Colony Picking Assistant (CFA)
The CFA is a tool for trackable colony picking into a 96-well flat bottom plate. This wiki page details the recommended steps for utilizing it.
Just venting, please feel free to skip the entire section.
Colony picking to screen candidate clones is an indispensable part throughout my PhD. Colony picking robots are not affordable for us though, and DIY automated solutions require heavy investment into image processing + developing customized hardwares for the OT-2. These are not justifiable when a PhD student is just picking around 2 to 6 96-well plates (~100 to 500 something colonies). The repetitiveness of a boring task was without question excruciating, yet it was nothing compared to the nightmare of making mistakes when traversing the rows and columns of a plate. Like most other microbiologists, I used to pick E. coli colonies using autoclaved wooden toothpicks, disposable loops or tips. Pick a colony, dip it into fresh medium, pull it out, and then discard the consumable. This works well for a small amount of test tubes, but when applying to plates, two mistakes frequently happen:
- Skipping a well without knowing it
- Picking two colonies into the same well without knowing it
To counter that one has to slow down considerably so as to mentally track the process. I later cut up toothpicks into halves so they could be dropped into 96-well deep well plates. That improved the experience, but the toothpicks strongly absorb liquid and caused a small amount of wells to rapidly dry up after overnight growth in a shaking incubator. And then sometimes, I thought I had autoclaved enough of those only to find a shortage for the last few wells. On the n-th occasion of these issues, I figured that I need a better solution, and it must:
- Free the user from the mental torture, whether by counting silently or by looking at the partially used tip box, of keeping track of which well already received a colony.
- Inherently avoid the disaster of picking two colonies into the same well.
- Require no preparation of customized consumable, like toothpicks halved in lengths.
- Work with 96-well flat bottom plates
- Allow mistakes, if any, to be rectified before the colony goes into the plate
- Minimize the effort when moving on from one plate to another
The CFA do all of the above.
The CFA requires autoclaved TipOne 200 µL tips and the accompanying iconic yellow inserts to operate.
Prepare 96-well flat bottom plates filled with an appropriate amount fresh media. 200 µL per well is strongly recommended and 150 µL should be the minimum. Sterilize a clear bench by wiping with 75% ethanol. Autoclave an empty, yellow TipOne tip rack insert.
- Put the CFA on top and insert an empty, sterile yellow TipOne tip rack insert into the slot.
- Get a tip from an autoclaved tip box, pick a colony, and then drop the tip into an empty slot on the yellow tip rack insert. Repeat the process until the rack is full.
- Take the lid off from a 96-well flat bottom plate. Align the CFA with tips over the 96-well plate, and slowly lower the CFA such that the bottom of the tips are submerged into the media.
- Orbitally shake the entire assembly to facilitate the inoculation. Theoretically the step can be skipped but my OCD always force me to do so.
- Slowly, gently and carefully lift the CFA + tips complex from the plate. Take note that no liquid is dripping from the tips as you move the CFA away. So long as the medium is not too vicious, e.g. LB, liquid spills or drips should not happen at all.
- Take the CFA complex with tips to the nearest biological waste bin, and rotate it so as to empty the tips. Remove the used yellow tip rack insert.
- Return to Step 1. For environmental-friendly labs, used yellow insert can be sterilized by leaving them overnight in Virkon, followed by rinsing with water and then autoclaving. For my project though cross-contamination cannot be tolerated and so I dispose the yellow insert together with the tips in Step 6.
I thank Dr. Logan Mackay (Mass Spectrometry Facility, University of Edinburgh) for providing access to a 3D printer, and Yang Liu (Wang lab, University of Edinburgh) for demonstrating the procedures while I film the actions.