Plaque Assay Titration of Vesicular Stomatitis Virus on BHK-21 Cells with Avicel Overlay and Crystal Violet Staining

Six serial 10-fold dilutions (1e-4 to 1e-9) of the VSV stock are each plated in duplicate wells of 6-well plates on confluent BHK-21 monolayers. Each assay includes mock (medium-only) wells and a previously titered reference VSV stock as a positive control. Two technical replicate plates per dilution and 3 independent biological replicates (separate cell passages/days) are performed. Only dilutions yielding 20-100 plaques/well are used for titer calculation. Plaque counting is performed by an operator blinded to dilution identity via coded plates.

BSL-2; VSV is a Risk Group 2 zoonotic pathogen that can cause flu-like illness and is hazardous to mucous membranes/eyes - wear eye protection and avoid aerosols/splashes. Work in a Class II biosafety cabinet with lab coat and gloves. Decontaminate with 10% bleach (10 min) or 70% ethanol; bleach all liquid waste to 1% final and discard formalin-overlay into compatible hazardous waste (do not mix formalin with bleach). Autoclave solids. Crystal violet/methanol and formalin are toxic/carcinogenic - handle in the BSC with secondary containment. IBC approval required.

Positive control: reference VSV stock must titer within +/- 0.3 log10 of its historical value, validating cell permissiveness and overlay restriction. Negative/mock control: medium-only wells must show an intact, uniformly stained monolayer with zero plaques (excludes contamination or overlay toxicity). A no-overlay (liquid medium) comparison can confirm that the Avicel overlay properly restricts spread to discrete plaques. Highest-dilution wells anchor the countable range; over-confluent or detaching monolayers invalidate a plate.

Countable wells (20-100 plaques) typically appear at 1e-7 to 1e-9 for high-titer VSV. Plaques are round, clear, and well-separated, 1-3 mm diameter at 24-48 h. Expected titer 1e8-1e10 PFU/mL. Adjacent dilutions should differ ~10-fold in count, confirming linearity. The monolayer outside plaques is uniformly purple; diffuse clearing or background staining indicates overlay or fixation problems.

• BHK-21 cells (ATCC CCL-10), passage 5-25, in DMEM + 10% FBS + 1x pen/strep
• VSV Indiana test stock, single-use aliquots at -80 C
• Reference VSV stock of known titer (positive control)
• Infection medium: DMEM, 2% FBS, 1x pen/strep
• Avicel RC-591 (microcrystalline cellulose), 2.4% (w/v) autoclaved stock in water
• 2x MEM or 2x DMEM for overlay preparation
• DPBS without Ca2+/Mg2+
• Crystal violet 1% in 20% methanol, or 0.1% crystal violet in 10% neutral buffered formalin
• 6-well tissue culture plates
• 10% neutral buffered formalin for fixation

To quantify replication-competent VSV (Indiana serotype) as plaque-forming units per mL (PFU/mL) by infecting confluent BHK-21 cell monolayers with serial dilutions, restricting viral spread with an Avicel (microcrystalline cellulose) semi-solid overlay, and counting plaques after crystal violet staining at 24-48 h. This yields a direct, single-event measure of infectivity for stock characterization and antiviral plaque-reduction assays.

1. Seed BHK-21 at 6e5 cells/well in 2 mL into 6-well plates; incubate ~18-24 h at 37 C, 5% CO2 to a confluent monolayer.
2. Thaw a VSV aliquot on ice; prepare 10-fold serial dilutions (1e-4 to 1e-9) in cold infection medium, changing tips each step.
3. Aspirate medium; wash each well once with 1 mL DPBS.
4. Add 400 uL of each dilution per well (duplicates). Add medium only to mock wells. Incubate 1 h at 37 C, rocking every 10-15 min for even adsorption.
5. Prepare overlay by mixing equal volumes of 2.4% Avicel and 2x medium (final ~1.2% Avicel, 2% FBS) warmed to 37 C.
6. Aspirate inoculum; gently add 2 mL overlay per well down the side to avoid disturbing the monolayer. Incubate 24-48 h at 37 C, 5% CO2.
7. Fix by adding 1 mL 10% formalin directly on top of overlay for 30 min; discard overlay+formalin into bleach waste, rinse wells with water.
8. Stain with 1 mL 1% crystal violet for 10 min, rinse with water, air-dry, and count clear plaques against the stained monolayer.

Independent variable: log10 virus dilution. Dependent variable: plaque count per well and derived PFU/mL. Controlled variables: BHK-21 passage and seeding density (6e5/well), monolayer confluency, inoculum volume (400 uL), adsorption time (1 h) and temperature, Avicel overlay concentration (~1.2%) and volume (2 mL), and incubation duration (24-48 h).

If the stock contains infectious VSV at a quantifiable concentration, then discrete plaques will form in proportion to the inoculum such that counts in the 20-100 plaque range scale linearly across adjacent 10-fold dilutions, yielding a reproducible titer (expected 1e8-1e10 PFU/mL) within +/- 0.3 log10 between replicate plates.

Count plaques only in wells with 20-100 plaques. PFU/mL = (mean plaque count) / (dilution factor x inoculum volume in mL). Average duplicate wells, then average across replicate plates; report mean log10 PFU/mL +/- SD. Use a plaque-counting aid (gridded plate back, or automated counting in ImageJ/CTL ImmunoSpot) and record raw counts and the dilution used. Exclude merged/edge plaques per a predefined rule and note any wells outside the countable range.

1. No plaques even at low dilution: monolayer not permissive, virus inactivated, or overlay too toxic - check BHK-21 health, use a fresh aliquot, reduce overlay incubation. 2. Comet/diffuse plaques (no discrete edges): overlay too thin/liquid - increase Avicel to 1.4%, ensure full coverage. 3. Monolayer detachment during overlay addition: add overlay slowly down the wall, pre-warm to 37 C. 4. Counts not scaling 10-fold across dilutions: pipetting/carryover error - change tips, mix each dilution. 5. High background or faint staining: insufficient fixation or over-rinsing - extend formalin fixation to 30 min, stain 10 min, rinse gently.

Report titer as mean log10 PFU/mL +/- SD across n=3 biological replicates. Compare two stocks or treatments by unpaired t-test on log10 titers (Welch's correction); for plaque-reduction neutralization (PRNT), fit percent reduction vs antibody dilution to a 4-parameter logistic and report PRNT50. Acceptance: inter-replicate SD <= 0.3 log10. Power: detecting a 0.5 log10 difference (SD 0.2 log10) at 80% power, alpha=0.05, needs n=3 independent assays per group.