De Incredibles

SIP posting : week 20

2008-11-09T11:46:00.009+08:00

YES! Finally it’s the end of SIP. But it also means submission for reports and all are coming. Hmm.. Anyway, I was posted to blood bank for the last week. But I am going to post on another test done in the Haematology lab, which I was in for 3 weeks before blood bank lab.

In Haematology lab, it is actually one of the busiest lab. There are constantly a lot of samples sent to the lab to be tested. There are only two types of specimen sent; blood and CSF samples. I am going to share on Erythrocyte sedimentation rate test. In the lab, they make use of the method Sedi-rate P4-Micr System to test for ESR.

Principle

Erythrocyte Sedimentation Rate is the rate of sedimentation of red cells in the first 50minutes. This will happen when anticoagulated whole blood is allowed to stand for a period of time, which the red cells will settle from the plasma. The increase in ESR shows indicates some abnormalities in the body.

Sedimentation of the red cells actually occurs in 3 stages.
1) Preliminary stage: In the first few minutes, rouleaux occurs and aggregates forms.
2) The sinking period: This is where the sinking takes place at a constant speed.
3) Last stage: This is where the rate of sedimentation slows which is due to the aggregates settling at the bottom of the tube.

Materials

Ø Aquisel tube with stopper filled with 0.8ml of Trisodic Citrate. (Available commercially)
Ø Pipette (for ESR)
Ø Rack/Aquisel tube styoform holder
Ø Mechanical pipette
Ø Pipette tips
Ø Timer

Methods

1. The EDTA blood samples sent to the lab should be mix well.
2. 320µl of blood were pipetted from the EDTA sample to an Aquisel tube containing Trisodic Citrate.
3. The Aquisel tube is mixed well.
4. The pipette is introduced into the Aquisel tube. The pipette is pushed downwards until the blood fills the whole pipette. The blood level should be at “0” on the markings of the pipette.
5. The timer is set to 50minutes and time starts.
6. A small LIS generated sticker is to be stick over the Aquisel tube.
7. Another small LIS generated sticker is to be pasted on the ESR record book. (For record purposes)
8. A “miscellaneous testing” is stamped on the request form and initiated.
9. After 50minutes exactly, the values on the pipette is read and results are taken down in the ESR record book and the request form, which is also to be signed for traceability.
10. The results are to be entered and verified manually in the LIS system.

Reference value

Neonates 1 to 2 days old : 0 to 4mm/50minutes
Neonates 3 days to 1 month old : 0 to 10mm/50minutes
Paediatrics 2months to 12years old :0 to 10mm/50minutes
Adults more than 12years old :0 to 10mm/50minutes

IF the ESR values are higher than the reference value, it can means:
Ø Increase in fibrinogen and/or globulin
Ø Decrease in albumin concentration
Ø Decease in cell pack volume
Ø Tuberculosis, Rheumatic fever, kidney disorder, systemic Lupus Erythematosus (SLE)
Ø Anemia

*Things to take note
The specimen tubes should be checked, there should not be any blood clots or platelet clumps, if there are clumps present, the results would not be vaild. The test should also be done in 4 hrs after the blood collection as the red cells would become spherical after that and it affects the results. The ESR pipette should be left standing on a rack upright, if there is any tilting, it accelerates the ESR. If the blood is lysed, the results will also have error.

That's all!

Zhenling
TG02

Sip posting

2008-11-04T15:31:00.005+08:00

Hey guys, this is my last posting for our SIP.

I was posted to Histopathology lab for 2 weeks and everyday was packed with things to be done due to low manpower. Anyway, in the lab everyone has to be well rounded in all aspects such as immunology, sectioning, trimming/passing and so on.
The day starts with blocking/embedding of fixed specimens into paraffin wax that were prepared overnight. Next, the lab technicians will shave the blocks and immersed them in softeners for around ten minutes to achieve a smoother and softer cut to get the ribbons for fixing onto the slides. (Note that we have learnt these during pathology so I won’t go in detail, but you guys can ask questions about it)

The most interesting task that I look forward to everyday would be the passing part in the afternoon when we will retrieve all the specimens for the day and sort them accordingly. Passing is done by lab technicians whereas trimming is done by pathologists and the dictations are assisted by the technician. The difference is that the pathologists trim/section the larger kinds of specimens such as colon, breast tissue, stomach, etc and frozen section specimens (specimens received from the operating theater) whereas the technicians will pass specimens such as breast lumps, appendix, prostatic cores, etc. In our lab, appendix, intestines and breast tissues are common and it is quite rare to receive specimens such as aborted fetus, nose (however we did received it for the first time 3 weeks ago), leg, etc. I helped out daily with the dictation part by writing the measurements of the specimens received, their description and comments given by the technicians.

The thing that I would like to further touch on would be the retrieval of prostatic cores.
It is done through needle biopsies for the diagnosis of prostate cancer by the pathologist. A minimum of 8 cores are required and it is made up of 4 from each side of the prostate gland and the 4 cores are from the apex, mid, periurethral and base. The whole procedure is done using a biopsy gun which is inserted through the wall of the rectum into the prostate gland area. I’m not clear of the whole procedure as I don’t get to see it.
Next, during passing of the prostate cores, the length are noted and inked blue before being wrapped in a filter paper and place into a cassette for fixing. Note that the cassettes are to be placed in formalin as soon as possible to prevent the cores from drying causing shrinkage leading to inaccurate diagnosis.

Photos retrieved from http://www.upmccancercenters.com/cancer/prostate/biopsyneedle.html

These are examples of stained prostate core slides:

Retrieved from http://www.prostate-cancer.org/education/staging/img/Dowd_GleasonScoreFig1.jpg

Retrieved from http://www.hopkinsmedicine.org/hmn/F02/feature2.html

Yup, that is all, I’m not sure are the information sufficient cause in the histology lab, although I get to observe the procedure of the immunology staining process, but we are not allowed to do anything and there is not much learnt there. =)

Debbie

TG02

SIP sharing: LC-MS/MS

2008-11-02T21:56:00.001+08:00

This will be my last post.1 more week and internship is completed. I am still in chemistry lab (or to be more specific is chromatography lab). Last few posts have shared thin layer chromatography and column chromatography. This post will be on liquid chromatography tandem mass spectrometry (LC-MS/MS). LC-MS/MS is the last step that will be carried out for my MP. It is used for chemical analysis of samples.

Chromatography means separation of components of sample based on chemical or physical properties of sorbent. (e.g. hydrophobic interaction, hydrogen bonding etc)

(Image of LC-MS/MS: taken from www.agilent.com)
What is LC-MS/MS?
LC-MS/MS instrument consisted of high performance liquid chromatography (HPLC) coupled to mass spectrometer (MS). HPLC will separate the sample and detect by UV and MS carry out mass analysis.

Since LC-MS/MS consists of 2 part, HPLC and MS, I shall explain them separately.

HPLC
HPLC has an injector that will inject the sample into the column.
A pump then force sample through column.
It is based on the same principle as column chromatography (mentioned in my second post). The difference is that in column chromatography, the sample flows through sorbent by gravity and is more manual while in HPLC, the sample is forced through the column by pressure and is more automated.

After separation of sample, the components are detected by UV detector of HPLC. Different components can be detected at different UV wavelength. Therefore, it is important to set the UV detection at a wavelength which the analyte of interest can be detected. Other than UV detection, retention time also differs for different compounds since different compounds have different affinity for the sorbent. So, different compounds remain in sorbent for different duration.

Sorbent (stationary phase) often used are reversed phase and normal phase. An e.g. of sorbent for reversed phase is C18 and e.g. of sorbent used for normal phase is silica gel. In normal phase, compounds are eluted in increasing polarity (i.e. least polar to most polar) while reversed phase is vise versa. Of course, elution of compounds depends a lot on mobile phase used. (e.g. methanol, acetonitrile water etc)

As mentioned in alex’s post (http://codec-5.blogspot.com/2008/07/attachment-experience-from-weeks-i-ii.html), parameters that affect results can include injection volume, stationary phase, mobile phase, UV wavelength, flow rate, column diameter etc.

(Example of HPLC chromatogram: Image taken fromhttp://www.ualberta.ca)
X-axis is the time and Y axis is absorbance (AU). Absorbance is proportional with the concentration. Each peak represents one fraction.

The above is just explanation on HPLC…..now is moving on to mass spectrometry instrument.

MS
Why need mass spectrometry?
UV detection can only detect compounds with double bonds or ring structure. Compounds without those properties will not be detected by UV. Thus, mass spectrometry is needed since all compounds have mass.

Mass spectrometry instrument consists of a ion source, mass analyzer and mass detector.

Ionisation is required to forms ions for detection by mass spectrometry. Two main ionization modes that are often used are the electrospray ionization (ESI) and the atmospheric pressure chemical ionization (APCI).

Mass analyzers will store the ions and eject them through mass filter to be detected by mass detector. The ions are ejected by varying voltage.
(will not be elaborating more on this part since lots of physics (voltage, direct current (DC), alternating current (AC) etc) is involved and the entry will become several times longer)

A mass spectrum shows the fragmentation pattern of the analyte.i.e it shows the quantity of ions at different mass to charge (m/z) ratio. By comparing mass spectrum of sample with a database containing mass spectrums of thouasands of compounds….the identity of the compound in a sample may be figured out.

Image of a mass spectrum. Taken from http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/MassSpec/masspec1.htm
X-axis is the m/z value and y-axis is the abundance.

Lim Xin Ni

TG02

0607325H

SIP last entry-Heamatology

2008-10-26T19:12:00.004+08:00

Hellllooo! This is the last blog entry for SIP! 2 more weeks for SIP to end!

I'm currently attached to heamatology lab now.

This blog entry would be on cytospin.

I'll try to keep it concise:D

Introduction

For oncology patients, CSF is usually taken for testing. This is so as infiltration of blast cells into the CSF can occur for oncology pateints. To confirm such findings, CSF smear is done to examine blast cells which is important.

To make a CSF smear, a cyto-centrifuge is used. This centrifuge is specially designed for CSF and fluid sample.
A normal healthy individual should not have any blast cells in the CSF. This is used as a reference for screening of blast cells in the CSF.

How is it done..

1. Firstly CSF is collected in the EDTA tube.

2. 300 microlitre of CSF sample is then pipetted into the sample chamber of the cytopad.

3. A glass slide is labeled with a small LIS label and initialed by the medical technologists.

4. It is then loaded right behind the chamber frame

5.It is balanced by placing a empty chamber on the opposite side.

6. The lid is then placed and the whole rotor is placed on the drive hub and spinned for 5 mins.

After centrifugation, cells and the sediments in the CSF is being deposited on the glass slide.The glass slide is taken out for staining and the cytopad is discarded into the biohazad bag.

Staining...

The glass side is air-dried and stained manually using Leishman's stain.

1. The glass side is flooded with Leishman's stain
2. Then, twice the amount of phosphate buffer is added on top of the stain
3.The glass slide is left to stain for 5 mins
4. The slides are them washed and left to air dry on the rack
5. Slides are then viewed under the microscope under high power (40X, 100X)

If cells appear to be disintegrated, then the whole process have to be repeated. But if the cell population is too high, then the sample may be eluted in saline.

This whole process only takes about 15 mins! That's it for this blog entry:D
Hope u guys have enjoyed ur SIP...cya back in sch!:D

Neela
TG02

Week 16: SIP sharing

2008-10-12T12:00:00.003+08:00

Hi, this week i will be sharing on virus amplification.
Similar to what Shu Hui has previously shared in one of her entry on how to cultivate virus by infecting cells in a flask, my lab also uses this method for viral amplifcation.

Introduction
To amplify (or multiple or to increase in the number of) a specific virus, we need to have cultures of the host cells specific to the virus. The virus is used to infect the host cells, so that it is able to replicate and increase in number using the host cell machinery. We make use of this mechanism to amplify our virus stocks, once the volume is running low, when the virus titer (pfu/ml) is too low or when the virus stock is too old for usage.

For the type of virus we are using, the virus has 3 phases in its infection cycle.
The 3 phases can be classified into the early infection, late infection and very late infection.
Also, this virus we are using has two unique morphology, one is the budded form and the other is a polyhedron form.

During each phase of the NATURAL infection cycle, certain event takes place in the host cell as follows:

Early infection (0-6 hours) : Virus enters the host cells and decodes itself
Late infection (6- 24 hours) : Replicates and buds out of host cells without lysing the cells; this give rise to the budded form of the virus
Very late infection (18-24 to 72 hours) : Single or a few budded virus comes together and gets encapsulated in a polyhedron coat; this give rise to the polyhedron form of the virus.
When the host cells eventually lyses during this phase, the polyhedron form of the virus will be released.

Note
*The virus we use in the lab is genetically modified for research purposes. The polyhedron gene is deleted from the virus, because only the budded form of the virus is useful to our research. The polyhedron form of this virus is not able to infect any cells and thus cannot be manipulated for research purposes such as protein expression or gene delivery
Therefore, even til the very late infection cycle, only budded virus will form and buds out of the host cells we infect, when this genetically modified virus is use.* However, the host cells will still lyse after >72 hours.

Method
We use tissue culture flask as shown in (picture 1)to seed the host cells. These tissue culture flask are the same as those used to culture mammalian cells which has a inner flat treated surface for easier cell attachment.

We use different sizes of the flask, depending on the scale of our amplification.

There are 3 types of flask:
T25 (Yellow) : Smallest flask (25cm square)
T75 (Green) : Medium flask (75cm square)
T175 (Red) : Largest flask (150cm square)

T25 flask requires 5 million host cells
T75 flask requires 10 million host cells
T175 flask requires 20 million host cells

Picture 1: T25, T75 and T175 Tissue culture flask, Taken from www.wwmponline.com/cart/images/T/flasks-01.jpg

Before the required number of host cells is seeded into the flask, medium has to be first added into the tissues culture flask.
This volume of medium, is just enough to cover the surface of each flask:
T25 flask requires 5ml
T75 flask requires 10ml
T175 flask requires 18ml

Then the required volume of cells (depending on the concentration no. of cells/ml) is added to the medium in the flask. The flask is held horizontally and rocked back and forth at a gentle angle of about 30 degrees. It is rock for a few times to evenly spread the cells so that the cells covers the whole surface of the flask as it begins to attach to the treated surface of the flask. These flask are incubated for 1 hour (at 27 degrees celcius, 5% carbon dioxide incubator) for the cells to attach.

The cells are incubated at 27 degrees celcius because it is a non-mammalian cell.
The flask cap has a unique 0.22micrometers membrane, which allows carbon dioxide to diffuse into flask.

After the cells attach it looks like this:
This is just one small part of a T175 flask under 10x magnification

Picture 2: Virus Host cell (Taken with permission)

After the cells has attached to the flask treated surface, a certain volume of the medium is pipetted out to concentrate the cells in a smaller volume. This would allow the virus to attach to the cells more easily later, when the virus is added.

T25 leave behind 1ml

T75 leave behind 3ml

T175 leave behind 5ml

Add ~100microliters of the virus stock (the current one that we have) to the concentrated cells. The volume to add can vary, depending on how old the virus stock is. A older virus stock may have a lower virus titer (pfu/ml), so we can add more volume such as ~200microliters .

These flask are then put on a rotator (for 2 hours) which continuously rock back and forth to allow the virus to find and attach to the cells.

After the virus attach to the cells (assuming, because we can't see any changes yet), we add more medium to each flask to maintain the infected cells in the flask.

T25 add 5ml
T75 add 10ml
T175 add 18ml

The infected cells are incubated for 72 hours (at 27 degrees celcius, 5% carbon dioxide incubator), so that the cells are now in the very late infection cycle.

These are how the cells look like during the very late infection cycle: under 10x magnification with 1.6x aperture

Signs to take note of: Cells look swollen, has black dots in the centre of the cells, uneven surfaces of the cells and some cells have even fuse together.

Picture 3: Infected virus host cells after 72 hours (Taken with permission)

When these signs are observed in the very late infection cycle, we assume that majority of the viruses has bud out of the infected host cells, (virus are too small to be seen under our phase contrast microscope. It can only be seen unless a high power electron microscope is used) and it is very likely that the cells are going to lyse soon. Virus has to be collected before the cells lysed. When the cells lysed, enzymes released can cause break down of proteins found on the surface of the virus and lowers the virus titer greatly.

Virus collection

Since the cells are attached to the flask surface, as the viruses buds out of the infected cells, it is released into the medium. Therefore, virus can be collected by pouring the medium from the flask into a 50ml tube. The infected cells remains attached to the flask surface and are discarded.

This tube is spin down at 1000rcf for 10minutes to remove any cells debris (from a small proportion of infected host cells that may have detached). The supernatant is collected in a new 50ml tube without disturbing the pellet of cells debris. The new tube is wrapped with non-shiny side of the aluminium foil (because viruses are light sensitive), and stored at 4 degree celcius for future use.

Jean Leong
TG02

SIP sharing - Week 15

2008-10-05T00:00:00.000+08:00

Okie, time to blog again. I am attached to the biochemistry lab for this two weeks. During the campus discussion week, i've shared about G6PD deficiency testing. Now, i am going to blog on another test done in the biochem lab.

Except G6PD, most of the things done here make use of machines for testing. Neonatal Total Bilirubin is one of them that uses machine to analyse the results.

Background infor

We've learned how bilirubin is formed in school already. In case some of us forget, here is brief explanation of the process. Haemoglobin in the body is broken down into heme (iron and porphyrin) and globin (protein). Iron and protein are then re-utilized by the body while heme goes through a metabolic degradation where the porphyrin ring opened up to form biliverdin. Biliverdin is then further reduced to bilirubin, where it is transported to the liver via the plasma. It is in the liver where it conjugates with glucuronic acid to form bilirubin diglucuronide and excreted into the bile and then goes into the intestine. It will then reduced to form urobiliogen by the action of intestinal bacteria.

So, in this test, the sample that is collected is blood. The blood has to be spin down such that the plasma could be obtained to test for the bilirubin level.

Clinical Significance

When blood contains excessive bilirubin, it gets deposited in the tissues and cause the shade of yellow. It is also referred as jaundice or icterus. This condition may be due to 3 reason.
1) The bilirubin produced is more than the kidney can process and excrete. ( Hemolytic disease)
2) Having liver damage which resulted in inability to excrete normal amount of bilirubin produced.
3) The liver’s excretory ducts are obstructed and blocked.

Jaundice in the infants are quite common as their liver are immature or inefficient. Due to the deficient liver enzymes, the liver could not conjugate the bilirubin. The condition gets better when the liver is more matured and the enzymes are more functional. Possible treatment for the babies are phototherapy.

The bilirubinometer is used to measure the total bilirubin in the plasma of newborns. By measuring the absorbance of the plasma at 460nm, the concentration of bilirubin can be determined.

Methods

1) A heel prick is done and blood is collected via the capillary tube through capillary action.

2) A capillary tube is used to collect the blood of the newborn. When it is received in the lab, make sure that one end of the tube is properly sealed.

3) The capillary is placed in a plastic tube in the centrifuge in spin down at 3000rpm for 3 minutes. (make sure that one end of the tube is sealed and sealed end placed facing down)

4) Plasma should be visible at the top of the tube.

5) Spring-lock the capillary into a dispenser and slowly inject the plasma into a clean cuvette. ( Make sure there is no air-bubbles)

6) The capillary tube is then discarded and the cuvette is loaded into the bilirubinometer.

7) The measuring chamber will rotate downward to align the plasma of the patient to the optical path and results will be shown.

8) The results are to be recorded on the request form for entering into the system later on.

* If the reading exceeds the critical value of more than 300µMol/L, the test have to be repeated.

That's all folks.

Zhenling
TG02

SIP sharing---week 14

2008-09-26T14:16:00.004+08:00

It is the 14th week!!!…..left 6 more weeks to go. Jiayou pple. As I am still in a chemistry lab for the last 14weeks and for the future 6 weeks, this week post will continue from my first post. (week 4) which is on my major project which is on phytoplankton (diatom and dinoflagellate are some e.g. of phytoplankton.)

Here is a recap:
1) Seed phytoplankton cells
2) Harvest (centrifuge) when culture is ready
(Centrifuging takes 1 whole day. Why? Cause got about 20 litre of culture to spin down and there is only 1 rotar available----1 rotar can only carry 6 bottles, each bottle has a volume of 250ml, so after calculating…figure out !?!)

3) On the supernatant, carry out SPE
a. Methanol loaded first to condition the cartridges and wash away any impurities
b. Then load DI water to remove methanol
c. Next, load the supernatant (this take many days)
d. Then, load DI water again to remove salts (cause seawater contains salts)
e. Spin down the cartridges to ensure all DI water is removed from cartridges
f. Lastly, load methanol to elute out the extract.

So, now I have the eluent which contains the solvent (methanol) with the extract I want.
To get the extract and remove the methanol, evaporation is carried out.

Technique: evaporation
Used for removing solvent
Equipment used: rotary evaporator

(Image taken from http://www.eyelausa.com/category.php?cID=1)
It is a very easy process but take very very long for large quantity of eluent.
The bottle on the right is the evaporating flask and on the left is the receiving flask.
Water aspirator is connected to the hook-like thing on the condenser. Condenser pump is placed in a beaker with water covering the pump and the remaining space in the beaker is filled with ice.
So first switch on the condenser (the tall tube with coil inside) and water bath (it take time for the water bath to reach the temp set).

What temp to set for the water bath?
Temp set is around 40Cto 50C as long as it is below methanol boiling point.
Depending on the compound want to obtain, if it is stable and does not react easily (meaning it has few hydroxyl(OH) groups), then it is alright to set at higher temperature but the temp must be below the boiling point.

Eluent is poured into the evaporating flask, filling around 1/3 of the flask. If the flask is filled too full, eluent might spill out while evaporating and the whole process need to be repeated again.

After everything is set, the water aspirator is on. Water aspirator provides the vacuum required. Next, the pressure is gradually reduced. The eluent in the evaporating flask had to be monitored constantly in case there is lots of bubbles (‘boiling”). When this occurs, the pressure is released a while then reduced again. If violent boiling occurs, the desired compound may spill over to the receiving flask.
When evaporating, methanol from the evaporator become vapours and travels up to the condenser. The vapours then condense on the cool surface of the condenser. The methanol in liquid form then dripped into the receiving flask.

This whole process continue until the eluent in the receiving flask left a very small quantity, then I top it up with more eluent. When all eluent has been poured into the evaporating flask, then I evaporate the remaining eluent till dryness.
And the residue in the evaporating flask is the extract that I want to get. The residue is of a very very small quantity. To get sufficient amount for LCMS (discuss about it in next post) …. I need to accumulate about 3 times of SPE eluent.
After accumulating, I use methanol to dissolve and suspend the accumulated dry extract. Then I transfer the extract into a small tube using a dropper. As there may be some extract then cannot be removed from the walls of the flask, sonication using a sonicator is carried out to detach the extract from the walls.
Next I dry the extract in the tube using stream of nitrogen gas. (Why nitrogen? Actually nitrogen is a common gas used for drying.)

For the cells,
Methanol is used to remove the cells from the centrifuge bottles.
The methanol containing the cells is transferred into a conical flask.
The cells is left in the flask for about 1 week. Meanwhile, the flask is sonicated.
Methanol can lyse the cells to get the extract from the cells.
Sonication helps to enhance the process of cell lysis because high frequency sound waves can shear cells.
After that, the mixture (cells in MeOH) is filtered to remove the cells.
Methanol extract is evaporated as mentioned above.

LCMS on the extract will be discussed in the next post.

6 more weeks to go.
Hope my explanation is clear.
Any doubts, questions or uncertainty, juz ASK. =D
Lim Xin Ni
TG02
0607325H

Campus discussion :D

2008-09-26T13:39:00.002+08:00

Department: Biochemistry
Topic: Processing of specimens and LX PRO Analyser

Hey all, i'm in biochemistry lab now.
I'm going to talk about the processing of biochem samples and about the LX PRO Analyser.

Firstly when samples are reeceived in the biochem lab, the med tech has to check a few importatnt things.
1. Patient's name
2. Patient's IC no
3. Accession no
4.Tests ordered and collection priority: Urgent or routine

Samples are being spinned down as the serum is the one tested by the Analyser.
Samples that are in the primary tube could be directly placed on the rack with the adaptor. While the samples in the microtube should be proceed differently. The serum should be pipetted into the sample cup and a LIS acession label should be put on the sample cup to allow the analyser to read it.It must be checked that there are no bubbles in the sample cup as it will affect the results.Then only should it be placed on the rack with the adaptor.Rack is then placed on the autoloader of the Beckman LX PRO analyser.

Beckman LX PRO analyser is a machine that conducts all kinds biochemical tests.
It includes urea, glucose, creatinine, albumin, uric acid, total protein, Phospurous, magnesium, gentamycin etc.
Before patients' samples could be run, QC have to be done every morning.
The level of the modular chemistry agents should be checked. The ones with low levels should be loaded.

Next calibration has to be done.The analyser analyzer performs the calibrations. It automatically checks the calibration and produces the data at the end of the calibration. Error codes will be printed out against failed calibrations. Troubleshooting has to be done against failed calibrations and only if calibration passes,we can proceed to run controls.

QC schedule plan has the chemistries to do the QC run. Controls should be mixed well and then dispensed at the required volume into the specified control into the sample cup. Analyzer runs all the chemistries for that control. Troubleshooting has to be done for flagged chemistries. Control check has to pass before proceeding to run patient samples.

That's it.It's not very difficult, pretty easy.
Cya all:D

Neela
TG02

Campus discussion week's blog

2008-09-26T13:30:00.000+08:00

For this week, we are supposed to post an entry on any test that we have done. Therefore, I will share with all of you a test to detect for G6PD deficiency.

Introduction

G6PD (Glucose-6 Phosphate dehydrogenase) is a key enzyme in needed for the formation of NADPH in the hexose monophosphate pathway. HADPH is important for maintaining the integrity of the RBC(erythrocyte) membrane and functioning properly.

G6P-DH
Glucose-6-P + NADP+ <======> Gluconate-6-P + NADPH + H+

The lack of G6PD is the cause of hemolytic disease in newborn. It is an inherited condition where the body does not have enough G6PD. It can be also be transmitted as X-linked recessive. It can cause hemolytic anemia, usually after exposure to certain drugs (e.g. some oxidant drug) or food etc.

Principle of test

The name of this test is called the Fluorescent spot test. It is basically mixing the working substrate with the blood sample. After that, it is pipetted onto a filter paper. If NADPH is produced in the reaction, when the filter paper is placed under long wave UV light, the spot of blood will fluorescence.

Methods

1) G6PD working stock is removed from the refrigerator
It contains
- 1 tube of positive control
- 1 bottle of working substrate

2) One micro tube of intermediate control and one deficient control is taken out from a
-20degree Celsius freezer and then thawed.

3) For every specimen, checked that the name and the IC number on the form and the specimen bottle tallies. Also, check that the test ordered is correct as indicated on the sticky label.

4) The tubes were to be prepared for loading of specimen according to the number of specimen and label accordingly. Also, label 4 other tubes; blank, intermediate, deficient and positive

5) 100ul of substrate was pipetted into each tube at room temperature.

6) 5ul of the patient’s specimen is added into the specific tubes. Also, 5ul of intermediate, deficient and positive is allocated into the specific tubes. The mixture is pipetted up and down several times to mix it.

7) Allow 10minutues for the reaction to take place. Time is taken using a timer.

8) The filter paper that is provided in the kit is to be labelled with the number
9) After 10minutes is up, 10ul of the mixture from each tube of the patient’s samples are added to the filter paper. Also, 10ul of the blank, intermediate, deficient and positive are also added to the filter paper.

10) The filter paper is then placed into the incubator to allow it to dry completely (approximately 15mins)

11) After 15mins, take out the filter paper and view it under UV light.

12) Patient’s sample with normal G6PD should show strong fluorescence while patients. Samples that fail to fluoresce may indicate marked deficiency or total lack of G6PD enzyme.

13) It is reported as positive for samples that fluoresces and test is repeated for weak or no fluorescence.

For the specimen that test is needed to be repeated, check the specimen tube to see if there are any clots present, if there are no clots, then the test repeated over again to confirm. If there are clots, then inform the ward for a repeat of test with a new specimen.

That's about all =)

Zhenling
TG02

Attendance.

2008-09-26T10:26:00.003+08:00

Erm, Hi. haha. Anyway, I decided to talk about HBeAg that is done in the lab using Architect machine at the Immunology sector.

HBeAg determination is used to monitor the progress of hepatitis B viral infection.
Hepatitis B viral infection could be contracted via contact with infectious blood, body fluids from having sex with an infected person, sharing contaminated needles to inject drugs/ reused during treatment, or from an infected mother to her newborn.

HBeAg can be detected in the early phase of Hep B infection after the appearance of Hep B surface Ag. Both increases significantly during the viral replication in acute infection. HBeAg's presence correlates with increased no. of the infecious virus aka Dane particles, the occurence of core particles in the nucleus of the hepatocytes (liver cells, presence of hep B viral specific DNA and DNA polymerase in the patient's serum.

The virus reproduces transporting into the liver by first attaching to the membrane. The cpre particle then releases its DNA and DNA polymerase contents into the liver cell nucleus. Once it enters within the cell nucleus, the Hep B DNA causes the liver cell to replicate the DNA and produce more copies of the viral DNA. Then it produces copies of the virus within the cell before being released into the blood stream.

Architect uses Chemiluminescent microparticle Immunoassay (CMIA) technology and the presence/absence of HBeAg is determined by comparing the chemiluminescent signal in the reaction to the cutoff(CO) signal. If it is lesser than the CO signal, the sample is considered to be nonreactive for HBeAg.
Usually blood collected from plain tubes are used and the interpretation of the results are as follow:
Specimen with S/CO values < 1.000 = Non-reactive
Specimen with S/CO values >or= 1.000 = Reactive

Yup, thats about it for now. I'm open to questions, but pls don't make things too hard for me. Haha.

Debbie
TG02

SIP week 13-DNA diagnostic and research

2008-09-21T21:06:00.004+08:00

Helllloooo to everyone!
For the past one week i've been in DNA diagnostic and research lab.
it was quite interesting over there. I learnt about different kinds of genetic diseases, mainly thalassemia.
In DDRL lab, alot of DNA extraction is done and majority of their samples are blood.
Hence, i did DNA extraction and that is what i'm going to post this week.

Firstly cell lysis has to be done.

1. 3 ml of whole blood has to be added to 9ml of RBC lysis solution.It is inverted to mix well and incubated for 5 mins at room temperature.
2. it should be centrifuged at 2000rcf for 2min to pellet the white blood cells.
3.The RBC lysis supernatent is then poured off and the tube is then inverted on a clean absorbent paper for 10 seconds to drain the residual liquid.
4. The tube is then vortexed vigourously to resuspend the cells in the residual liquid.
5. 3ml of Cell lysis solution is added to resuspend the cells in the tube.
6. To lyse the cells, the tube is vortexed on high speed for 10 seconds.

Next protein precipitation has to be done.
1. 1ml of protein precipitation solution is added to the cell lysate.
2. The tube is vortexed at high speed for 20 seconds to mix the protein precipitation solution uniformly.
3. It is then centrifuged at 2000rcf for 5 min. This is to precipitate the proteins
to create a tight, brown pellet.

Following that DNA precipitation has to be done.
1. The supernatant containing the DNA ( leaving behind the precipitated protein pellet) is being poured into a 15ml centrifuge tube containing 3ml 100% Isopropanol
2. the sample are mixed by inverting until the white threads of DNA are being seen.
3. The tube is centrifuged for 3min at 2000rcf, where the DNA will be visible as a small white pellet.
4. The supernatant is poured off and the tube is drained by inverting the tube on a clean absorbent paper. 70% ethanol is added and inverted several times to wash the DNA.
5. The tube is then centrifuged at 2000rcf and then the ethanol is poured off carefully.
6. The tube is then inverted to drain on a clean absorbent paper, and allowed to air dry for 15 mins.

The last step is DNA hydration.
1. 250 microlitre of DNA hydration solution is then added.
2. Sample could be rehydrated by storing at 65 degree celcius for 1 hr or room temperature overnight.
3. the sample is then transferred to a ependorff tube and stored at 4 degree celcius.

This is the whole process of DNA extraction from blood.Its quite technical. If u guys want more explanation, pls do ask me.
Thanks and have fun!

Neela
TG02

Week 12

2008-09-15T09:59:00.013+08:00

Hey to all,

for the past 2 weeks, i was posted to biochemistry sector. Initially it was very boring as it was mostly observation work cause i wasn't allowed to do anything else besides loading and unloading of the samples into the centrifuge. But from the 4th day onwards, i was capable of identifying the which machine or sector to send the samples to according to the requested test.

In biochemistry, we use Beckman Coulter Synchron, LX20 PRO on closed tube samples. Anyway, we carry out a series of tests like liver panel, renal panel I, renal panel II, chemistry tests - Magnesium, Phosphate, Potassium, etc.

I will be touching on one of the tests done in renal panel, the glucose test. Glucose (Glc), is a monosaccharide (or simple sugar) used as a source of energy and metabolic intermediate.

http://science9.files.wordpress.com/2007/04/diabetes-glucose.gif

Both hyperglycemia (increase in blood glucose level) and hypoglycemia (decrease in blood glucose level) could be caused by renal dieases.

ie) Hypoglycemia - characterized by a drop in blood glucose level (<4.0>

In LX20, glucose test is used for the diagnosis and monitoring of carbohydrate metabolic diseases, neonatal hypoglycemia, pancreatic-islet cell tumours, etc. The principle used is the determination of the glucose concentration by an o2 rate method employing a Beckman Coulter Oxygen electrode. The ratio used is 1:76, which is 10 ul of sample to the glucose reagent from Beckman. Usually the samples are collected in EDTA tubes or flouride tubes (glycolysis inhibitor) and tests are carried out within 2 hrs from the time of collection. GLUCm assay must be calibrated every 48 hrs / with each new bottle of reagent.

The rate of O2 consumption is directly proportional to the concen of GLUCm in the sample.

As O2 consumption is measured instead of peroxide formation, peroxide must be destroyed without causing release of oxygen.

Therefore ethanol is added in the presence of catalase.

To ensure complete destruction of peroxide, iodide and molybdate are added to the enzyme reagent, causing the following reaction:

Yup, thats it for all. =)

Debbie

TG02

week 11- SIP sharing

2008-09-03T21:38:00.012+08:00

Last week review...

Result
Picture 1

This week I am going to share on how to get a recombinant Bacmid and how to package this recombinant Bacmid into a virus.

This will require 2 steps, Transformation into a special cell call 10a and Transfection into host cells respectively.

Method is just some common protocol, whats more impt is the principle =)

Transformation

Method

1. Obtain the 10a competent E.coli cells from -80ºC freezer (1 vial = 100μl) and carry it in ice box. Thaw the cells on ice.

2. Aliquot the appropriate amount of recombinant plasmid DNA into an eppendorf tube.

3. Place the tube onto ice.

4. Aliquot 25μl of 10a competent E.coli cells into the tube ON ICE, and incubate for ½ hour.

5. Meanwhile, check that antibiotic plates (Kanamycin, Getamycin, Tetracycline, Bluo-gal, IPTG) are warmed up in the 37°C incubator (plates are inverted to prevent contamination) and ensure that water bath is heated up to 42°C

6. After the incubation for ½ hour, heat shock the bacteria cells for exactly 45 seconds in 42°C water bath without shaking.

7. Immediately transfer it onto ice for 1 to 2 mins

8. In the LAF hood, add S.O.C medium at room temperature (similar to LB medium) in 1:9 ratio to competent cells. è therefore add 225μl of S.O.C medium into each tube.

9. Shake the tube at 37°C at 300rpm in an incubator for 4 hour.

10. Obtained the transformed cells from the incubator and place it in the LAF hood.

11. Using a sterile disposable spreader, plate the whole volume (250 µl) of the each tube of cells onto the pre-warmed antibiotic plate.

12. Allow both plates to stand in the LAF hood with covers fully covered for 1 to 2 mins to allow E.coli cells to attach to agar.

13. Invert the plates (to prevent contamination) and put it into the 37ºC incubator. (incubate for 24 hours to see colonies and 48hours to see blue and white colonies)

14. After 48 hours, obtain the plate from the incubator, parafilm it and store in 4ºC fridge.

Principle

The gene of interest in the recombinant plasmid is flanked by the left and right arms of Tn7 and contains ampicillin and gentamycin resistance gene for selection, and other features to form a mini Tn7. (Shown in picture 1-above)

Host cell for this plasmid, 10a cells, contains a bacmid with a mini-attTn7 target site and a helper plasmid. When recombinant combinant plasmid was transformed into the 10a cells, transposition between the mini Tn7 on the plasmid and mini-attTn7 target site on the Bacmid occurred to produce a recombinant Bacmid. (transposition is something like “transfer” the recombinant plasmid into the Bacmid).

Recombinant bacmid was selected out on specific antibiotics plates containing (tetracycline, gentamycin, kanamycin, IPTG and bluo-gal), restreaked on new antibiotic plates, PCR and analyzed, followed by extraction and purification using the Miniprep kit.

Tetracycline – because of tetracycline gene in the helper plasmid
Gentamycin- because of gentamycin gene found in recombinant plasmid
Kanamycin- because of kanamycin gene found int the Bacmid
IPTG & bluo-gal- For blue-white selection. Presence of recombinant plasmid in bacmid (white). No recombinant plasmid in bacmid (Blue).

Transfection

Method
*transfection can cause alot of cells to die, as the DNA:lipid complexes complex that are to be taken up by the cells are toxic, therefore to ensure we get as much transfected cells as possible we have to be very careful and ensure the conditions are optimal.

1. In a 6-well culture plate, seed 4 x 105 host cells/ml per well in 2ml of supplemented medium (medium suitable for culturing the host cells)

2. Allow the cells to attach at 27°C for at least 1 hour.

3. After about 15 mins later, obtain the unsupplemented medium (free of serum) from the fridge and a new 50ml tube and place it in the BSC.

4. In the BSC, aliquot about 12ml of unsupplemented medium into a new sterile 50ml tube, and return the original bottle back into the fridge.

5. We have to prepare 2 eppendorf tubes, 1 eppedorf tube for the cellfectin reagent and the other for the recombinant Bacmid DNA.

6. In the BSC, prepare as follows in 1.5ml eppendorf tubes:

Tube 1: Dilute 1µg (~0.7µl of our sample) of each purified bacmid DNA in 100µl of unsupplemented medium. (Important: ONLY pipette 1 time)

Tube 2: Mix cellfectin® reagent thoroughly before use by inverting the main tube 5-10 times. Remove 6µl of cellfectin® reagent and dilute in 100µl of unsupplemented medium.

Tube 3: Combine 100µl of the diluted Cellfectin® Reagent to tube of sample ( ~210µl). Mix gently by just tapping the bottom of the bottle, and incubate for 30minutes at RT in the BSC.

7. By now the cells should have incubated for about ½ hour. Obtain the cells on the 6 well plate from the incubator and check under the phase contrast microscope that the cells has already attached to the surface of the 6 well plate.

8. While the DNA:lipid complexes are incubating, remove the medium from the cells in the 6 well plate and wash once with 2ml of unsupplemented medium. Do this by pipetting away the old medium and replace it with 2ml of
unsupplemented medium and swirl the plate to make sure that the cells are washed. Then return the plate into the incubator.

9. After the ½ hour incubation of the Bacmid DNA: Cellfectin® Reagent complex in the BSC, take out the 6 well plate from the incubator and introduce it into the BSC.

10. Pipette out the 2ml of unsupplemented medium and add 0.8ml of unsupplemented medium to each well, followed by 200µl of the Bacmid DNA: Cellfectin® Reagent complex, swirl and rock the 6 well plate after adding.

11. Label the wells according to what was added and incubate in the 27°C incubator for 5 hours.
After about 4 hours of incubation, prepare unsupplemented medium containing antibioticsà 50units/ml penicillin and 50µg/ml streptomycin final concentration

12. After 5 hours, remove the Bacmid DNA: Cellfectin® Reagent complex from the well and add 2ml of supplemented medium to cells.

13. Incubate the transfected cells in the 27°C incubator for 72 hours or until we start to see signs of viral infections.

Principle
Purified recombinant bacmid were complex with cellfectin® reagent and transfected into host cells. (how cellfectin reagent work is already shared in week 1). This complex will cause formation of liposomal structure that causes host cells to uptake the complex.

The original bacmid before transformation was done, is actually a modified genome of the virus (got lac Z and antibiotic genes etc). The recombinant bacmid is the genome of the virus, now consisting of the recombinant plasmid in its Tn7 site.

After about 4 days, signs of viral infection start to appear, and viruses start to bud out.

Signs of infect:
1. Black dots in cell nucleus
2. Cells look swollen
3. Rough surfaces of cells
4. So cells will look fused together

Preparation of the supplemented medium containing antibiotics

The purpose of adding antibiotics to this medium that the cells are going to be incubated in for 72 hours is to prevent bacteria contamination. Bacmid is a piece of very large DNA, and since it is left as a DNA in the naked form to be incubated with the cells, it can easily be contaminated by Bacteria, to prevent this from happening we added this 2 antibiotic as recommended by the manufacturers.
we usually do about 200 folds of dilution
Therefore, since in total we prepare about 40ml of the supplemented medium in this case, and antibiotic mixture (200µl) and pipette up and down to mix thoroughly.

Supplemented and unsupplemented medium
For supplemented medium, we have to add supplement such as FBS (Fetal Bovine Serum). Serum is expensive and also needs to be purified from the medium if the DNA is to be used for transfection, as the serum can interfere with transfection and results in lower efficiency. But serum allows the cells to grow better as it provide more nutrient than unsupplemented medium. Unsupplemented medium is only use for transfection.

Viruses are collected in an eppendorf tube and stored at 4 degree celcius fridge. These viruses are amplified to produce second and third batch of viruses to be used (will be shared in next post). To check for the titer of the viruses constructed, plaque assays are done. (shared in post 1)

Thanks for reading this very long post n not v nice pics i noe.. hahas..cos its all molecular stuff, i cant really show u guys the real thing =/ Hope u all understand what i have shared.

Feel free to ask qns. Enjoy ur attachment =)

Jean Leong
TG02

Week 10 - SIP sharing

2008-08-31T15:01:00.005+08:00

Hi all,
for the past two weeks, i was attached to the histology lab. It was really a eye-opening experience i saw a lot of interesting things that i had never seen before, like the pathologist sectioning different organs and also observed post-mortem by the professor there. It was rather scary esp for the post mortem done on pre-mature babies.

Yup, but for this entry, i am going to share about frozen section.

What is it?
For frozen section, it is only done for emergency cases. It is done when the patient in the operating theatre and the doctor needs to know if that particular part of the tissue/organ is cancerous or if they have any benign tumors. The doctors would need to know if the margins pf tumors are clear. So what usually happens is they will take out a small section of the part of the tissue and sent to the lab for diagnosis. And as I mentioned they are still in the middle of the operation, they will need the results fast. Therefore, the whole process from the moment the pathologist starts to section to reading the results under the microscope.

When the tissue/section is cut out by the doctors in OT and ready for collection, the staff in the lab will go to the OT to collect the specimen. The section is sent fresh, which means no formalin is used to fix it etc. Tissues are collected in an ice box.

Specimen received must not be sent in formalin, but sent fresh. It will also not be performed for tissue from patients suspected of tuberculosis and other high risk infection like HIV, HBV and HCV etc.

All the patients name, accession number, specimen type, diagnoses are to be recorded down in a handbook for referring purposes. The pathologist will be the one sectioning and there will be a medical technologist to assist them in recording the morphology and dimensions of the specimen sent down. Specimens with history of infection will be handled in the BSC.

All types of tissues can be sent down as long as they are from the human body, usually the more common ones sent down are sections of breast tissue, uterus, gynaecology, cervix and etc.

Procedures

Preparation
-A small amount of O.C.T (optimum cutting temperature) compound is added onto a semi-cold specimen disc. (It is to hold the tissue on the disc and acts as an embedding medium)
- The tissue will be placed on the O.C.T compound
- The tissue will be covered with more O.C.T compound until the entire section is covered.
- It is then quickly placed in liquid nitrogen to freeze the specimen rapidly
- The section disc is inserted into cryocut and the heat extractor cylinder is used to flatten the surface for easier sectioning.

Sectioning
- The frozen section is performed using the Leica Cryocut 1800. The interior of this machine is around -20 to -30 degrees Celsius
- The tissue sections are cut at 5micrometers
- The sectioned specimen were then picked up by using a warm glass slide, the specimen will be attached to the glass slide by itself when it touch the surface of the slide.
- It is then to be stained with Rapid H & E. (used usually as when viewed under the microscope, this stain will give enough contrast and allow better diagnosis)

Staining
- The section fixed in
- 10% formal alcohol for 20s
- Rinse in water
- Haematoxylin for a min
- Rinse in ammonia water
- Wash
- Eosin for 15s
- Dehydrate in 2change of alcohol and 2 change of xylene.
- Mount in DPX

Lastly, the pathologist will view the slides under the microscope and interpret the results. ( to see if there are tumors seen etc.)

That is all =) Hope u enjoy reading

Zhenling
0606970B
TG02

SIP Sharing-Week 9

2008-08-23T20:10:00.009+08:00

HI, everyone, it’s my turn to blog again. As I am in a research chemistry lab and am not rotated anywhere, thus, for the past 9 weeks, I have been carrying out the same procedures mentioned in my first post (week 4). Thus, for this week’s post, I will be sharing on a procedure that I have observed in my lab.

Technique: Column chromatography
Principle: For separation of a mixture of compounds and isolation of particular component from a mixture
Solvents commonly used (in increasing polarity): Hexane (CH3(CH2)4CH3), dichloro-methane (CH2Cl2), ethyl acetate (CH3COOCH2CH3), acetone (CH3COCH3), methanol (CH3OH).

Column Chromatography: Image taken from http://orgchem.colorado.edu/hndbksupport/colchrom/colchromproc.html

In column chromatography, the vertical column is first packed with silica gel (SiO2) that is mixed with hexane. The gel is loaded above a plug of wool. The wool is for preventing solid materials from contaminating the products. The silica gel in the column is known as the stationary phase (also known as the sorbent). After packing the column with the sorbent and loading the sample, another plug of wool is placed above the sample and the sorbent. This is so that later when pouring the solvent in, there is minimum disturbance to the stationary phase and the sample as disturbance to the surface will result in poor separation.

The basic principle is quite similar to the SPE that I have mentioned in my first post. Except that in the SPE I have mentioned in my first post is use C18 (reversed phase) while the column chromatography I have observed use silica gel (normal phase) thus is based on increasing polarity. This means the least polar component of the mixture will be eluted first and the most polar one will be eluted last.

For column chromatography, a very important factor is the choice of solvent for elution for better separation of components. Solvent to use is determined by Thin Layer Chromatography (TLC).

What is TLC?

TLC is also a method for separation based on polarity but on a much smaller scale than column chromatography thus sample required is also much smaller, TLC is also faster than column chromatography. The stationary phase used in TLC is a thin layer of silica gel on a flat sheet (usually glass). The whole process is similar to paper chromatography (what is that?: Recall from sec chemistry), just that TLC is based on polarity instead of molecular weight.

Thin Layer Chromatography: Image taken from http://www.chemistry.adelaide.edu.au/external/soc-rel/content/tlc.htm

Using a capillary tube, a drop of the loading solvent containing the sample is applied onto the plate. Then, the plate is placed into the solvent submerging only the bottom of the TLC plate (the location where the sample is spotted.) The solvent will then slowly migrate up the plate. It is a matter of trial and error of different kinds of solvents with different ratio to find the ideal solvent to be used for column chromatography. The desired outcome is for the desired spot (the compounds we want) to be as near to the baseline as possible. Baseline is the location where the drop of sample is placed at.

Why is this the desired outcome?

Because on silica gel, the spot nearest to the baseline is the most polar and the spot furthest away is the least polar. So the spot nearest to the baseline in TLC is going to be the last to be eluted in column chromatography (Recall: Elution using silica gel is in increasing polarity). Being the last to be eluted will result in better separation and purification of the compound we want because the desired compound will travel over a longer distance thus is more purified cause more time exposed to the silica gel.

Why in TLC the spot nearest the baseline is the most polar?

Cause there is stronger hydrophilic interaction between the more polar compounds with the silica. Thus the more polar compounds are more attached to the silica (the stationary phase) and the less polar compounds will be carried further away by the solvent.
So in the case of the desired spot being too far away from the baseline, a less polar solvent is used to bring the spot closer to the baseline.

How to get a less polar solvent?

By increasing the ratio of the less polar chemical since usually a mixture of two chemicals are used for the solvent.

If the compounds are colourless, iodine vapour is then used to visualise the spots. This is done by dipping the plate into a bottle of iodine (powder form). Why use iodine in solid form? If recall from secondary school chemistry, pure iodine at room temperature is in solid state, iodine can sublimes at room temperature.

After finding out what solvent to use as the eluent, next we had to check whether the loading solvent is more or less polar than the eluent. If the eluent is more polar than the loading solvent, the eluent can be used directly in the column chromatography to remove the loading solvent. If the eluent is less polar than the loading solvent, a less polar solvent such as hexane is loaded first before loading the eluent. This is because if the loading solvent is more polar than the eluent, it will function as the eluent and push the sample forward, thus the sample will travel over a shorter distance, which will result in less separation.

In column chromatography, since the compounds are colourless, we will not know to collect till which mark, thus the eluent are collected in a series of small tubes. Thus TLC is carried out on each of the tubes with a drop of the desired pure standard beside. A spot that is at the same height as the pure standard with no other spots elsewhere indicates the particular tube where the sample is taken from is pure with only the desired compound in it. Next, all the samples which are pure with only the desired compound in it are combined.
Ta-ta…..pure desired compound is obtained from the mixture.

Feel free to ask questions.

Lim Xin Ni (0607325H)

TG02

Neela's SIP =) - Cytogenetics

2008-08-17T16:31:00.005+08:00

Hello folks! My turn 4 posting again:D
I'll keep it short and sweet k :D
Past 4 weeks i've been in cytogenetics and i learnt alot on chromosomes and their abnormalities.
In this posting i'll talk bout Wolf Hirschhorn Syndrome.

Wolf Hirschhorn Syndrome is causes by a genetic error in the 4th chromosome. The short arm of chromosome 4 has a partial deletion. This could be inherited from the parent chromosome where there is a translocation. Wolf hirschhorn is also known as 4p- syndrome because the deletion is in the p arm of the chromosome.

Retrieved on 17 August 2008 from, http://www.slh.wisc.edu/cytogenetics/cases/gifs/com_karyotypes/CoMMar97karyo.gif

Wolf hirschhorn syndrome affects fetal growth and developement, hence causing malformations in most body parts.

Facial features are described ad the "Greek warrior helmet" features. Their forehead is usually prominent with wide eyes and broad beaked nose. They are short in stature and have malformations of hands, feet and spine. Likely to have heart defects and malformations or underdevelopment of organs(urinary and genitals).Profound mental retardation, small head and seizures(50% of the individuals are affected) are the brain and muscular features of wolf hirschhorn symdrome.

Retrieved on 17 august 2008, from, http://medgen.genetics.utah.edu/photographs/diseases/high/17_mod.jpg

Wolf Hirschhorn syndrome could be detected by fluorescence in-situ hybridisation(FISH). It detects the deleted portion of the 4p chromosome by molecular probes which fluoresces. Addditional tests could include x-rays to look for bone and internal malformations, renal ultrasonography to examine the kidneys and magnetic resonance for the imaging of brain.

Thats all for this posting. i guess i've explained very briefly, so don't hesitate to ask questions.

Thanks:D

Neela

TG02

2nd posting!

2008-08-12T22:12:00.002+08:00

hmm, okayy, i'm next! haha. anyway, i know i posted late again, thats cause i was in jakarta. sorry to all.

Anyway, this is about my experience that things that i have learnt in processing for the past 2 weeks and hopefully more to come for the next 2 and a 1/2 weeks. haha.

Processing - the 'general office' in the lab as every sample received from the clinics or the wards are processed by the lab clerks first before being dispatched out to the respective sections such as routine / haematology.
I had to be the 'runner' when the canister tubing system was being upgraded. haha. great exercise for the legs. haha. But luckily, our supervisor decided to make the porters (not sure bout the spelling) come in the lab instead and i got my own 'coffee table' where i got to sort out the awaiting and non awaiting samples, the micrology, histology and routine samples. It can get really busy and messy at times.
Next, i had to be a paster, by sticking the ID labels on the order forms and the blood/urine /stool samples. It is crucial to check that the name on the specimen tallies with the order form as errors do happen. At times, empty or insufficient specimen bottles and unlabelled specimens are received and its is quite troublesome. However, as a student, i won't be able to be the one to call the nurse/wards to correct the issue. haha. (Its a good thing though.)
Then, for the most days of the past 2 weeks, fatin, elyn and i were up to our necks with the preparation of samples for the Health Awareness Week that is currently ongoing. On one of the days, we had to work till 7.30 p.m as we had to send the forms and the tubes to the respective wards. It was quite fun though.

Anyway, my job in processing is quite self explanatory but nevertheless, feel free to ask me anything. =)

Debbie.
Tg02

week 6- SIP sharing

2008-08-02T17:20:00.011+08:00

Hi everyone, it is my turn to share again =) Remember the first posting on the construction of the virus? Well, that is just a brief outline of what I have been doing for the past 6 weeks and will continue to be doing for the next few weeks.
*(Later I will share with you why it takes such a long time) ==> remember this one of the big question for this post, if you can answer this, then you did a *good job in understanding this entry =) heex. Those who cannot..(no such thing k) lols.jk. can ask me =)

Basically, because I am attached to a research lab, I planned to share more of some of the techniques the labs are using nowadays for molecular biology research work and some cell culturing techniques in the later posts.

I will break down the procedures into a few separate postings so that more detailed learning of each of the techniques can be shared.

For the procedure in the construction of virus it involves two MAJOR components.

1. Cloning
2. Packaging into the virus (I haven reach here! so this will be shared next next time k, because I foresee first step will take very long==> why? Find out by continue reading..)

1. Cloning
Cloning in this context refers to isolating a defined DNA sequence and obtaining multiple copies of it. Cloning is use to amplify (by using living cells such as bacteria E.coli) the specific DNA sequence containing the (gene of interest)*secret.

E.coli is often used because of the easy manipulation and it can produce high copies of plasmid.

(the gene of interest)*
E.g. Gene A ==> has to be cloned==> packaged into the virus==> get virus A==> introduce gene into mammalian cells
E.g. Gene B==> has to be cloned==> packaged into the virus==> get virus A==> introduce gene into mammalian cells

Each (gene of interest) has to be clone separately because each gene size is very big: up to 2 to 2.5kb.The longer the sequence of the gene the easier/ higher possibility of getting mutation in the gene when we are doing cloning. This is because during the procedure of cloning, it involves a lot of manipulation of the gene, especially PCR which can introduce single bp mutations. Even if very high fidelity (means accuracy of the copy to the source) PCR mix is used, the PCR process is not 100% perfect.

Bacterial cells containing the gene of interest is provided by the manufacturer in a very interesting form. It is in a small transparent glass bottle (cylinder shape) which has ¼ filled agar inside, and one very big white colony is seen sitting on the agar. (The bottle is only about 4cm in height and 1.5cm in diameter). The manufacturer provided it in the bacteria form on agar for easy usage, because we can just pick and use the bacteria.

Cloning can be further broken down into 4 main parts- this is called the “Cloning Strategy”
1(a) Isolation of DNA insert
1(b) Ligation
1(c) Transformation
1(d) Screening (Blue-white) / Selection strategy (antibiotics)

According to the procedures shared in post 1, this is a summary map of the procedures. (Construction of virus: cloning and packaging into the virus).
You CAN click on the picture

Picture taken from http://www.invitrogen.com

I am currently still in the green box, which means I have gone through these steps:

1(a) Isolation of DNA insert ==> Original (gene of interest) purchased from manufacturer, that comes in the form of plasmid contained in bacteria cells.

1(b) Ligation ==> of the (gene of interest) into Vector Backbone containing 7nTR (Facilitate later steps)==> to get a recombinant plasmid.

1(c) Transformation==> of recombinant plasmid into first type of E.coli cells (lets call it 5a)

1(d) Selection strategy ==> of the transformed cells (5a) (using amphiciliin antibiotic)==> then pick a one colony randomly *clue and culture it in broth *(15hours) ==> extract the recombinant plasmidè restriction digestion to cut out gene of interest and run on gel to confirm presence of gene of interest ==> and send for sequencing (using the fluorescence labeled ddNTP) to check for any mutation of the (gene of interest) IN THE recombinant plasmid ==> if there is mutation of the bp (seen in sequencing results) ==> go back to the plate and *re pick the colony, re grow them and sequence again. (thats partly why my lab people always say "pray hard!"==> cos its like kinda 'chance' thing even when optimal conditions are provided.)

Cloning process can take up to 1month if every goes well, but more than 1 month if we keep re picking the colonies. Usually, we will number the colonies on the agar plate, so that we know if that colony has already been picked before.

Sequencing process is done by sending it to an external company which does sequencing, E.g. The company 1st base. We have to provide the samples (picked from colony) and primers specific to the gene we want to sequence. Usually sequencing results using the fluorescent ddNTP method starts to become less sensitive when it reaches the later part (~700bp), therefore one primer for every 700bp we want to sequence should be provided). Sequencing *results usually takes about 3 to 4 days to arrive and usually a soft and a hard copy will be provided. The soft copy shows both the sequence in e.g. “actgactg form” and the electropherogram, while the hard copy shows us the electropherogram results with different coloured peaks but the different bases (a, t, g, c) only.

In the green box, it shows a recombinant plasmid which I am supposed to get to proceed to transformation of second type of E.coli cells (lets call it 10a) ==> shown in the picture named as “competent E.coli cells”.

Steps for :
Isolation of DNA insert
Therefore once I received the bacteria cells (gene of interest inside) ==> plate them on LB +amp plate to get single colony==> pick one colony==> grow in broth (15hours) ==>extract the DNA è PCR to amplify the gene of interest and introduce restriction sites è restriction digestion ==>

Ligation, transformation and selection
Ligation==> transformation ==> selection==> pick colonies ==> grow them in broth (15hours) ==> extract the DNA ==> restriction digestion to cut out the insert (confirm insert is inside recombinant plasmid) ==> sequencing (using the fluorescence labeled ddNTP)

Most of the techniques are just recapped from the molecular genetics and molecular Biology and culturing of bacteria from basic microbiology.

One thing which I found very interesting is this technique: Cloning of PCR product, I ask the other students and a lot people never heard of this before in school! so it’s a good thing to know =)

PCR refers to polymerase chain reaction, it is a process which allows the amplification of any region or even very complex genomes in just a few hours. (for details refer to link)

http://bcs.whfreeman.com/lodish6e/default.asp?s=&n=&i=&v=&o=&ns=0&uid=0&rau=0
go to this website, click on animation and then PCR

Cloning of PCR product

PCR product can be directly used for coloning? How?
A restriction enzyme recognition site can be added to the 5’ end of the oligonucleotide primers used for the PCR reaction. These sequences will also be into corporated into the amplified PCR product as the PCR cycles continue, it can later be digested to produce the specific sticky or blunt ends for ligation. The primer will bind to the complementary part of the DNA template, but the restriction enzyme recognition site does not match the template. However, since the direction of synthesis occurs in the 5’ to 3’ direction and specificity depends mainly on the 3’ end of the primer, the DNA still can be amplified efficiently and the product will contain the restriction sites at its end. Extra bases are also eventually and normally added to the 5’ end of the restriction site to ensure that the restriction enzymes functions efficiently.

Taken from PCR by (1997) second edition from C.R. Newton & A.Graham

Hope you learnt new things! =) Take care!

Jean Leong
TG02
0607991G

Reference

1. C.R. Newton & A.Graham.(1997) PCR (2nd edition). BIOS Scientific Publishers Ltd, USA.

Week 5 - SIP sharing

2008-07-27T12:51:00.007+08:00

Hello all!

Its my turn to share my SIP with you all Finally! Attachment is rather fun here and yup, i had learned alot of things from the staff working here. Till now, it has been a good experience. =)

In this hospital, there are alot of department. But for the 1st month of my attachment here, i am attached to the microbiology department, so basically what they do here is to culture and/or use different test to identify the pathogens that are causing infections. In this post, i shall describe one interesting reaction i saw in antibiotic susceptiblity test(which all of us should be very familar to). Susceptiblity test is done here to allow the doctors to know what antibioic to use base on the sensitivity of the strain of the bacteria. The method they make use of is called CDS method(invented by a group of people) to obtain better and more accurate results and reaction.

The method is the same as the one we do in micro lab. (See below)

Image Taken from http://web.med.unsw.edu.au/cdstest/GTF_CDS_site/WebPages/HomeLevel/ManualFrames.htm

ESBL(Extended Spectrum Beta-Lactamases) reaction which can be observed on the agar like this(below). Notice the reaction between cefotaxime(CTX), Augmentin(AMC) and ceftazidime(CAZ). There are keyhole like shape produced at CTX and CAZ. Cool right.!

Image taken from http://web.med.unsw.edu.au/cdstest/GTF_CDS_site/WebPages/HomeLevel/ManualFrames.htm

Usually after a patient's samples being cultured on agar shows significant isolates, it will be tested against beta-lactam antibiotics. Usually, Escherichia coli and Klebsiella especially can be resistant to these anti-biotics.

Beta-lactamase enzyme is a common bacteria resistance mechanism that is able to break down beta-lactam ring of penicillin-like drugs. They are able to hydrolyse antibiotics like CTX, AMC and CAZ. What lactamase enzymes do is that they break down the structural ring and make the molecules's anti-bacteria properties no longer functional. Therefore, when key-hole reaction can be seen, it shows that this bacteria strain is resistant to these antibiotics. The expression of the enzyme differs depending on the genetic control of the beta-lactamase production. TEM-1, TEM 2 and SHV-1 are the common plasmid-encoded enzymes that are resistant to penicillin.

The picture above shows Enterobacter being resistant.

So that is about all for this post. Hope that it is not too boring. Smiles =)

Zhenling TG02

reply to comments for week 1 =) by Jean

2008-07-22T10:48:00.005+08:00

Picture 3: Shows a general overview of how the virus can infect the cell and causes cell lysis. When the cells lysed, it forms a clearing in the plaquing medium. Each clearing can usually be visualized with the naked eyes or using some other techniques such as staining, microscopy, hemadsorption or immunofluorescence. Stain agarose is shown in picture 1 (above)
http://www.flutrackers.com/forum/attachment.php?attachmentid=326&d=1148840493

Picture 2: Schematic diagram of plaque assay.
Retrieved from http://homepages.strath.ac.uk/~dfs99109/BB211/phageinfect.jpg

Picture 1: Shows a Plaque assay on a 6 well plate. Agarose (or also known as plaquing medium) has been stained with neutral red for easy visualization of plaques. Staining is optional and our lab usually don’t stain it, we just check the number of plaques by holding the plate against the light to check for clearings/plaques.
Retrieved from: http://pathmicro.med.sc.edu/mhunt/plaque.jpg

To Han Yang

Thanks for reading my entry =) very sorry for the late reply also, didn’t check that there were comments posted for my entry.

1) The type of mammalian cells I’m using is fibroblast but I’m not sure if I can tell you the strain. Sorry about that.

2) The cells that contain the gene I want from step 1 are actually from a company called invitrogen. The purpose of culturing these cells is only to get the gene which was supplied to us as cell cultures, because only these cells is known to contain such genes. The medium we use is according to what was stated in the protocol supplied by the manufacturer. Which was to use normal DMEM with 10% FBS supplemented. I believed the DMEM is a complete medium which already contains essential amino acids, vitamins and salts. Essential amino acids (i.e. those synthesized by the body) are required by most of the culture cells together with cysteine and tyrosine. However, this requirement will vary from one type of cell to another. Probably the amino acids in the DMEM is already sufficient. (I’m not sure but I will confirm with my mentor.)
As for antibiotic, the common and original use of antibiotics is to reduce the frequency of contamination. However, since we are already handling most of our medium in the LAF (laminar air flow hood) and BSC (Biosafety cabinet), there is no need to add antibiotics. We chose not to add antibiotic because the presence of antibiotic in the medium may “hide/mask” the presence of low level contaminations such as bacteria. If there is contamination, we would want to detect it and not use that batch of cultured cells.

3) I do not know why. LOL. =X And my mentor is not around to answer me also. (I will confirm with him when he’s back). Hmmz, but I do have my own answer to this questions. I think the virus titer drops over time because it is being stored in the cold room and used quite often. Although we wrap the tubes with the dull side of the aluminium foil to prevent exposure of the viruses to light (because they are sensitive to light), the opening of the cap still exposes it to light. As for the temperature, I really don’t know. =)

4) Well, he did test the virus efficiency earlier on when he first got the batch of virus produced, but the virus titer can drop over time and we may need to measure it again after about 3-4 weeks. For more detailed understanding of how the lab works read below =)
Batch 1 we call it P1, batch 2 we call it P2, and batch 3 we call it P3 etc. The very first batch of virus we get by following the outline given in the blog is called batch 1 or P1. This batch of virus titer is quite low and if we want to increase/ amplify the titer, we use P1 virus to infect a newly cultured 6 well plate of cells and harvest the virus. This is known as P2 viruses. P2 is supposedly quite concentrated and we do not use this P2 directly. Instead, we do amplify another batch from P2, called P3. This P3 virus is the virus that will be kept in the cold room and used frequently.
We have to produce new P3 stock once P2 virus titer is low. P3 can be used for quite a long time as it has about 20 plus ml, and usually we only need a few hundred µl. Whenever we get a new batch of virus, be it P1, P2 or P3 we will do a plaque assay to get an estimate of the virus titer and then these virus are kept back in the cold room. Without plaque assay we can not know the virus titer.(it takes about 7 days for the results of the plaque assay to be out). Also, the virus titer can drop over time, but sometimes we may have already used the viruses for some of our experiments and if we realized that we did not get the expected results, we may troubleshoot and do plaque assays to check if the virus titer has dropped. However, plaque assay requires quite a lot of cells and since our whole lab share only one flask of cells, maintained in a spinner flask, we can only do plaque assays when we have cells. These cells will only be split on Monday, Wednesday and Friday.

5) Well, since my mentor has dealt with this type of cells before, he already knows roughly the doubling time of the cells. Moreover, the manufacturers who provided the cells also provided the protocol that we should incubate roughly half to one hour to get 50% confluency. The confluency of the cells can be checked under the inverted microscope. 50% confluency means that the cells occupies about 50% of each well of the 6 well plate surface. If it is not 50% confluence yet, we can always return it back into the incubator for the cells to continue to grow. Usually, it should not take more than 1 hour to get 50% confluency for this particular type of cell we are using.

6) Well, the goggle is actually not necessary for the handling of the viruses. We handle the virus in the BSC and should not come into contact with our eyes. The goggle is actually given by the safety department for everyone working in the lab and is recommended that we wear it when we handle with large bottle of chemicals/ liquids, probably TAE buffer when preparing the agarose gel, because of the potential spillage. So I wear my goggles when I am preparing the agarose gel for running my sample. The goggles is required also when we are doing bench work that is handling with a lot of eppendorf tubes, the water droplets on the inner side of the cap may accidentally come into contact with the eyes as we pop open the cap. Also, since I do not wear my specs in the lab, goggles is required.

Jean Leong
TG02

To Ms Chew and Peers

Thanks for reading my entry =)

Plaques are visible structures (usually a clearing as seen in the picture 1) formed in a cell culture contained within a nutrient medium. (e.g the plaquing medium). These clearings has no cells in it because the virus infect the cell and eventually lysing it. (lytic cycle shown in picture 3). The cells that were initially seeded in the well and allow the virus to infect it (from step 1 to 4), then overlay with plaquing medium for the cells to attach to the agarose and grow. (if you use bacteria then a bacteria lawn will be formed;but a different method is used, on agar plate instead of 6 well plate. Done Before in Basic Microbiology. However we do not use bacteria in this case). Just like how each colony is derived from a single E.coli cell in an agar plate, each clearing here is derived from one virus attaching to one cell, and when the virus infects the cell and multiple it continues to lyse the other cells around and forms a clearing around that area. As that clearing, also known as plaque, arise from one virus, we are able to determine the number of viruses (which is also known as titer which is also = no. of plaque forming units/ ml) by counting the number of plaques. =)

1) Harvest the cells that the virus can infect on the 6 well plates and incubate til cells are 50% confluence.
2) Perform serial dilution of the virus such as 10-2, 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8. in eppendorf tubes.
3) Remove medium from the cultured cells and add the diluted virus to the 6 wells and incubate to allow viruses to infect cells.
4) Remove the fluid (containing virus) in each of the well, as the viruses has already attached to the cells.
5) Add plaquing medium, which is a type of agarose gel containing medium to each of the well after the fluid is removed, and allow agarose gel to harden before incubating the plate of cells.
6) The function of the agarose gel is to immobilize the viruses so that it forms a clearing as it infects the cells.
7) These clearings (plaques) can be counted as plaque forming unit/ml, which is the titer of the virus.

Jean Leong

TG 02

To liyanah

2008-07-20T00:12:00.003+08:00

The carboys used in the research here are 20 litres plastic containers.

(Picture taken from http://www.nalgenelabware.com/products/productList.asp?search=3&category_id=135&brand_name=Labware&category_name=Carboys)

Week 4: SIP sharing

2008-07-18T23:13:00.023+08:00

Subject Title: Lab Techniques

Hi, Everyone. I am the fourth to post here and I am posted to a research lab. In the lab, there is a lot more chemistry processes than biology.

The lab I am attached to deals with phytoplankton…..a single-cell organism that can photosynthesize.

For this first post, I will be discussing on these:

1)Preparation of culture
2)Solid Phase Extraction (SPE)

The rest of the steps shall be discussed in future posts =P

For the preparation of culture, several carboys of sterilized seawater from an institute on St. John Island were collected personally. (take a ferry there =D). Why the seawater is collected from there and not other places? My supervisor say because seawater at that place is much more clean….less pollution. The seawater has been filtered through several different size filters and UV-sterilised at the institute.

Nutrients are added according to a given secret recipe.

The medium is then filtered using a filter unit.
The type of filtration carried out is pressure filtration.
This means gas is used to force the content from a cylinder tank to pass through the filter unit. The gas will travel from the cylinder, enter the pressure cylinder tank and force the medium from the tank to pass through the filter unit.

This is then followed by the addition of the seed cells. Then, the cells are left to grow in optimal conditions-optimal light intensity and optimal temperature.

After growing, we harvest the culture by separating the medium (supernatant) and cells (pellet) by centrifugation.
Time to harvest depends on the growth rate of the cells…….there is no standard amount of time to grow the cells…….if a lot of cells die then it is time to harvest.

The supernatant is then filtered.

Then SPE (solid phase extraction) is carried out on the filtered supernatant.

(Picture taken from www.biotage.com/DynPage.aspx?id=54871)

Principle of SPE: Process used to extract desired compounds from the medium according to the chemical or physical characteristics of the compounds.

In the lab I am working in, the SPE used is reversed phase SPE. Other types of SPE include normal phase and ion-exchange SPE. I am not very sure about these others types of SPE since only reversed phase is carried out in our lab. Reversed phase SPE extract compounds base on polarity.
Reversed phase SPE use cartridges that contains C18 silica-based sorbent (stationary phase) which retain compounds by hydrophobic interaction. C18 means the silica is linked by a link that consists of 18 carbon atoms.
So, these cartridges are placed on top of a tank.

Picture taken from www.biotage.com/Print.aspx?id=35833)

1)Then, methanol (organic solvent) is loaded first to remove any impurities in the cartridges. Next, DI water is passed through these cartridges to wash away the methanol otherwise the desired compounds cannot be retained on the sorbent.

2)Then, the sample is passed through the cartridges, and the compounds will be bounded onto the sorbent by hydrophobic interaction.

3)DI water is then used to wash away impurities that may be present in the medium. It must be ensured that no DI water is left in the cartridges after washing otherwise it will inhibit the elution step.

4)To get the compounds from the sorbent, methanol is used as solvent to elute the compounds. The methanol will disrupt the hydrophobic interaction resulting in the compounds to detach from the sorbent and flow out of the cartridges with the methanol.

Actions carried out on the eluent and how to get the compounds from the eluent shall be explained in future posts. =)

Since, throughout the SIP, I will be dealing with a lot of methanol, PPE such as goggle, lab coat, and gloves are necessary. Also, the experiments are carried out in fume hood whenever possible. (Methanol is toxic when inhaled/consumed and can cause blindness.)

Hope everyone is enjoying your SIP. ^_^

Lim Xin Ni (0607325H)
TG02
Group 9

2008-07-15T21:37:00.002+08:00

From the left(seen under microscope)

Parainfluenza-40x, Influenza A-10X, Adenovirus-20X

Neela's SIP =)

2008-07-13T20:11:00.004+08:00

RSV under microscope( 20x)
Picture courtesy of Molecular Micro lab(KKWCH)

Hihihihi! This is the 3rd week of SIP and its my turn to post! Hope all of you guys are enjoying your SIP!
Well I’m attached to the Microbiology department for 4 weeks. It has 2 laboratories, Microbiology itself and Molecular microbiology. One easy and interesting method we learnt back in school to detect virus/bacteria is immunofluourescence. I had the opportunity to practice it daily in Molecular microbiology lab.

In the lab I was attached to, tests were mostly done to detect respiratory viruses in children and infants. Such viruses include Respiratory Syncytial Virus, commonly known as RSV, Adenovirus, Influenza and Parainfluenza. Among these, RSV is considered the most common virus affecting children, especially below the age of 6. It rarely affects adults. RSV infects the lungs and the breathing passages causing wheezing, whooping cough and other breathing difficulties in children.

Usually when the doctor diagnoses pneumonia, bronchitis, bronchiolitis or other respiratory illnesses, they would send for tests to detect what virus has affected the child.
Samples sent to the lab are nasopharyngeal swabs or aspirates. Most common ones are swabs.
This is how it is being processed:

1.Firstly, samples should be checked with patient’s test request form to confirm that the identity is similar to the sample.
2.About 0.5-1.0ml of PBS should be added into a clear tube and the swab is placed into the tube.
3.A wire cuter is being used to snip the swab into the tube and capped. It is then vortexes until the suspension becomes turbid enough to be placed on the slide.
4.The slide is contains small wells and 2 wells are allocated for 1 sample. 1 drop in being placed on each well. This step is quite crucial as the size of the drop depends on the turbidity of the cell suspension. Have to make sure the size of the drop is as similar as possible on each well.
5.The slides should be air dried and fixed in cold acetone for 10 mines. It should be air dried after the slide is taken out from acetone.
6.10 µl of respiratory screen reagent should be added to one well. The respiratory reagent contains the seasonal reagent to detect the respiratory viruses.( RSV, influenza, Para influenza and adenovirus)
7.10 µl of RSV reagent should be added to the other well to detect for RSV as it is the most common virus affecting the children.
8.Slides should be incubated at 35-37 degree Celsius for 15-30 mins. After incubation slides should be rinsed with PBS for 5 mins to wash off the screening dyes.
9.They should be air dried and mounting fluid should be added followed by cover slips to be read under microscope.
10.If respiratory screen turns out positive and not the seasonal viruses, then the procedure is repeated again.

How to read the slides and recognize the viruses?

It is quite beautiful to see the slides under microscope. In fact, they are quite glaring when they turn out to be positive. Positive results mean that the cells fluoresce, which indicates that the patient is infected by that virus. Usually if the screening reagent and the RSV turn out to be positive, it proves that that patient is infected by RSV. However, if RSV turns out to be negative and screening reagent turns out to be positive, it means that the patient is infected by other viruses. It could be predicted by the morphology of the cells.

Parainfluenza would have a granular cytoplasm and the cells would look a little ‘bloated’.
Influenza virus would have a green cytoplasm.
RSV would have a crescent like shape at the edge of the nucleus.
These are the typical characteristics of the cells. However, this is not the case in all situations. Sometimes, it very difficult to identify them unless you are very used to looking at them
Hence, most of the time confirmatory screening has to be done using the specific screening reagents (like influenza, parainfluenza or adenovirus). If Influenza or parainfluenza is being identified, they are being sub grouped into influenza A or B, Parainfluenza 1,2 or 3.

So this is how immunofluourescence is done in Molecular microbiology! I know it’s quite lengthy, but sorry, I have to add all the details. Have fun!

BY NEELA
TG 02

References

http://kidshealth.org/parent/infections/lung/rsv.html

Oops.

2008-07-12T02:06:00.001+08:00

Sorry to all. To view the picture, please visit the website given.
Sorry for the inconvenience caused. =)

Debbie.

2nd attempt.

2008-07-12T02:05:00.000+08:00

Hmm.. i'll try again.

Picture taken from:

http://diagnostics.siemens.com/webapp/wcs/stores/servlet/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1015867~a_langId~e_-111~a_productId~e_172988~a_storeId~e_10001.htm

Week 2.

2008-07-12T02:02:00.000+08:00

HI all, sorry for the late submission, computer was down and all. In any case, for my first 2 weeks, I was posted to the Urinalysis/ Routine section. As most of the tests done are through automation, I was required to learn how to operate the machines, the quality control tests made at the start of the day or at specfic hour of the day. There were times when the urine FEME tests are required to be done manually using dipsticks due to insufficient amount of sample. My job was to load up the specimen into a tube, ensuring that it has been labeled correctly before being placed in a rack for loading into the Atlas machine.

Picture taken from:

http://diagnostics.siemens.com/webapp/wcs/stores/servlet/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1015867~a_langId~e_-111~a_productId~e_172988~a_storeId~e_10001.htm

Clinitek Atlas is a fully automated reflectance spectrophotometer used to perform urinalysis testing. The various results retrieved are the glucose level, bilirubin, ketone, specfic gravity, blood, pH, protein, urobilinogen, nitrie. Leukocytes, the color and the clarity of the urine. It is capable of performing up to 200 samples at one go and that each roll is made up of 490 reagent strips. The samples(minimum volume ≥ 5ml) are loaded onto the sampler tray (maximum 10 tubes) and placed onto the infeed area.

Firstly, the bar code reader would scan the sticker on the tube for identification purposes and the automatic pipette will come forth to collect the specimen via the syringe pump and then dispenses a specific amount of sample onto each of the reagent strip pads and into the SG well for determination of the specific gravity and clarity. The remaining sample are dispensed into the rinse well followed by a larger volume of rinse solution that rinses both the inside and outside of the pipette. Finally, the pipette dispenses the rinse solution into the SG well to ensure complete rinsing of the well.

Quality controls are done every morning by the morning shifts. The CLINITEK ATLAS Control solution(s) are prepared by soaking CLINITEK ATLAS positive and negative control strips in 12ml of deionised water each for 30 minutes before being run under ‘ Control analyze mode’. The results should tally with the reference range provided by bayer healthcare.

Yup, that’s all for now. Any questions, feel free to ask me. Haha. I will do my best to answer them. =)

Debbie.

Reply to Johan

2008-07-08T19:29:00.003+08:00

To Johan

Hi Johan, thanks for reading my entry.
The method we use to transform the cells is: Heat Shock at 42 degree Celcius

This is how we do it:

1) Use 50-100µl of competent cells (E.coli), and add 0.5- 1µl of recombinant vector and incubate tube on ice for ½ hour. (keep the cells cold so that transformation efficiency will remain high).
2) Warm up agar plate that has amphicillin antibiotic, in a 37 degree Celcius incubator
3) Ensure water bath is warm up to 42 degree Celcius, ready for the heat shock process. (heat shock the cells so they open up their pores and DNA enters)
4) After ½ hour incubation, heat shock the mixture in step 1 for 45s in 42 degree celcius water bath, and immediately transfer it onto ice for 1 to 2 mins. (cool it immediately so that the pores closes back).
5) Add S.O.C medium (something similar to LB broth) in a 1:9 ratio. E.g. 50µl cells: 450µl of S.O.C .
6) After adding the S.O.C medium, put into 37 degree celcius incubator with shaker for 1 hour.
7) Obtain the transformed cells form the incubator, do a 50x dilution and plate it on the amphicilin plate.
8) Do 2 spread plate, one with the 50x dilution and an undiluted spread plate.
9) Allow both plates to stand for 1-2 mins then invert the plate and incubate it at 37 degree celcius for 1 day.
10) The following day, observe for any colonies formed. These cells are resistant to amphicilin, because of the amphicilin gene in the vector, acquired through the transformation process.

Blue White selection strategy (use to determine the presence of insert)

The blue white selection strategy makes use of alpha-complementation, which means the alpha and the beta part of beta-galactosidase is “completed”.

The diagram (above above) (duno why it appear at the top) is a lac-operon: lac operon is only switched on when glucose is absent and lactose is present.
IPTG (analog of lactose) is a gratuitous inducer of the lac operon.
Grow the colonies in the presence of X-gal, which is a colourless compound that can be cleaved by ß- galactosidase to produce blue colouration.

The alpha and the omega peptide must be “completed” for ß- galactosidase to be functional to cleave X-gal.
The lac Z alpha gene is found in the vector, and only when the vector is introduced into the lac Z alpha mutant (no alpha peptide) during the process of transformation, then there will be alpha complementation and produce functional ß- galactosidase and cleave X-gal to produce blue coloration. (Blue Colonies).

However, if there is an insert in the vector, because of successful ligation, the presence of the insert will cause disruption to the lac Z alpha gene and there will not be alpha complemention > no functional ß- galactosidase> no cleaving of X-gal >colonies remain white.

Jean Leong

TG02

Week 1: SIP sharing

2008-06-30T22:04:00.011+08:00

Hi everyone! I will be the first in my group to share about what I have learnt during my first week of attachment. Attachment HERE is very fruitful because I get to learn from my mentor, we have a comfortable environment for work and the laboratory is very well equipped. =)

I am attached to a research laboratory which deals with delivery of proteins, genes and drugs into cells. The particular laboratory I am working in focuses on delivery of genes into mammalian cells, hence we are required to carry out a lot of Molecular Biology and Mammalian Cell techniques. My MP would also be on the delivery of certain genes into mammalian cells to get a certain desired outcome.

The delivery of genes is not as straight forward as I thought, because we have to keep constructing new viruses for each new type of gene we want to deliver! The virus vector is constructed to carry the desired gene so that it can be transferred to the mammalian cells via transduction.

For the whole of the first week, I have been learning how to construct viruses and how to measure the titer of the viruses that my mentor has already constructed. I am still learning how to construct the virus as it involves many steps. So far, I have only performed up to the digestion of the vector and the insert (step 6).

Outline on how to construct a Virus (The main points are in red, the details are for those who may be more interested in how the step goes about).

1) Culture the cells containing the desired gene that we want to package into the virus, on a tissue culture flask. Cells will adhere onto the treated surface of the flask, spread and grow.

2) Maintain the cells in healthy condition and prepare a suspension culture (by trypsinization) for counting and DNA extraction. Trypsin detaches the cells from the surface of the flask by breaking down protein that binds cells to the flask surface. Counting of cells is required as part of the DNA extraction protocol for maximal DNA extraction yield.

3) Use DNA extraction kit such as DNeasy® Tissue Kit to extract DNA from cell suspension and measure the concentration of DNA extracted.

4) Run the extracted DNA on agarose gel to confirm that the DNA is pure and check what is the kilo base pair of the DNA by comparing it with the DNA ladder marker.

5) Excise the band of DNA and dissolve gel to extract DNA using a gel extraction kit.

6) Obtain a suitable vector backbone to be ligated with the insert (gene) and perform
restriction digestion and ligation.

7) Transform cells with recombinant vector and select out the cells via antibiotic resistance and blue white colonies screening strategy.

8) Extraction of recombinant DNA using mini-prep kit.

9) Send the constructed recombinant DNA for sequencing to confirm the gene of interest is in the correct orientation for expression.

10) Tranfection of cells (to be infected by virus) with recombinant DNA. Transfection is a process of transferring DNA into the cells that are to be infected by the virus. The DNA is complex with some salts (calcium phosphate) from the transfection reagent that facilitate transport into the cells.

11) Virus is used to infect the transfected cells, and viruses will package these genes into their capsid as they replicate.

12) The virus titer is measured using plaque assay. (pfu/ml).

13) If the titer of the virus constructed is too low, we would need to amplify the virus.(another protocol).

14) Store these constructed virus stock in 4 degree Celsius cold room.

On the first day of my attachment, my mentor is already testing the efficiency of 2 types of viruses he has constructed months ago. He tested the efficiency of the viruses by measuring the virus titer (pfu/ml) using plaque assay. (Refer to below). Measuring the titer of the virus can be used for 2 purposes:

Determine the titer of virus constructed.
To determine the titer of virus stock that has been stored for a long time.

He needs to do this because the viruses are stored in 40ml centrifuge tube in the cold room at 4 degree Celsius, and overtime the virus titer will drop from 1 x 10^7pfu/ml (initial) to as low as 2.5 x10^6pfu/ml after 3 to 4 months. Measuring the titer is very critical because if the virus titer is too low, it will not be effective in infecting the cells and gene delivering efficiency consequently will be low.

Outline on how to perform Plaque Assay in a 6 well plate format

1) Harvest the cells that the virus can infect on the 6 well plates and incubate til cells are 50% confluence.

2) Perform serial dilution of the virus such as 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7 and 10^-8. in eppendorf tubes.

3) Remove medium from the cultured cells and add the diluted virus to the 6 wells and incubate to allow viruses to infect cells.

4) Remove the fluid (containing virus) in each of the well, as the viruses has already attached to the cells.

5) Add plaquing medium, which is a type of agarose gel containing medium to each of the well after the fluid is removed, and allow agarose gel to harden before incubating the plate of cells.

6) The function of the agarose gel is to immobilize the viruses so that it forms a clearing as it infects the cells.

7) These clearings (plaques) can be counted as plaque forming unit/ml, which is the titer of the virus.

That’s all for now! Thanks for reading my long entry! =)

Jean Leong
TG02

2008-06-16T23:10:00.004+08:00

Subject: Laboratory Management & Quality Assurance

Topic: Safety Aspects in Clinical Laboratories & ISO 14K

Area: Bloodborne Pathogen (BBP) Standard

Aim: Minimise exposure to BBPs found in body fluids

Requirements to ensure compliance

Effective Training Program 1
Exposure Control Plan 1,2
Form task categories to identify exposure potentials
HBV Vaccination
Develop procedures to handle exposure circumstances
Plan is reviewed & updated at least annually
Post-exposure Evaluation with Follow-Up 1
Documentation
Identification, testing, evaluation of source after consent taken
Engineering & work practice controls 3
Engineering: Biohazard hoods
Work practice:
PPE: gloves, laboratory coats, face protection 1,2
Handwashing procedures
SOPs. E.g. specimens secured in containers, disinfecting & decontamination, double gloves when handling body fluids 1
Respiratory Protection 1
Labelling
Recordkeeping 1

(Number of words: 100)

References:

Book
1.Montgomery, Lynn.(1995).Health and safety guidelines for the laboratory. USA: Chicago, III: ASCP Press.

Websites
2.Occupational Safety & Health Administration. (2008). Bloodborne pathogens. - 1910.1030. Retrieved June 15, 2008, from
http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=10051
3.Lab Safety Supply Inc. (2008). Complying with OSHA's Bloodborne Pathogens Regulation, 29 CF 1910.1030. Retrieved June 15, 2008, from http://www.labsafety.com/refinfo/ezfacts/ezf105.htm

Lim Xin Ni

TG02

2008-06-16T23:00:00.003+08:00

Area: Hazardous Chemical Standard

This is a requirement by OSHA; standard aims to minimize exposure of personnel to hazardous chemicals. 1

Goal: educate workers on proper handling, storage and disposal. 1

-Scope and application
All employees who use Hazardous Chemical. 1
-Chemical hygiene plan
Protects employees from health hazards of chemicals and keeps exposure below specified limit. 1
-Employee information and training
As much literatures and trainings on methods of protection to chemicals. 1
-Medical consultation
Medical attention to signs and symptoms of potential exposure. 1
-Hazard identification; MSDS
MSDS: identify properties of chemicals produced by laboratory. 2
-Recordkeeping
Personnel medical consultations and accidents records. 1

(100 words)

Bibliography

Website
1. Environment, Health Ernest Drlandd Lawrence Berkeley National Laboratory. (2008) Chemical Hygiene and Safety plan: Chemical hazard: defination. Retrieved June 15, 2008 from http://www.lbl.gov/ehs/chsp/html/hazard_eval_.shtml

Book
2. Montgomery, Lynn.(1995).Health and safety guidelines for the laboratory. USA: Chicago, III: ASCP Press.

Jean Leong
TG02

Fire saftey concerns and disaster plans

2008-06-16T22:43:00.000+08:00

-Fire codes
Explains the minimum requirements for fire prevention and safety. Adopted at facilities to ensure maximum prevention of fire.

-Signs
Let the people know where the escape routes and fire exits are(1)

-Storing flammables
Away from potential sources, by distance or by fire-resistant enclosures(2)

-Fire extinguishers and saftey equipment
Appropriate fire extinguishers should be placed at prominent places (E.g. exits) to act during fire. Inspection is important, check for any deficiencies and should be changed.(2)

-Fire drills and safety lectures
Should be practiced to train the laboratory staff and workers on various fire drill procedures. Preventative measures should be lectured(2)

-Disaster plans
Know the outline of the building and emergency exits(2)

(100 words)-info only

References

1.Wikipedia(2008), Fire codes, Retrieved on 16 June 2008
http://en.wikipedia.org/wiki/Fire_code

2.World Health Organization (1997), Safety in Health care laboratories, pg 33-38, Retrieved on 16 June 2008

Done by
M.Neela
Tg02

2008-06-16T22:35:00.004+08:00

Area: ISO 14000 - ISO 14001 and ISO 14004

ISO 14000 environmental management standard1 helps minimize environmentally harmful waste produced by an organization, namely 14001 and 14004.

ISO 14001 is a management tools1 that helps find out and manage the environmental impact1 of its activities, products or services1, to constantly improve its environmental performance1, and to systematically achieve environmental objectives1 and targets set providing evidence of it to protect the environment.2

ISO 14004 provides detailed guidelines on development and implementation3 of the of environmental management systems (EMS)1. It describes the principles, systems and supporting techniques3 on EMS to fulfill the requirements and operate effectively according to standards.1

( total:100words)

Reference

1. International Organization for standardization. (2008). ISO 14000 essentials. Retrieved June 15, 2008 from http://www.iso.org/iso/iso_catalogue/management_standards/iso_9000_iso_14000/iso_14000_essentials.htm

2. Praxiom Reseach Group Limited. (2008). ISO 14001 2004 ENVIRONMENTAL MANAGEMENT STANDARD TRANSLATED INTO PLAIN ENGLISH. Retrieved June 15, 2008 from http://www.praxiom.com/iso-14001-2004.htm

3. David Sheppard. (2003) What is the ISO 14000 Series?. Retrieved June 15, 2008 from http://www.bsu.edu/web/dcsheppard/ISO/iso14000series.htm#_top

Teo Zhenling
TG02

2008-06-16T22:22:00.000+08:00

Area: ISO 14000 Series

How does it work?

Provides a general framework of multiple standards to tackle the organization's environmental policy, plans and actions.[1]

Establishing a common reference for communication about environmental management issues between organizations and the public.[1]

What it achieves?

Internal objectives:

Providing assurance to management that controls the organization’s processes and activities.[1]
Providing assurance to the employees
Minimize negative effects of operation.

External objectives:

Providing assurance on environmental issues to the community by complying with environmental regulations that supports the organization's claims about its policies, plans and actions.[1]
Demonstrating conformity via several assessments

(Number of words: 99)

Reference:

1.International Organization for standardization. (2008). ISO 14000 essentials. Retrieved June 15, 2008 from http://www.iso.org/iso/iso_catalogue/management_standards/iso_9000_iso_14000/iso_14000_essentials.htm~

Debbie Tan
TG02