Angels n a Demon

Angels n a Demon

Monday, November 10, 2008

20th posting~~

Hi again. This will be the last posting for my group about our SIP experience. This time, I will be talking about a particular new shift that I was assigned to a few weeks ago. It was called Corporate and Polyclinic section or CP for short. The shift starts at 10am and will finish at 7pm, with my lunch break at 2pm instead of the normal 1pm.

As I was new in doing that shift, I was asked to observe how the workflow is. For the poly samples, I was told that there are a total of 3 batches of samples that is sent every day. The first batch usually comes before 11am, the second batch after 1pm and the third one before 5pm. And for each batch, we would receive samples from 3 polyclinics. They are Bukit Batok, Chua Chu Kang and Jurong. Now let me explain the workflow.

The moment the samples arrive at the lab, the deliveryman will tell the poly clerk in-charge the maximum and minimum temperature of the samples. The poly clerk will take down the temperatures together with the time delivered. The poly clerk will then check that the samples sent matches with the ones that are in the form. After that, the clerk will have to ‘ech’ the specimens into our lab system. ‘ech’ is simple a method we use to manage the samples sent by the poly. It is necessary to ‘ech’ all the samples first before running them as if not, the machine would not recognize the sample and it would not run. After that, the samples are then passed on the med tech doing the CP shift.

The poly usually sent the samples either in plain, fluoride or edta tube. The plain tubes are usually used for the routine biochemistry tests. Thus, we just need to load the tubes into the MPA where all the pre-analytical processes like centrifuging will take place. It will then automatically run the samples into the adjacent machine called the swa or cobas. However, if the sample is insufficient, we will have to spin it down first using a centrifuge and then load it into mpa or swa. As for the fluoride tubes, we have to spin all of the tubes first before loading it into the machine. This is because if we load it into the mpa, the system is not able to transmit the result. For the edta tubes, we have to check if it is for type and screen or just for blood count. If it is for type and screen, we need to spin it down first before giving it to the blood bank section. If it is for blood count, we will straightaway pass it to the hematology section. The tubes are then archive into special racks. This is to make identification easier.

After each batch, the med tech needs to check the incomplete log for any incomplete test. If there is any not run tests, we need to locate the tube and run it again. That’s basically it for poly samples.

For corporate samples, it is only slightly different. Instead of ‘ech’, we need to order it as corporate screening samples. So, we are going to order the tests like how we order our in-house test. We just need to change the location to indicate where it came from. For example, those samples that came from the Institute of Mental Health, the location code is 1IMH. The corporate samples usually order the test in different packages. Therefore, instead of ordering each test one by one, we have a code that will include all the tests for that package. Thus, each package will have different code. We just need to make sure that we are ordering the correct packages as stated in the form. After ordering, the workflow from then on is similar to poly samples.

Then once again, the med tech needs to check the incomplete log for any miss out tests. After all the result is out, we need to print it out and sent it back to the companies together with the request form. And also, corporate samples will not be sent every day. It will only be sent on certain days as requested by the companies.

Hmm.. that’s basically the job of the CP person.


Done by: Nur Sofieyana
Class: TG02

Sunday, November 2, 2008

Week 19 entry

Seems like just yesterday we've started the first of SIP and now we're left with one more week to go. YAY! =D

So now, i would like to share with all of you my experience of being posted into a Cytology Laboratory. The Cytology Laboratory in the Hospital that I am attached to makes use of Liquid-Based Cytology for the diagnosis of cervical cancer.

Collection of Specimen for Pap Smear Test

First, a broom-like device is inserted into teh endocervical canal of patient to allow the shorter bristles of the device to fully contact the ectocervix. teh device is gently pushes iand rotted in a clockwise direction5 times. then, the broom is rinsed into the PreserCyt® Solution by pushing the broom into the bottom of the vial for 10 times (Figure 1), at the same time forcing the bristles apart. Finally the broom is swirled vigorously to furthe release any material into the vial. The collection device is discarded and the vial is tightly capped, corrected, labeled with the sticky label and sent down together with the patient’s form, to the Cytology Laboratory.

Figure 1(Image taken from

The Thin Prep Process

In the Cytology Laboratory, Pap Smears are prepared via Liquid-Based Cytology. Patients samples that comes in vials are processed using the ThinPrep 2000 Processor(Figure 2.).

Figure 2. (Image taken from

The process involves 3 steps:
1. Dispersion of cells in the vial.
2. Cell collection onto the filter.
3. Cell transfer from the filter onto a glass slide to produce thin layer of cells onto the glass slide. (shown in Figure 3)

Figure 3. (Image taken from

After the Pap Smear is obtained on the glass slide, it is fixed in 70% alcohol for 20 minutes minimum and then stained using the Pap stain. Basically, the stains involved in the Pap stains are the Haematoxylin stain (used to stain nucleus of cells), Orange-G and EA-60 (cytoplasmic stains). after staining, teh slides are mounted with Depex, dried and then read by Cytologists.

Comparison of Conventional Method and Liquid Based Cytology

Originally, Pap Smears are prepared using the Conventional Method which had many disadvantages compared to Liquid Based Cytology. This is shown in the table below.

This is my last blog post for SIP and i hope all of you have benefitted from reading my blog posts =D.



Monday, October 27, 2008


Hello everyone! A few weeks more and we're all back at school. This will be my last post for our SIP so, here it goes...

Before a person is allowed to donate blood, he or she has to undergo certain physical examinations. The donor centre representative evaluates the prospective donor with regard to general appearance, weight, temperature, pulse, blood pressure, pulse, blood pressure, haemoglobin, and presence of skin lesions.

In this post, I will be talking about donor haemoglobin testing.

One of the methods used at the blood bank to estimate the level of haemoglobin in a donor is by the copper sulphate method.


i. The method of haemoglobin estimation using copper sulphate method is based on the principle of specific gravity. If a drop of blood is dropped into copper sulphate solution, it becomes encased in a sac of copper proteinate which will prevent the specific gravity from changing for at least 15 seconds. If the specific gravity of the blood is higher than that of the solution, the drop will sink within 15 seconds. If not, the drop will hesitate, remain suspended, or rise to the top of the solution.

ii. A specific gravity of 1.053 corresponds to a haemoglobin concentration of approximately 12.5g/dL

iii. False positive results are rare and donors whose drop of blood sinks nearly always have an acceptable haemoglobin level. However, false negative reactions occur fairly commonly and can cause inappropriate deferral unless the haemoglobin level is checked using another method.

Sample of blood obtained by finger prick.

i. Copper Sulphate Solution (specific gravity: 1.053)
ii. Glass beaker
iii. Cotton swabs, with 70% alcohol or spirit
iv. Sterile disposable lancets
v. Capillary tube
vi. Biohazard sharps box
vii. Disposable gloves
viii. Alcohol swab (individual pack)


i. Make sure that the glass beaker (250ml) is clean and free of debris. Fill the beaker with copper sulphate solution until it is slightly above 200ml marking.

ii. Put on a pair of gloves.

iii. The donor’s middle or ring finger is normally chosen for the finger prick procedure. Avoid fingers with rings on. Clean the chosen finger using a piece of alcohol swab. Allow the alcohol to dry or wipe dry with a new piece of dry cotton swab. Discard the swabs in the biohazard waste bin. Do not re-use the swab.

iv. Prepare a disposable sterile lancet by pressing and turning the knob into the lancet until there is a ‘click’ sound.

v. Use your thumb to slightly press the donor’s finger from the top of the knuckle towards the tip. Puncture the finger firmly, near the end but slightly to the side, with the sterile disposable lancet. Ensure that there is a good free flow of blood. DO NOT SQUEEZE the puncture site repeatedly as this will dilute the drop of blood with excess tissue fluid and lower the specific gravity. Dispose off the lancet in the sharps box.

vi. Wipe away the first drop of blood from the punctured site with a new piece of cotton swab. Collect the second drop of blood in a capillary tube until it is at least three quarters full, without allowing air to enter the tube.

vii. Hold the capillary tube about 1cm above the surface of the copper sulphate solution in the glass beaker, and let one drop of blood fall gently by unassisted gravity from the tube into the solution. Dispose off the capillary tube in the sharps box. DO NOT FLICK the drop of blood out of the tube as this will lead to in accurate haemoglobin measurement.

viii. Observe the drop for 15seconds. If the blood drop has a higher specific gravity than the copper sulphate solution, it will sink within 15 seconds. If not, the sinking drop will hesitate, remain suspended or rise to the top of the solution.

ix. Discard the copper sulphate solution after every 20 tests or every 2-3 hour intervals or when the solution is turbid. If there is any floating blood residue on the surface, it should be removed with an applicator stick.


i. If the drop of blood sinks within 15 seconds, the haemoglobin is greater than 12.5g/dL which is acceptable for blood donation unless there are other conditions which require a higher pre-donation haemoglobin level.

ii. If the drop of blood does not sink or sinks very slowly, the haemoglobin is most likely less than 12.5g/dL, and the result must be rechecked using quantative haemoglobin estimation by the HemoCue haemoglobin test.

iii. Because the copper sulphate method is not a quantitative test, the quantitative method by the HemoCue haemoglobin test is necessary to check the exact level in donors who fail the test as it suggests that the haemoglobin is low and the donor may need medical advice


i. This is not a quantitative test and it shows only whether the potential donor’s haemoglobin is below or above 12.5g/dL

ii. The copper sulphate solution must be stored in tightly capped containers to prevent evaporation. The solution should be kept at room temperature to brought to room temperature before it is used.

Rusydiana binte Kusni
TG 02

Monday, October 20, 2008

WeeK 17!!!

Oh my its Week 17 already!!!

Hello friends!! How are you guys doing?? I hope everything is fine especially with the projects. Well, this week Im doing on Quality Assurance. Theres not that much work to be done actually. We carry out QA tests on media and agars used in the laboratory. Here are 2 of the tests that I did for the week,

A. Citrate Test

· To determine the ability of an organism to utilize citrate as a sole source of carbon for metabolism and growth

· Certain organisms are able to utilise citrate, an intermediate metabolite in TCA (Kreb’s) cycle, as the sole source of carbon.
· In bacteria, cleavage of citrate involves an enzyme system without coenzyme A involvement
· Bacteria break the conjugate base salt of citrate into organic acids (formic acid and acetic acid) and carbon dioxide.
· An organism that can use citrate as its sole carbon source also uses ammonium salts as its sole nitrogen source.
· Simmon's citrate media contains ammonium salts and a pH indicator, bromthymol blue
· Bacteria extract nitrogen from ammonium salts with production of ammonia leading to alkalinisation of medium which causes the indicator, bromthymol blue to change to an intense blue or a Prussian blue colour when pH is above 7.6 showing a positive result

· Simmon’s citrate media
· Disposable inoculating loops
· Enterobacter aerogenes W 3/3/90 (positive control)
· Escherichia coli (E.coli) ATCC 25922 (negative control)

1. Streak the surface of the Simmon’s citrate slant
2. Incubate at 35⁰C overnight (usually 18hrs)

Left: Negative, Right: Positive Picture taken dddddddddddddddddddddddddddddddfrom:
Results and Conclusion
· Positive result (Enterobacter aerogenes): Deep blue colour of agar
· Negative result (E.coli): Absence of growth or no colour change
· Citrate test can be use to differentiate members of the Enterobacteriaceae family and other Gram negative organisms

Possible errors
· Too heavy inoculums may give a false-positive result
· Preformed organic compounds from dying bacteria may release carbon and nitrogen which may result in false positive results

Urease Test

· To test the ability of an organism to break down urea by the action of the enzyme urease

· Urea which is often referred to as carbamide, a diamide of carbonic acid
· Due to the presence of amides, urea is readily hydrolyzed by a specific enzyme, urease
· Urease is an important microbial enzyme which is used in the decomposition of organic compounds
· Urease breaks the carbon-nitrogen bond of amides to form carbon dioxide, ammonia, and water
· Urease is detected by plating bacteria onto an amide containing medium, specifically urea
· Urea will be hydrolyzed into ammonium carbonate as the end product,
· Formation of ammonia alkalinises the medium causing a change in the phenol red indicator
· Urea agar is slanted with an adequate butt portion to allow gradation of positive reactions
· Proteus, Providencia and Morganella species produce urease in large quantities

· Christensen’s urea agar slant
· Disposable Inoculating loops

1. Streak the surface of urea agar slant
2. Incubate aerobically at 35⁰C overnight (usually 18hrs)
Left:Negative, Right: Positive Picture taken from:
· Positive result: urea slant change to an intense pink-red to red-violet colourchange Positive result
· Negative result: no colour change; urea slant remain yellow
· Some organisms like Pseudomonas aeruginosa may produce ammonia by breaking down peptones present in the agar slant à False positive results
· Presence of glucose in the Christensen’s medium prevent organisms from using ammonia produced as its sole source of nitrogen which will lead to inaccurate results
· Organism like Helicobacter pylori are able to hydrolyze urea rapidly, producing a positive reaction within 1 to 2 hours
So thats all! Any questions just ask me aites!! Bye! Take care!!

Saturday, October 11, 2008

week 16...!!!!

Well..... For the past 2 weeks i was attached to the Specimen Reception (SR)department. It is the pre-analytical part of the whole laboratory, where the specimens are received and labelled.

It is here, in this department where I found out that mistakes are simply not tolerated. The personnels must be constantly on their toes, fast and at the same time, do their work accurately. A simple mistake of not tallying up the names on the specimens to the name on the form, can spell disaster.

There's no tests involved, but I am to include the workflow of the SR department.

  • The dispatch crew will arrive with the samples from the various clinics. The samples are received by the personnels there, where the number of samples and specimens are counted and tallied with those counted by the dispatch crew.

  • The samples are then passed over to another group of personnels who will open the samples. Here, they will check that the names on the specimens tally with those in the request form, and write down the number and type of specimens that they receive for that request form only.

For example: 2 long plain tubes, 1 EDTA tube and 1 urine will be written as 'LP x 2, E, U' in a vertical fashion.

Then, they would sign their initials before seprating the samples into single profile (includes only HIV, VDRL and tests for malaria parasites) and profile (which is all the other tests other than those previously named).

  • The seperated samples are then subjected to primary barcoding, where the request forms and specimens are labelled with their own individual barcode, and allocated into baskets according to the tests that are requested (such as HIV samples are placed into the HIV basket, and so forth). Plain tubes, fluoride tubes, and certain EDTA tubes and urine (which are already poured into 5ml test tubes) are centrifuged. The rest are collected by the respective departments, such as Microbiology collecting the urine, stool and swab samples.

  • The centrifuged samples then goes through secondary barcoding, where certain specimens' serum are required to be aliquoted out due to the type of tests involved. The specimens are then loaded onto a sample tray, where they will be placed in the Sample Manager (explained in my earlier posts). The sample manager will then sort the specimens according to the type of tests to be run, and send them to the various machines via the transportation track.

  • At the end of the day, the specimens will undergo allocation and be placed in the cold room, where the will be kept for a week to 10days. This is so that if there is any additional tests that is to be run, we can still use the specimen and not bother the patient to go for another blood-taking session.

Sunday, October 5, 2008

Hello everyone!! Before i start talking about my attachment, i would like to wish all my Muslim frenz Selamat Hari Raya!! We shall go visiting one day alryte??

So this week, i am going to talk about the stool occult blood test and OC Light test.

Stool occult blood test

Introduction – To detect occult blood in stool

Principle of analysis - When stool specimens containing occult blood are applied to Hemoccult SENSA test paper, which is impregnated with guaiac, the hemoglobin portion of the occult blood comes in contact with the guaiac. When the Hemoccult SENSA peroxide developing solution is added, a guaiac peroxidase like reaction occurs. Oxidation of guaiac becomes visible by the appearance of a blue color within 30 seconds. A food that contains substances with peroxidase activity may cause false positive results.

Specimen – Stool


Hemoccult SENSA test slides
Hemoccult developing reagent

Retrieved from :


1) Apply a very thin smear of stool inside section 1 and 2 with an applicator stick.

2) After 3-5 minutes, open the perforated section on the back of the slide.

3) Apply 1 drop of Hemoccult SCREEN developing reagent to the positive and negative well each and 2 drops to the reaction wells.

4) Read results after 30 seconds and before 1 minute.


Read the results only when the controls give the correct color after the addition of the developing reagent.

  • Positive – Traces of blue colors in either one or both of the reaction wells
  • Negative – No indication of blue color in either one or both of the reaction wells

Positive stool samples are confirmed using the OC Light Occult Blood Method.

Limitations of procedure

Hemoglobin is not usually found in the stool of healthy individuals. Its presence indicates bleeding in the gastrointestinal tract. Causes include tumors. Tumors are common in the elderly and this test is used as a screen to select patients for further invasive investigation.

Stool OC Light

Introduction – The OC Light method is used to confirm the positive cases by the Hemoccult SENSA method.

Principle of analysis – The OC Light is designed to measure human hemoglobin in fecal samples. The OC Light immunochromatographic test has a test detection range from 10µg to 100mg/g of feces. The test strip reacts only with human hemoglobin and does not react with hemoglobin of other animal origin.

Specimen - Stool

Reagents -

OC Light Sampling Bottles

OC Light Strips

Retrieved from :

Method -

1) Remove the green cap from the sampling bottle and dilute the stool inside the bottle.

2) Remove the white cone nozzle.

3) Insert the strip into the sampling bottle from the dip slide.

4) Read test result, presence of a blue line at the lower and upper center of the strip after 5 minutes.


Read the result only when the controls give the correct color after the addition of the developing reagent.

Positive result – Presence of blue line at the lower and upper center of the strip may be considered positive.

Negative result – Presence of blue line only at the upper center of the strip is negative.

Equivocal result – No blue in line in both the lower and upper center of the strip or at the lower center of the strip only should be considered equivocal. Such samples should be retested by changing OC Light test strip.

That's all for now peepz!!

Before i sign off, i would like to say sorry if i have not answered any of your questions regarding my last two posts.. It's been hard for me to get access to the internet. I will try my best to reply to all the questions ASAP.. Really sorry especially to Ms Chew. I hope i have not been penalized by my lack of participation. I will try to participate more often once i got my laptop soon.. Sorry once again..


** Sofie **

Friday, September 26, 2008

week 14th.

I am going to explain a test which I had the oppotunity to view for myself during my placement at Haematology Laboratory.

Bleeding Time using Surgicutt

This test is to estimate the integrity of the hemostatic plug. It is a screening test for
qualitative and quantitative platelet functions and generally helps to diagnose hemostatic defects and platelet dysfunctions.


When a minor standardized incision is made on the forearm, time between the infliction of the incision and the moment the bleeding stops is taken. A hemostatic plug is formed after the incision to stop the bleeding. This plug formation depends on adequate platelets to adhere to the subendothelium to form aggregates.
For this test is a screening test, an increased bleeding time would not qualitatively or quantatively diagnose an underlying platelet disorders. However, it is indicative of the need for more quantifiable testing such as platelet counts, APPT, TT testing etc.

Image of Surgicutt Bleeding device taken from


1. First, a sphygmomanometer is used to cuff the upper hand of the patient. The pressure is raised to 40 mm Hg.This is the standard pressure for children and adults. For infants, the pressure would be dependant on the weight. Patient’s blood pressure was to remain at such throughout the duration of the test.
2. The volar surface of the forearm, where the incision is to be made, is rubbed with alcohol swab.
3. The incision is made using Surgicutt bleeding time device and the timer is started simultaneously. There are three different devices to suit different age group, mainly infants, children and adults. This is shown below.

Age group :1 - 4 months
Device:Surgicutt Newborn
Depth of blade (mm) :0.50

Age Group:5 months to 15 yrs
Device: Surgicutt Junior
Depth of blade (mm): 1.00

Age Group: 16 years and above
Device: Surgicutt
Depth of blade(mm): 1.00

4. Every 30 seconds, a blotting paper was used to wick the blood close to the incision without touching the incision itself.
5. The timer is stopped when the bleeding stopped.
6. Reading on timer is recorded down.

The following are the reference values for normal bleeding time:

Infants (1-4 months) :
0.85- 1.65 mins

Children (5 months to 15 years) :
1.3- 8.99 mins

Adults (16 years and above) :
2-8 mins

For neonates whose bleeding does not stop after 5 minutes, time is reported as >5 mins. For adults whose bleeding does not stop for more than 20 minutes, time is reported as >20 mins.


Sunday, September 21, 2008


Hope you guys are doing well.

This week, I will be touching on a topic which may be foreign to some of you. It will be on glycerolization/freezing and deglycerolization/thawing of red blood cells. This process is done in the cryopreservation lab of the blood bank.

Freezing RBCs with glycerol dates back to the 1950s. Frozen RBCs can be stored for up to 10 years for:
1. Patients with rare phenotypes
2. Autologous use
3. In case of national emergencies in which blood cannot be dispositioned out to hospitals quickly enough to prevent expiration.

The resulting deglycerolized product is free of leucocytes, platelets, and plasma due to washing process. Cryoprotective agents can be categorized as penetrating and nonpenetrating. A penetrating agent involves small molecules that cross the cell membrane into the cytoplasm. The osmotic force of the agent prevents water from migrating outward as extracellular ice is formed, preventing intracellular dehydration. An example of a penetrating agent is glycerol. An example of a non-penetrating agent is hydroxyethyl starch (HES). This comprises large molecules that do not enter the cell but instead form a shell around the cell, preventing loss of water and subsequent dehydration.

Two procedures used for glycerolizing RBCs are high glycerol and low glycerol methods. The methods differ in the equipment used, the temperature storage and the rate of freezing. Most blood centres practice the high glycerol method.

High Glycerol (40% w/v)
This method increases the cryoprotective power of the glycerol, thus allowing a slow, uncontrolled freezing process. The freezer is generally a mechanical freezer that provides storage at -80oC. This particular procedure is probably the most widely used because the equipment is fairly simple and the products require less delicate handling. It does however require a large volume of wash solution for deglycerolization. RBCs are frozen within 6 days of collection when the preservative is CPD or CPDA-1 and up to 42 days when preserved in AS-1, AS-3 and AS-5. AABB Standards states RBCs must be placed in the freezer within 4 hours of opening the system. It is advisable to freeze a sample of donor serum in the event additional testing is required for donor screening.

Low Glycerol (20% w/v)
In this method, the cryoprotection of the glycerol is minimal, and very rapid, more controlled freezing procedure is required. Liquid nitrogen (N2) is routinely used for this method. The frozen units must be stored at about -120oC, which is the temperature of liquid N2 vapour. Because of the minimal amount of protection by the glycerol, temperature fluctuations during storage can cause RBC destruction.

Red cells that have been frozen require thawing and deglycerolization before they can be safely transfused. Glycerol will be removed from RBC to avoid in vivo and/or in vitro heamolysis.

The thawing process takes approximately 30 minutes and involves immersion of units into a 37oC water bath and washing the RBCs with solutions of decreasing osmolarity (eg. 12% NaCl, 1.6% NaCl, 0.9% NaCl, + 0.2% dextrose). An exception to this rule is a donor with sickle cell trait in which RBCs would haemolyze upon suspension in hypertonic solutions; in this case the cells would be washed in 12% NaCl and then 0.9% NaCl with 0.2% dextrose, omitting the 1.6% solution. Automated continuous-flow instruments can be utilized for washing. In our blood bank, a machine called the Haemonetic 215 is used. Once the RBCs have been deglycerolized, the unit is considered an open system with an expiration date of 24hrs and is stored at 1oC to 6oC.

Hope my post this week has enlightened you on some of the processes that occurs in a blood bank. Till next time..... Adios!!!

Rusydiana binte Kusni

Sunday, September 14, 2008

AMir's back...and its week 12!!!

How is everyone doing?.. wah 3 months of attachment already..thats fast.. 2 more months and we can meet up already..ok what shall i say about this week.. hmmmmmm


For last week I was posted at parasite lab.. In this lab, they mainly deal with protozoans and helminths(i hope you guys remember what we learn in BMic).. if not nvm i shall give a brief intro...

Parasite are organisms that live on or in a host organism, usually causing it some harm. They are relatively smaller in size and are dependent on their host for nourishment. There are many forms of parasites like, arthropod parasites (ticks), plant parasites (mistletoe), single-celled protozoan (amoebas) and even viruses. Numerous parasites live in the gastrointestinal tract, known as intestinal parasites which are common human pathogens. Parasite are subdivided into protozoans (unicellular) and helminths ova/larva (multicellular)

Eg of protozoans include: Amoeba histolytica, Giardia lamblia, Cryptosporidium
Eg of helminths include: Roundworms (Ascaris lumbricoides), tapeworm, flatworms

Parasite infections in Singapore is not as common as bacterial infections. So, we do not recieve much specimens. There are many different tests carried out: Ova and Parasite Exam, Silver stain for Pneumocystis jirovecii (PCP), leukocytes examination and occult blood. For this blog i will focus more on Ova and Parasite exam

Ova & Parasite exam

Ova and parasite exam is basically analysis of stool to check for the presence of a parasite or worm-like infection of the intestine. Most of the specimens, or i shall say ALL of the specimens are stool specimens, as intestinal parasites there will be released in the stool of infected humans. There will be a large population of parasites in the stool, there it will be easier to detect the presence of this parasites.


  • Ova and parasite exam is the most common performed procedure in diagnostic parasitology
  • For identification of intestinal protozoan parasites, it is based on the recognition of their cyst or trophozoites stages or even both
  • Cysts are spherical with smooth uniform walls formed during the dormant resting stage
  • Trophozoites have thin limiting membrane with variations in size and shape, they are formed during active feeding stage

  • image taken from:

  • For Helminthic infections, it is based on identification of eggs, larvae or proglottids in faeces
  • Eggs or ova are usually found in stool specimen as it is passed out in faeces
  • Ova and parasite exam consists of 3 separate protocols: Direct wet mount, Concentration and Permanent stained smear
  • Direct wet mount allows detection of motile protozoans tryphozoites and helminth worms
  • Concentration is designed to facilitate recovery of protozoan cysts, coccidian oocysts, microsporidial spores, and helminth eggs and larvae.
  • Formalin-ethyl acetate sedimentation method is used to prepare concentrated specimen for direct wet mount.
  • Permanent stained smear is used to identify intestinal protozoa, using the common Trichrome stain method
  • It is the most important procedure as it aids in the confirmation of any suspicious objects seen in the direct wet mount


  • Para-Pak® ULTRA Zn-PVA fixative

  • Para-Pak® Macro-Con® Kit
    - Surfactant bottle (30ml)
    - Conical 50ml centrifuge tubes with filtration units and screw caps

  • Disposable ice-cream stick and applicator sticks

  • Disposable Pasteur’s pipette

  • Paper towel

  • Microscopic slides

  • Timer

  • 10% Formalin

  • Ethyl acetate (Clearance)
  • Disposable cotton swabs
  • Normal saline

  • Trichrome stain
    · For destaining: 10% acetic acid in water, 50% and 70% ethyl alcohol


A. Preparation of material for Trichrome staining

1. Using a disposable ice-cream stick, place fresh faeces into the PVA fixative in the
Slides ready to be stained ratio of 3 parts PVA fixatives to 1 part faeces.
2. Mix thoroughly so that it appears homogenous
3. Allow the faeces to fix for a minimum of 30mins; overnight fixation is acceptable
4. Pipette out some of the well-mixed PVA-faecal mixture onto a paper towel using a disposable Pasteur’s pipette and allow to stand for 3mins to absorb out the excess PVA (This step is critical to obtain the best possible stain)
5. With an applicator stick, apply some of the faeces from the paper towel onto a clean slide (Spread the material onto the edge of the slide for better adherence)
6. Dry the slides for 3 hours or overnight at room temperature (Slide must be dried thoroughly to prevent material from being washed off during staining)

B. Concentration procedure
1. Add 10 drops of surfactant into the ParaPak specimen vial
2. Insert the filtration tightly into the specimen vial
3. Invert assembled unit, tapping sharply to force the solution into the conical tube
4. Unscrew the filtration unit and discard appropriately
5. Add 10% Formalin to the dotted line, plus 5 ml of Ethyl Acetate (Clearance)
6. Tightly screw the cap and shake vigorously for 1min
7. Centrifuge at 1500xg for 10mins
8. After centrifugation the specimen is clearly separated into four layers
9. Using an applicator stick, rim the debris layer. Pour off the debris and supernatant fluid, leaving the sediment
10. With the tube still inverted, use a cotton swab to clean and remove the remaining debris
11. Return the tube to upright position and add a few drops of saline to mix the sediment

C. Wet Mount
1. Using an applicator stick, prepare a coverslip preparation from the sediment
2. Examine microscopically

D. Trichrome Staining
1. Place slide in Trichrome stain solution for 10mins
2. Destain by placing the slide in acidified alcohol for 1-3sec (Do not allow slides to remain in this solution)
3. Dip slide several times in 100% ethyl alcohol
4. Place slide in 2 additional changes of 100% ethyl alcohol for 5mins each
5. Place slide in 2 changes of xylene substitute for 5mins each
6. Mount in immersion oil, using a No.1 thickness cover glass
7. Examine microscopically


In Trichrome stain, cytoplasm of cysts and trophozoites will stain blue-green tinged with purple. Parasite eggs and larvae usually stain red. All ova, pathogenic protozoan trophozoites or cysts seen must be reported. The reports will be sent to the doctors, and they will decide whether to do any further confirmatory tests depending on the patient's clinical symptoms. image taken from:,%20trophs%20(2)%20in%20mucus%20string,fixed%20great.jpg

So, thats about all!!!.. I shall be back sooon!!.. take care peeps!!



Saturday, September 6, 2008

week 11 already people!!

Know what? I'm really having a hard time finding what to blog about, considering most of the tests have already been posted. Haha.

Well anyways, I've finally decided to write about a test to detect anti-Mycoplasma pneumoniae antibodies, using SERODIA-MYCO II (also known as MPA), since now I'm currently in serology department. Alot of manual tests, if I may say.

First of all, Mycoplasma pneumoniae is a very small kind of bacterium that is usually associated with upper respiratory tract infections, together with fever, headaches, cough and malaise. It may lead to tracheobronchitis, which is a common respiratory infection characterized by inflammation of the trachea and bronchi. Its mode of transmission is person-to-person transmission by direct contact with the infected respiratory secretions, and it will usually take about 1-4 weeks before the symptoms will develop.

SERODIA-MYCO II is used in the detection of antibodies to Mycoplasma pneumoniae. It uses gelatin particles which are sensitized with extracted cell membrane components of Mycoplasma pneumoniae. Serum containing specific antibodies will react with the sensitized couloured gekatin particles to form a smooth mat of agglutinated particles in the microtitration tray.

SERODIA-MYCO II kit, which includes:

  • Sensitized particles
  • Unsensitized partiles
  • Positive control
  • Sample diluent


6 wells are required for a patient's sample

4 wells are required for control

  1. Label the wells (ID number or Control)
  2. Add 100ul of sample diluent to the first wells
  3. Add 25ul of sample diluent to the rest of the wells
  4. Add 25ul of positive control to the first well and mix well
  5. Bring 25ul of the mixed solution to the next well and mix well
  6. Repeat Step 5 until the last well, where 25ul will be discarded after mixing
  7. Repeat Steps 4-6 with patient's sample
  8. Add one drop of unsensitized particles into the second wells
  9. Add one drop of sensitized psricles into the rest of the wells starting from well 3
  10. Tap at the sides of the microtitration plate gentle to ensure proper mixing
  11. Cover the plate and incubate at room temperature
  12. Results to be read 3 hours later


Negative (-):

Definite compact button in the center of well with smooth round outer margin

Positive (+):

Definite large ring with firmly agglutinated particles spread within the circle

Positive (++):

Agglutinated particles spread out to cover th bottom of the well entirely