Working Dilution Determination
1.
Make ten fold dilutions (10 -1 through 10 -10 ) of the stock virus in MEM medium with 5% FBS added.
Two wells of 6-well plate are used for each virus dilution.
2.
Serially dilute each virus in 1.5mL tubes according to the tables below. Vortex each dilution and change tips between each tube.
(µL) 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10
Virus 70 70 70 70 70 70 70 70 70 70
Diluent 630 630 630 630 630 630 630 630 630 630
3.
Transfer 0.4mL of each virus dilution to 1.5mL tubes containing 0.4mL of MEM with 5% FBS. For negative control, prepare 0.8 mL of MEM with 5% FBS in a 1.5mL tube.
4.
Incubate the tubes at 37 o C for 1 hour.
5.
Label the Vero cell culture 6-well plates with the virus dilutions 10 -1 through 10 -10 in duplicate.
6.
Remove the cell culture media from the 6well Vero plates by gently aspirating the liquid using a 1mL pipette and a vacuum.
7.
Immediately after removing the cell culture media, inoculate the Vero cell culture plates with
300µL of virus/diluent mixture. Add the virus mixture to the sides of the wells, not directly on top of the cell/layer. Gently rock the plate after inoculating the wells to ensure that the virus mixture fully contacts the cell/layer.
8.
Incubate the inoculated plates at 37 o C with 5% CO
2
for 1 hour . Rock the plates every 15 min to keep the Vero cells from drying out.
9.
During 1 hour incubation make 1st overlay solution (for 4 plates) according to : a.
In one 250mL bottle combine i.
750mg Agarose agar ii.
37.5mL sterile DI water iii.
Microwave for 45 seconds b.
In separate 50mL conical tube combine (in order) i.
26.25mL sterile DI water ii.
7.5mL Earle’s BSS iii.
75µL gentamicin iv.
1.5mL FBS v.
2.25mL sodium bicarbonate
Each plate typically will need 18 mL of overlay solution. You may adjust the ingredients based on how many plates proportionally.
Depending on the type of microwave used for heating the agar, we can say the agar is ready (is cooked) when all of the bubbles (during heating) are gone.

10.
Underneath a Biosafety cabinet, after 1 hour incubation add 1 st overlay to 6-well plates at 3mL per-well and allow to sit on counter for 10 minutes to allow the agar to solidify, then place in incubator upside down. Make sure the agarose in the 250mL bottle is warm (45 o C – 47 o C) before adding the agarose on the wells. Add the overlay to sides of the wells, not directly on top of the cell/layer . Gently swirl the plate after adding the overlay to incorporate the inoculum into the overlay media.
11.
Incubate the 6-well plates at 37 o C with 5% CO
2 for 3 days.
12.
Prepare the 2 nd overlay according to : a.
Make Neutral Red stock solution in a bottle:
151 mg Neutral Red crystal/powder + 100mL
Autoclave at 115 o C for 10 minutes and store at 4 o C. The neutral red is light
sensitive, cover the bottle with aluminum foil or store it in the dark. b.
Prepare the neutral red solution for 2 nd overlay:
Mix 3 mL neutral red stock solution (from step a above) with 100 mL PBS (you can also make the solution in 2 batches : 1.5 mL neutral red stock solution (from step a above) + 50 mL PBS, and put the mix of each batch in 50 mL conical tube)
13.
Add 3 mL the neutral red solution (from step b above) into each well of the 6-well plates. Incubate the 6-well plates at 37 o C with 5% CO
2
for 6 hours. Before adding the neutral red solution into the
wells, turn off the biosafety cabinet light since the neutral red is light sensitive.
14.
After the staining is complete, aspirate the neutral red staining solution, ensuring the agarose plug is not disturbed, and then proceed to incubate the plates at 37 o C with 5% CO
2
for overnight.
15.
The wells are then examined for total number of plaques observed. This number is recorded for each dilution tested. For wells the desired number of plaques is approximately 50. To obtain the dilution required to produce 100 plaques per 0.3 mL of inoculated sample, select the well with the number of plaques closest to 50. Then calculate the dilution of virus that will yield approximately 50 plaques.
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:
(plaques counted) (diluted used) = (plaques desired) (dilution required)
(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).