0.00236) over a range of diameter sizes between 2.77 and 6.12 mm.

 

Figure 2

Solutions