Mar 18, 2025

Public workspaceCalculating Multiplicity of Infection (MOI)

DOI
dx.doi.org/10.17504/protocols.io.rm7vzj1wrlx1/v1
  • 1Icahn School of Medicine at Mount Sinai;
  • 2ASAP Discovery Consortium
Briana L McGovern
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.rm7vzj1wrlx1/v1
Protocol Citation: Briana L McGovern, Kris White 2025. Calculating Multiplicity of Infection (MOI). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj1wrlx1/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
We use this protocol and it's working
Created: August 06, 2024
Last Modified: March 18, 2025
Protocol Integer ID: 104826
Keywords: MOI, infection, antiviral, analysis
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Abstract
Multiplicity of infection (MOI) is the ratio of viral particles to cells. The ideal MOI for antiviral screenings will depend on several things - the cell type, the cell passage, the virus type, the amount of cells, etc.
Before start
The appropriate MOI for antiviral screenings will depend on several factors - the cell type, the cell passage, the amount of cells, the virus type, etc.

We always run an MOI test, which is an MOI dilution series, when we create fresh cell or virus stocks. From this MOI test we select the MOI that achieves approximately 30% infectivity.
Download Virus Dilution Calculator (1).xlsxVirus Dilution Calculator (1).xlsx10KB
Use this excel sheet for quick MOI dilution calculation.
Or if you would prefer to perform by hand:

1. desired MOI x (# cells / well) = Plaque Forming Units (PFU)/well
2. (PFU/well) x (1000/50) = PFU/ml needed
3. (stock titer) / (PFU/ml needed) = Dilution Factor
4. ((5ml x # plates)(1000)) / DF = total volume of virus stock needed
5. (5ml) - (volume of virus stock) = amount of 2% media for the dilution

For example,
our current stock of SARS-CoV-2 WA1 has a titer of 3.5e6 PFU/ml
we want to infect the wells with MOI 0.05
we have 5 plates and we want to infect wells with 50ul volume (~estimate 5ml volume needed per plate)

0.05 (1.6e4 cells/well) = 800 PFU/well
800 (1000/50) = 16,000 PFU/ml
(3.5e6)/16,000= 218.75
((1000(5ml x 5 plates)) / 218.75 = 114.29 ul SARS-CoV-2 WA1 virus stock needed
5ml - 114.29ul = 4,885.7 ul 2% Media