Oct 07, 2021
Full plate & spot test plaque assays + PFU/mL calc. - aerobic bacteria
- 1Depart. of Food Science, University of Copenhagen, Denmark
- FOOD Micro UCPH
Protocol Citation: Torben Sølbeck Rasmussen 2021. Full plate & spot test plaque assays + PFU/mL calc. - aerobic bacteria. protocols.io https://dx.doi.org/10.17504/protocols.io.byurpwv6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: October 07, 2021
Last Modified: October 07, 2021
Protocol Integer ID: 53873
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Protocol for plaque assays - either a full plate plaque assay that are more laborious but with high accuracy, or a spot test plaque assay that are for higher throughput but with less accuracy.
Materials
Materials needed: Broth, agar plates, soft agar, bacterial culture, phage culture, 1M CaCl2, 1M MgCl2, 1x SM buffer, centrifuge tubes, 1000 µL and 200 µL filter tips, heating block/bath.
Initial preparation of bacterial culture
Initial preparation of bacterial culture
Spread bacterial culture on an agar plate and incubate at required temperature until clear colonies appear.
Inoculate with a single colony of the bacteria to prepared media, incubate at required temperature, and wait until exponential phase has been reached. Preferably an OD600 between 0.2 – 0.5. Then continue to either Full plate plaque assay or spot test plaque assay - depending on the purpose
Full plate plaque assay
Full plate plaque assay
Prepare a 10-fold dilution series of phages in SM-buffer.
Prepare test tubes with 3-4 mL melted soft agar cooled down to 50°C in a heating block
Add 300 µL of bacterial culture
Add 50 µL of 1M CaCl2 and 50 µL of 1M MgCl2
Add 100µL of the 10-fold dilution series made of the phage lysate.
Do not vortex, but mix by shaking carefully
Immediately after pour on an agar plate. Let it solidify for 15-20 min or until the lid is clear for condensate water
Incubate plates at 37°C or other required temperatures.
Count the plaques and choose the plates with 30-300 plaques to calculate PFU/mL
(PFU / 0.1 mL) * (1/dilution) = PFU/mL.
Spot test plaque assay
Spot test plaque assay
Prepare a 10-fold dilution series of phages in SM-buffer.
Clearly mark on each plate the area for each dilution.
Prepare test tubes with 3-4 mL melted soft agar cooled down to 50°C in a heating block
Add 300 µL of bacterial culture
Add 50 µL of 1M CaCl2 and 50 µL of 1M MgCl2
Immediately after pour on an agar plate. Let it solidify for 15-20 min or until the lid is clear for condensate water
Deposit 10 µL of phage suspension on top of the marked area. The lid can be slightly opened to speed up the drying.
Dry plates until the phage solution have evaporated - may take 30-90 min.
Incubate plates at 37°C or other required temperatures.
Count the plaques lysis and choose the plates with 10-50 plaques to calculate PFU/mL
(PFU / 0.01 mL) * (1/dilution) = PFU/mL