Saturday, July 07, 2007

Lab Techniques and Microbiology work

ok! This week is my turn to blog what I had done for the past weeks..

Well, I am doing my SIP in house and during the first week there is nothing much to blog about as our TSO in-charge was not assigned to us yet and also, I wasn't quite sure about what to do for my MP yet..So basically, during the first week, I just tagged along with my partner, observing what she was doing and taking notes at the same time. On my own, I did subculturing of human lung cancer cells. This is carried out in the BSL lab of AS4. The laminar flow hood that was use was of class 2 category as human lung cancer cells ( NCI-H460) or known to us as 177 was rather pathogenic, thus proper preventive measures had to be taken care of. The laminar flow hood that I used has a alarm system, and it will sound if the shield is raised above the protective level to inform the user to lower the shield down for personal protection purposes. Subculturing is done when the cells had reach about 60% confluency. Confluency refers to the space that the cells had occupied, and the cells viality will decrease with increase confluency, thus it is crucial to monitor the cells' growth under the inverted microscope and carry out subculturing when required. Trypsin is required to help digest the cells to enable the cells to be detached from the flask surface. After which, media is added to the flask containing the trypsin and cells to help stop the trypsin action, thereby preventing excessive digestion of the cancerous cells.

Apart from subculturing, I was also introduced to the various machines preset in the labs and one of them is the Beckmen Avanti J251 Centrifuge, also known as the standing centrifuge. This centrifuge is able to centrifuge at a very high speed of about 20,000 rpm or even higher. One very troublesome about this machine is that it is balanced based on weight and thus before putting the centrifuge bottles into the centrifuge, it is a must to measure the bottles and the difference of each pair can only be +/- 0.05g. Anything more than this tolerance will cause imbalance and the centrifuge will vibrate. Similar to the usual centrifuge, the bottles of similar weight are placed opposite one another to allow balancing. The bottles used are also round bottom as conical flasks or others non-round bottom flask might break and result in spillage.

As my project requires herb extraction, I had to make use of the rotary evaporator to enable the evaporation of the 50% ethanol in which my herb ( S. barbata) is dissolved in. This evaporator allows the round bottom flask to be rotated when it is being heated so as to enable uniform heating and evaporation. Connected to the heater is also a condenser and vacuum pump. The condenser is there to allow the evaporated ethanol to condense and fall into the other attached flask and the condensed ethanol is labeled as waste and thrown into the waste bottle. The vacuum pump will enable heating to be carried out at a lower temperature of about 35 degrees. At this temperature, ethanol (boiling pt of 78 degress) is usually unable to evaporate so quickly, thus the vacuum can lower the boiling point and quicken the heating procedure.

During the 2nd week, I was already assigned to a TSO and had to do pour plate of Potato Dextrose Agar (PDA) and Nutrient Agar (NA). At first I thought a pipette had to be use to aliquot the agar into the culture dish, but my supervisor told me that all I had to do was to pour the agar from the bottle into the dishes. This is because, by using a pipette, there will be alot of unwanted bubbles present in the agar and it will affect the state of the agar. When I first poured, it was relatively difficult to estimate the required volume so as to get the agar of the best thickness, and i spilled some of the agar. However, after some practise, it became easier. In addition to that, I was told to prepare 5L of NA. This is done by dissolving 23 g of NA powder in 1L of DI water. Its just as simple as that. Then the 5 bottles was autoclaved.

These are basically what I had experienced for these 2 weeks. Nothing much, mainly just some operating of the big machines which I thought was complicated but doesn't turn out to be as difficult as I thought it was, and the ongoing subculturing of cells. Feel free to ask any questions and hopefully I am able to answer all of them.

Have Fun during your SIP guys! Take Care!

Charmaine
0503186I
TG 01

21 comments:

VASTYJ said...

harlow~! wow that was a very clear post on your sip. haha seems that we are the same, nothing to do much through the first week. can only observe and learn abit here and there.

hmm, dun think i have any qn. jus drop by say hi. do check my blog for updates! cya soon

Regards,
Chaur Lee
TG01

VASTYJ said...

HIHI cHarmaine ~!
lung cancer cells sounds dangerous. hahas. Anyway , what is PDA and NA for arhx ? thx thx ~ do take care arhx ~! have fun ~!
vaLerie =)

royal physicians said...

heya charmaine...woah..the centrifuge seems irritating...aniwei wanna ask u..y do u use PDA and NA??is there any special reason to it?? and y do u use 50% ethanol to dissolve the herb??..aniwei njoy ur SIP girl and all da best

from:
nur zahirah tg02

J.A.M.M.Y.S said...

Hey Azhar here

Just wondering, you mentioned that you prepare your own media, so are there any Quality Control procedures that you need to do for the media?

Thanks,

Azhar TG01

J.A.M.M.Y.S said...

hiya,
I would to know briefly how do u do subculturing of the cells?
Thanks ya!
Michelle

MedBankers said...

Hi Charmaine, u mentioned :

"Trypsin is required to help digest the cells to enable the cells to be detached from the flask surface."

Could it be instead of digesting the cells, it digests certain proteins that causes the cells to adhere to the surface of the flask? Because digesting the cells would mean to break the cells into many pieces literally.

If so, (just curious to know), what protein does the trypsin digests?


Thanks,
Yeng Ting

VASTYJ said...

Hi Charmaine,

You have a very clear description of your assigned tasks in your post and I can understand them very easily just by reading them once.
Cheero!

However, out of curiosity, what specimens are used and for what purpose to use the centrifuge that spins at 20 000 rpm or higher?

Is it a very huge centrifuge?

Loh Sharon , Tg 01

The Lab Freaks said...

Haha..Clear post so that no qns to ask mah..But seems that still got qns to be answered..

Okie! PDA is actually an agar that is used to culture and enumerate moulds and yeasts. Acid is also added to PDA to help lower the pH, thus inhibiting other unwanted organisms from growing. NA is the general agar that we normally used in our labs, it allows majority of cells to grow. It does not inhibit or enhance any particular growth.

To zahirah, the agars that i prepared are for staff use i guess, I'm not to sure either. 50% ethanol is usd for greater dissolving purpose. At a increase %, the solution will evaporate too far and the herbs could not be completely dissolved.

With regards to the QC part, there's not much issue about it as we basically follow the protocol and whatever we used were actually sterilised by autoclaving or filter sterilisation.

Subculturing is actually very simple. Just discard the used media, then wash the cells with PBS for 2x. After which, trypsin is added and incubated at 37 degrees for about 4 min. After the cells detached, the suspension is pipetted to a tube and centrifuge. Supernatant is thrown away then the pellet is resuspended in 6ml of media. 0.5ml or the required amount is pipetted to a new flask containing about 14ml of media. Thats all for the subculturing.

Erm, with regards to Yeng Ting, trypsin is also used to digest the proteins in the cells. But yes, for subculturing, it only digest the adherence matter. Sry if i confused u. Trypsin cleaves proteins at lysine and arginine amino acids.

Sharon, what do u mean by specimens? Only human lung cancer cells are used in my experiment. Centrifuging at 20,000rpm will allow the herb to settle at the bottom and sort of harden so that we can elute the supernatant out easily, and also due to the greater amount of substance to centrifuge, this high speed is required.

Hopefully, this clear your doubts! Take Care and Have Fun!

Charmaine

The Lab Freaks said...

oops..yah sharon..its quite a huge centrifuge..much much much bigger than the usual ones..

The Lab Freaks said...
This comment has been removed by the author.
The Lab Freaks said...

hey charmaine... i observed something similar about the growth of yeast and mould... do u further isolate the different species and do a write up or any microscopy drawings? because i realise there are many different types of species...

anyway... enjoy yr SIP!

~Jeremy~

VASTYJ said...
This comment has been removed by the author.
VASTYJ said...

Yoz Charmaine,

Because in your post you said apart from subculturing you also learn about the gigantic centrifuge, so I thought that was not related to your project since you didn't get your MP title in the first week.

Nevermind .. So your post wasn't as clear as I thought?! haha. Not bad actually!
=X

Loh Sharon, Tg 01

The Lab Freaks said...

jeremy! erm..I din do anything to the culture, so i duno what happened to it actually. My TSO also din specif the actual use.

erm sharon. i dun get what you mean.I did get my MP title already and the centrifuge had to be used for herb extraction. So if your specimen refers to whats being centrifuged, then thats the herb extracts. Maybe you mixed up me din get my TSO with not getting the MP title. SRy if i confused you yah..

:) charmaine

BloodBank.MedMic.Haematology said...

hey.. what is the difference between culturing and subculturing? haha. hmm.. thanks for answering my question.. :P

doreen (tg 01)

VASTYJ said...

hello!
for the agar preparation, do u have to dissolve the agar first in a heated water bath first before autoclaving? my lab does. and my lab does QC everytime a test is done, which is making a separate blank pour plate with only agar in it.

Ying Ying
TG01

ALsubs said...

I had to make use

Hi charmaine..

You mentioned that you have to make use of the rotary evaporator to evaporate the 50% ethanol in the herb ( S. barbata). Why do you have to do this step?? U need the herb to perform some other test? whats the use of the herb?

Vinodhini =)
TG02

Star team said...

Hey,

Do you have to carry out any QC procedures in the lab?

Yong Yang
TG02

The Lab Freaks said...

hi ppl..

To Doreen:I think rite, subculturing actually means u culture the cells that was already passaged. but for culturing, its like a new vial of cells.

To Ying Ying:erm, I didn't heat the agar at all. All I did was to swirl the bottle to dissolve the agar powder.

To Vino: actually the rota vap is for uniform evaporation and also to decrease the evaporation time by using the vacuum. and yes i nid to use the help to treat my lung cancer cells. the herb is actually used to induce apoptosis in the lung cancer cells to enable us to detect and deduce the route of apoptosis.

To Yongyang: To check the quallity for the herbs that I nid to use rite, I actually make use of the high performance liquid chromatod=graphy machine. This thing actually separates the vairous components in the herb and shows a chromatogram. thus allowing comparison between the 2 batches of herb that i extracted to be done. This is also compared to a standard chromatogram.

Charmaine

royal physicians said...

Hey Charmaine

Just want to ask are there any rules of not bringing your cells out of the lab (as in the BSL itself)? Are the cell pathogenic to everyone or only to immunocompromised individuals?

Johanna, TG02

The Lab Freaks said...

johanna, actually we can bring our cells out of the lab, but gloves must be worn to hold the flask and also the cap must be tightly screwed. Cos the inverted microscope is located at the TCM lab, and we nid to use it. actually, if i'm not wrong rite, i think it's pathogenic to all. like if there's a cut, we need to plaster it and wear double gloves.

:) charmaine
TG01