We present a detailed protocol describing: (i) Plating of no more than 200-500 colonies/ 10cm plate (1 hour/ 100 plates) (ii) Incubation of plates until colonies form (at 30°C for 5-7 days); (iii) Imaging of plates (30min/ 100 plates); and (iii) running the automated pipeline described in (Carl et al; bioRxiv, doi: https://doi.org/10.1101/801845) on the obtained images (20min/100 images). Although we established this protocol for Schizosaccharomyces pombe, implementation for other microbes should be straight-forward.
YE plate preparation (1l / 50 plates)
1. Mix 1.0 l ddH2O, 5.0 g Yeast Extract, 30.0 g α-D-Glucose and 20.0 Agar with a magnet until all powder clumps disperse.
2. Autoclave at 121°C for 20 min.
3. Cool down under constant stirring to 50°C.
4. If desired, add antibiotics and stir for 2 min to ensure a homogeneous mixture.
5. Pure 20 ml of autoclaved agar media into each 10 cm plate.
6. After solidification (c.a. 20 min at room temperature), transfer plates upside down into plastic bags and store in a cold room until use or up to two months.
Note: For consistent results keep autoclaving time the same for all plates within an experiment. Reduced autoclaving time can lead to slightly increased accumulation of the red pigment.
Note: When working with PMGc plates, low thiamine concentration reduces epigenetic ade6+ silencing phenotypes in S. pombe.
Plating of yeast cells
From liquid culture:
1. Measure optical density (OD).
2. Dilute samples to 100 cells /ml.
3. Pipette 1 ml onto a cover slip and recount under a microscope.
4. Dilute to 500 cells / 100 ml (per plate).
5. Homogeneously distribute 100 ml / plate with glass beads.
From plate:
1. Transfer cells from a plate with a pipette tip into a 1 ml H2O containing Eppendorf tube. Mix well by vortexing.
2. Dilute 1:100.
3. Pipette 1 ml onto a cover slip and count cells under the microscope.
4. Dilute to 500 cells / 100 ml (per plate).
5. Homogeneously distribute 100 ml / plate with glass beads.
Note: When working with mutants that impact viability or selecting for a fraction of cells that are resistant to antibiotics, estimate how many cells will survive beforehand and plate accordingly.
Incubation of yeast plates
Incubate plates upside down at 30°C for 5-7 days.
Note: If working with growth defective mutants or incubating plates at different temperatures, grow cells until colonies reach a diameter of at least 1 mm.
Note: If colonies are overgrown (e.g. 10 days at 30°C), i.e. the center protrudes and a ring forms around the center of the colony, many white colonies are misclassified as pink.
Note: Storing plates in the fridge enhances the red color. Treat all plates within an experiment the same.
Imaging of plates
1. Place plate on a dark black background.
2. Adjust camera height or zoom that the entire plate fits into one image.
Note: If plates were previously stored in the cold, let them warm up to room temperature before imaging to avoid classification artefacts caused by condensation.
Note: Different colored backgrounds can possibly be used, however a black background is recommended.
Note: Set color balance to manual and not automatic to avoid increased red in images with only white colonies.
Running the automatic classification
Run the classification pipeline as described in (Carl et al; bioRxiv, doi: https://doi.org/10.1101/801845):
1. Download and install the required python packages as detailed on the github page.
2. Download the classification pipeline script.
3. Place all images that you would like to classify into the same folder.
4. Run the pipeline as described on the github page.
github link: https://github.com/fmi-basel/buehler-colonyclassification