Overdue answers to all your questions..Sorry.

Eloo guys. Sooooooooooo Sorry that I took such a long time to answer all your questions. I totally forgot about them until Ms Chew reminded me. Sorry again. Below are the answers to the questions. Hope it will help. All your postings are very interesting and I learnt a lot form all the experiences that you guys blog about.

To Randall
For my lab, if there is any contamination, I would just check the microbes responsible for the contamination. If it is a bacterial infection, I simply add sodium hypochlorite (NaClO) before disposing them as general waste. Fungal infections are the most dangerous among all, as the spores are very hardy and could spread easily. Therefore for a fungal infection, the CO2 incubator will be disinfected.
In my lab, there are a lot of frozen cell lines. Lab stuff also frequently freezes cells. Therefore if there were any contamination, the cultures will be disposed into the biohazard bin and new cell line will be thawed.
If there are still contamination occurring, I will culture the media without any cells or add PBS to the media to check if the media contains any microbes.

To Charmaine
Yup I wash twice with PBS so that for the second wash, all the trypsin will be removed from the media. Like I said in my post, washing with PBS for the second time will decrease the possibility for the cells to be resistant towards the trypsin as trypsin is totally removed in the second washing step. Yup I pipette the cells in the centrifuge tube.

To Eugene
I have 8 cancer lines and they all need different types of media. Each media contains different components. Usually I will just go to the webpage where my lab bought the cells and refer to the recommended media for the particular cell line. The media used in my lab are pre-made by the supplier. Then before using the media, I have to add 10% FBS and antibiotics to the media. I will elaborate on which cell line gets which media during my next post.

To Yong Young
For my lab, there are no limits to the number of passages as I am using cancer cell lines. Cancer cell lines are more hardy and have the machinery to over come cell death. (immortal cell lines). But there’s always a but, even though I can subculture as many times I want for cancer cell lines, I have to check for its morphology as they might revert back to the normal, uncancerous state through series of mutations. I havent encountered any mutations though. If such things happen, I have to discard the particular cell and thaw new cells.

To Shu Hui
Trypsin is an enzyme that will help to dislodge the cells that are attached on the surface of the culture flask. It helps to re-suspend those adherent cells from the culture flask. With this, we are able to subculture and count the cells for other experiments or assays.

To Lizzie
For me, if there is a colour change in the media, I will check the culture under the inverted microscope. If there are cells floating (dead cells), I will change the media as soon as possible. Sometimes media colour changes are also due to contamination. Usually, I will change the media once the colour in a way that is very different form the normal colour of the media. For example I will change media if the media turns orange yellow or yellow. I will not change if the media is orangy-red and there are no floating cells found.

To Zahirah
There are other dyes that can be used for cell counting. I will give you an example of one that I researched on. It is know as a Fluorescent dye. This method indirectly counts the number cell by counting the nucleus with the help of a machine known as the NucleoCounter (an integrated fluorescence microscope designed to detect signals from the fluorescent dye). The dye, propidium iodide (PI) bound to the DNA of cell nuclei. The NucleoCounter counts the total cell count and viability of cell samples such as mammalian, yeast, somatic, fish and sperm cells.

To Ye Tun
I will elaborate more on the cell lines I use for my next post.
In my lab, I use the CO2 incubator system. To me manually adding buffers (done in your lab) to maintain the pH of the culture risk more contamination as you have to expose the culture when adding the buffers. In my lab I use ventilated caps for my culture flasks. Which means, I do not need to unscrew the cap a little to allow gasses to exchange in the CO2 incubator thus minimize the risk of contamination. The ventilated caps pores are made specially small so that no microbes could enter.

To Suat Fang
I will elaborate and explain on MTT assay on my next post.


Have fun ppl.

Najib Bin Hamid
(0503217B)