Evaluation of a Liquid Overlay System for Viral Plaque Assays

Abstract

Source: Baer, A. and Kehn-Hall, K. Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems. J. Vis. Exp. (2014).

This video demonstrates the use of a liquid microcrystalline cellulose-based overlay in a plaque-based assay by comparing its effectiveness in viral plaque formation with that of a traditional semi-solid agarose overlay.

Protocol

1. Preparation of cells and reagents

1. The day prior to the assay, plate appropriate host cells for the virus in question (Table 1 and Table 2) at 90 - 100% confluency.
2. Prepare a fixing solution of 10% formaldehyde in H₂O. In example, mix 5.56 ml of 36% stock formaldehyde with 14.44 ml distilled H₂O (dH₂O).
   NOTE: Use appropriate safe handling practices and ventilation when using formaldehyde.
3. Prepare crystal violet stain: 1% crystal violet (CV) in 20% ethanol and dH₂O.
4. Prepare 2x plaque media (cell type dependent at 2x concentration; see Table 2). Filter using a 0.2 µm filter if any reagents are not sterile.
5. Preparation of immobilizing overlays (Table 3)
   1. For liquid overlays, make a sterile 2.4% solution of Avicel in dH₂O. To prevent clumping, add the powder to a flask containing water that is rapidly mixing with a stir bar, pour Avicel in slowly and stir rapidly at room temperature (RT). When the solution proves too viscous for a stir bar to homogenize, switch to a flask shaker for >30 min with heavy agitation to ensure even homogenization.
      NOTE: Stock solutions can be made at lower concentrations, but the solutions may separate and will need to be remixed to ensure homogenization. Working solutions of Avicel can be used, ranging from 0.6-3%.
   2. After homogenization, autoclave the solution and store it sealed at RT.
   3. For agarose and carboxymethyl cellulose (CMC) overlays, prepare a stock solution in dH₂O of 2% CMC or 0.6% agarose. Mix with a stir bar and autoclave or microwave to bring into solution.

2. Dilutions and infections

1. The day after plating, visually check the confluency and viability of the cells prior to starting the assay. Ensure standard cellular morphology and that a ~90% confluent monolayer is present.
2. Perform a tenfold serial dilution of the infectious samples. Use the cellular growth media for viral propagation as the diluent (Table 2). Vary the number of dilutions required based on the expected titer of the virus in question, and always use an uninfected control sample to independently ensure cellular viability and aid in plaque identification.
3. From the serial dilutions, infect the cells for 45 min to 1 hr (Table 1). Use a sufficient volume of inoculum to cover the cells, while keeping the volume as low as possible to maximize viral contact with the monolayer (Table 4). Gently rock plates every 20 min to ensure even coverage and prevent the cellular monolayer from drying.
4. After the infection, overlay an appropriate volume of immobilizing medium directly to the inoculums in the well (Table 2) using a 1:1 mix of 2x plaque media and the immobilizing overlay of choice (CMC, agarose or Avicel). Gently rock to mix.
   1. For liquid overlays, mix 1:1 with warmed 2x plaque media and 1.2 - 2.4% RT stock Avicel to obtain a working solution of 0.6-1.2% Avicel overlay medium.
   2. For an agarose overlay, use a 1:1 mixture of warmed 2x plaque media and a stock solution of heated 0.6% agarose, place in a 56 °C water bath for 30 min to equilibrate the temperature obtaining a final agarose/overlay concentration of 0.3%.
   3. For CMC, prepare a 2% stock solution and treat as described for the agarose overlay.
   4. When applying the overlay to the monolayer, always equilibrate hot agarose or CMC in 56 °C water to prevent damage to the monolayer. Ensure the solution is warm, but not hot to the touch to prevent cell death and reduced viral titers. Most agarose overlays will begin to solidify below 42 °C, work quickly and/or prepare small batches to prevent solidification while handling.
5. After addition of the overlay, incubate plates to produce distinct plaques that are clearly countable. Plaque formation can take 2–14 days depending on the virus being analyzed (Table 1).
   NOTE: Once liquid overlaid plates are placed in the incubator, do not move them. Movement of the liquid overlays during the incubation period will result in smeared plaques. Agarose and CMC overlays are semisolid and can be moved or checked periodically under a light microscope to monitor plaque development.

3. Fixing and staining cells

1. To fix the cells, pour off or aspirate the Avicel overlay, and fix cells using the 10% formaldehyde solution for 30 min to overnight (<1 ml per well for a 6 well plate).
   1. For agarose or CMC, directly add the formaldehyde solution to the overlay for 1 hr overnight.
      NOTE: Samples can be kept for extended periods of time in the fixative provided it does not evaporate and dry out as this can distort the monolayer.
2. Prior to staining and after fixation, discard the formaldehyde and remove the semisolid plugs for the agarose and CMC with either running water or manually with a spatula. Rinse Avicel plaques with water to remove residual overlay/fixative prior to staining.
3. For staining, cover the cells with a minimal amount of crystal violet solution for ~15 min. Rock plates if necessary to ensure even coverage.
4. Gently wash off the crystal violet stain with water. Once fixed, stained, and dried, store plaques indefinitely for future analysis.

Table 1. Plaque Assay Inoculation Conditions and Cell Types

	RVFV	VEEV	Influenza B
Cell type	Vero	Vero	MDCK
Infection period	1 hr	1 hr	45 min
Incubation time	3 days	2 days	3 days

 

Table 2. Plaque and Viral/Cellular Growth Medias

	RVFV	VEEV	Influenza B
Cell Type	Vero	Vero	MDCK
Growth Type	DMEM1	DMEM1	DMEM2
Plaque Media	2xEMEMA	2xEMEMA	2xEMEMB

1. Dulbecco's Modified Eagle Medium supplemented with 10% Fetal Bovine Serum, 1% L-Glutamine, 1% Penicillin/Streptomycin.

2. Dulbecco's Modified Eagle Medium supplemented with 1% L-Glutamine, 1% Penicillin/Streptomycin, 0.2% Bovine Serum Albumin, 0.025% HEPES, DEAE-Dextran 50ug/ml.

A. 2x Minimal Essential Media (500 ml) supplemented with 5% FBS (25 ml), 1% Minimum Essential Amino Acids (5 ml), 1% Sodium Pyruvate (5 ml), 1% L-Glutamine (5 ml), 2% Pen/Strep (10 ml).

B. 2x Minimal Essential Media (500 ml) supplemented with 0.2% Bovine Serum Albumin, 1% Minimum Essential Amino Acids (5 ml), 50ug/ml, 0.025% HEPES, DEAE-Dextran, Trypsin-TPCK*

*Just prior to preparation, add 1 µl per 25 ml of 2 µg/ml stock of TPCK-Trypsin to the aliquot you will be using to mix with agarose for plaques.

Table 3. Overlays Stock

	RVFV	VEEV	Influenza B
Cell type	Vero	Vero	MDCK
Infection period	1 hr	1 hr	45 min
Incubation time	3 days	2 days	3 days

 

Table 4. Plate Formats

	6 well	12 well	96 well
# of cells/well	5 x 105	2.5 x 105	3 x 104
Volume of inoculum (μl)	400	200	50
volume of overlay (ml)	3	1.5	0.100

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Crystal violet	Sigma Aldritch	C3886-25G
Formaldehyde solution	Sigma Aldritch	F8775-500ml
Avicel RC-591 NF	FMC BioPolymer USA	RC-591 NF	Shake vigorously when reconsitiuting for >30 min to homogenize
UltraPure agarose	Invitrogen	16500-100
Carboxymethyl cellulose, sodium salt medium viscosity	Sigma Aldrich	C4888