Specification Sheet
Innovative Bioanalysis LLC. WAC LIGHTING UVF7IN-120V-R1-MOD AEROSOL Page 7 of 11
TCID50 PROCEDURE:
Materials and Equipment:
- Certified Biological Safety Cabinet
- Micropipette and sterile disposable aerosol resistant tips – 20uL, 200uL, 1000uL.
- Inverted Microscope
- Tubes for dilution
- Hemocytometer with cover slip
- Cell Media for infection
- Growth Media appropriate for cell line
- 0.4 % Trypan Blue Solution
- Lint Free Wipes saturated with 70% isopropyl alcohol.
- CO
2
Incubator set at 37°C or 34°C or other temperature indicated.
Procedure:
1. One day prior to infection, prepare 96 well dishes by seeding each well with Vero E6 cells in
DMEM plus 7.5 % fetal bovine serum, 4mM Glutamine, and antibiotics.
2. On the day of infection, make dilutions of virus sample in PBS.
3. Make a series of dilutions at 1:10 of the original virus sample. First tube with 2.0 mL PBS and
subsequent tubes with 1.8mL
4. Vortex Viral samples, transfer 20 uL of virus to first tube, vortex, discard tip.
5. With new tip, serial dilute subsequent tips transferring 200 uL.
Additions of virus dilutions to cells
1. Label lid of 96 well dish by drawing grid lines to delineate quadruplicates and number each grid
to correspond to the virus sample and label the rows of the plate for the dilution which will be
plated.
2. Include 4 Negative wells on each plate which will not be infected.
3. Remove all but 0.1 mL of media from each well by vacuum aspiration.
4. Starting from the most dilute sample, add 0.1 mL of virus dilution to each of the quadruplicate
wells for that dilution.
5. Infect 4 wells per dilution, working backward.
6. Allow the virus to absorb to cells at 37°C for 2 hours.
7. After absorption, remove virus inoculum. Start with the most dilute and work backwards.
8. Add 0.5 mL infection medium to each well being careful to not touch the wells with the pipette.
9. Place plates at 37°C and monitor CPE using the inverted microscope over a period of 1 to 4
weeks.
10. Record the number of positive and negative wells.
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