Week 9 - Virology

Hello people! For the 4th to 8th week, I was at the virus isolation section of virology.
For this area, it is mainly responsible for specimen processing, virus inoculation, maintenance of culture tubes and repassaging (Repassaging is to subculture or split cells into new culture tubes to prevent overgrowth and degeneration.)

Basically, after the specimen is processed, it is inoculated into respective culture tubes and incubated. The tubes are examined for cytopathic effect (changes in appearance of caused by the virus that is growing) everyday and results are recorded. If any cytopathic effect is observed, then the specimen will be sent for confirmatory tests – immunofluorescence, acid test and neutralization test [This will be elaborated in the next post :)]

The culture tubes are maintained by changing their media on every alternate day. This is done to maintain the viability of the cells - as nutrients will deplete and waste products produced by the cells are detrimental to growth. However, this will still depend on the condition of the cells. If the cells are going to be repassage the next day or they are still healthy, then there is no need to change media on that day itself. As for repassaging, it is usually done on the 7th and 14th day after inoculation when degeneration of cells is observed.

Changing media for culture tubes

1. Remove media in culture tubes by using a 1ml disposable plastic pipette.
2. Discard media in disinfectant, sodium hypochlorite.
3. Add in 1ml of new media using a new pipette.
4. Cap tightly and arrange it back into roller drum.
5. Incubate in respective incubator, depending on the type of virus.

ROLLER DRUM (Picture taken from http://www.nbsc.com/rollerdrums.aspx )

Repassaging for culture tubes

1. Select the culture tubes according to the original cell type (Eg, Hela tube needs to be repassage, the new tube chosen must also be hela.)
2. Check the selected tubes under microscope to ensure cells are present and confluent.
3. Label the newly selected tubes with its original lab number and the date of repassage.
4. Loosen the caps of the tubes [Done in BSC-II].
5. Scrape the cells in the original tube by using a bent pipette.
6. Flush the cells up and down to ensure that it well mixed.
7. Inoculate 0.2ml of the cell suspension into the newly selected tubes.
8. Cap tightly and arrange it back into roller drums.
9. Incubate in respective incubator, depending on the type of virus.

In this section, we will receive many different specimens. Each of them will come with a request form, indicating what virus is suspected and needs to be isolated. Hence, we will only perform that particular test.

Here are some of the commonly received specimens and the virus that is suspected of…

ETDA Blood -- Enterovirus/ Neurotropic/ Cytomegalovirus

Lung tissue -- Respiratory virus

Heart/intestine/brain tissue -- Enterovirus/ Neurotropic virus

Urine -- Cytomegalovirus /Mumps /Adenovirus

Stool -- Enterovirus/ Neurotropic virus

Vesicles/ ulcer / genital swab -- Herpes virus



Now, let me share with you guys on specimen processing.

Urine Processing

1. Centrifuge the urine sample for 10minutes at 200rpm.
2. Add 1.2ml of media to a clean sterile container.
3. Pipette 1.2ml of supernatant and mix it with the cell deposit, thereby forming a cell suspension.
4. Transfer the cell suspension to the container with media.
5. Inoculate 0.3ml of cell mixture into respective shell vials and tubes.
6. Preadsorb for 1 hour before incubation (preadsorb will help in virus penetration)

Stool Processing

1. Add 10 glass beads into a sterile plastic tube.
2. Add 10ml of Dulbecco PBS, 1ml of antibiotic and 1ml of chloroform to the tube above.
3. Take a pea-sized amount of stool and add it in.
4. Seal the tube with parafilm to prevent leakage.
5. Vortex the tube and place it on the horizontal shaker at 200rpm for 20 minutes.
6. Centrifuge at 2600rpm for 20 minutes.
7. Transfer 5ml of the supernatant into a clean sterile 5ml tube.
8. Select the tubes and label them with date of inoculation and lab number.
9. Inoculate 0.2ml of the supernatant into each tube.
10.Preadsorb tubes before incubation (preadsorb will help in virus penetration)

Glass beads- serve as stirring purpose
Dulbecco PBS- contains calcium ions that will help in stabilizing the virus
Antibiotic- prevent bacteria contamination
Chloroform- helps to disperse virus aggregate and kill any bacteria or fungus present

Tissue processing

Lung tissue (to test for respiratory virus)
1. Transfer the lung tissue into a mortar and cut it into small pieces using a scissor.
2. Add in alumdum powder and grind it with a pestle.
3. Transfer the contents into a sterile centrifuge tube and centrifuge at 3500 at 20 minutes.
4. Separate the supernatant from the deposit ( supernatant into a sterile 5ml tube while deposit back to the original container)
5. Select the tubes and shell vials before labeling them with date of inoculation and lab number.
6. Inoculate 0.3ml of supernatant into the tubes and shell vials.
7. Centrifuge the shell vials at 1800rpm for 30minutes.
8. Preabsorb the tubes at 33±0.5̊̊ C for 1 hour.
9. Incubate both tubes and shellvials overnight and observe for cytopathic effect the next day.

Alumdum- To provide sharp edges for grinding the tissue


That’s all for now. Hope that you guys can understand.

Shihui
0607135A