This is a protocol to dilute and spot bacteria for colony forming unit (CFU) quantification. Bacterial samples are passed through a series of 10x and 2x serial dilutions with each dilution spotted onto a rectangular agar plate such as the Nunc OmniTray or something similar. These plates are incubated for some duration of time, which needs to be optimized by species, and then imaged using an instrument like the Evos m7000 to count the colonies at the different dilutions. The protocol can process up to 40 bacterial samples at a time (5 plates x 8 wells (1 column) per plate). This protocol was strongly inspired by the protocol available here.

• Agar media (usually PPY), sterile
• M9, sterile

• Standard lab 1-well reservoir (the kind that can be autoclaved) for M9 dilutant (e.g., axygen_1_reservoir_90ml)
• 96 well plates for the serial dilution
• Rectangular tray (e.g., the Nunc OmniTray or something similar) with 30 ml of the selective agar of choice
• Opentrons P20 8 Channel Electronic Pipette (GEN2)
• Opentrons OT-2 96 Tip Rack - 20 µL

  Optional: if using OT2 to distribute dilutant

• Opentrons P300 8 Channel Electronic Pipette (GEN2)
• Opentrons OT-2 96 Tip Rack - 300 µL

  OR: if manually distributing dilutant to plate

• A multichannel pipette that can pipette 50 to 100 µl.

If you need to change defaults in these scripts you will need to edit the scripts directly before importing them into the Opentrons app. The only things that would possibly need to be changed are:

1. If the p20 or p300 are on the left or right side of the robot. This can be reviewed/changed at line 26-27 of 01_cfu_distribute_dilutant.py and line 68-69 of 02_cfu_plate_spotting.py
2. If using fewer than the default 5 plates/40 samples then you need to change the plate numbers at line 34 of 01_cfu_distribute_dilutant.py and lines 76 and 85 in 02_cfu_plate_spotting.py
3. If some columns of tips have already been used in your tip box then you can specify the tip position you want the robot to start from. The default is 'A1.' This can be changed at lines 45 and 92 in the two scripts. NEVERMIND! This works for a single channel pipette but not the multichannel. Thus, we will always need to start the protocol from column A of the tip box.

1. Gather the required reagents and disposables. Agar plates should be prepared in advance by filling the Rectangular trays with 30 mL of melted agar. These should be dried on as flat a surface as possible to ensure consistent heights across the plate.
2. Distribute dilutant. Importantly, columns 8 and 10 are a 2-fold dilution of the prior column and not a 10-fold dilution. This is done to optimize the dynamic range of CFUs for counting, but it will probably take some trial and error to select which dilutions in the series to use. The dilution series is presented in the table below.
   • If you are using the 01_cfu_distribute_dilutant.py protocol, the only work that needs to be done outside of the OT-2 is to load 100 µL of your samples into columns 1 and 12 of a 96 well plate inside of a biosafety cabinet.
   • If you do no wish to use the robot to add dilutant to the plate, then you can use a multichannel pipette to add 90 µl to columns 2-7, 9, and 11 and 50 µl to columns 8 and 10 in a biosafety cabinet.

3. Next the 96-well plates with bacteria and dilutant should be loaded on the OT-2 deck into positions 4, 5, 6, 10, and 11 as in the figure below. Not all positions need to be used at once (e.g., you could use only positions 4 and 5 or just position 4), but this will need to be changed from the default in the protocol 01_cfu_distribute_dilutant.py. The default is to use all 5 plates/trays and process the maximum of 40 samples at once.

   

4. If using the robot to add dilutant to the plate, import and run the protocol 01_cfu_distribute_dilutant.py. The OT-2 will distribute M9 dilutant to columns 2-11 as described above.

5. If you have already added dilutant to the plate manually, then you can skip running 01_cfu_distribute_dilutant.py and instead continue to loading the rectangular trays onto the OT-2 deck as in the figure below. Use the same number of trays as you have 96-well plates.

   

6. Import and run the protocol 02_cfu_plate_spotting.py. Here it is important to perform the "Labware Position Check" which will allow you to set offsets specific for the rectangular trays with agar. MOST IMPORTANT is to set the "z" offset for the agar plates so that the OT-2 considers the "top of the well" as when the pipette tips are just barely touching the agar.