TITLE: Plaque Assay for Microcystis aeruginosa NIES-298 and phage Ma-LMM01
AUTHORS: Laura E Smith, Steven W Wilhelm

[DESCRIPTION]
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.

[STEPS]
SECTION: Prepare CT Agarose
1. 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.  
SECTION: Prepare CT Agarose
2. 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. 
SECTION: Concentrate Microcystis aeruginosa cells
3. 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.
SECTION: Concentrate Microcystis aeruginosa cells
4. 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.
SECTION: Dilute Viral Lysate
5. 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. 
SECTION: Plate
6. 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. 
SECTION: Plate
7. 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.
9. 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. 

SECTION: Incubate
8. 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.