Culturing MRC-5 cell lines

Hello, i am Stanley and i will be doing my very first blog posting. I am sorry for the delay as i have only started my experiment this week. I have managed to perform some simple experiment today and i will like to share my experience with you guys. These experiments were done in a bio safety level 2 laboratory and are related to Mammalian cell technology (MCT).



The aim of major project is to evaluate the effect of green tea on large lung cancer cells. I am given two different cell lines which are namely the MRC-5 cell line ( healthy lung cell) and the NCI-H460 ( large cell lung cancer). Both cell lines are previously purchased from ATCC and are both in passage number 2 when they are given to me. The objective for today's experiment is to culture both cells in their respective media therefore allowing healthy cell growth and expansion.



First of all, i will have to prepare the respective media for each cell lines. I will be stating the steps involved in preparing the modified Dulbecco's modified eagle media (DMEM), which will be use to culture the MRC-5 cells.


The steps to prepare 1 litre of DMEM are:

1) Thawing of the Fetal bovine serum(FBS), L glutamin, penicillin streptomycin, sodium pyruvate
in a 37 Degree Celsius water bathe.

2) Dissolve 1 packet of DMEM powder and 37g of sodium bicarbonate into of 1 litre ultra pure
water.

3) Add 10ml of penicillin streptomycin

4) Add 10ml of L- glutamin

5) Add 10ml of sodium pyruvate

6) Add 50ml of FBS

7) Filter sterilize



Although the above steps might seem simple and rather straight forward. I made a mistake of introducing too much bubbles which resulted in overflowing of media into the electric vacuum. Although, i had learnt how to filter sterilize DMEM media in Mammalian cell technology. The modified DMEM media is completely different as it contains 10% FBS and many other constituents. I later learnt from my supervisor that the presence of FBS is the main reason behind the large production of bubbles.

Following the preparation of DMEM media, i am ready to culture my MRC-5 cells. The steps are as followed:

1) Pipette a small amount of DMEM media into a centrifuge tube

2) Pipette 2 tubes of MRC-5 cell line into the centrifuge tube

3) Centrifuge it at 1000 rpm for 5mins

4) Pipette out the supernatant

5) Add 20ml of DMEM media to resuspend the cells

6) Pipette the resupended cells into a T75 flask
There are a few key points to note while culturing cell
7) Incubate the culture under 5% Co2 level at 37 Degrees Celsius



While culturing fragile cells for example the MRC-5. We will have to ensure that the media is properly warmed to around 37 Degrees Celsius which is near to our body temperature before it can use to culture the cells. Similarly, no bubbles should be present in the culture as this might lead to cell death.


Strict aseptic techniques have to be observed throughout the whole experiment as any contamination will results in the discarding of media or materials. It is rather scary as my supervisor had previously ordered the materials in week 2 but the it only arrive on week 6 of my major project.

Here are how both cells look like under an inverted microscope.

Mrc-5 cells--------------------->

H460 cells ----------------->

Thank you

Islet isolation (:

Hi! I'm back for episode 2!

Isolation of pancreatic islets from mice

Retrieved from BBC (2008). Scientists make weight loss claim. Retrieved on 01st July 2009, from: http://news.bbc.co.uk/2/hi/health/7372495.stm

For this experiment you will need these few things:
White mice, Anaesthesia, Surgical Scissors, surgical wire, Forceps, Syringe, Needle, Tube, Collagenase solution.

When I first stepped into the animal room, a strong smell engulfed me. At first I thought it smelt nice (maybe because it was before lunch), but as I babysit the mice longer, they started to stink. The mice were stored in a styrofoam with holes as they were transported from a facility (which I shall not name).

A total of 4 mice were used in this experiment. The mice were making a lot of noise and they kept moving around! They almost had the strength to push open the styrofoam cover and even tried to escape.

My senior picked up the mouse by its tail, and tried to hold up its head by its ears in between her thumb and index fingers and held the tail using her last finger. Basically, the mouse was immobilized by a hand. The other hand was used to inject the anaesthetic into the mouse’s abdomen. This has to be done carefully. If injected wrongly, this may cause the mice to die earlier, which will be no good for the experiment.

As you guys know, mouse is very small. Therefore my mentor used a binocular microscope for easier visualization. To ensure that the mouse is really asleep, she will pinch its legs to check for reaction. No reaction would indicate the mouse is asleep. Next, she used a surgical scissors to cut open the abdomen, and exposed its contents.

Do you know that a mouse’s liver is really big? At first, I thought the liver was the lung! Below the liver is the pancreas. First, my mentor skillfully tied up the bile duct with the surgical wire. Tying up the bile duct before injecting collagenase into the mice ensures that collagenase will only be injected in the pancreas. If not, collagenase solution will flow into the stomach and intestine, therefore the pancreas will not swell up. Next, she carefully inserted a small tube which contains collagenase into the bile duct. Collagenase is used to digest the external cells. Once collagenase is pumped into the pancreas, the pancreas starts to swell up. I once thought that the pancreas is green in colour, but actually it is quite yellowish-transparent. Once the entire amount is injected in, the tube was carefully removed and pancreas was removed and placed in a tube containing collagenase. The tube was then placed in 37*C waterbath. After the removal of pancreas, the mouse would die a few minutes later.

However, my observation did not stop here. As my senior needed to collect blood for her experiment, I watched as she inserted the needle into the mouse’s really small heart and took the small little amount of blood. It was really weird, as during the procedure, the mouse would have reflex action, almost like the mouse is having hiccups.

Lastly, after we have gotten what we needed, the mice were covered in aluminium foil for disposal.

after putting pancreas in collagenase, you'll incubate at 37oC for 17-18 minutes. after that, you'll shake the cells, to ensure homogenicity and check that there is no remnants. next, we will pour the solution into a petri dish. using a binocular microscope, islet cells are handpicked.the cells will be transferred into a petri dish containing RPMI (Roswell Park Memorial Institute) media which is suitable for islet growth. the cells. the handpicking of islet cells are repeated onto subsequent petri dishes to remove all the epithelial cells.
Although I wouldn't have a chance to ever cut the mouse open, but this has been a great experience, though this isn't and will not be my first and my last.

Lim Jia Hui (JOEY)
0703605F

Microbiology - questions asked

hihi.. sorry for the late reply. i didnt know the response was so overwhelming. hahaha..

please read on.... =)

to Jess

Question: does Salmonella appear black on all agar

No. it appear black only on XLD (Xylose lysine deoxycholate) plate.

Question: how does E.coli and Acinetobacter baumanii appears on the agar that differs from Salmonella

Both Salmonella and Acinebacter baumanii is non-lactose fermenter. Whereas E.coli is a lactose fermenter. Therefore Macconkey plate can be used to differentiate the lactose fermenter from the non-lactose fermenter. Inorder to differentiate the three different types of bacteria, perform biochemical test.

to Siewming

Question: what is Tip specimen

when patient with dextrose drip, central venous catheter, or any form of needle attaching to body, shows signs and symptoms of infection (eg. fever), blood FEME is usually done. when the blood FEME shows that there is bacteria growth or high amount of WBC present,

it means positive for infection. Hence it might be due to tip contamination, which is contamination of the needle or even the tubing. Thus, part of the tube is cut and send for comfirmation.

Question:is there a need to culture different type of stool on different type of agar plate.

Yes. different form of stool formed is due to different bacteria. Hence, with a standard protocol in the lab, different type of agar plate is used so to select and maximise the growth of the target bacteria.

to Jordan

Question: is it a must to streak on TSA plate then Macconkey plate for urine specimen.

Yes, Macconkey agar is a selective agar which may inhibit the growth of certain organism. thus if we streak on Macconkey agar first, we might transfer some of the nutrient in the Macconkey to the TSA plate which may inhibit the growth of certain organism. this may lead to inaccurate result as there might be more than 1 organism present.

to Jeremy

Question: what does XLD stands for and its uses in stool culture

XLD stands for Xylose Lysine Deoxycholate agar. Basically it is use to isolate Salmonella and Shigella.

Question: why does Hemophilus Influenza (HI) unable to grow on blood agar and is there any way to establish growth of HI on blood agar

HI does grow on blood agar only with the presence of Staphylococcus streak. this is bacause, Staphlococcus streak contains the X and V factor with enhance the growth of HI.

Question: what is the significance of detecting MRSA in people.

The only antibiotic that can treat MRSA is vancomycin. Given that vancomycin will damage the kidney function of neonates, it is very crucial.

to Lok Pui

Question: why do we need to do cell count for urine specimen

FEME is done to support the report. For instance if there is bacteria growth on the agar plate but there is no WBC observe under the microscope, it could be due to contamination of the agar plate. Hence urine FEME serves as a countercheck for the results.

Question: how to culture solidfy stool

Solid stool doe not undergo any dilution in broth. A stick is use to scrap abit of the stool, then it is smear on the agar plate.

Question: what kind of specimen is used for ear culture

For ear culture, ear swab is obtain.

(extra info: ear culture is done more commonly in newborn to screen for Group B Streptococcus that may be pass on from the mother.)

to Liyana

Question; how germ test is performed to confirm for Candida Albicans

In germ test, rich plasma is supplied. Only Candida Albicans is able to grow germ tube within 24 hours.

this is a picture of germ tubes of Candida Albicans

(adapted from http://en.wikipedia.org/wiki/Germ_tube)

to Janice

Question; do we use different plate for different form of stool

Yes, we do use different plate for different form of stool. (for more information, can refer to the post that i wrote)

Question: do we put ear specimen on cook meat

Nope. we only use CDC and Macconkey without salt agar plate.

Question; for Salmonella found on plate, how do we identify

we do a serotyping to confirm which group of Salmonella it belongs to. In this test, Salmonella Antisera is used. There is a total of 9 different group of Salmonella. In order to perform the serotyping, ensure that bacteria looks homogenous and no autolysis occurs. Next, one colony is treated with PO and PH, a bacteria antigen. PH is a test against flagella . Both PO and PH must be positive to ensure that the bacteria is a Salmonella species. Commercial reagent is then used to test for the group.

for those questions on the chat box, so sorry, i didnt realise there were questions over there. hence its not included in this post. will reply soon !! =)

bye bye..

JOCELYN =D

Building your very own bubble trap

This post is posted by Alvin Tan, 0703829B.


I have learnt to build my own bubble trap, instead of buying pre-made ones available on the net. My bubble trap design looks something like the bottom diagram.







Principle:

The inlet will allow water to flow into the trap itself. With the air space in the vial, any bubble will will join with the air and be eliminated. Water will drop and exit via the outlet.
The filter has a pore size of 0.45um. It will block off most bacteria but not viruses. However, a filter of 0.45 pore size is enough for my experiment. This filter can be changed according to your preference.



Things needed:

1. Filter

2. Cryovial

3. Connectors

4. Forcep

5. Needle



Steps:
1. Using the needle, punch 3 holes on the cryovial. 1 on the top of the cap, 2nd at the bottom of the vial and 3rd at the side.


2. The holes wont be big enough for you to fit the connectors and filter in. Therefore, use a forcep to enlarge it manually. Which means, just dig and dig till the hole becomes big enough to fit.

3. Fit the filter and connectors and then the tubings and you get your own bubble trap.

Alvin
0703829B
14 July 2009

Microbiology Lab

JOCELYN YEO 0703359J



I was attached to microbiology lab for three days.



Upon receiving specimen, all request form is needed to clot in time of arrival in the lab. This is for tracibility and also to track the turn around time. Specimens received are sort according to high vaginal swab (HVS), ear swab, methicillin-resistant staphylococcus aureus (MRSA), urine specimen, blood specimen, Tip specimen and stool specimen. Before processing the specimens, we are suppose to check patient information labelled on the specimen against the request form. Different specimen will require different condition, procedure and agar plate for incubation. After incubation, medical technologists will read and type the result in a computer system.



Urine processing



Macconkey without salt and TSA blood agar plate is used to culture.

Shake the bottle of urine. Dip the 1µl disposable innoculation loop into the bottle. The loop should not be dipped all the way to the bottom. Then streak on the TSA blood agar first. Dip the same innoculation loop into the urine specimen again and streak on the Macconkey agar. Next, use a capillary tube to draw the urine and insert it into the disposable hemocytometer for cell counting.















Method to streak for urine specimen.

Stool specimen

Different form of stool specimen is needed to culture on different agar plate.
1. Formed or loosed stool : XLD, campylobacter plate and selenite F broth
2. Watery stool : XLD, campylobacter plate, selenite F broth, TCBS, TSA blood agar and alkaline peptone water
3. Bloody stool : (select mucus or blood flecked material to culture) XLD, campylobacter plate, selenite F broth, CIN, Macconkey sorbitol agar.
4. Stool from BMT or oncology patient : XLD, campylobacter plate selenite F broth, blood with gentamicin, blood agar, Macconkey plate and VRE plate.
5. Request for aeromonas or pesiomonas culture : XLD, campylobacter plate, selenite F broth, blood plate.
6. Request for VRE and ESBL screening : VRE plate and gentamicin blood agar plate.
(Macconkey sorbitol is to look for E.coli 0157 and campylobacter plate is to look for campylobacter.)
7. Rotavirus tested by using a commercial kit. Before test procedure, ensure test kit is at 20-25 degree celsius for optimum result. Add 1µl of diluent to 100µl of stool specimen and mix it. Allow sedimentation for 3 minute. Pipette 4 drops of mixture into the cassette. Read the result after 5 minutes. Inorder for the result to be valid, the control must be positive.



Ear culture

Culture on CDC and Macconkey without salt agar plate.



Wound specimen

Blood agar and Macconkey agar plate is used for superficial wound specimen. An additional CDC plate is needed for deep infection to check for anaerobic bacteria.



Tip specimen

Only blood agar plate is used as the aim is to check for any bacteria growth.



Upper respiratory system

Streak on blood plate and chocolate agar.
(chocolate agar is an enrichment media, hence Hemophilus influenza is able to grow.)



Endotrachel Aspirate (ETTA)

Culture on blood agar and Macconkey agar.



Subchronic specimen

This include specimen such as placenta, womb, etc.
Blood agar, Macconkey agar plate and CDC plate is used.



Sterile specimen

For all sterile specimen, gram staining must by carried out and analyze before plate reading.

1. CSF : Culture on blood agar, chocolate agar and CDC plate. (specimen is kept 3 days)
2. Pleural fluid : Culture on blood agar, chocolate agar, CDC plate and Macconkey agar.
3. Tissue (eg. lymph node) : Culture on blood agar, Macconkey agar and CDC plate.
4. Endocervical : Culture on Thayermartin media (MTM)















Method to streak for miscellanous specimen (all specimen except urine)



Results


Urine
1 colony is approximately 1000cfu/ml. If there is less than 10 colonies, report as no significant growth.
However, if bacteria is suspected to be yeast (candida) , perform wet mount and look under microscope. Germ test is then carry out if yeast is observed under microscope. Germ test is a test to confirm for Candida albicans.
Typical morphology of E.coli is flat, and lactose fermenter (pink on Macconkey plate). If bacteria is suspected to be E.coli, perform spot indole test by putting a drop of indole reagent on a strip of filter paper and scrap a colony. If colour changes to green, it is indole positve. Indole negative remain pink, hence send for identification of bacteria and susceptibility test.
Spot indole test should use colonies from the TSA plate as colonies on the Macconkey plate might be stained with colours thus affecting the result.



Stool
black colony may suggest Salmonella species. Other bacteria to look out for are E.coli and Acinetobacter baumanii.



Wound specimen
common bacteria are S. aureus, Group A Streptococcus, Pseudomonas aeruginosa and GMV.



Upper respiratory system specimen
look out for Streptococcus pneumonia, Hemophilus influenza and S. aureus.



Endotrachel aspirate (ETTA)
if Macconkey shows orange, it means the bacteria is a non-lactose fermenter, thus perform indole and oxidase test.
Colonies that look flat, spreading and metallic may suggest pseudomonas species. Thus perform oxidase test .
Catalyst test is to differentiate between Staphylococcus and Steptococcus species.



Ear specimen
look for Group B Streptococcus. There might be GMV and enterococcus.



Subchronic
look for gram negative bacteria.



MRSA
methicillin resistant organism will appear maurve colour (purplish pink) on MRSA select media.
If suspect methicillin resistant organism present, perform agglutination test with Bactistaph reagent. If Bactistaph reagent forms agglutinates with the colony, S. aureus is present.

Cytology Lab

Posted by: Tan Siew Ming 0702862D

Hey people! I'm here to share about my SIP experience of the 1st week! (:

As for the first 3 days of the first week, I was attached to the Cytology Department. Cytology is the analysis of cells to diagnosis diseases. The lab deals with 2 types of specimens : 1) Gynaecological Specimens and 2) Non-gynaecological Specimens.
Gynaecological specimens refer to Pap Smear of the cervix, while non-gynaecological specimens refer to fine needle aspirations (FNA) and bodily fluids collected.
Examples of the bodily fluids collected can be: Urine, Pleural, Pericardial and Peritoneal washings. And, FNA can be from the thyroid, lymph nodes, breast, bones, etc.

The routine process of the lab upon receiving the specimens is:
Maintaining of the specimens on the computer (that is to generate an access code/individual code for each specimen) -> generate the label sticker for labelling of the specimens-> processing ->staining-> mount-> screening of cells under the microscope.

1) Processing of Gynaecological specimens (Pap smears)

Pap smear is a screening test that determines any abnormalities of the cervical cells that may lead to cervical cancer. The aim of a pap smear is to detect any Cervical Intraepithelial Neoplasia (CIN) caused by the Human Papilloma Virus (HPV).
Basically, there are 3 different grades of CIN: CIN I- mild dysplasia, CIN II- moderate dysplasia, CIN III- severe dysplasia.
There are 2 ways if preparing and processing pap smears: 1) Conventional method, and 2) Liquid-based cytology.

The conventional pap smear method is carried out by collecting the cervical cells using a spatula or endobrush, then smearing the cells directly onto a glass slide. Next, the slide is fixed immediately with alcohol spray. This immediate fixing is to prevent drying artifact from happening. However, the conventional method is not widely use anymore because the smear may contain other debris like blood, mucosal or inflammation cells. Also, it produces a thick layer of cells that are overlapping each other, thus, affecting the efficiency and accuracy of the screening process.

Instead, the Liquid-based cytology is being adopted now. This method employs the use of a machine known as ThinPrep processor. The processor will cleverly differentiate and separate the cervical cells from any debris, blood, mucous and inflammation cells. Another advantage of this processor is that it creates uniform and even layer of cells.

These are the instruments involved in a ThinPrep Pap Test:











From left: Spatula, endobrush
Picture taken from: http://www.imvs.sa.gov.au/tissuepath/graphics/spatula_conventional.jpg











Broom-like Brush
Picture taken from: http://www.cervexbrush.com/images/CombiLong.jpg












Vial with preservative fluid
Picture taken from: http://www.labnews.co.uk/cms_images/Image/Prod-Dec-07/31-NOV.jpg












Inside of the ThinPrep Processor
Picture taken from: http://cyto.igabinet.pl/data/user_files/image/TP%20Processor%202.JPG









Slide produced from ThinPrep
Picture taken from: http://www.muliabrothers.com/Picture1TP.jpg










Difference in the quality of the slides produced by Conventional and ThinPrep method
Picture taken from: http://imaginis.com/graphics/cervical-cancer/pap_smear.gif

An endobrush, spatula or broom-like brush will be used to collect the cervical cells. After which, the brush will be rinsed vigorously in a vial containing preservative fluid. The vial will then be loaded into the ThinPrep machine for processing.
A suction filter will be inserted into the vial to filter the cells, separating the cervical cells from any debris, blood, mucosal or inflammatory cells. The cervical cells will then be imprinted on the glass slide. After which, the machine will gently drop the slide into 95% xylene for fixing. After fixation, the slides will go through pap staining in the autostainer, Leica Autostainer XL. The slides will then be mounted and screened under the microscope for any abnormalities.

2) Processing of Non-gynaecological specimens

The processing of non-gynae specimens is quite different from gynae specimens.
First of all, the specimens will undergo cytospin to obtain the pellet. To the pellet, cytospin collection fluid (light green colour) will be added to the cells. The amount the fluid to be added depends on the size of the pellet, i.e the fluid added must be at least twice the amount of the size of the pellet. A pipette was then used to mix up the mixture.

After these, a cytofunnel chamber is prepared. A glass slide, together with a filter card are attached to the cytoclip and cytofunnel. The whole cytofunnel chamber is then loaded into the cytospin. Due to the pressure from the spinning motion in the cytospin, the cells will be imprinted in the slide, according to the size of the filter card. Similarly, the slides will be stained in an autostainer.
Although both gynae and non-gynae specimens are stained with the same stains, they are stained in different autostainers. This is to prevent contamination of the different types of specimen. After staining, the slides will be mounted and screened under the microscope for abnormalities.














Process of assembling the cytofunnel chamber
Picture taken from: http://www.thermo.com/eThermo/CMA/PDFs/Various/File_24579.pdf


In the cytology lab, all equipments used will be disinfected with reagent CIDEX OPA. For the other disposable equipments like pasteur pipette and supernatants, they will be disinfected and discarded into a small bin of dissolved chlorine tablet.

Research: Nanodrop

Hi, I'm JIAHUI (:

These 1 week and a half, I've been really busy!! With all new things that I need to learn, there's so much to blog about!

Nanodrop (ND-1000 V3.1.2)

(Picture taken from: https://www.pasteur.fr/recherche/genopole/PF2/images/nanodrop.gif)

NanoDrop is a low-volume spectrophotometer that is used to measure concentrations of cDNA, proteins and RNA. It does not require the use of curvettes. It is also capable of using just 1μl of sample to measure a wide range of concentration, from as low as 5ng/μl to as high as 3000 ng/μl. (1)

Method:
1. Wash the metal tip with ethanol. Wipe clean with Kim wipes by dabbing.
2. Wash the metal tip with RNase-free water. Wipe clean with Kim wipes by dabbing.
3. Choose program ND-1000 V3.1.2.
4. Click on nucleic acid
5. Sample type: other (for cDNA)
6. Constant: 33
7. Add 1.5μl of RNase-free water and blank it.
8. After each addition of sample, wipe clean with Kim wipes.
9. Add 1.5μl of sample.
10. Record 3 results.
a. 260/280 (RNA/DNA ratio)
b. 260/230 (RNA/protein ratio)
c. Concentration (ng/μl)


RNA/DNA ratio and RNA/protein ratio = ideally more than 2

Depending on your sample type, the constant changes.
cDNA: constant = 33
RNA: constant = 40

1. University of Texas (2009). NanoDrop. Retrieved on 26th June 2009, from: http://www.icmb.utexas.edu/core/DNA/NanoDrop/Nanodrop.htm

It's that simple! (:

Done by, Lim Jia Hui

0703605F