Jul 07, 2022

Public workspaceMS2 Plaque Assay V.1

This protocol is a draft, published without a DOI.
  • 1College of Engineering, Department of Civil, Environmental and Geodetic Engineering, The Ohio State University
  • Daniel Ma: PhD Student
Daniel Ma
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Protocol Citation: Daniel Ma 2022. MS2 Plaque Assay . protocols.io https://protocols.io/view/ms2-plaque-assay-b944r8yw
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: May 27, 2022
Last Modified: July 07, 2022
Protocol Integer ID: 63356
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Abstract
MS2 plaque assay based on EPA Method 1601 and modified as a spot plating assay (Beck et al., 2009).
Materials
  • Sterile Tryptic Soy Broth (3%, 30 g/L)
  • Bacto Agar
  • Tryptic Soy Broth powder
  • P10, P100, P1000 pipets and tips, sterile
  • 125 mL and 250 mL flasks
  • 1L bottles
  • Auto-pipette and serological pipet tips
  • 100 mm Petri dish
  • Water bath
  • Autoclave
MS2 Plaque Assay
MS2 Plaque Assay
1d 7h 15m
1d 7h 15m
Sterilize: Autoclave pipet tips and growth media at least two days before the plaque assay.
E. coli Famp Overnight Culture: Add Amount10 µL E. coli Famp (ATCC 700891) cyrostock to Amount10 mL sterile Tryptic Soy Broth (30 g/L). Incubate Shaker180 rpm, 37°C, 16:00:00. to obtain stationary phase.

E. coli Famp Morning Culture: Add Amount0.75 mL E. coli Overnight Culture to fresh Amount100 mL sterile Tryptic Soy Broth (3%) in a 250 or 500 mL flask. Incubate Duration02:30:00 Shaker180 rpm, 37°C. to exponential phase.
Note
Remove Morning Culture after the incubation period elapses and leave flask at room temperature. Use host cells within 4-6 hours.


2h 30m
Prepare Soft Tryptic Soy Agar: Prepare fresh agar on the morning of the plaque assay. Add 30 g/L Tryptic Soy Broth and 7.5 g/L Bacto Agar to distilled water. Mix vigorously. Sterilize in autoclave at Temperature121 °C Duration00:45:00 . Remove molten agar from autoclave and place in Temperature48.5 °C water bath.
Note
Agar must be cooled before adding host cells to prevent thermal inactivation when adding cells to the molten agar.


45m
Sample Preparation: Prepare MS2 samples by ten-fold serial dilution in micro-centrifuge tubes with Amount900 µL 1X PBS. Vortex samples between dilutions.

Spot Plating Assay: After MS2 samples are diluted, prepare agar plates (10-15 mL) per plate to be used for spot plating.

  1. First, prepare negative control plates containing only agar (no host cells).
  2. Add Amount1 mL of host cells per Amount50 mL agar. Mix gently.
  3. Add 10-15 mL of molten agar to bottom of Petri dish. Swirl to spread agar evenly across the bottom of the dish. Dry for 5-10 minutes or until the agar is solidified.
  4. Label bottom of Petri dishes with sample information and draw grids for applying spots.
  5. Spot MS2 samples onto the surface of the agar without touching the agar with the pipet tip. Apply the spots in labeled areas to keep track of sample information. Spot volumes can be anywhere from Amount1 µL to Amount50 µL . Incubate plates for Duration12:00:00 to Duration16:00:00 at Temperature37 °C .
  6. Remove plates from incubator and count plaque forming units (PFU) per spot for each sample and dilution. Record plaque forming units, dilution, sample identification, spot replicates, and spot volume.
  7. Calculate PFU/mL for each sample by aggregating total PFU across dilutions and accounting for the total undiluted volume:

PFU/mL = (Total PFU across dilutions) / (Total Undiluted Volume of Sample across dilutions).
Note
Important: Provide enough spacing between spots to avoid spots running. Avoid moving plates before spots dry. Smaller volume spots dry faster than larger volume spots.

Note
WaterTEAM: Spot 10 technical replicates of Amount10 µL spots per dilution.


Note
Pour Plating: As an alternative plating method, aliquot agar with host (e.g. in sterile culture tubes or centrifuge tubes) and add MS2 sample (record volume), swirl the tube gently in-between palms, and pour into the bottom of a 100 mm Petri dish. Swirl gently to evenly cover the bottom of the dish and dry completely.



1d 4h
This spreadsheet set up can be used for recording data and calculating concentrations.

Screenshot of Spreadsheet for Plaque Assay Calculation. Yellow = Data Entry, Blue = Calculation or Formula.
Download Plaque Assay Calculation.xlsxPlaque Assay Calculation.xlsx