Virus needs living cells to grow; hence in order to isolate and grow these viruses we need different types of cell culture. In this section, we will prepare 11 types of cell lines, as there is no single cell line that can grow all the virus. The examples are as follows:
| Cell line | Type of virus that is grow in |
| Hela (cervical cancer cell) | Adenovirus, rhinovirus, polio virus |
| Hep (throat cancer cell) | Respiratory syncytial virus, polio virus |
| Hel (human embryo lung fibroblast) | Herpes, varicella, cytomegalovirus |
| Vero/slam (genetic engineered cell from vero) | Measles virus |
| Vero (African green monkey kidney cell) | Adenovirus, enterovirus, SARs virus |
| L20B (mouse cell) | Polio virus |
| C636 (mosquito cell) | Dengue virus |
| LLC (African monkey kidney cell) | Influenza virus |
| MDCK (Dog kidney cell) | Influenza virus |
| ML (mink's lung cell) | Herpes virus |
| RD (muscle cancer cell) | Enterovirus |
The things that i have learnt so far are as follows. Firstly, Let's start from media preparation.
Minimum essential medium (MEM) preparation
1. Add the consituents (NAHCO3, water, hepes and MEM powder) in their respectives volumes and amount into a sterile flask.
2. Mix well by placing a magnetic stirrer into the flask and place it on a hotplate.
3. Switch on the hotplate to stir the contents for 1 to 2 minutes.
4. Sterilize the media by using peristaltic pumps (0.2micrometer pore size membrane).
5. Filter the media through filter membrane into different sterile bottles inside the BSC II ( biological safety cabinent class two).
Sterility test for the media prepared ( from above):
- Add 1ml of the media prepared to both nutrient and sabouraud broth respectively.
Nutrient broth is to test for growth of bactera while sabouraud is for fungus growth. This serves to check that there is no contamination in the media ( indicated by turbidity) after 1 -2 weeks before the media is used to grow cells.
Next, retrieving the cells from liquid nitrogen.
Cell recovery from liquid nitrogen
1.Check for the type and location of cells that sholud be taken out.
2. Put on cryogenic gloves and face shield.
3.Take the respective cryotube out.
4. Thaw the cryotube quickly in 37 degree waterbath.
5. Disinfect the outside of the cryotube in 70% alcohol.
6. Pipette the thawed cells into a culture flask containing 5ml of fresh growth media.
7. Change the media the next day.( as it contains DMSO- cryoprotectant, that is added to the cells during freezing. It serves to lower freezing point and minimize the effect caused by freezing )
8. Alternatively, centrifuge the thawed cells to obtain the pellet (discard the supernatant).
9. Transfer the pellet into fresh growth media in culture flask. ( In this case, there is no need to change the media the next day as DMSO have been removed.)
Lastly, storage of cells in cell bank.
Freezing of cells for storage
[ Cells to be stored in cell bank must achieve the standard concentration of 5 x 10^6 cells/ml]
1. Trypsinise the cells that are to be freezed ( Wash 2 times with Phosphate buffered saline and then add trypsin solution)
2. Prepare the freezing media(0.3ml DMSO and 2.7ml fresh MEM) and prechill in fridge.
3.Add cells to media in a plastic centrifuge tube.
4. Take 1 drop from the centrifuge tube and place in on a parafilm.
5. Add a drop of trypan blue to this drop and mix well - 1 drop : 1 drop, so gives dilution factor of 2. ( Trypan blue will stain the dead cells , thus allowing the viable cells to be quanitified)
6. Load it into the hemocytometer
7. Count the number of cells under microscope.( count 4 squares then take avarage and mutiply by dilution factor)
8. Determine the volume of cells to be loaded into the cryotube by microsoft access. ( Consist of a formula that is already set up for calculation of the volume needed to load in cryotube - to achieve the standard concentration of cells)
9.Add the desired volume to the cryotube and seal it with masking tape parafilm.
10. Place the cryotube in Mr frosty ( Freezing container with isopropanol - to allow the cryotube to come to temperature of freezer at about 1 degree/minute)
11. Place Mr frosty in -85 degrees freezer before transfering it to liquid nitrogen.
That's all for now. I will be moving on to virus isolation section soon. Hopefully, i can share with you guys more interesting things.
For more information on the viruses above, you may want to visit these websites:
http://www.emedicine.com/med/byname/adenoviruses.htm
http://www.emedicine.com/ped/topic2707.htm
http://www.dhpe.org/infect/rsv.html
http://www.dhpe.org/infect/cytomegalo.html
http://pathmicro.med.sc.edu/virol/picorna.htm
Goh Shihui
0607135A
TG02
6 comments:
hihi friend
ur sip seems cool, dealing with virus...haha
here are some questions that i wanna ask you...
why there is a need to grow those virus ? after growing them what did your lab do to them ? study them under microscope and... ?
how do you determine what type of virus grows well in what type of cell line ?
for the sterility test, what will you observe when it is sterile or when it is not sterile? by looking at the appearance- clear or not clear?
hope you have fun at you sip !! haha C YA on fri!!
TING-JIE
TG02
0608495H
Hi..
Would like to know...what are the type of specimens usually rcved which are suspected to have viral infections? since u dont really know initially which type of virus it is..do you grow it in all the 11types of cell lines?
thanks a lot =)
Nur Farhana
0604834B
Hello Shihui (:
Let's say if one patient's sample are inoculated into all the 11 cell lines, how do you determine which virus is growing on the medium? such as the Hela, Hep, Hel and Vero whereby a few viruses can be culured. Do you observe for the different morphology just like the bacteria medium?
Thanks (: see you on friday!
-Yvonne Teo
Hi tingjie,
Actually it is to isolate the virus from the patient's specimen. We will receive request form from the doctor, which will indicate what type of virus is suspected of. Then we will try to see if we are able to isolate the suspected virus to confirm the diagnosis.
We will then observe under the microscope to look out for cytopathic effect (CPE)- which is changes in appearance of the cell culture caused by the virus that are growing. Each virus will have their own particular CPE.
As for what type of virus grows well in which cell line, that is actually determined by all the research that was done in the past. The researcher who discover the cell line or virus had conducted many experiments to identify the best combination.
For the sterility test, it is actually to test whether there is contamination. So we will just observe the appearance.
Hi farhana,
The type of specimens received are usually nasal,throat,genital swab, blood, urine, stool, lung and heart tissue.
The specimen will come with request form, indicating what virus that it is suspected of. For example, A person suspected of having herpes. A genital swab will be done and sent here, requesting for herpes virus isolation.
Therefore, we will then see if we can isolate herpes virus from the specimen, by inoculating the specimen into HEL and ML(cell line).
Hello yvonne,
We won't inoculate a patient sample into all 11 types of cell line. This is because the patient's specimen will come with a request form, which will indicate what type of virus it is suspected of. Then we will only perform the particular test that is requested for. For example,If person suspected for having polio so only polio virus will be tested-done by inoculating patient's specimen to HELA,HEL and L20B cell lines.
We will observe under the microscope to look out for cytopathic effect (CPE)- which is changes in appearance of the cell culture(cell line) caused by the virus that is growing. Each virus will have their own particular CPE, hence it can be distinguished from one another.
Hope that u can understand..
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