WEEK 19 - Virology

Hello everyone, for this post I will be sharing with u guys on my major project. My project is to evaluate the effectiveness between the conventional culture tube method and the shell vial method, in culturing of human Metapneumovirus(hMPV).

This study is implemented to compare the two methods. This is because shell vial is considered as a rapid method as results are seen in just a few days unlike tubes, which might take up to weeks. As for conventional tube culture, it is slower as there is no centrifugation. However, its advantage is that it can be maintained up to 28 days by repassaging, and thereby allowing a high level of virus to be harvested. But for shell vial, only a small amount of virus can be quantified. Thus, there is a need to evaluate both methods to determine the best way to isolate and culture HMPV. The evaluation will be based on the earliest date of detection and strength of fluorescence (the strength of the fluorescence will indicate how well the cells is infected by the virus). Therefore, a stronger fluorescence will show that the method is better.

Basically for this project, I will inoculate isolate into both shellvials and tubes. Shellvials will be monitered for cytopathic effect ( refer to my 2nd post) and they will be screened by IF each day, for a period of 7days. Whereas for the tubes, they will also be examined for CPE and screened when necessary, for 21 days. Changing of media will be done as required. Repassaging will be done every 7days, or when degeneration occurs.

Diagram taken from
http://www.sks-science.com/images/358606LRG.jpg
http://www.aname.es/microscopia/ems/preparation/219ems.GIF


Materials
Phosphate buffered saline (PBS)
hMPV isolate
Serum free- minimum essential media( with crystalline trypsin)
Vero cells
LLc cells
Hep cells
RD cells
hMPV monoclonal antibody
Conjugate

Equipments
37 ̊C Water bath
Centrifuge
Incubator
5ml plastic tube
1ml disposable pipette
Forceps
Micropipette
Pipette tip
Wastebin
Moist chamber
Slide warmer
Shaker

Inoculation of isolate into shell vial and tubes

1. Thaw the frozen isolate in water bath for 10 to 15 minutes( ensure that it is fully thawed)
2. Centrifuge the isolate in the 4̊C centrifuge at 2000rpm for 10 minutes.
3. Remove the supernatant from the tube and transfer into a sterile 5ml plastic tube.
4. Discard the media in the shell vials and wash 2 times with PBS.
5. Inoculate 0.2ml of the supernatant into all the shell vials and tubes( except for the control), using a micropipette.
6. Centrifuge the shell vials at 1800rpm for 30minutes.
7. Preadsorb the tubes in rack (stationary state) for 1 hour.
8. Add 1ml of media (containing crystalline trypsin) into the shellvials and tubes
9. Incubate shell vials in 36±0.5 ̊C incubator.
10. Arrange the tubes in roller drums and incubate in 36±0.5 ̊C incubator overnight.

Screening of coverslips by IF method

1. Vortex coverslip to dislodge the cells.
2. Add appropriate amount of PBS to the vial and centrifuge at 2000rpm for 10 minutes.
3. Discard PBS and repeat step 2 again.
4. Discard PBS, leaving a small amount behind.
5. Mix the remaining PBS and the cell pellet with a dropper, to form a cell suspension.
6. Label the slide that is used for spotting.
7. Spot the well in slide adding a appropriate amount of suspension to the well.
8. Dry the slide on a slide warmer.
9. Fix the slide in acetone for 10 minutes.
10. Add monoclonal antibody using a micropipette, ensure that it is well spread (Vortex mab before use).
11. Place the slide into moist chamber and incubate at 37 ̊C incubator for 35 minutes.
12. Immerse the slide in PBS to wash for 10 minutes, before placing it on a shaker.
13. Dry the slide on the slide warmer.
14. Add conjugate to the slide.
15. Repeat step 11-13.
16. Add mounting fluid to the well and mount it.
17. View the slide under fluorescence microscope and record the results.

That’s all for my last post. Happy reading!

Shihui
0607135A

Week 18 - Medical Microbiology

After the urine specimens have cultured onto Blood Agar plates and Cysteine Lactose Electrolyte Deficient agar plates, the plates will be incubated at 35°C overnight. The next morning, the plates are read and results are reported. [1st post recap]

Some of the most common Urinary Tract Infection (UTI) causing agents are:

Escherichia coli
- Flat, size varies

Klebsiella spp.
- Mucoid and spready colonies

Proteus spp.
- Swarms in blood agar plates

Pseudomonas aeruginosa
- Spready, irregular shape
- Grape-like smell

Plates are grouped into further and no further tests carried out.

1. No further identification / biochemical tests carried out:

- For plates that do not have any growth -> Report as No bacteria growth.
- For plates that do not have significant number (less than 50,000 cfu/ml) of bacteria growth:
a. Check whether the plate is a pure or mixed culture.

b. For mixed cultures (2 or more organisms present), Report as No significant bacteria growth.

c. For pure cultures, check the colonial morphology and presume the type of microorganism present. If the microorganism is not one of the common UTI causing agents -> report as no significant bacteria growth.

d. If it is one of the common UTI causing agents, check the FEME results. If the WBC count is less than 5, the patient may not be suffering from UTI. Therefore it is reported as no significant bacteria growth.

e. If the WBC count is more than 5, it may suggest UTI and the results will be reported as less than 50,000. But still, identification test is not done because the viable count is less than 100,000 cfu/ml.

**Viable count of more than 100,000 cfu/ml will then be considered as having UTI**

- For plates with mixed bacteria growth:
f. If the viable count is 50,000 cfu/ml containing 2 or more types of organisms -> Report as Mixed 50,000.

g. If the viable count is 100,000 cfu/ml containing more than 2 types of organisms -> Report as Mixed 100,000. These may suggest a contamination or mid stream urine is not collected. It is unclear as to which organism is responsible for UTI therefore the viable count is reported without any further tests done.


2. Further tests carried out:

- For plates with significant bacteria growth,
a. Containing 100,000 cfu/ml of pure culture -> Report as 100,000.

b. Containing 100,000 cfu/ml of 2 types of organisms present -> Report as Mixed 100,000 with doubtful significant.
[Doubtful significant is included because there are 2 types of organisms present and the organism responsible for UTI is uncertain]

c. Containing 50,000 cfu/ml of pure culture -> Report as 50,000 and doubtful significant.
[In this case, even though the viable count is only 50,000 cfu/ml, it is a pure culture that may or may not suggest having UTI. Therefore further biochemical tests are still carried out and doubtful significant is reported together with the results.]

In summary, only plates with more than 100,000 cfu/ml [both pure and mixed cultures (2 types of microorganisms present)] and also pure cultures with 50,000 cfu/ml will be sent to the Investigation Laboratory to conduct biochemical / identification tests.


LeeJin
TG02

Week 17 - Hb Electrophoresis (Haematology)

I was posted to the Hb electrophoresis lab for 3 weeks. Samples ranging from EDTA blood tubes, plain blood tubes and cord blood are sent here. Tests such as Kleihauer test and Sickling tests are doone here. Since quite a number of them have posted on the Kleihauer test, I shall post on the Sickling test. =)


Sickling test


USE:
Sickling test is used to demonstrate the sickling phenomenon in test samples. The technique detects both the heterozygous (AS) and homozygous (SS) forms of sickling and is a rapid screening test for the Hb S status of an individual. It is also done as a confirmatory test after alkaline and acid haemoglobin electrophoresis. Haemoglobin electrophoresis is able to differentiate between homozygous and heterozygous forms of sickling.

ADDITIONAL INFO: From the haemoglobin electrophoresis, heterozygous (AS) sickle cell trait will show 60% HbA and 40% HbS. In homozygous (SS) sickle cell anaemia, there will be a higher percentage of HbS (more than 50%) as compared to heterozygous sickle cell trait.

For sickle cell trait (AS), one of the genes is HbA, the other is HbS. In other words, the person is a carrier of the sickle haemoglobin gene but do not display symptoms of sickle cell disorder. However, one has to be careful when doing activities that require less oxygen such as scuba diving. For sickle cell anaemia, both the genes are HbS and the person will display symptoms of sickle cell anaemia such as anaemia and pain.


PRINCIPLE:
Sickling phenomenon occurs with low oxygen tension as HbS has a decreased oxygen affinity. A small drop of blood added to a reducing agent (sodium metabisulphite) to induce sickling in susceptible cells. It is then sealed between slide and cover glass and incubated at 37degrees for the sickling phenomenon to occur.


PROCEDURE:

1. 5 drops of freshly prepared reducing reagent (0.2g of sodium metabisulphite dissolved with 10mL of millipore water) is added to 1 drop of EDTA blood on a slide.
2. The reagent and blood is mixed with the pipette tip on the slide and covered immediately with a coverslip.
3. It is then sealed with petroleum jelly-paraffin wax on all 4 sides of the coverslip. It must be properly sealed to prevent false negatives.
4. The slide is then placed in a black box to ensure moisture when it is placed in the incubator at 37degrees for 2 hours or overnight. This is to prevent the preparation from drying out and obtaining false positives.
5. The slide is then examined under the microscope.


That's all for my last post for this 20-week long SIP! 3 more weeks! Yay! JIA YOU everybody. lol.


MALERIE
TG02

Oh my god. How time flies. This is the last 5 weeks of our SIP already. Before we know it, we’ll be back in school tackling tutorial and lecture quizzes and term tests. Ok then, let me cut the chase and get on with my post.

Anyway, for the past 2 weeks, I have been attached to the Serology department. Within this department, there are 2 labs: STD lab and Serology lab. In the STD lab, tests for syphilis and gonorrhoeae are done. But I think the gonorrhoeae section will be moved to the Bacteriology department at the beginning of November. In the serology lab, tests for autoimmune and infectious disease are done. But for this post, I shall just concentrate on the tests for syphilis.

Let me give a brief introduction to syphilis. I am sure you all know that syphilis is a sexually transmitted disease. It is caused by bacteria known as Treponema pallidum. This organism belongs to the order Spirochaetales and is a member of the Treponemataceae family. It is a thin, delicate organism with tapering ends. Its size can vary from 6 to 15 microns in length with a thickness of around 0.25 microns. The organism also has 6 to 14 spirals with pointed ends that have finely spiral terminal filaments. Syphilis can be divided into several stages: early syphilis, late syphilis and congenital syphilis. Early syphilis is the initial stage of infection while late syphilis occurs only 10 to 20 years after initial infections. Congenital syphilis is not connected to early or late syphilis. It is syphilis infection in infants.

Tests for syphilis can be divided into non-treponemal and treponemal tests.


Non-treponemal test
Non-treponemal tests are screening tests as they screen patients for non-specific reagin antibodies that are produced during an infection. The antigens used for screen are lecithin and cardiolipin. When treponemas is present, the lipid from the cell surfaces would cause the host to produce anti-lipid IgG and IgM antibodies. So the lecithin and cardiolipin antigens will bind to these antibodies, forming complexes, which will stay suspended in the tests via flocculation. The type of non-treponemal test used in the lab is the Venereal disease research laboratory (VDRL) test.


Venereal disease research laboratory (VDRL) test

Principle:


VDRL test is a slide microflocculation test for syphilis. When treponemas is present in the host, anti-lipid IgG and IgM antibodies known as reagin will be produced, in response to the lipid on the treponemal surface. These antibodies will then react with substances on the mitochondrial membrane. The antigen used in this test is mixed with buffered saline and consists of cardiolipin, cholesterol and lecithin. These substances will react with the reagin present in the serum of syphilitic patient.

A reactive test means that flocculation, a combination of principles of precipitation and agglutination, has occurred when the antigen reacts with antibody, thus forming antigen-antibody complexes. These complexes can be viewed as big clumps under the microscope. A weakly reactive test means that the clumps are smaller than that of a reactive test. A non-reactive test means that no clumps are observed under the microscope.

The test is divided into qualitative test and quantitative test. For qualitative test, it just indicates whether the sample is reactive or not. For the quantitative test, dilution of the serum will be done. So the highest dilution with a reactive result can be known.

However, as reagins may be produced in other treponemal diseases and in response to a non-treponemal disease, which results in tissue damage, a reactive VDRL test does not confirm a T. pallidum infection resulting in syphilis. But for a reactive VDRL test, a treponemal test (e.g TPPA) would follow to confirm the reactive result.


Procedure:

Qualitative test


1. Spin the blood at 3000 rpm for 10 minutes.
2. Transfer 50 uL of serum onto 1 ring of the ceramic slide.
3. Dispense 1 drop (17 uL) of antigen suspension onto the same ring using a syringe.
4. Place the slide onto a rotator for 4 minutes at 180 rpm.
5. Immediately read the slide at 10X objective under the microscope.


Quantitative test
1. Dispense 50 uL of 0.9% saline onto 5 separate ceramic rings.
2. Transfer 50 uL of undiluted serum into the first ring to make a 1:2 dilution and mix the solution.
3. Transfer 50 uL of diluted serum into the second ring to make a 1:4 dilution and mix the solution.
4. Repeat this step for the 3rd (1:8), 4th (1:16) and 5th (1:32) ring.
5. Add 1 drop of antigen suspension into all the rings.
6. Place the slide onto a rotator for 4 minutes at 180 rpm.
7. Immediately read the slide at 10X objective under the microscope.


Results:

Qualitative test


Medium/ large clumps - Reactive (R)
Small clumps - Weakly reactive (WR)
No clumping/ very slight roughness - Non-reactive (NR)


Quantitative test
- The last dilution titre that produces a reactive result will be reported.
- E.g If 1:16 is weakly reactive and 1:8 is reactive, 1:8 dilution will be reported.




Treponemal test

For treponemal tests, instead of non-specific antigens being used, antigens specific for T. pallidum is used to detect antibodies against T. pallidum. These tests are used as a confirmatory test and can detect all stages of syphilis, excluding the first 3 to 4 weeks after initial infection, as humoral antibodies specific for T. pallidum have not been produced. In secondary, latent and congenital syphilis, the treponemal tests would be 100% reactive.

However, these treponemal tests cannot be used to determine the efficacy of treatment or the presence of a re-infection as the tests are qualitative tests.

One of the treponemal tests carried out in the lab include the Treponema pallidum particle agglutination assay (TPPA).


Treponema pallidum particle agglutination assay (TPPA)

Principle:

The TPPA test is used to detect antibodies specific to treponemas, which causes syphilis. For this test, coloured gelatin particles carriers sensitized by T. pallidum antigens are used. So the patient’s serum is first diluted with sample diluent in microplate wells. Then, the sensitized gelatin particles will be added to the diluted serum. If antibodies specific to T. pallidum are present, it will bind to the particles, forming a smooth mat of agglutinated particles. For non-agglutinated particles, they will gather to form a button at the bottom of the wells. [8] This will indicate a negative test. As a control to ensure non-specific reactions have not occurred, unsensitized cells will also be used. If there is agglutination with the unsensitized cells, it means that there is non-specific agglutination, thus rendering the test as not accurate.


Procedure:


1. Label 4 wells on the microtitre plate wells 1 to 4.
2. Add 100 uL of sample diluent (provided by the test kit) into well 1 and 25 uL into wells 2, 3 and 4.
3. Add 25 uL of serum into the first well and mix thoroughly.
4. Transfer 25 uL of diluted serum from the first well to well 2 and mix thoroughly.
5. Repeat this step for wells 3 and 4 and discard the last 25 uL taken from well 4.
6. Add 25 uL of sensitized cells into well 3 and 25 uL of unsensitized cells into well 4.
7. Place the plate on an automatic shaker for a few seconds and incubate the plate at room temperature for 2 hours.


Results:

- Particles settled in the centre of the well in the shape of a button: Non-reactive (-)
- Particles concentrate in the shape of a compact ring with a smooth round outer margin: Indeterminate (±)
- Definite large ring with a rough multiform outer margin and peripheral agglutination: Reactive (+)
- Agglutinate particles spread out covering the bottom of the well uniformly: Reactive (++)

The results

So, this is a description of what I did in the STD laboratory. Hope that this post has been ‘understandable’.

5 more weeks to go. So let’s carry on working hard for our SIP and MP =).

- Li Ping-
0607498C
TG o2

In my project, PCT is analyzed on two different platforms, COBAS 6000 and KRPTOR to evaluate the parameters of COBAS 6000.
The evaluation parameters are:
Linearity- Linearity testing is the assessment of useful analytical range of a laboratory method. The stating the upper and lower limits of the range are usually stated by the manufacturers’ as their reportable range of their methods. A linear response produced by the analytical method is generally assumed and the upper and lower limit is then reportable.

Analytical sensitivity- Analytical sensitivity is the evaluation of the lowest concentration of procalcitonin that can be measured. It is critical to have accurate information on the lower concentration limit to have an indication on the measuring range

Method comparison- Using linear regression and correlation, Method comparison is carried out to investigate the degree of association between two analytical methods. for this project, BRAHMS KRYTOR and COBAS 6000 PCT assays would be the analytical methods being compared. When the replacement of a current method with a new method is considered, Comparison of methods is often performed as it can determine if the new method has better operational qualities than the former one.

Interferences- The interference/limitations experiment is carried out to calculate the amount of interference caused by other materials that may be present in the samples being analyzed. The limitations of the procalcitonin assay must be well understood as interfering materials would deviate the result and thus reducing the accuracy. The concentration and type of interfering material also plays a part in the degree of deviation. Typical interferences include haemolysis samples, icteric samples and lipemia samples.
Imprecision- The imprecision experiment estimates the random error caused by varying daily operations of the method, such as the pippetting of samples, the reaction conditions that depend on timing, mixing, temperature, and heating. With automated systems, such as COBAS 6000, small variations may occur due to the lack of uniformity and the instability of instrument and reaction conditions.

There are two types of imprecision experiments. Within-run imprecision involves the testing of samples within a run (1 day) while between-run imprecision involves the testing of samples conducted over a longer period of twenty days. When samples are analyzed within a single analytical run, the "within-run" random error observed will generally be low because the results are affected only by varying factors in that single run as compared to an between run experiment conducted over a period of twenty days, which is expected to provide an even more realistic estimate of the variation that will be seen in patient samples over time

yuxuan

Week 14 - Virology

This is the 14th week already. For the past 4 week, I was at the Immunofluorscence section. This area is mainly on antigen and antibody detection. For antigen detection, it is to detect the particular antigen that is present on the cell. There are 2 methods: direct and indirect.
For direct method, a fluorescent-labeled primary antibody is added and it will react with the antigen. Thus, fluorescence is observed.
As for indirect method, it will employ the use of a specific primary antibody (unlabelled) and a labeled secondary antibody (labeled with fluorescence dye). Therefore, antigen present will bind to primary antibody. And when labeled secondary antibody (also known as conjugate) is added, it will then bind to primary antibody and thereby staining the positive cells. The positive cells will then fluorescence under the UV microscope.
This method is more specific, thus reduce non-specific binding.

As for the antibody detection, it is usually to detect the presence of antibodies in serum.
Commercial antigen slides will be used, which means that the slides are already fixed with the specific antigen. If the corresponding antibody is present, then it will bind to the antigen. Following that, conjugate labeled to secondary antibody is added and it will bind to the primary antibody. Fluorescence will be observed when viewed under the microscope.

Now, I will know talk about the detection of Cytomegalovirus antigen by using cytospin. This is the most common test that is requested for in IF section. The purpose of the test is to identify the lower matrix protein pp65 of cytomegalovirus in the white blood cells. The principle of this test is mentioned above (antigen detection by indirect method).

Cytospin processing for CMV antigen

Processing the blood

1. Transfer the blood (in EDTA tubes) into sterile plastic centrifuge tubes
2. Add separation solution or 6% dextran, ¼ of the total volume of blood to the blood.
3. Place the tube in 37 ̊ C waterbath to allow the separation to take place.
4. Transfer the upper layer of suspension ( contains serum, plasma and leucocytes) into another sterile plastic centrifuge tubes.
5. Centrifuge the tube at 300g for 10 minutes.
6. Discard the serum and add cold water to lyse the red blood cells.
7. Vortex to mix well before adding phophate buffered saline (PBS).
8. Centrifuge at 300g for 10 minutes again.
9. Discard the supernatant, leaving the cell pellet behind.
10. Add an appropriate amount of PBS to the cell pellet and mix well.
11. Determine the white blood cell concentration using the coulter counter.
    
    Generating the cytospin slides
12. Vortex the tube before inoculating 0.2ml into the cytospin funnel.
13. Balance the funnel in the cytocentrifuge container and spin at 800rpm for 5 minutes.
14. Remove slides and dry it on slide warmer before fixing.
    
    Fixing the slides
15. Fix the slides with in fixative solution for 10 minutes.
16. Rinse the slides 2 times with PBSA.
17. Immerse the slides in permeabilization solution for 5 minutes.
18. Rinse the slides 3 times with PBSA again.
19. Dry the slides on slide warmer.
    
    Staining the slides
20. Add monoclonal antibody using a dropper, ensure that it is well spread (Vortex mab before use).
21. Place the slides in a moist chamber and incubate at 37 ̊C incubator for 35 minutes.
22. Immerse the slides in PBS to wash for 10 minutes, before placing it on a shaker.
23. Dry the slides on the slide warmer.
24. Add conjugate to the slides using a dropper (Vortex before use).
25. Repeat step 21-23.
26. Add mounting fluid and mount it with a coverslip.
27. View the slide under fluorescence microscope and record the results

Separation solution or 6% dextran - Acts to separate out the plasma and red blood cells.
Fixative solution- Fix the cells on the slides
Permeabilization solution - Permeabilise to allow antibody to access to intracellular structures of the cells and maintain the morphological characteristics.
Moist chamber- to maintain the humidity condition for incubation


Possible problems encountered:

• The cytospin funnel and chamber were not assembled well. Hence, the cells were not properly fixed as a circle on the slide. This may interfere with the interpretation of results.
• White blood cell concentration is too low, due to high dilution with PBSA. The resolution is to centrifuge the tube again to obtain cell pellet. Then, thus time a smaller volume is added to dilute the cell pellet.
• Cross contamination with other sample. Hence, to minimize this, always work with 1 cell line. And keep the other chambers away when loading cell suspension for 1 sample (As 1 or few drops may splash into other chamber).

For more information on cytomegalovirus, please refer to this website;
http://www.dhpe.org/infect/cytomegalo.html (same as my previous 1st entry)
http://www.cdc.gov/cmv/

That’s what I have for now. Thanks for reading!!

Shihui
0607135A

Week 13 – Medical Microbiology

Topic: Histoplasma capsulatum

This week I will be sharing about Histoplasma capsulatum. Histoplasma capsulatum is a yeast type of fungus that primarily infects the lungs and may spread to other parts of the body. It causes an infectious disease known as Histoplasmosis. Some of the signs and symptoms are fever, headache, chest pain, dry cough and chills.

Immunodiffusion method is used to detect histoplasma capsulatum antibodies in patients’ serum. Patients’ serum and the control antigen are placed in opposite wells and diffuse outward into the medium. To a point of equivalence concentration of the antigen and antibody, two visible precipitin lines (M and H) are formed between the two wells. M line forms near the antigen well while H line forms near the serum well.

Materials and Reagents used:
1. Glycine buffered agarose agar: a diffusion medium that does not support the growth of molds.
2. Positive control: antiserum that contains H and M antibodies specific to histoplasma capsulatum.
3. Negative control: antiserum that does not contain any specific antibodies.
4. Control antigen: Contains H and M antigens from histoplasma capsulatum.

Specimen: Serum

Methods
1. Spin down the specimen at 3100rpm for 15mins.
2. Place the template underneath the Petri dish.


3. Using a hollow pin to punch the agar and a pointed stick to pick out the unwanted agar to obtain 7 wells.

4. Add about 8ml of each reagents into the respectively wells in the following order:

a. Firstly, add positive control into wells #1 and #4.
b. Negative control into well #6.
c. Patients’ serum into wells #2, #3 and #5. (Depending on the number of specimens requested for this test)
d. Lastly, add control antigen into the centre well.

[Add the reagents in this order so as to prevent any contamination that may lead to false results.]
[Prevent overflowing from one well to another as this may also lead to false results.]

5. Place the Petri dish in a moist condition at room temperature for 23-25 hours.
6. Examine the agar over a light source. (Eg. Under a microscope)


Results


Positive controls: Two lines formed between wells #1 & 4 and the centre well. [This test is valid]
Negative control: No lines formed between well #6. [This test is valid]
Patient 1 (well #2): Positive reaction as two lines are formed between well #2 and the centre well.
Patient 2 (well #3): Negative reaction as there are no lines formed between well #3 and the centre well.
Patient 3 (well #5): Positive reaction as M line is formed between well #5 and the centre well.


LeeJin
TG02