WEEK 5 & 6

It’s been a month already, Li Jun and I have now moved on to the Isolation Section of the Virology Lab. The Isolation section consists of the Tissue Culture Lab and the Viral Isolation Lab. The Tissue Culture(T/C) Lab is where the med techs grow and prepare cell lines such as Hela and MDCK that act as hosts for viruses in shell vials, flasks and tubes. The Isolation Lab is the site where the med techs would inoculate samples of patients with suspected viral infections into the cell lines and culture and observe the cultures for over a period of time. Cultures grown in the tubes would be viewed for cytopathic effect(CPE) and tests such as the neutralization test or acid tests would be done to confirm the results. Culture tubes for some viruses such as HSV are sent to the Immunofluorescence Section for Ag IF detection.

Quality Control
In the Tissue Culture Lab, when we prepare the media and reagents, etc. We need to record down the batch details in the Quality Control Record for Media and Reagents. Details recorded include the date of preparation & expiry, result of sterility check, date of use, initial of the med tech responsible for the preparation of the reagent or media.

Sterility test
For every batch of media or reagent prepared, a sterility test must be done.
1) Inoculate 3ml of prepared medium/solution to 1 tube of Nutrient and 1 tube of Sabouraud’s broth.
2) Incubate Sabouraud’s tube at Room Temperature and Nutrient Broth tube at 37C for 7 days, observe daily.
- Discard any aliquot corresponding to tube which shows signs of growth.
- The sterility test is done to ensure that the reagents prepared are not contaminated with bacteria(Nutrient broth) or fungi(Sabourad’s broth) so that the cell lines would not be contaminated.

During our attachment in the Isolation Lab, Li Jun and I were given a sample each to isolate and observe. I was provided with a throat swab sample from a patient who was suspected of having Hand, Foot and Mouth Disease(HFMD). The test required for this particular sample is the Enterovirus culture. Before we start inoculating the specimens, we first need to process the sample by allocating a lab number for the sample and recording down the time and date the specimen was received and also include the initials of the person who was responsible for the receipt of the specimen. We then selected the tubes/shell vials of cell lines needed for inoculation of the sample. For the throat swab sample, a pre-adsorption is done. Pre-adsorption is believed to enhance entry of viruses into the cells. The medium is first removed from the cell line tubes and the 0.3 ml of the specimen is inoculated in to the tube and the tubes incubated at 36C for 1 hour after which, the tubes would be re-fed with medium and incubated and observed daily over 21 days and the results recorded in a specimen worksheet.

The tubes are read daily so as to detect any Cytopathic Effect(CPE) that may be present. When viruses multiply in the cell lines, they usually produce changes in the growing cells. This change is called the CPE of the virus and may be characteristic of the particular virus. A preliminary identification can be made by the appearance of the CPE. However, we must always confirm the result by performing a more specific test such as an immunofluorescence assay.

WEEK 7 & 8
During the period of observation, the tubes need to be maintained by regularly changing the media when it has become too acidic(yellow) and also the tubes(except for diploid cell lines) need to be repassaged every 7 days.

Repassaging of culture tubes
1) Scratch the cells off the inside wall of the tubes using a bent pipette.
2) Vortex the tubes to break up clumps of cells.
3) View the tubes under the microscope to ensure that most of the cells have detached.
4) Incoulate 0.2ml of the cells into a new cell line tube.

Repassaging is done to prevent overgrowth and overcrowding of the cells.

During the last week of observation, I got a shock when I saw there was fungi growing in the media and the inner walls of 2 of my culture tubes. I immediately informed the med tech there and she taught me how to filter the contaminated tubes.

Filtering of contaminated tubes
1) Freeze and thaw the tubes 3 times.
2) Filter the culture using a 0.2 Um millipore filter.
3) Inoculate 0.2ml of the filtered cells into a new cell line tube.

Eventhough my tubes got contaminated, I wasn’t upset about it because I realized that I actually benefited from it. Before the incident, I had observed a med tech there perform the filtering of contaminated tubes. However, she was too busy to explain the steps in detail. This incident has allowed me to perform the filtering myself and thus, I get to learn and remember the steps better and I also understand the rationale behind each step.