Feb 08, 2024
Plaque Assay for Microcystis aeruginosa NIES-298 and phage Ma-LMM01
- 1The University of Tennessee, Knoxville
Protocol Citation: Laura E Smith, Steven W Wilhelm 2024. Plaque Assay for Microcystis aeruginosa NIES-298 and phage Ma-LMM01. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd1j7lmk/v1
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
Created: August 29, 2023
Last Modified: February 08, 2024
Protocol Integer ID: 87114
Keywords: plaque assay, cyanophage, Microcystis aeruginosa, harmful algal blooms
Funders Acknowledgement:
The Simons Foundation
Grant ID: 735077
Abstract
This protocol describes a plaque assay for the enumeration of phage infecting Microcystis aeruginosa NIES-298. It is a single agarose layer assay and reproducibly quantifies infection by the Microcystis-infecting phage Ma-LMM01. This protocol has been adapted from work by Moore et al. (2007) as per Lindell (2014).
Guidelines
The protocol has been designed to work with cultures / lysates in a laboratory setting. If phage that will infect this host are present in natural samples, there is no reason the protocol should not detect them.
Materials
Microcystis aeruginosa NIES 298 is available from the National Institute for Environmental Studies (Japan)
Ma-LMM01 was kindly provided by Professor Professor Takashi Yoshida (Kyoto University).
Prepare CT Agarose
Prepare CT Agarose
Make 0.56% agarose in CT medium base by adding 0.56 g low melting point agarose / 100 mL CT medium prior to autoclaving. You will need 12 mL of CT agarose per plate. Note: the final agarose concentration in plates will be ~ 0.45% once culture and lysate are added (see below). CT medium is modified from Watanabe and Hiroki, 1997 by adding phosphorus of equal concentrations using sodium phosphate in place of Na2*beta-glycerophosphate.
After autoclaving, place in water bath at 34 °C . Add vitamins once agarose has cooled but not solidified. Make 12 mL aliquots and keep each in a water bath at 34 °C until ready to plate. Alternatively, CT agarose (without vitamins) can be prepared in advance and microwaved prior to the water bath step.
Concentrate Microcystis aeruginosa cells
Concentrate Microcystis aeruginosa cells
Concentrate log-phase M. aeruginosa NIES-298 cells (culture should be no more than 3-4 days old and ~ 1-2 * 106 cells/mL) by centrifugation at 8000 xg for 10 min (x2). The first centrifugation step will collapse the gas vacuoles; the second will form the pellet. Remove excess medium.
Resuspend cells in fresh CT medium. Cells should be concentrated to ~2-3 * 107 cells/mL. You will need 2 mL of concentrated cells per plate.
Dilute Viral Lysate
Dilute Viral Lysate
Make a serial dilution of virus containing solution to be tested using CT media. Fresh Ma-LMM01 lysates generally contains ~ 2-6 * 107 pfu/mL, so 10-5 -10-7 dilutions will get plates in the countable range of ~ 20-200 pfu (plaque forming units) per plate. PFUs outside this range are difficult to quantify accurately.
Plate
Plate
Add 1 mL of diluted sample to 2 mL of concentrated cells. For negative controls, add 1 mL of CT medium to 2 mL concentrated cells.
Pour the 12 mL aliquot of CT low melt agarose into the plate. Add the 3 mL combination of lysate and cells next to the agarose. Swirl gently to mix together to form one homogenous layer.
Incubate
Incubate
Plates can be carefully moved to an incubator adjusted for Microcystis growth immediately after pouring, or once they are solidified. Note: plates will take 1-2 hours to solidify.
Important: unlike standard bacterial plaque assays do not flip plates. Plates will not be firm enough for flipping for at least one day.
Incubate at 26 °C and 30-40 µmols photons m-2 s-1 continuous light. Plates can be flipped on day 2. Plaques should start to become visible ~day 3, and can be counted through day 5.
Protocol references
Lindell, D. (2014). The Genus Prochlorococcus, Phylum Cyanobacteria. In: Rosenberg, E., DeLong, E.F., Lory, S., Stackebrandt, E., Thompson, F. (eds) The Prokaryotes. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-38954-2_125
Moore, L.R., Coe, A., Zinser, E.R., Saito, M.A., Sullivan, M.B., Lindell, D., Frois‐Moniz, K., Waterbury, J. and Chisholm, S.W., 2007. Culturing the marine cyanobacterium Prochlorococcus. Limnology and Oceanography: Methods, 5(10), pp.353-362.
Watanabe, M. M. and Hiroki, M. 1997. NIES-Collection List of Strains, Microalgae and Protozoa, 5th ed. Microbial Culture Collection, National Institute for Environmental Studies, Tsukuba, Japan. Available at https://www.nies.go.jp/kanko/gyomu/pdf/f097-1997.pdf (last access Feb 7, 2024)