Date Issued: Jan 15, 2014
SOP: Plaque Assay
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
SOP: Plaque Assay
One Health Center of Excellence
Emerging pathogen Institute
University of Florida
I.
PURPOSE:
This SOP provides detailed information on performing plaque assay which is primarily
used to determine the number of infectious viral particles in a sample as plaque-forming
units (pfu/mL). The procedure would be used to identify viral titer in pfu/ml units for
each of West Nile virus (WNV), dengue virus (DENGV), Chikungunya virus (CHIKV) or
Japanese encephalitis virus (JPV). Please read carefully all the below steps prior to
processing any of your samples.
Consult your supervisor or University of Florida before varying any part of this protocol
II.
PRECAUTION:
This procedure involves working with viral isolates of life threatening pathogens. You
must be familiar with University of Florida biological safety regulations. Use the
precautions for BSL-2/3 agents. Always wear gloves and a lab coat. Do all work in a
biological safety cabinet (BSC). Properly disinfect the work area with 2X CiDecon, 10%
bleach or SaniWipes® followed by 70% ethanol to remove any residual cleaning residue.
Dispose all materials that contact specimens in the biohazard waste containers and
autoclave all infectious materials before final disposal.
III.
PROCEDURE:
The four viruses of interest all grow on the same cell line/s, please refer to the cell
culture SOP for preparing 6 well plate with 90% confluent monolayer of VERO cell line.
A. Plan to infect cells when they are 90% confluent.
B. Remove growth media by gently aspirating from the side of the well with a thintipped pipette using a pipet aid.
C. Wash the cells with 100µl of media in each well, rock the plate gently to allow
maximum coverage of the media to all cells then aspirate one more time.
D. Prepare Virus Stock Dilution:
1. Prepare a 10-fold serial dilution of viral-stock with 10 different dilutions(figure1)
2. Inoculate a 300µl of each dilution into each well.
3. Accurately label the cover of the plate with the corresponding well dilution.
4. Incubate at 36 oC and 5% CO2 for an hour, make sure to rock the plate every 20
minutes to avoid cell drying.
Figure (1) http://www.virology.ws/2009/07/06/detecting-viruses-the-plaque-assay/
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
SOP: Plaque Assay
Date Issued: Jan 15, 2014
5. Prepare overlay by mixing Avicel with 2XMEM + 20% FBS at a 1:1 ratio, then
vortex vigorously until fully dissolved.
6. Add 1ml of the previous overlay mixture to each well.
7. Incubate at 36 oC with 5% CO2 for 4-24 hours depending on the virus type.
(Hint: please mention incubation time per virus)
Working dilutions of the stock virus in MEM medium with 5% FBS will be
prepared according to the below table. Each dilution will be used in two wells
and the average count of the two wells will be used.
(µL)
10-1
Virus
70
Diluent 630
10-2
70
630
10-3
70
630
10-4
70
630
10-5
70
630
10-6
70
630
10-7
70
630
10-8
70
630
10-9
70
630
10-10
70
630
E. Agar Overlay
1. Prepare a sterile solution of 4% agarose in ddH2O by autoclaving at 121 oC for 30
minutes as follows:
a) in a 250 mL bottle combine the following:
i. 1g Agarose agar
ii. 50 mL sterile deionized water (DI water)
iii. Microwave for 30-45 seconds, avoid allowing the flask to boil in the
microwave, open frequently and rock to allow maximum mix and reduce
evaporation time.
iv. The agar is completely melt if no observed bubbles are seen in the bottle.
b) in a separate 250 mL bottle combine the following (in order)
i. 35mL sterile DI water
ii. 10mL Earle’s BSS
iii. 100ul gentamicin
iv. 2mL FBS
v. 3mL sodium bicarbonate
2. Warm MEM in a 37◦C water bath. Give the media enough time to be completely
warmed up into 37◦C before adding it to your cells.
3. Place the mix into a 37◦C water bath all the time.
4. Gently remove media from the infected monolayer cells using fine pipette. Avoid
touching the monolayer with the pipette to prevent cell shearing. (you may use an
aspirating pipette attached to a vacuum)
5. Mix the media with the agar under sterile conditions.
6. Underneath a biosafety cabinet, add 3ml of 1st overlay at the side of each well.
Figure (1) http://www.virology.ws/2009/07/06/detecting-viruses-the-plaque-assay/
-Ctrl
0
630
Date Issued: Jan 15, 2014
SOP: Plaque Assay
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
7. Allow the plate to sit in the hood for 15 minutes at room temperature until the
agar overlay solidify.
8. Move the plate into a humidified incubator at 360C with 5% CO2 upside down.
9. Incubate for 72 hours.
F. Second Overlay:
1. Prepare the 2nd overlay as follow:
a) Preparation of Neutral Red Stock Solution:
i. 151 mg neutral Red Crystal/powder + 100mL DIH2O
ii. Autoclave at 121 0C for 10 min and store at 4 0C
iii. Since neutral red dye is photosensitive, always cover the bottles with aluminum
foil before storing at 40C
2.
3.
4.
5.
6.
b) Prepare neutral red solution for 2nd overlay:
i. Mix 3mL of neutral red stock solution with 100mL PBS
Add 3mL of neutral red solution for 2nd overlay on the side of the wall of each
well. (turn off the hood light in this step to avoid stain degradation)
Incubate the 6-well plates at 370C with 5% CO2 for 6hrs.
Aspirate the neutral red staining solution without disrupting the agar overlay.
Continue to incubate overnight at 370C.
Examine the plates for plaques.
G. Plaque Visualization:
1. Plaque forming unit (PFU/mL): a virus or group of viruses that cause a plaque.
2. Count the number of plaques formed in each well (remember to look on the
plate from the bottom of the wells i.e. inverted) over a light box.
3. Calculate the concentration of the initial viral solution inoculated on the single
monolayer to determine PFU/mL
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑃𝑙𝑎𝑞𝑢𝑒𝑠 (𝑃𝐹𝑈/𝑚𝑙) = 𝑝𝑙𝑎𝑞𝑢𝑒𝑠 ∗ 𝐷 ∗ 𝑉
D = Dilution Factor
V= Volume of diluted virus added to the well.
Example:
If you inoculated 100µL of dilution 10-6 in a well and you counted 44 plaques. The titer
from that well is 106 X 44 = 4.4X107 PFU/100µL (the initial inoculum), you may multiply
the this number by 10 to obtain PFU/mL to be 4.4X10-8PFU/mL
4. If plaques are above 60 consider them too numerous to count (TNC) and avoid
using this concentration to determine your plaque.
5. To perform your own plaque forming dilution use the following equation:
Figure (1) http://www.virology.ws/2009/07/06/detecting-viruses-the-plaque-assay/
SOP: Plaque Assay
Date Issued: Jan 15, 2014
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
(𝑷𝒍𝒂𝒒𝒖𝒆𝒔 𝑪𝒐𝒖𝒏𝒕𝒆𝒅) ∗ (𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝑼𝒔𝒆𝒅) = (𝑷𝒂𝒍𝒒𝒖𝒆𝒔 𝑫𝒆𝒔𝒊𝒓𝒆𝒅) ∗ (𝑫𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝑹𝒆𝒒𝒖𝒊𝒓𝒆𝒅)
Example:
1 : 10,000 virus dilution produced more than 100 plaques, and the 1 : 100,000 virus dilution
produced 30 plaques, the estimated virus dilution needed to produce 50 plaques can be
calculated as follows
(30) ∗ (100,000) = (50) ∗ (𝑋)
X = 3,000,000 divided by 50
X = 60,000
A 1 : 60,000 dilution of virus should yield about 50 plaques. This dilution, when used as the
stock virus to prepare PRNT, is referred to as the “working dilution” (wd).
Appendix I:
Equipment:
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Incubator with CO2 gas chamber 37oC
Biosafety Cabinet
Water Bath
Microwave 37oC/56oC
Refrigerator
100 – 250 mL bottles and flasks
Tissue Culture Plates (Polypropylene)
CO2 cylinder
Pipet aid
Micropipette tips : 0-200uL, 0-1000uL
Sterile dilution tubes
Vortex
Ice Maker machine
Appendix II:
Reagents:
1. Complete Growth medium for VERO cell-line (Minimal Essential Media)
2. Agarose Gel
3. Neutral Red
Figure (1) http://www.virology.ws/2009/07/06/detecting-viruses-the-plaque-assay/
Date Issued: Jan 15, 2014
SOP: Plaque Assay
Figure (1) http://www.virology.ws/2009/07/06/detecting-viruses-the-plaque-assay/
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida