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

Answers (Bone Marrow Lab)

To Lyn,

Not all 3 smears have to be done. It depends on what the doctor orders. There are 2 parts to the bone marrow aspiration. The first is to get the aspirate and the second is to get the trephine sample. If the doctor orders for just the aspirate, the med tech only has to do the aspirate and the squash smears. If the doctor orders for both the aspirate and the trephine sample then the med tech will have to do all the 3 smears. Below are the pictures of the 3 different types of smears. (taken with permission from my supervisor)

Picture 1:
aspirate smear (with fragments)


Picture 2:
imprint smear (of trephine sample)


Picture 3:
squashed smear (from aspirate with fragements)


To Leslie,

Trephine is just the name of the instrument used to obtain the bone marrow biopsy. Items include:
- a 10 cc/mL syringe to inject the local anaesthetic.
- a 20 cc/mL syringe is used to withdraw the aspirate.
- 2 needles, 1 blue and 1 green. Blue is to withdraw the local from the bottle. Green is to inject the local into the biopsy site (hip bone area)
- Iodine and septanol to clean the biopsy site
- 2 tubes of local anaesthetic (1% xylocaine), in most cases. Some patients with low threshold of pain, in other words, unable to tolerate pain as well as others, will need more tubes.
- 1 dressing set
- 1 aspiration set which contains a special large-bore needle which is inserted into the hip bone area, the extraction cannula (trephine), a marked probe is used to check the length of the sample and also to expel the trephine sample from the trephine (extraction cannula).


To Yumei,

Few particles as in the fragments present in the aspirate. As shown is picture 1 above, those round things are known as fragments.
ADDITIONAL INFO: When the doctor withdraw the aspirate, there must be fragments present, or else it is rejected and the doctor will have to re-withdraw the aspirate again. If there really are no fragments present, just the blood or the doctor is unable to withdraw any aspirate from the patient, the doctor has no choice but to get another trephine sample and put it in the tube containing lithium heparin (for cytogenetics) and add 0.9% NaCl to act as a medium.


To Ben,

Myelodysplasia is a disorder of bone marrow stem cells due to ineffective hematopoiesis. Patients suffering from myelodysplasia may progress to acute myeloid leukaemia (AML) within months to a few years. It can be detected by doing a blood test. In most cases, there is a great decrease in RBCs, causing fatigue and pallor (anaemia). Rarely, there is a decrease in platelets which will lead to bruising and bleeding or a decrease in WBCs which will lead to fever and infection. At times, nonspecific symptoms like loss in weight and appetite may occur. It is because of the abnormal CBC results that lead to the decision of performing a bone marrow aspiration on the patient.


To cornelyus,

Yup, maygrundwald-giemsa stain contains methanol. Methanol is used to fix the smear to the slide so when it is washed, it will not be washed off.


To quan jun,

1) yup, I got to observe the bone marrow aspiration LIVE. =)
There are 2 parts to the procedure. The first part is to get the aspirate. The second part is to get the trephine sample. First, iodine followed by septanol is used to clean the biopsy site. Local anaesthetic is administered to numb the site. Then, a special large-bore needle is inserted and rotated. A syringe is then attached to the needle and the aspirate is withdrawn. For the second part, the needle is inserted further inside. A marked probe is inserted to check the length of the sample and removed. An extraction cannula is inserted into the needle cannula. The needle is then rotated and the extraction cannula is removed from the needle. The marked probe is used to expel the trephine sample from the extraction cannula.
As for the reagents used, I’m not sure what u meant. Or do u mean items used to carry out the aspiration? If yes, do refer to my answer to leslie above.

2) as for bone marrow fragments, do refer to the picture 1 above. They are fragments found in the aspirate from the bone marrow.

3) for this job-related question, I’m not sure which section u are referring to. But since u mentioned about processing samples, I’ll presume it’s the routine and coagulation section. For the routine section, we can process up till 250 to 400 samples a day for normal working hours (8am to 4pm). Monday is always the busiest day. After 4pm, the samples are left for those who are doing payback or night shift. There isn’t really a time limit given to process each sample. As long as there are samples received, we will just process it. During working hours, if the result of the sample is required by the doctor urgently, the samples are sent to the STAT lab.
For the coagulation samples, we can process up till 100 to approximately 200 samples a day during working hours (8am to 4pm). Urgent and non-urgent samples are run together. However, if the sample is really urgent like from the A&E section or so, we will try our best to run it first.


MALERIE (not malarie) =)
TG02

Week 12 - Bone Marrow (Haematology)

I was attached to the Bone Marrow Lab for 3 weeks. Basically, the job of a medical/lab technologist is to get the aspirate and trephine sample (bone marrow biopsy) which the doctor extracted from the patient’s bone marrow and prepare the smears on the spot. Blood taken from the patient’s finger, pricked using a lancet is also smeared on the spot. The technologist is also responsible for letting the doctor know how much aspirate or trephine sample is needed and place it in the respective tubes and bottles. How much aspirate needed depends on the tests ordered by the doctor on the request forms.

After that, the slides are brought up to the lab where they are stained, dried, labeled, mounted and examined under the microscope. Patients’ information such as the name, hospital, ward, bed no., diagnosis, gender, age, etc. are recorded into the record book and also entered into the LIS.

During my 3 weeks there, I was able to observe bone marrow aspirations and also helped the doctor prepare the items needed for the aspiration when the nurse is busy with something else. I also helped my colleague place the aspirate and trephine sample into the respective tubes and bottles and ensured that all the required forms and tests requested by the doctor are ticked. After about 2 to 3 patients’ procedures are completed, I brought the slides up to the lab where I stained, labeled and mounted the slides and also recorded the required information into the record book and forms. During my free time, I practiced doing the various types of bone marrow smears except for the imprint smear as we do not have any extra trephine sample.

Preparation of smears

The 3 types of bone marrow smears to be prepared are:

1) Aspirate smears:
- Aspirate must contain bone marrow fragments.
- Same way as preparing a PBF smear.
- Accept only when marrow fragments are found mostly at the tail of the smear and free marrow cells can be seen in stained films.

2) Squashed smears:
- Few particles are placed on the centre of a clean slide.
- Another slide is used to gently compress to spread and disperse the particles as the slides are pulled apart.

3) Imprint smears:
- Bone marrow trephine biopsy sample is gently touched and rolled along between two clean slides.

The purpose of doing a bone marrow examination by preparing these 3 types of smears is to be able to show the different cells present in the patient’s bone marrow in order to diagnose certain conditions, assess the progress or stage of the patient’s disease and also to monitor treatment of certain conditions. Conditions include multiple myeloma, leukemia, lymphoma, anemia, amyloidosis, polycythemia vera, myelofibrosis, etc. Take for example leukemia, the patient could be suffering from acute or chronic myeloid or lymphoblastic leukemia and which type of ML or LL the patient is suffering from.

Staining of slides

1) The slides (2 aspirate, 1 squash, 1 imprint and 1 blood smear) are stained with Maygrundwald-Giemsa stain for the morphological classification of hematopoietic cells.

Maygrundwald-Giemsa stain
The Romanowsky stain consists of methylene blue and its oxidation products and eosin Y or eosin B. The combined action of these dyes produces the Romanowsky effect, yielding purple colouration to the nuclei of leucocytes and neutrophil granules and pinkish colour to the erythrocytes.


2) A separate slide (usually the aspirate smear) is stained with iron stain for the classification of anaemia associated with defective haemoglobin synthesis such as thalassemia, leukaemia, myelodysplasia and sideroblastic anaemias. The identification of ringed sideroblasts is used for the diagnosis of sideroblastic anaemia and myelodysplasia.

Iron stain
Cellular iron exists in the form of ferritin or hemosiderin (storage iron) in the bone marrow. Prussian blue iron stain is for the presence of hemosiderin (storage iron) in bone marrow and stainable iron in erythroblasts (sideroblasts) and/or erythrocytes. Hemosiderin stains blue-green; nuclei of cells stains red. Iron-containing granules will be demonstrated in siderocytes and normally in many of the normoblasts (precursor of erythrocytes) of human bone marrow. Extracellular iron deposits will be demonstrated in bone marrow. This siderotic material (hemosiderin) is distinct from ferritin, which is water-soluble and not detectable by Prussian blue reaction.


Examination under the microscope

Aspirate smears are able to show RBCs, WBCs and platelets. Squashed smears will be able to show whether the bone marrow is hypocellular or hypercellular. Imprint smears will be able to show cells that are present on the surface of the bone marrow core (bone).

Types of cells one will be able to see in the smears under the microscope: blasts such as myeloblasts, promyeloblasts, monoblasts and lymphoblasts, myelocytes, promyelocytes, promonocytes, eosinophils, basophils, erythrocytes, megakaryoblasts, megakaryocytes, prolymphocytes, etc.

Malerie Goh

TG02

More answers::

To Xin Yi:
Upon consulting my mentor there, I found out that it is not possible to tell you the 'common' colours of moulds as different moulds express a different colour. Even as the mould grows, the colour may change. For e.g Aspergillus spp. may first appear white, then as it grows, turn to any shade of green, yellow, oange, brown or black, depending on its species. Thus, colour of the mould is used as a guide and not a confirmatory test for identification of mould.

To Leslie:
Err.. yes. In that fungus lab, only fungus identification is carried out. Should there be any bacteria present, they will report 'bacterial growth' and let the doctor decide whether a seond specimen should be sent for bacterial culture etc. The fungus lab will not undergo the entire process of identifying what type of bacteria it is.

As for what plate to use, it depends on what type of specimen it is. For example:
- Nails, skin scrapping: SDC + AA plates are used.
- Fluids, brain and oral cavity: SDA + SDC tubes are used.
- Tissue: SDA + SDC + BHIA + BHIB + CG tubes are used.

To Farhana:
Pseudohyphae are long branching filamentous cells of fungus, that are separated by cross-walls known as septa. These are normall(Look at the long strands of cells. watch out for the cross-walls)


http://www.uni-duesseldorf.de/WWW/MathNat/mikrobio/ernst/interest-Dateien/intere2.gif

Blastoconidia are yeast cells, thus they are not found in moulds. These cells reproduce by budding. Budding means that the parent blastoconidia separates to form a daughter cell.


http://bugs.bio.usyd.edu.au/Mycology/images/Topics/StructureFunction/buddingYeastCells.jpg

To Jean:
OOpps. should have defined the terms. These terms actually describes the gram-morphology of the organism.
- GNB: Gram-negative bacilli
- GPC: Gram-positive cocci


All righty. Hope all questions are answered in the best possible way =)!

Hope it helped!

-Li Ping-
TG 02

Answers::

To Ka Hang and Yvonne (your second question):
The germ tube test is carried out by:
1. Inoculate the yeast into the horse serum.
2. Incubate the serum at 35 – 37oC for 2 - 3 hours.

So, no subculture is needed. We can just do a wet mount directly from the tube. No gram stain will be needed.
3. Do a wet mount using the serum.
4. Observe the slide under the microscope.

To Yvonne (first question):
API is kit that consists of a combination of biochemical test used for identification of organisms. There are different types of API avaliable. For example, for identification of Candida, the API 20C AUX is used. So in conclusion, different types of API kit are used to identify different types of organisms.

To Andika:
To differentiate these Candida species, the same type of API method will be used. But this API intepretation is different from the API method for bacteria.
- For yeast, a change of turbidity will be noted.
- For bacteria, a change in colour will be noted.

To Xin Yi:
About the colour of moulds, i've gotta check it out cos' different moulds have different colours. But i'll find the more common colours for you.

About the cornmeal agar, there is 1% Tween 80 added as a basal medium. This will help to encourage the growth of chlamydospores.


All righty. Hope that I've helped make understanding easier =)!!

-Li Ping-
TG o2

Week 11: Microbiology

Another 5 weeks passed and it's my turn to post again. Anyway, 5 weeks gone by and I'm shifted to another lab. Fungus lab. A little F.Y.I: In Singapore, the lab must have a special license to operate a fungus lab. Also, fungus cultivation is also a slow process (with incubation time of at least 2 weeks), with low profits and high risks. So many labs may not have a fungus lab, preferring to send their specimens out to a lab with a fungus lab.

Cultivation of fungus is like, as my mentor says, maintaining a garden. Fungi are pretty interesting to look at under the microscope. Instead of the normal GNB and GPC, we can see beautiful formations of conidia and hyphae shaped like flowers.



As different fungi has different morphology, it is possible to observe the fungi under the microscope, note the formation of conidia and hyphae, then identify what type of fungi it is. So unlike bacteria identification, very little biochemical tests are carried out.

Filamentous fungi (moulds) can be differentiated by observing:
- Colour of the mould
- Formation of the spores
- Growth rate
- Temperature differences - some mould grow better at higher temperatures, while others may
grow better at lower temperatures.

Candida spp. are another type of fungi known as yeasts. There are 2 strains that can be differentiated from the other strains. They are Candida albicans and Candida dubliniensis. These 2 strains can be differentiated from the other strains using these 3 tests:
- Germ tube test
- Use of cornmeal agar
- Urea test

Germ tube test: Candida albicans and Candida dubliniensis will produce germ tubes when inoculated into horse serum. These tubes supposedly contributes to the pathogenecity of the organism



The long tubes at the end of cells are germ tubes. (http://upload.wikimedia.org/wikipedia/commons/1/12/C_albicans_germ_tubes.jpg)

Cornmeal agar: It is a medium made up of cornmeal and agar with added Tween 80. After inoculating the organism on the agar, incubate the agar for 18 - 24 hours. Then observe under microscope. These following features should be noted:
-Pseudohyphae
- Blastoconidia
- Chlamydospores

Candida albicans and Candida dubliniensis will have these 3 features while the other Candida spp. will not have chlamydospores. Chlamydospores are lollipop-like cells.


Chlamydospores (http://www.forestresearch.gov.uk/website/FCPicLib.nsf/LUImagesByFilename/43887.jpg/$FILE/43887.jpg)

Urea test: It is basically the same as the one used for bacterial identification. Candida albicans and Candida dubliniensis are urease positive while the other Candida spp. are urease negative. Positive test = pink agar.

To differentiate between Candida albicans and Candida dubliniensis: use API method.

To differentiate the other Candida spp.: use API method.


The medium and agar used in the fungus lab are also different. Instead of the conventional blood agar and macConkey agar, other types of agar are used. I have summarised their abbreviations, functions into the table below. Hope it helps make understanding better.


* Click on picture for bigger view. Sorry for inconvenience =).

So, this is the end of my stint at the fungus lab. Next week, it'll be to the anaerobic lab, where i'll be using this interesting-looking incubator thing.

Hope my post have been coherent enough for understanding!

11th week gone. 9 more weeks to go =).

-Li Ping-
TG o2

PCT

Hello, for my SIP I have already started on my Major project which is on the evaluation of a machine that analyses on PCT. I will describe the PCT in detail in this post.

The prohormone precursor for CT is a 116-amino acid polypeptide which has three component peptides, a 57-amino acid peptide at the amino-terminus, named aminoprocalcitonin (aminopro-CT), a centrally placed 33-amino acid immature CT and a 21-amino acid CT carboxyl-terminus peptide-I. its expression is induced several fold in the systemic bacterial infection and sepsis to elevated levels found in blood.
PCT has been demonstrated to be the best marker for differentiating patients with sepsis from those with systemic inflammatory reaction not related to infectious cause. Procalcitonin provides support for early diagnosis and allows critical decision making which could direct an effective treatment at the proper timing and also to save unnecessary costs in the use of appropriate antibiotics for critically ill patients. Produced in numerous neuroendocrine cells such as the thyroid glands and present in very low concentrations in blood, it has been shown that systemic bacterial infection causes a marked increase in PCT expression (encoded in the Calc-1 gene), hence its clinical utility as a indicator of sepsis of bacterial origin. Very significant concentrations of procalcitonin (up to 1000 ng/ml) can be detected in the patient sample with severe bacterial infection/sepsis

The inflammatory and sepsis markers present now are IF-2, IF-6, IF-8, PCT, CRP and TNF-α. PCT has currently show in recent studies that it has better specificity and sensitivity over the classical sepsis markers in systemic sepsis, moreover, it has a higher and earlier increase during sepsis, and the level of PCT present usually correlates with the severity of the disease.

PCT compared to CRP

In this diagram PCT is shown to have a higher pooled sensitivity and specificity over C reactive protein, which is a classic inflammatory marker.

References: http://www.procalitonin.com/
http://www.jle.com/

yuxuan

Week 9 - Virology

Hello people! For the 4th to 8th week, I was at the virus isolation section of virology.
For this area, it is mainly responsible for specimen processing, virus inoculation, maintenance of culture tubes and repassaging (Repassaging is to subculture or split cells into new culture tubes to prevent overgrowth and degeneration.)

Basically, after the specimen is processed, it is inoculated into respective culture tubes and incubated. The tubes are examined for cytopathic effect (changes in appearance of caused by the virus that is growing) everyday and results are recorded. If any cytopathic effect is observed, then the specimen will be sent for confirmatory tests – immunofluorescence, acid test and neutralization test [This will be elaborated in the next post :)]

The culture tubes are maintained by changing their media on every alternate day. This is done to maintain the viability of the cells - as nutrients will deplete and waste products produced by the cells are detrimental to growth. However, this will still depend on the condition of the cells. If the cells are going to be repassage the next day or they are still healthy, then there is no need to change media on that day itself. As for repassaging, it is usually done on the 7th and 14th day after inoculation when degeneration of cells is observed.

Changing media for culture tubes

1. Remove media in culture tubes by using a 1ml disposable plastic pipette.
2. Discard media in disinfectant, sodium hypochlorite.
3. Add in 1ml of new media using a new pipette.
4. Cap tightly and arrange it back into roller drum.
5. Incubate in respective incubator, depending on the type of virus.

ROLLER DRUM (Picture taken from http://www.nbsc.com/rollerdrums.aspx )

Repassaging for culture tubes

1. Select the culture tubes according to the original cell type (Eg, Hela tube needs to be repassage, the new tube chosen must also be hela.)
2. Check the selected tubes under microscope to ensure cells are present and confluent.
3. Label the newly selected tubes with its original lab number and the date of repassage.
4. Loosen the caps of the tubes [Done in BSC-II].
5. Scrape the cells in the original tube by using a bent pipette.
6. Flush the cells up and down to ensure that it well mixed.
7. Inoculate 0.2ml of the cell suspension into the newly selected tubes.
8. Cap tightly and arrange it back into roller drums.
9. Incubate in respective incubator, depending on the type of virus.

In this section, we will receive many different specimens. Each of them will come with a request form, indicating what virus is suspected and needs to be isolated. Hence, we will only perform that particular test.

Here are some of the commonly received specimens and the virus that is suspected of…

ETDA Blood -- Enterovirus/ Neurotropic/ Cytomegalovirus

Lung tissue -- Respiratory virus

Heart/intestine/brain tissue -- Enterovirus/ Neurotropic virus

Urine -- Cytomegalovirus /Mumps /Adenovirus

Stool -- Enterovirus/ Neurotropic virus

Vesicles/ ulcer / genital swab -- Herpes virus



Now, let me share with you guys on specimen processing.

Urine Processing

1. Centrifuge the urine sample for 10minutes at 200rpm.
2. Add 1.2ml of media to a clean sterile container.
3. Pipette 1.2ml of supernatant and mix it with the cell deposit, thereby forming a cell suspension.
4. Transfer the cell suspension to the container with media.
5. Inoculate 0.3ml of cell mixture into respective shell vials and tubes.
6. Preadsorb for 1 hour before incubation (preadsorb will help in virus penetration)

Stool Processing

1. Add 10 glass beads into a sterile plastic tube.
2. Add 10ml of Dulbecco PBS, 1ml of antibiotic and 1ml of chloroform to the tube above.
3. Take a pea-sized amount of stool and add it in.
4. Seal the tube with parafilm to prevent leakage.
5. Vortex the tube and place it on the horizontal shaker at 200rpm for 20 minutes.
6. Centrifuge at 2600rpm for 20 minutes.
7. Transfer 5ml of the supernatant into a clean sterile 5ml tube.
8. Select the tubes and label them with date of inoculation and lab number.
9. Inoculate 0.2ml of the supernatant into each tube.
10.Preadsorb tubes before incubation (preadsorb will help in virus penetration)

Glass beads- serve as stirring purpose
Dulbecco PBS- contains calcium ions that will help in stabilizing the virus
Antibiotic- prevent bacteria contamination
Chloroform- helps to disperse virus aggregate and kill any bacteria or fungus present

Tissue processing

Lung tissue (to test for respiratory virus)
1. Transfer the lung tissue into a mortar and cut it into small pieces using a scissor.
2. Add in alumdum powder and grind it with a pestle.
3. Transfer the contents into a sterile centrifuge tube and centrifuge at 3500 at 20 minutes.
4. Separate the supernatant from the deposit ( supernatant into a sterile 5ml tube while deposit back to the original container)
5. Select the tubes and shell vials before labeling them with date of inoculation and lab number.
6. Inoculate 0.3ml of supernatant into the tubes and shell vials.
7. Centrifuge the shell vials at 1800rpm for 30minutes.
8. Preabsorb the tubes at 33±0.5̊̊ C for 1 hour.
9. Incubate both tubes and shellvials overnight and observe for cytopathic effect the next day.

Alumdum- To provide sharp edges for grinding the tissue


That’s all for now. Hope that you guys can understand.

Shihui
0607135A

Week 8 Microbiology

This week I will be sharing with you about ‘Motility’. In the Investigation lab, motility test is one of the biochemical tests carried out. It is a characteristic to classify and identify the type of bacteria. Examples of motile bacteria: Proteus vulgaris and Pseudomonas aeruginosa. Examples of non motile bacteria: Staphylococcus aureus and Streptococcus pyogenes.
There are two ways to determine motility.
1. Through the use of a medium.
2. Microscopic examination.

1. Using a medium
The medium used is OF (Oxidative-Fermentative) medium.
[Oxidative-Fermentative is another biochemical test.]
OF medium has an agar that is in semisolid form which can also be used to determine if the bacteria are motile or non motile. Semisolid agar (soft agar) allows the bacteria to be diffused throughout the medium by the presence of flagella. Since one tube of OF medium can be used for 2 biochemical tests (Oxidative-Fermentative test and motility test), there is no need to use the motility medium which can only be used for motility test.

Methods:
a. Pick a few colonies and mix with a tube of saline to obtain a McFarland turbidity standard of 0.5.
b. Use a disposable inoculating needle to obtain a drop of the suspension and stab into the medium.
c. Incubate at 35°C in O2 conditions for 18 – 24 hours.
d. Examine the medium.

Results:

Motile bacteria: The inoculation line cannot be clearly seen as the bacteria are able to grow away from the inoculation line and diffuse into the surrounding medium.

Non motile bacteria: The inoculation line [stab] is clearly seen whereas the surrounding medium is clear. (The bacteria remain localized at the inoculation line).












(non motile bacteria)

2. Microscopic examination
This method is known as the hanging drop. It is used when the results of the previous method is not significant (Not sure whether it is motile or non motile).

Methods:
a. Prepare a glass slide with a ring of plasticine.






b. Use a wooden stick to obtain a drop of suspension from the tryptophan broth containing the bacteria and place it on the cover slip. [Tryptophan broth is used for another biochemical test. The OF medium used in the previous method cannot be used as it is not in liquid form.]

c. Invert the glass slide and press it against the cover slip. The drop of suspension should be hanging inside the ring of plasticine.

d. Observe under the microscope at 40X objective.

Results:
Non-motile: The bacteria appear to be stationary.

Motile: The bacteria are moving in forward or in spinning direction.


LeeJin
TG02

Week 7 - Special Coagulation (Haematology)

For this week, I was attached to the Special Cogaulation Lab where there are many SPECIAL tests carried out. These tests require reagents that are expensive thus I do not really have any hands-on. All I can do is just observed. Some tests done in the lab are automated while some are done manually. For those tests done manually, timing is very important as reactions take place constantly.

For this entry, I shall share with all of you about the soluble fibrin monomer complexes test which is done manually in the lab.

Soluble fibrin monomer complexes test

USE:
It is a rapid qualitative test for the presence of the monomer in the plasma by the hemagglutination technique.


PRINCIPLE:
Fibrin monomers are intermediate products between fibrinogen and fibrin. They are produced during the proteolysis of fibrinogen by thrombin. [Proteolysis: digestion of proteins by enzymes called proteases] When the thrombin concentration in plasma is low, fibrin monomers are in insufficient quantities to aggregate to form a fibrin clot. Thus, fibrin monomers associate themselves with fibrinogen/ with fibrinogen degradation products to form soluble complexes. The presence of small amounts of soluble fibrin monomer complexes in the plasma can quickly be visualized with the F.S test by the agglutination reaction of human erythrocytes coated with purified fibrin monomer which do not agglutinate with normal plasma.

TYPE OF SAMPLE: Blood in sodium citrate tube.

PROCEDURE:
1) 100mL of patient’s plasma, 100mL of positive control and 100mL of negative control are added to 3 separate tubes.
2) 50mL of F.S test reagent is added to each tube and mixed well.
3) The tubes are incubated for 10 minutes in the water bath.
4) The tubes are removed from the water bath and dried. The contents in the 3 tubes are transferred using a pipette to a circle on the hemagglutination plate.
5) The plate is rocked gently to make the liquid swirl around the circle for 6 minutes.
6) The hemagglutination patterns of the patient’s plasma are then compared with those of the negative and positive controls.


REFERENCE RANGE:
Normal plasma does not produce agglutination of the F.S test.


INTERPRETATION:
Positive F.S test may be found in the following clinical conditions:
-Liver disease/cirrhosis
-Angioma
-Leukemic cancer
-Infection/Septicemia
-Polytraumatic condition
-Obsteric complications
-State of shock
-Thrombotic disease
-DIC of other organs


DONE BY:
Malerie Goh
TG02

Hello i hope everyone is having a great time at SIP now. its been so long now. For today i'll be blogging on osmolality. and my lab uses a different equipment from our school's. it seems to be more advanced. well on to it =)

Osmolality is an expression of total concentration of dissolved particles in a solute without regard to the particle size, density or electrical charge. The measure of total solute concentration can only be made indirectly by comparing one of the solution colligative properties with corresponding cardinal property of pure solvent.
Vapro osmometer is based upon a measurement of vapor pressure depression made possible by thermocouple hygrometry. This sensitive temperature sensor operates on the basis of a thermal energy balancing principle to measure the dew point temperature within the chamber.
10 micro liter of specimen is aspirated into a micropipette tip and then inoculated into a solute free paper disc. The paper disc is then pushed into the instrument and sample chamber is locked. The sensing element is a fine wire thermocouple hygrometer. This is suspended in a unique, all metal mount, which forms a small chamber enclosing the specimen when joined with the sample holderAs vapor pressure equilibrates in the chamber airspace, the thermocouple then seeks the dew point temperature within the enclosed space, giving an output proportional to the differential temperature. The difference between ambient temperature and dew point temperature is the dew point depressionSample used- serum or urineStepwise.push sample chamber level upwards and pull the sample slide out from the instrument.
use forceps supplied to place a single sample disc in the central depression of the sample holder. If more than two discs are used, results will slightly elevated

Using a micropipette, pipette 10ul of sample

Rest pipette tip on the notch of pipettor guide. It should be about 5mm away from center of sample discsmoothly depress micropipette plunger to release sample onto sample discwith plunger still held down, lightly touch the pipette tip to sample disc, then lift it away. Tip must briefly contact sample disc to press the disc flat against the holder

Sample disc should appear fully saturated, with a slight liquid meniscus on surface.

Note: when cleaning the sample chamber, use only lint free tissue. Facial or soft tissue will produce excessive lint residue that will contaminate the thermocouple sensor

Yuxuan

Week 5: Investigation lab (Microbiology)

Oh my, 5 weeks of our attachment have already passed. I'm have also moved onto another lab. From the central processing area (CPA), I'm now in the investigation lab (IV lab). In CPA, we 'prepare' the patients' specimens for testing. Then, these prepared specimens will be sent to the IV lab so that the bacteria present can be identified, so that a diagnosis can be obtained. Afterwhich the appropriate treatment can be given.

Having said so, it also means that the IV lab plays an important role as to whether the patient will receive the correct treatment. So as interns, we are not given much investigation work to do. Instead, we are being tasked with simpler and 'save-able' jobs. 'Save-able' as in if we do anything wrong, they can repeat the test again.

So far, I have done these few things:

- Subculture cooked meat and brain heart infusion broth (BHIB)

- Perform latex agglutination for the identification of Staphylococcus aureus

- Perform antibiotic susceptibility testing for urine cultures.

Subculture of cooked meat and BHIB

Cooked meat is an enriched media used to cultivate anaerobic bacteria. But according to my mentor there, cooked meat is also used as back-up. In some cases, there may be too little bacteria present in the specimen to show up on the agar plates. As the specimens are submerged into the cooked meat media, any bacteria present cannot 'escape'. After incubating the cooked meat for 1 day at 35°, the media will be subcultured onto blood agar and anaerobic plates. Blood agar is used as most bacteria can grow on blood agar. Anaerobic plates are used to detect the presence of any anaerobic bacteria.

Cooked meat medium [1]

BHIB is a general-purpose medium normally used to cultivate fastidious, non-fastidious, aerobic and anaerobic bacteria. But this BHIB has added antibiotics, oxacillin, to detect methcillin-resistant S. aureus (MRSA). So all bacteria, excluding MRSA, sensitive to oxacillin will be killed. MRSA will not be killed by oxacillin as it is resistant to oxacillin. So this type of BHIB is used for MRSA screening. The BHIB will be incubated for 1 day at 35°, and after 1 day, the broth will also have to be subcultured onto MRSA plates.

MRSA plates are actually made up of either blood agar or mannitol salt agar with added methicillin or oxacillin. It is selective media used to detect MRSA. Presence of MRSA can be indicated by purple colonies present on the agar.

MRSA growing on MRSA plate [2]

Latex agglutination for identification of S. aureus.

This test works by detecting 3 components:
- Fibrinogen affinity factor, also known as bound coagulase or ‘clumping factor’
- Protein A, which has an affinity for crystallisable fragment (Fc) of the gamma

immunoglobulins (IgG)

- Capsular polysaccharides of S. aureus

The reagent used in this test contains latex particles that have been sensitized with fibrinogen, IgG and specific monoclonal antibodies specific against capsular polysaccharides of S. aureus. A combination of these 3 components allows both highly and poorly encapsulated strains of S. aureus to be recognized

For highly encapsulated strains of S. aureus, antibodies specific to the capsular polysaccharide will be used to agglutinate the bacteria. For poorly encapsulated strains of S. aureus, the fibrinogen and IgG will be used to agglutinate the bacteria.

Positive result = agglutination observed with loss of'red background

Negative result = no agglutination with no loss of red background

False-positive results may occur when there is a cross-reaction with Streptococcus spp. as some strains of streptococcus contains a protein with an affinity for the Fc portion of the IgG. So it may react with the latex reagent, thus causing a false positive result.

False-negative results may also occur as some S. aureus do not produce fibrinogen affinity factor (clumping factor), protein A, or capsular polysaccharides. Without the capsular polysaccharides, no specific antibiotics will produced.

Antibiotics susceptibility testing

This form of testing is used as a guide to the doctors as to which type of antibiotics would work best to kill the bacteria present in the patient. So tests are performed in vitro and how well the organism grows in response to the antibiotics will be measured. [6] The antibiotics susceptibility testing is normally done using Mueller-Hinton agar. Mueller-Hinton agar is used for this test because of a few reasons:
- Good reproducibility of the medium
- Simplicity of its formula
- A lot of experimental data are available regarding the usage of this medium [7]

Type of antibiotics to be used for the test is determined by a few factors:
- Target location of the antibiotics (e.g Antibiotics meant to treat bacteria in the brain

must be able to pass through the blood brain barrier)

- Target bacteria, as some antibiotics work better for gram-positive bacteria, while other

antibiotics work better for gram-negative bacteria)
- Avaliability of the antibiotics, as some antibiotics may be more commonly used than others.

As mentioned above, different types of bacteria will require different groups of antibiotics to be used. The groups of antibiotics are described as ‘lines’.

Staphylococcus = A line

Streptoccus = E line

Enterococcus = BE line

Pseudomonas aeruginosa = E line

*Enterobacteriaceae = F line

*Enterbacteriaceae refers to any other bacteria not mentioned in the previous lines. They include Escherichia coli, general Pseudomonas and so on.


Procedures
1. Inoculate single colonies of bacteria into saline. The turbidity of the mixture should match

the 0.5 MacFarland turbidity standard.

2. Dip a cotton swab into the mixture and drain off excess mixture.
3. Swab the entire surface of Mueller-Hinton agar with the cotton swab by turning 60 degrees

after each swab.

4. Stamp the appropriate group of antibiotics onto the Mueller-Hinton agar.
5. Incubate plates at 35° for 1 day.

Result

Bacteria susceptible to the antibiotics = zone of inhibition will be observed around the disk.
Bacteria resistant to the antibiotics = zone of inhibition will be absent around the disk.

Antibiotics susceptibility test [3]

So these are basically what I'm doing in the IV lab. I will be starting on biochemical tests next week. Yes, all the urease, oxidase, catalase tests and so on. So wish me luck!

Picture references:

1. http://www.oxoid.com/uk/blue/press/press.asp?art=Y&arch=Y&pRef=PR023605&c=uk&lang=en&yr=2005

2. http://www.chemoquip.com/

3. www.med.unsw.edu.au/pathology-cds/

-Li Ping-

TG 02

Pictures of urine cultures

To Benjamin


For Q1


This is how the urine specimen on blood agar plate looks like after incubation..












(Picture taken in lab. Permission given by supervisor.)



And the urine specimen on CLED plates..













(Picture taken in lab. Permission given by supervisor.)




LeeJin
TG02

Hello I hope that you guys are there are enjoying your SIP at ur individual places. I’m in the clinical biochemistry department for my SIP. And I am going to talk about bilirubin measurment for neonates. In my lab we use a bilirubinometer that is a direct spectrophotometer that measures total bilirubin in neonates( babies ) by using their serum samples. These samples are usually collected in capillary tubes as neonates do not have much blood to be drawn out for analysis.
Mechanics of the bilirubinometer

1. Light passes through the cuvette and is split by a dichroic mirror and directed to 460 and 550 photo detector

2. Output of the photometer is calculated by control electronics to measure total bilirubin

3. bilirubin and oxyhaemoglobin’s peak absorbance is 460nm. It is necessary to measure oxyhaemoglobin at 550nm The difference in reading at 460 and 550nm is the total bilirubin value.

4. usually haemolyzed samples do not affect the result as the bilirubinometer in my lab substracts the oxyhaemoglobin value from the total value that is collected by the photometer .

5. this bilirubinometer only measures neonate sample as interfering substances become more prevalent and will affect results for adults

Interfering substances
Pigments that are absorbed at 460nm will affect the reading of the sample in the bilirubinometer. Some examples are lipochromes and carotenoids. Neonatal samples that are collected usually lack such interfering substances to affect the reading

Certain drugs also interfer with readings, like for example sulfonamides, phenols and amines.

Intravenous fat emulsions and lipaemic samples are also know to affect the reading. Such an example is Lyposin that has affected direct spectrophotometric measurements

Daily Controls
2 assayed galss cuvette is provided by the manufacturer, it is usually done between runs, at the start of each day.

EQC
my lab has taken part in certain EQC programmes for bilirubinometer for neonates. Some examples are CAP, neonatal bilirubin assay programme





Materials
Sample
test tube
special pipette
centrifuge machine
cuvette
bilirubinometer

Methods
1.Place capillary tube sample into test tube affixed with plastic stopper to prevent blood from leaking out during centrifuging. This will cause of loss of serum which is precious as neonates are involved

2.place in centrifuge and counter balance

3.spin for 5 mins at 3500 rpm

4.using a capillary tube pipette, fill cuvette with serum. The cuvette volume is 20 uL but there’s no need to measure the sample as the exact volume is determined by the cuvette. Cuvette needs to filled full.

5.the outside surface of the cuvette must be free of serum, dust and smudges as it will affect the machine’s capability of interpreting the result

6.bubbles will affect the reading, thus the rectangular area of the cuvette must be cleared of bubbles. Tap lighting on cuvette to remove bubbles.

7.place cuvette in bilirubinometer and press start button.

Yuxuan

picture reference: www.reichertai.com