WEEK 19 - Virology

Hello everyone, for this post I will be sharing with u guys on my major project. My project is to evaluate the effectiveness between the conventional culture tube method and the shell vial method, in culturing of human Metapneumovirus(hMPV).

This study is implemented to compare the two methods. This is because shell vial is considered as a rapid method as results are seen in just a few days unlike tubes, which might take up to weeks. As for conventional tube culture, it is slower as there is no centrifugation. However, its advantage is that it can be maintained up to 28 days by repassaging, and thereby allowing a high level of virus to be harvested. But for shell vial, only a small amount of virus can be quantified. Thus, there is a need to evaluate both methods to determine the best way to isolate and culture HMPV. The evaluation will be based on the earliest date of detection and strength of fluorescence (the strength of the fluorescence will indicate how well the cells is infected by the virus). Therefore, a stronger fluorescence will show that the method is better.

Basically for this project, I will inoculate isolate into both shellvials and tubes. Shellvials will be monitered for cytopathic effect ( refer to my 2nd post) and they will be screened by IF each day, for a period of 7days. Whereas for the tubes, they will also be examined for CPE and screened when necessary, for 21 days. Changing of media will be done as required. Repassaging will be done every 7days, or when degeneration occurs.

Diagram taken from
http://www.sks-science.com/images/358606LRG.jpg
http://www.aname.es/microscopia/ems/preparation/219ems.GIF


Materials
Phosphate buffered saline (PBS)
hMPV isolate
Serum free- minimum essential media( with crystalline trypsin)
Vero cells
LLc cells
Hep cells
RD cells
hMPV monoclonal antibody
Conjugate

Equipments
37 ̊C Water bath
Centrifuge
Incubator
5ml plastic tube
1ml disposable pipette
Forceps
Micropipette
Pipette tip
Wastebin
Moist chamber
Slide warmer
Shaker

Inoculation of isolate into shell vial and tubes

1. Thaw the frozen isolate in water bath for 10 to 15 minutes( ensure that it is fully thawed)
2. Centrifuge the isolate in the 4̊C centrifuge at 2000rpm for 10 minutes.
3. Remove the supernatant from the tube and transfer into a sterile 5ml plastic tube.
4. Discard the media in the shell vials and wash 2 times with PBS.
5. Inoculate 0.2ml of the supernatant into all the shell vials and tubes( except for the control), using a micropipette.
6. Centrifuge the shell vials at 1800rpm for 30minutes.
7. Preadsorb the tubes in rack (stationary state) for 1 hour.
8. Add 1ml of media (containing crystalline trypsin) into the shellvials and tubes
9. Incubate shell vials in 36±0.5 ̊C incubator.
10. Arrange the tubes in roller drums and incubate in 36±0.5 ̊C incubator overnight.

Screening of coverslips by IF method

1. Vortex coverslip to dislodge the cells.
2. Add appropriate amount of PBS to the vial and centrifuge at 2000rpm for 10 minutes.
3. Discard PBS and repeat step 2 again.
4. Discard PBS, leaving a small amount behind.
5. Mix the remaining PBS and the cell pellet with a dropper, to form a cell suspension.
6. Label the slide that is used for spotting.
7. Spot the well in slide adding a appropriate amount of suspension to the well.
8. Dry the slide on a slide warmer.
9. Fix the slide in acetone for 10 minutes.
10. Add monoclonal antibody using a micropipette, ensure that it is well spread (Vortex mab before use).
11. Place the slide into moist chamber and incubate at 37 ̊C incubator for 35 minutes.
12. Immerse the slide in PBS to wash for 10 minutes, before placing it on a shaker.
13. Dry the slide on the slide warmer.
14. Add conjugate to the slide.
15. Repeat step 11-13.
16. Add mounting fluid to the well and mount it.
17. View the slide under fluorescence microscope and record the results.

That’s all for my last post. Happy reading!

Shihui
0607135A