Day Nine!

HI! We are back again, to continue with what we left off ytd as well as continue with some new techniques. We did PCM, plasma treatment for some polymer film, went to the animal house (RATS!! and mice too) and SEM preparation!

PCM isn't a complex procedure. All we did was view the cells that we cultured yesterday and take a picture of it using a programme in the computer. It is a great way of storing images of cells.

The picture of the fibroblast. This is taken using a light microscope. Tomorrow we would be using an electron microscope.

We moved on to plasma treatment for polymer film.The purpose is to change the polymer film from hydrophobic to hydrophilic nature using plasma treatment. There are various ways of doing it and we are using vacuum to do this. Dr Wen Feng shared that in some cases, this process can take up half a day because there was a lot of waiting time. For example, if a different gas is used from the previous one, we would have to wait for all of the gas to be removed from the chamber.

1. We picked out some pre-prepared glass slips with a thin film of polymer on it.
2. We used this machine to do the plasma treatment. The mechanics are pretty complicated, something like different voltahe between the metal plates.
3. Before we did the actual treatment, we did some trial runs in the plasma surface treatment machine. We first ran argon into the machine, due to the splitting of molecules, the energy used correspond to the visible light spectrum and a pretty pink colour was observed in the vacuum container. Then the argon gas was removed using a vacuum and replaced with oxygen gas. A white colour was observed instead. Some bits of pink can still be observed because some argon gas is left.
4. For the actual runs, we placed the glass slips into the vacuum container, ran argon into it for about a minute. Although it is supposed to be pink, it looks a bit purple-ish. This is because not all the oxygen gas was removed from the trial runs. A layer (not really visible) of plasma was formed.
5. We tested the nature by placing a drop of water on it and the water would spread out. The control cover slips reamined hydrophobic and the same drop of water did not spread out. Hence, this proves that the nature of cover slips was changed from hydrophobic to hydrophilic.
6. To have a more accurate result, we could do a WCA test to test the angle of the water on the surface. A smaller angle would mean more hydrophilic as the water would spread. Although we didn't get to do the test, we observed someone else doing it.

Glass cover slips with a thin layer of polymer. We had fun taking them out. HEHE

Plasma surface treatment machine

The different gases used for the plasma treatment. We are using argon gas and oxygen gas.

Before

After argon gas is added

After oxygen gas is added

We used argon in the actual treatment. It looks a little purple-ish though.

Testing the nature of the cover slips with a drop of deionised water.

The control remains hydrophobic. 

After treatment, it becomes hydrophilic. (The drop of water spreads out!)

This is actually a product video, it doesn't show the full process. But it shows the before and after of this procedure. Start from 0:11.

This is the WCA test.

And so we went to the ANIMAL HOUSE. Probably the highlight of the day. A lot of caution is taken before entering the place. We had to put on mask, hair cap, surgical gown, shoe covers and gloves. Also, in general, only people with the license are allowed to interact with the animals in the house. We saw mice and rats. The mice looked like grey hamster except they have slightly bigger ears and a much longer tail. Dr. Wen Feng anathesise a big fat rat (RAT, not mouse) for about 3 mins. The rat was about one and half hands long and had RED eyes (so rats with gleaming red eyes do exist). The rat kinda fainted and electrodes were fixed to the limbs to measure the heartbeat using a ECG machine. We spent only a short time inside and we spent more time outside wearing all the necessary things (mask, hair cap, surgical gown, shoe covers and gloves). Being our first time in an animal house, it was definitely a cool experience. We didn't see any being operated on, that would have been pretty gruesome. But the rat Dr Wen Feng worked apparently went through a heart surgery before. We couldn't take pictures of the procedure, but here is picture of the rats in one of the holding rooms. They live a pretty cosy life.
(We saw test tubes with the hearts of the rats.... We are not going to post pictures.)

A lot of things need to be put on. 

Lab rats.

After lunch, we came back to start on the preparation for the scanning electron microscope (SEM) we are going to use tomorrow.

1. After removing the culture medium from the culture plate we incubated earlier, we washed it with PBS.
2. 2.5% glutaraldehyde (GA) is added to fix the cells and left for an hour in room temperature. Actually, if you realised, we have used a variety of different chemicals for cell fixation. Ajay used paraformaldehyde with triton, while Ivan used ethanol. GA is also another chemical we can use for cell fixation.
3. Ethanol of increasing concentration is added with 10mins intervals. After 10mins, the previous ethanol solution is removed and replaced with another ethanol solution of increasing concentration. 50% ethanol is added first, followed by 70%, 95% and 100%. Ethanol is used to dehydrate the cells by slowly removing the water in the cells on the scaffolds.
4. MDS is added to replace the ethanol solution in the cells as ethanol can damage the cell surface membrane. A better alternative would be to use liquid carbon dioxide because MDS can also slight harm to the cells as well. However, we don't have it in the lab.
5. The cells and the scaffolds are incubated overnight at 37 degrees C.

Varying concentrations of ethanol

Dr Wen Feng allowed us to prepare the ethanol solutions ourselves!

Lucky Alethea also got the add the ethanol to the cells as well.

We learnt a lot from Dr Wen Feng! Thank you very much!

And that is the end of our ninth day! Alethea is flying off tonight, so she is going to post her reflections of the entire programme today. Tomorrow we would have our last mentor, Huaqiong, who would guide us on how to use the SEM. Tomorrow is our last day at MSE. ): We will continue to work hard tomorrow, learn more new things and end things off on a good note. See you all tomorrow! (: