Medical Microbiology - Streptex Test

Hi, there! Muna here XD

SIP is finally over. 5 months of hard work, blood (literally!), sweat and tears.

Good luck to all for the MP project reports.

Streptex is a rapid latex test used to determine the Lancefield grouping of streptococci. This test uses the principle of antigen-antibody binding. The antigen-antibody complex forms as a visible precipitate in a positive reaction.

Most of the Streptococcus sp. contain group-specific antigen. These antigens are actually carbohydrate structural components of the bacterial cell wall. It was shown by Lancefield that these antigens can be extracted in a soluble form and tested using precipitation reactions with homologous antisera.

In Streptex, the antigen extract is obtained using a simple enzyme extraction procedure. Polystyrene latex coated particles coated with group-specific antibodies are used to identify the antigen in the extract. Agglutination will occur strongly in the presence of the specific homologous antigen and remain smooth or milky in its absence.

The latex reagent contains a combination of fibrinogen, IgG and anti-capsular monoclonal antibodies to allow recognition of capsular polysaccharides of Staphylococcus aureus.



5 pure growth colonies of Streptococcus sp. are used to make a 0.5 McFarland suspension in 400ml of Extraction Enzyme.
Incubate at 35oC between 10 mins and 1 hour.
Re-suspend the test kit latex by shaking.
Add 1 drop of each test kit latex (A, B, C, D, F, G) to the test circles on the reaction card.
Add 1 drop of extracted sample to each test circle.
Rock card horizontally or use a disposable rod to mix for 1 minute.
Read the result immediately after 1 minute of shaking.

Positive
Visible clumps
Negative
No clumping/agglutination
Milky appearance

The main use of this test is to test for the more virulent beta-haemolytic strains of Streptococcus.


*Colonies should be taken from Blood Agar Plates (BAP), Chocolate Agar (CHOC) or CAN (Columbia agar) and not be more than 48 hours of growth.

References:
Taken from
http://www.remelinc.com/Clinical/DiagnosticTests/Streptex.aspx
http://www.dcss.cs.amedd.army.mil/field/FLIP%20Disk%2041/.\Documents\M403_Micro_Aug\Streptex.DOC
on 21 Sep 2009

Muna

0703791D

Pathogens found in stool.

Two out of all the organisms found in stools which are pathogens to our body are Vibrio cholerae and Campylobacter jejuni

Vibrio cholerae
Vibrio cholerae species belong to the gram-negative bacteria group. It is a facultative anaerobie and curved rod in shape. It is motile and moves by its single flagella. Vibrio cholerae is also identified by being oxidase positive and does not produce hydrogen sulfide and gas Kliger Iron agar.
Vibrio cholerae can be found water surface in many parts of the world.

Below shows a picture of Vibrio cholerae species.


Pathogenicity
Vibrio cholerae causes Cholera in humans. It is an acute intestinal infection and is transmitted to human through contaminated water and food, or through uncooked food such as shellfish and other seafood.
One of the symptoms that people with cholera have is diarrhea. It is caused by a toxin produced by the organism which targets our epithelial cells and that explains for the watery stool appearance in patients infected by Cholera.




Campylobacter jejuni

Campylobacter jejuni is a gram negative, spiral shaped and motile organism.It is a microaerophilic organism therefore, it cannot survive in an environment with high level of oxygen. This organism is usually in found in poultry and contaminated water or sometimes even unpasteurized milk.

Below shows a picture of a Campylobactor jejuni species.



Pathogenesis
The name of the infection caused by this organism is called Camphylobactoriosis. People who are infected by this bacterium have symptoms such as: bloodly, watery or sticky stool. Some people may experience headache, muscle pain and nausea.


So sorry that i forgot to indicate who i was. This is Jennifer here

Neonatal Bilirubin

Hello all~! Yan Ling here~! Sry for the late post. ><

Can u believe? We had finished 16 wks of sip le~! 1 more mth to go~! xD

Okay for this post, which should be my last post already, I'm going talk about neonatal bilirubin testing. I'm was sent of a medical center somewhere in sg where they do neonatal bilirubin testing as there are nurseries there. Believe everyone should still remember abt the incompability of mother-fetal bld group, which result in hemolysis of fetal's RBC by mother's IgG.

When the fetal's RBC are broken down, bilirubin as the by-product was produced and is to be removed by the fetal's liver. However, when the amounf of bilirubin exceeds the theshold of the liver to remove the bilirubin, it will remain in the body, and thus resulting in neonatal jaundice. And to treat the neonatal jaundice is by giving phototherapy so the light, usually blue in colour, will break down the bilirubin.

And so, measuring the amount of bilirubin in the baby's serum is important so to see if he/she requires any treatment or able to go home with his/her mummy.

First, the collection of baby's blood is using heel prick where the phlebotomist will use a lancet to prick the either side of the baby's heel and using a capilliary tube to collect the blood. After that, the tube is spin down to get the serum. The centrifuge used is different from the normal ones we see/uses in the lab, its especially for spining down capillary tube, however, using the normal centrifuge is also possible but it requires longer spining time compare to the special centrifuge which usually requires abt 3-4 mins to spin down.

So after the tube is been spun down, it will be separated into serum and RBC. Then, the serum part will be used to read for the neonatal bilirubin. The analyser used in my lab is reichert, which

uses a curvette to read the amount of bilirubin.

The principle of this test is spectrophotometer which measures the colour of the serum. The machine measures at 460nm and 55onm. At 460nm, the machine will measure the bilirubin and oxyhemoglobin and at 550nm, the machine will only measure the oxyhemoglobin. And thus the difference of 460nm and 550nm concentation will be the total bilirubin. Since oxyhemoglobin is measured, hemolysed specimens will not affect the result since the hemoglobin is measured.
The values of the bilirubin are then faxed to the clinics/nurseries where the doctors will decide whether to continue the treatment or not.

Here are some pics to show you about the machine. xD

The Reichert machine with curvette, and the syringe-looking thing is to add the serum into the curvette.


Capillary tubes with babies' blood after spun down.

Thats all. xD Feel free to ask me any questions. Will try to answer wor. ><

yaNLing xD

Biochemical Test

Hi!

I was posted to the Anerobic(Quality control)on my 16th week.And we have been doing Biochemical test for the identification of Bacteria. The bacteria identification test done for the post is for E.coil

Biocehmical test is used for the identification the bacteria, by using the biochemical propaty of the bacteria. It is done using the following basic media.

Kliger iron agar

It uses the ablity to utilise carbohydrate fermentatively & to librate H2S.It contains one part glucose (0.1%) and 10 part lactose (0.1%).Phenol red is used as pH indicator.H2S formation is by the reaction of bacterium with sodiumthiosulplate with will form ferrous sulphide when H2S reacts with ferric ion.


Simmons citrate agar

Organism utilise citrate as their sole carbon source which is a intermediate metabolite in the kerb’s cycle. Media contain inorganic ammonium salt which the organisum use as a sole nitrogen source. The presence of organisum utilise the media a creats a alkaline environment that turns the bromthymol blue to a intense blue when pH reaches above 7.6.

Typtophan broth

The indole test determines the ability of an organism to produce indole from the degradation of the amino acid tryptophan in the broth. Tryptophan is hydrolysed by tryptophanase to produce three possible end products which is the Indole, pyruvic acid and ammonia.A bright red color product is produced when the indole is combined with aldehydes group.

Oxidation Fermentation (OF) basal Medium

The medium contains a high concentration of added carbohydrate to avoid the utilization of peptone by an aerobic organism as it would neutralize slight acidity produced by an oxidative organism.Bromthymol blue is used as a indicator.The agar permits the determination of motility and aids in the even distribution of any acid produced at the surface of the medium



Inoculation Method and reaction

Kliger iron agar

Stab to the bottom of the tube and streak the slant
Acidic butt and slant: Glucose and lactose fermentation (E.coil)
Acidic butt and alkaline slant:Glucose fermentation,Lactose nonfermentation
Alkaline butt and slant: Glucose and lectose non fermentation
Black media for H2S production.
Bubble when other gas is produced


Simmons citrate agar

Streak the slant.
+ve: Growth on the slant or deep blue colour observed. (E.coil)
-ve: No growth and no clour change (remains green)


Typtophan broth
Inoculate organisum by dissolving the colony in the broth.
After 24 hrs of incubation add 5 drops of kovac to the inner wall to see the pink interface.
+ve: Bright fuschia red interface with in secs.(E.coil)
-ve: No change

OF basal Medium
Stab the organisum stright to till the ¾ of meduim.
+ve: Medium becomes yellow indicating acid production. (E.coil)
-ve:Remains green.
Motality is observed when organisum grow away from the stright inoculum line.(E.coil)

By

Shameema :)

Medical Microbiology [Coagulase Test]

Coagulase test is used to differentiate between Staphylococcus aureus and other coagulase negative species. This test is based on the coagulative properties of S. aureus with plasma. This test is usually performed if the latex agglutination test for S. aureus is invalidated or equivocal.

S. aureus produces 2 forms of coagulase – free and bound. Different types of tests are used to observe the coagulation reactions, depending on the form of coagulase targeted.

Bound coagulase, also known as “clumping factor”, is detected using the slide test. This form of coagulase is bound to the bacterial cell wall, hence its name. It reacts with fibrinogen in plasma to form visible clumps. However this test is not performed in the lab anymore.

The tube test is done on free coagulase. Free coagulase released by the bacterial cell is often found in culture filtrates. It exists as a free form, unlike the bound coagulase attached to the cell. The free coagulase acts on prothrombin, producing a complex with similar properties to thrombin - staphylothrombin. This substance then acts on fibrinogen to form a fibrin clot in plasma.

1. 2-5 staphylococcal colonies are suspended in 0.5ml of rabbit plasma w/EDTA.
2. Incubate aerobically at 35^oC for 4 hours.
3. Check for clot formation.
4. If negative, continue incubation overnight and check again after the extended incubation.

Positive (+ve)	Visible clumps suspended in plasma
Negative	Absence of clumping

NOTE: Care should be taken when examining the clot formation. If too much time has passed before examining the clot formation, it could produce false negative results. This is due to the ability of certain S. aureus to produce staphylokinase, which breaks down any clot that has formed.

References:

"http://www.hardydiagnostics.com/catalog2/hugo/CoaguStaph.htm"

Also taken from http://en.wikipedia.org/wiki/Coagulase on 27 Sep 2009.

EDIT: By the way this entry is from MUNA.
teehee. XD

Fecal Flotation

Hello!!! The test that i am going to talk about today is the fecal flotation test.

A fecal flotation test is normally used to detect the fecally-shed eggs (and sometimes the larvae) of various parasitic worm species as well as the oocysts shed into the droppings by various, replicating protozoan organisms.

The results will help the trainers to decide whether if it is time to deworm the horses.

The equipment that we are using for this test is the Fecalyzer®.

Steps:

• Lift cap and remove green insert. place 1 gram of fecal sample into valve can fill it the FECALYZER® Flotation Medium.
  

• Rotate green insert vial back and forth to separate ova from fecal sample. Mix thoroughly

• Seat green insert vial firmly in place.

• Fill holder completely with additional FECALYZER Flotation Medium to the brim to form a meniscus.

• Cover the float 22mm cover slip on meniscus for 15-20 minutes.
  
  
• Transfer the cover slip to a microscopic glass slide and examine it under the microscope under 40-100x magnification.

Here are some of the pictures or parasite eggs:

• Isospora oocyst (extreme close-up - 1000x) starting to undergo maturation to its infectious state

• A Spirometra tapeworm egg under 1000x oil immersion.

• a Whipworm or Trichuris vulpis egg under 1000x oil immersion.

pictures taken from: http://www.pet-informed-veterinary-advice-online.com/fecal-flotation.html

Regards,

Joash Ong(0702657H)

Medical Microbiology - idenification of bacteria

Hello all, Yan Ling here. First, I'm sorry for the late post, supposed to blog last wk. But here I'm now going to talk abt bacteria identification that I learnt during my 3 wks at microbiology at my company I'm attached to.

But let me explain the type of plates usually used for cultures. For urine culture, sheep blood agar, MacConkey agar, CLED agar and Mellur Hinton(MH) agar with E.coli lawn are used. MH agar with E.coli lawn is used to identify if the patient is taking antibiotics as if there is presence of antibiotics in urine, there will be clearing on the bacteria lawn. For genital swab, eg, high vaginal swab etc, sheep blood agar, MacConkey, GC Lect agar and Sabouraud Dextrose (Sab Dex) agar are used. GC lect agar is used to identify ,Neisseria gonorrhoeae and Sab Dex agar is used for identify presence of fungi at genital site. For wound swap, 2 blood agar and MacConkey agar are used. 2 blood agar are required as one is for aerobic bacteria and another is for anaerobic bacteria. And lastly for ear and eye swabs, blood agar with S. aureus streak, MacConkey and cooked meat medium are used. S.aureus streak is used as they haemolyse the RBC in the blood agar, and thus allowing H. influenzae to grow as H. influenzae requires Cooked meat medium is an enrichment broth which allows growth of any bacteria present in the swab. The sequence of which type of agar to streak first is: blood agar -> macconkey agar -> follow by the required agar for each type specimen. Blood agar is usually streak first as it is a universal agar which allows any type of bacteria to grow. MacConkey agar as you all know, selects only gram negative bacilli to growth and helps to differentiate between lactose fermenters (pink colonies) and non-lactose fermenters (non-pink colonies).

Streptococcus VS Staphylococcus
Streptococcus (gram-positive, catalase negative) appears translucent and shiny on blood agar. There are 3 types of haemolytic strepococcus. Alpha-haemolytic where it is only partial haemolysis, Beta-haemolytic which is complete haemolytic and gramma-haemolytic where there is no haemolysis. In alpha haemolytic, S. pneumoniae is clinically significant, and to identify S.pneumoniae, optocin is used as S.pneumoniae is sensitive to optocin. For beta-haemolytic, there different groups, namely Grp A, B, C, F and G. And for gramma-haemolytic, it is Grp D. The grouping are according to the Lancefield Grouping. Grama-hemolytic are identified as enterococcus sp. which is bile esculin positive which turns bile esculin black.

Staphylcoccis(gram-positive, catalase positive) appears creamy on blood agar and has larger colonies than strepotococcus. Coagulase is used to differentiate S.aureus and other types of staphylcoccus. S.aureus is coagulase positive.

Gram-negative bacilli
Gram-negative bacilli(GNB) can be oxidase positive or oxidase negative. Oxidase negative can be sub-divide into lactose fermenters. Identification of GNB is by using Microbact which is smth to the api 20 that we learnt during mmic pract, but it is used for GNB only. For oxidase negative GNB, only microbact 12A is used and for oxidase positive GNB, microbact 24E or 12A and B are used. Microbact is fairly easy to do, inoculate some colonies into saline to MacFarland 0,5 (remember that time when we all are doing the antibiotic sensitivity test, we use the MacFarland as the guide to how turbid the saline with colonies should be?). Then add 4 to 5 drops to each well of the microbact and add mineral oil to those wells with black rings as mineral oil prevents oxidation. Then it is incubate overnight and colour change is observed and noted down value of colour change using the table they provide. add up the values to form a 4-digit number, then using microbact system, key in the digit and yeah~ we got our bacteria. The system will give a list of bacteria with the probability, only probability of >75% is considered clinically significant. If is < 75%, it will be reported as no pathogen inoculated. Microbact is actually those biochemical tests like triple sugar test, hydrogen sulfate, indole test, urease test etc but in a smaller and more convenient way.

Here is a website that have some information about microbact: http://www.rapidmicrobiology.com/news/603h77.php?s=wells. Retrieved on Sept 13, 2009.

Okay, will say till here, these are the more important ones of bacteria identification. If i explain all, i think the post will be super long.

Feel free to ask any qn~! I will try to answer them. :)

yaNLing xD

Hi. This is Jennifer. Today i will be updating about catalase test which is done to defferentiate to identify organism species.

Catalase Test

Catalase is enzyme which breaks down hydrogen peroxide into oxygen and water. One of the oxidative end products that are produced by aerobic carbohydrate metabolism is hydrogen peroxide. If hydrogen level is too high for bacterial cells, it will kill them.
In this case, the catalase test that is uses to differentiate Neisseria gonorrhoeae and other Neisseria and related Species. ( Kingella denifrificans)
Catalase converts hydrogen peroxide into water and oxygen shown by the following reaction:

catalase
2(H2O2) --------> 2(H20) + O2 (Gas bubbles)


Method
1. A small drop of the hydrogen peroxide solution was dropped onto a plastic surface, eg. Small agar plate or microscope slide.
2. A small amount of cells were transferred from a well- isolated colony from the agar to mix with the solution.


Results
Positive: Explosive bubbling reaction should occur right immediately when the cells and solution were mixed.

Negative: No bubbling reaction.

Below is a picture showing the result of a positive and negative catalase test.
On the left shows a positive catalase test by having immediate explosive bubbling reaction.
On the right shows a negative catalase test by having no bubbling reaction.

Microbiology ( Faecal Occult Blood Test )

Fecal Occult Blood Test (FOBT) and Ova, Cyst and Parasite test are two of the most common tests done in the Parasitology bench.

FOBT is done to determine the presence of hidden (occult) blood in the stool. This may be caused by undetected gastrointestinal bleeding, which could be an indicator for colorectal cancers. The test is based on immunochromatography. It uses antibodies against haemoglobin, a main component of blood. . The presence of haemoglobin will cause it to bind to the antibody, and cause a colour change in the test strip.




The stool needs to be sampled at 6 places to allow for homogenous sampling of the stool.


After the sampling vial has been inoculated with the stool, a test strip is then inserted into the test solution in the vial. The result should be visible within 5 minutes. Any result obtained after the 5 minutes window period will be considered void, as there may be a chance of false positives occurring.




The control line must be working in order to obtain a validated test result. The control line ensures that all the procedures have been done correctly. If the control line does not show, it means that the test is equivocal and cannot be used. Thus, a retest needs to be done.


In ova, cyst and parasite testing, there are two main procedures – the fixed smear and the wet mount. Fixation of the stool sample helps to preserve the state of the parasitic organisms in the stool. The wet mount enables the visualization of motile parasitic organisms in the stool.

Fixed Smear
1. Mix 1 part stool to 3 parts of Zn Poly-Vinyl Alcohol (PVA) in the provided PARA-PAK tube.
2. Let stand for 30 minutes.
3. After 30 minutes, pour some of the Zn-PVA-fixed material onto a paper towel. Let most of the excess PVA be absorbed.
4. Once most of the excess PVA has been absorbed, spread some of the fixed stool material evenly onto a glass slide. The edges of the slide must be smeared with some of the fixed material to adhere better onto the slide.
5. Once dry, heat-fix for 3 hours on a slider warmer at about 35oC.
6. After which, perform a trichrome stain.

Wet Mount
1. 10 drops of surfactant is added to the remaining Zn-PVA fixed sample and shaken vigorously. The surfactant helps to break down the larger stool particles.
2. The Macro-Con conical filter tube is then attached to the PARA-PAK tube and inverted to allow filtration of the sample.
3. Once the sample has been filtered, the empty PARA-PAK tube and filter is discarded, leaving the MACRO-CON tube full of fixed sample solution.
4. Add buffered formaldehyde (formalin) (4.4 % w/w of solution) up until the indicator line on the tube.
5. Add 5 ml of clearene.
6. Spin at 2000 rpm for 10 minutes in the centrifuge.
7. Discard the supernatant and clean the sides of the tube with a clean cotton swab to remove excess clearene and formalin.
8. Resuspend residue in equal amounts of normal saline.
9. Place a drop of the resuspended solution onto a glass slide and mount with a coverslip.

View both the fixed smear and the wet mount under a microsope.


MUNA
0703791D

Retrieved from “ http://www.nagase.com/products/oclight/oclight.pdf” on 2 August 2009.

Retrieved from http://www.cancer.net/patient/Library/Cancer.Net+Features/Treatments,+Tests,+and+Procedures/Fecal+Occult+Blood+Tests+%E2%80%94+What+to+Expect
On 2 August 2009.

Mycology

Cryptococcal Antigen Latex Agglutination Test

Hi! I'm posted to mycology for this week and i''ll share with you guys the test that i get a hands on with.

Cryptococcal Antigen Latex Agglutination Test is a qualitative and quantitative test. The test is used to detect capsular polysaccharide antigens of Cryptococcus neoformans in patient’s serum and cerebrospinal fluid. It is done using commercial latex particles coated with anticryptococcal globulin (detection latex). In the presence of capsular polysaccharide antigen in the patient's serum or CSF, the latex particles coated with anticryptococcal globulin will react with the antigen and form a visible agglutination. Latex particles coated with normal globulin acts as the control (control latex).

This is the kit that is used CALAS®.

http://www.cardinalhealth.com/us/en/distributedproducts/ASP/140100.asp?cat=laboratoy

Procedure

1. First, when the blood sample is received it is centrifuged at 1000 rpm for 15 minutes.

2.Then, Transfer 100ul of patient’s serum to a test tube with 100ul of pronase.Pronase is an enzyme which degrades non specific macro globulin.

3. Incubate the serum-pronase solution in a water bath for 15 minutes at 56°C.

4. Immediately boil the solution for a full five minutes to terminate the enzymatic digestion.

5. Allow the solution to cool to room temperature.

6.Then remove enough cards (see the pic above) to run the test and label one ring as detection latex with the patient's lab number (DL) and another as control latex (CL).

7. Pipette 25ul of patient serum to both CL and DL rings.

8. Holding the detection latex bottle in a vertical position, squeeze one free-falling drop of reagent to the DL ring. Do the same for the control latex bottle and drop one free-falling drop of reagent to the CL ring.

9. Place card on a rotator and rotate at 125 rpm for 5 minutes.

10. Read the result immediately.

For CSF, it does not require steps 1-3. It begins by transfering about 0.5 ml of CSF to a test tube and boiling it for a full 5 mins. The subsequence steps are the same as the blood.

Result Interpretation

Negative: No visible clumping
1+: Fine granulation against a milky background
2+: Small but definite clumps against a slightly cloudy background.
3+: Large and small clumps against a clear background.
4+: Large clumps against a very clear background.

Shameema :) 0700486D

This is Jennifer. On the forth week of my SIP, I was posted to the blood culture station.



Blood Culture

The BACTEC Fluorescent Series
If bacteria or fungi grow in a medium that provides the essential nutrients which enhances their growth, they may cause haemolysis of red blood cells if the medium is blood. They may also cause the medium to be turbid or producing gaseous by product such as carbon dioxide production.
The BACTEC Fluorescent Series detects the production of carbon dioxide by the increasing numbers of bacteria in the blood.

If bacteria or fungi are present in the blood or other body fluids, they will break down the nutrients in these medium and release carbon dioxide. The dye in the sensor will react with the carbon dioxide produced. This reaction affects the amount of light that is absorbed by a fluorescent material in the sensor. The instrument’s photo detector measures the amount of fluorescence which responds proportionally to the amount of carbon dioxide produced by bacteria or fungi.

The instrument begins continuous automated testing and the vials’ fluorescent sensors are activated by the LED (Light Emitting Diodes) behind the vials that laminated the racks. The instrument’s detector would start taking the readings after a warm up period. The vials will be read by the detector every ten minutes and the cycle for all the racks inside the incubator will be finished after every ten minutes. If there is a positive culture, the indicator light will light up in front of the instrument and also shown on the monitor and the alarm will sound. After the positive vial was detected, the medical technologist in charge will remove the vial from the incubator and send them for blood culture for the isolation and detect the identity of the organisms present in the blood or medium.

Steps for the positive culture to be lighted up or identified:
1. Carbon dioxide was released by bacteria
2. Carbon dioxide reacts with dye in the sensor
3. LED, modulated by dye, activates fluorescent material in sensor
4. Increasing amount of fluorescent is read by photo detector
5. Raw data from detector is sent to rack microprocessor
6. To where positivity analysis is performed
7. Positive vial lamp illuminates, audible alarm sounds, positive stations are displayed


Usually, when blood specimens or other body fluids are received such as stem cells, peritoneum fluid from renal, and dialysis fluid, it comes in two bottles where one is to detect for present of aerobic bacteria while another is for anaerobic bacteria. But in some cases if there is a need to detect for the present of fungi, a third bottle will be sent together with the two bottles.

For aerobic bacteria, the cap of the bottle is in blue colour while gold colour for anaerobic bacteria.
For fungi or mycobacterium, it is in red colour.
Below is a picture that shows the BACTEC blood culture bottles with different cap colours for different types of purposes:



Procedures
The BACTEC bottles or vials are inserted into the rackets of the incubator and the racks constantly incubated at 35 degree Celsius and are agitated throughout the 5 days.
For aerobic and anaerobic bacteria, the vials are read every 10minutes by the detector and the bottles are discarded after 5 days if no positive are flag on the bottles.
In some cases such as patients with Endocarditis or Brucella, the bottles are tied with red or blue ribbons before incubated and cannot be thrown away even if flagged negative.
For positive vials, immediately being removed from incubator and sent to for blood culture.

medical mircobiology [serology] (part I)

Hello all~ yanling here.. its my turn to blog this wk~!

I'm going to talk about serology as im in that department for 3 wks (again). This week is my last week. xD

I will talk about one test in a post. So there might be quite a few posts at a go. But at least questions can be directed to each test~! xDDecided to write about some of the manual tests done in serology, because I think they require more skills, especially in pipetting. xD

Venereal Disease Research Laboratory Test

Its is to check if the person has syphilis.

The first test done is to screen for presence of syphilis/ Treponoma pallidum Ab using RPR reagent which consist of carbon particles coated with syphilis Ag. [I had made a mistake here, SO SORRY~!] screen for presence of Ab that are specific to the substance that were released by the cells that are attacked by Treponoma pallidum which causes syphilis. Since its about Ag-Ab, so the principle of this test is agglutination.

So how the screening test is done? First, 50ul of serum is dropped onto a circle of RPR card (will upload picture soon). Then a drop of RPR reagent will be added to each circle then rotate for 8 mins at 100rpm.

Then 8 minutes later, one has to check for agglutination. Here comes the difficult part as for weak positive, the agglutination will not be very obvious so there are chances that one may mistaken with the RPR carbon. So everytime in doubt, got to ask the staff for help.

So for the reactive samples, they will be keep aside for further tests. Then for the non-reactive samples, they will be scan and reported as "non-reactive".

Now I'm going to talk abt the further tests for reactive samples. The next test is venereal disease titre, which to find how strong the syphilis Ab is. There will be 6 circles required, 50ul of saline is pipetted onto second to sixth circles. Then 50ul of reactive serum is added to 1st circle (neat). Then another 50ul of the same serum will be pipette to 2nd circle. Then a 50ul serial dilution is done from 2nd circle to 6th circle. Then the serum is spread to the entire circle from the highest dilution to the neat. Then 1 drop of RPR reagent is added to each circle and rotate for 8 mins at 100 rpm. Then titre of the serum will then be recorded b4 proceeding to the next test. The titre from neat to the 6th circle is as follows: 1:1 1:2 1:4 1:8 1: 16 and 1: 32. And if there are still strong agglutination at the 6th circle it will be recorded as >1:32.

The next test done is Treponoma pallidum Haemagglutination (TPHA), is a more specific test for syphilis as VDRL test can be reactive if there is present of malaria parasite.

The principle for TPHA is haemagglutination, which uses cells with the Ag to check for agglutination whereas for VDRL, it uses particle coated with the Ag for agglutination, and for this case is carbon particle.

The steps are a little complicated, but i will try to explain to the best i can. The test uses 96 u-shaped well. For each sample, 6 wells will be used. So the max number that can be done in a plate is 15 as one row will be used for control. xD

So before adding anything to the plate, the side of each row of 6 wells must be labelled with the last 3 number of the barcode no. or for control, it will be labelled as C. Then 190ul of sample diluent will be added to the 1st well of each sample or control. Next, leaving the 2nd well empty, add 25ul of sample diluent into 3rd to 6th wells. As for control, the sample diluent is added till the 4th well. This because the control will show agglutination from 3rd well onwards, so in order to save the diluent, it is done till the 4th well. After that, 10ul of control/serum is added to the 1st well and mix well. Then take 25ul from the 1st well to then 2nd well and the 3rd well. Then a 25ul serial dilution is done from 3rd well onwards.

After the serial dilution, one drop of unsensitised cells will be added to 2nd wells of each sample and control. And 1 drop of sensitised cells will be added to 3rd wells onwards. The unsensitised cells contains cells without any T. pallidum Ag, so the results will always be negative. Then as for sensitised cells, it contains the T. pallidum Ag, so if there is any presence of T. pallidum Ab in the patient's serum, there will be agglutination. Then the sides of the plate is tapped to ensure the contents are mixed. Its like how we do our ELISA during AIMM i think? Then it will be incubate for minimum of 45 mins at room temp.

So how the TPHA negative and positive results will look like? For negative results, it will appear as a compact button or small ring at the center of the well as the cells will settle. Then for positive results, there will be agglutination and so there will not be any button or small ring in the center of the well. The titre of the test will be then recorded and reported. The titre is from 3rd well onwards, 1:80, 1:160 and 1:320. If there is still no button or small ring, it will report as >1:320.

yaNLing xD

medical mircobiology [serology] (part II)

Mycoplasma pneumoniae antibodies

if one is infected by M. pneumoniae, one will produce Ab against it, thus in this test, it is to see if the patient had been infected with the bacteria.

The principle is this test is also agglutination, where the results are similar to the results of TPHA i mentioned earlier.. if the result is negative, the sensitised particles will settle to the bottom of the well to form a compact ring or button.

the steps is more simpler here. 1st, add 100ul of sample diluent into 1st well, and add 25ul of sample diluent into 2nd to 4th well for control and 2nd to 6th well for each patient's serum. Then add 25ul of serum into 1st well and do a 25ul serial dilution down the row. Next, add i drop of unsensitised particle into 2nd well and add 1 drop of sensitised particles into the 3rd and 4th well for control and 3rd to 6th well for specimens. Pat the sides of the plate and incubate for 3 hours or overnight.

it is important that the 2nd well must be negative results, as the unsensitised particles are not coated with the bacteria Ag, thus there will not be any agglutination. If there is positive results for 2nd well, it might be due to cross contamination or added wrong reagent.

the results of each specimen are then recorded and report in terms of the titre as follow: 1:40(3rd well), 1:80(4th well), 1:160(5th well), 1:320(6th well) and >1:320(when there is still agglutination at 6th well).

yaNLing xD

*will add more tests soon when im free~!*

medical microbiology [serology] (part III)

Thyroglobulin & Thyroid Microsomal Antibodies (THYMA)

It is to test for presence of thyroidic autoantibodies, where the Ab attacks the thyroid. 2 tests are usually done, thyroglobulin antibodies test (ATG) and thyroid microsomal antiboides test (AMC). these 2 tests are usually ordered together. Presence of either Ab means that the person has autoAb to his/her thyroid.

The principle is agglutination and the steps are almost the same with MPA, only the volume of sample diluent and serum are different.

For both tests, the first and second well is added with 50ul of sample diluent and 75ul for 3-4th well for control and 3-6th well for each sample. Then, 10ul of control is added to the 1st well of control. Same goes to each specimen. After that, a 25 serial dilution is done down the row till the 4th well for control and 6th well for each specimen.

Same with MPA, 1 drop of unsensitised particles is added to second well and 1 drop of sensitised particles to 3 wells onwards. The sides of the plates are then patted and incubate overnight at room temperature.

The results is same with MPA, where unsensitised cells and negative results will show button in the center of the well, while positive results will show agglutination and absence of button in the center of well.

yaNLing xD

Clinical Biochemistry

Hello everyone, this is Joash here. The lab that i am posted to right now its just a very small routine diagnostic lab that only contains 3 main disciplines, which are Biochemistry, Hematology and Microbiology. i am going to talk about Biochemistry 1st.



The analyzer that we used in our lab is the Olympus AU400

This fully automated biochemistry analyzers contains 3 major components which includes robotic arms, called probes, controlled by pressure valves, next is the a photometer that measures the light from built-in cuvettes which are held at a constant temperature and lastly the microprocessor or software package which calculates the values from the signal and then displays the data in a user-defined format. The analyzer contains 2 probes, sample and reagent probe which has good accuracy and precision. The reagent chamber is refrigerated and accommodates 72 reagent bottles and can analyze up to 46 parameters at 1 time. The STAT table is also refridgerated and it is used for emergency analysis as it can hold other analysis 1st but run the samples on the STAT table. Routine parameters include liver function tests, renal function tests, glucose tolerance tests and special parameters such as free fatty acids, HbA1c, ketone bodies and much more. This analyzer can run samples such as serum, plasma and urine.

Daily, weekly and monthly maintenance is required for this analyzer and each involves different cleaning procedures such as cleaning and rinsing the sample and reagent probes. replacing the cleaning solotions and etc. QC have to be done daily before the analysis of samples can be done. This is to ensure that the results produced are accurate and reliable at that point of time. The QC menu of the software offers LJ plots, west guard rules, etc. for user review. The data can also be stored and printed. it allows us to review day to day control to monitor the QC for each components and it will flag if the QC results are out of the +/- 2SD off the mean.

After QC had passed, only that we can run our patient samples. The blood tube that we are using is the Red clot activator blood tube as we are analyzing the serum of the blood. We will 1st have to key in the required test into the LIS system for that particular patient and paste the barcode label along the blood tube that matches the patient's particulars. Then the blood tubes are centrifuged to spin down the clotted red and white cells with serum as the supernatant and after that, they are placed into the analyzer for processing. the analyzer will read the barcode and recognize what test to do for this patient. After the test is done, the results will be transmitted to the LIS system and the report is ready to be printed and summit to the vets for review.

Regards,

Joash(0702657H)

picture taken from:http://asia.olympus-global.com/lisg/diagnostic/au400e/index.cfm

Medical Microbiology(Cont')

Hi! This is Shameema. I was posted to Central Processing Area (CPA) in the Department of Diagnostic Bacteriology for the first 3 weeks. I worked on the respiratory, blood and stoot. On the forth week i was moved to parasite.

As Muna and Jennifer have done the posting about respiratory and urine, I will share my experience on stool in this blog.

Stool Culture

The stool culture test is used for the detection and identification of pathogenic bacteria in the stool. In the laboratory, the test is initiated by streaking small amount of a fresh fecal sample to a variety of media; details as follows:




The stool incubated in the selenite broth and alkaline peptone is cultured on to agar plates the following day:

Selenite --> MacConkey and SS agar plate

Alkaline peptone --> TCBS and blood plate

It is crucial that the subculture for alkaline peptone has to be done the following day. This is because the pH of the alkaline peptone which is set at pH 8 will decrease in the presences of vibro as acid is the end product of vibro. Decrease in the acidity increases the growth of other organism and decrease the concentration of vibro thus affecting the result. Selenite broth is the selective broth that will rule out the normal flora.


Vancomycin Resistance Enterococci Screening (VRE)

Enterococci are bacteria found in stool and some strains of the enterococci has become resistance to vancomycin antibiotic is referred to as VRE.


On the stool table I also had to do the widal weil- felix test. It is a diagnostic used for typhoid fever. It is an illness caused by the bacterium Salmonella enterica serovar typhi. The precense is detected by the aggultination of somatic (O) and flagellar (H) to Salmonella typhi in the patient's serum. Weil-Felix test is the detection of rickettsial antibodies thus indicating the presence of rickettsial species in the patient.

PROCEDURE

1. Blood sample is centrifuged at 2000rpm for 5 minutes to separate the serum from the blood.
2. Add 0.2ml of the patient serum to 7.8ml of normal saline in test tube to form 1/40 dilution.
3. Add 1ml of 1/40 dilution serum to 1 ml of normal saline to form 1/80 dilution.
4. Arrange Dryer’s tubes as demonstrated in table below and add one drop of commercial antigen in the Dryer’s tubes.
5. Add 0.8ml of the 1/40 dilution test serum to Dryer tube number 1-6 and 1/80 dilution serum to Dryer tube 7 and 8..
6. Shake the racks gently to remove trapped air.
7. Incubate overnight at 50 degree Celcius in water bath.

TO: Solmonella typhi Group D somatic antigen
TH: Salmonella typhi-H flagellar antigen d
AH: Salmonella parathyphi: A-H flagellar antigen a
BH: Salmonella parathypi B-H group B somatic antigen
OX19: Proteus OX19 antigen
OXK: Proteus OXK antigen

Will share my parasite experiance in my next blog.

Shameema :)

Medical Microbiology(Cont')

This is Jennifer from TG02. As what Muna had mentioned on the previous post, we were posted to CPA( Central Processing Area) in the Department of Bacteriology. I was rotated to the repiratory section during the third week of my SIP where i recieved specimens from patients from their upper or lower respiratory tract.

( Very sorry that due to some problems, i couldnt upload the pictures that i have downloaded. I will upload the pictures as soon as i have settled the problem.)


Upper respiratory tract infection

MRSA (Methicilin Resistant Staphylococcus aureus) Screening
MRSA can also be known as multidrug resistant Staphylococcus aureus as it is resistant to a huge group of antibiotics known as beta- lactams. This bacteria cause many infections which are difficult to treat since they are also resistant to cephalosporin and penicilins.

Usually, most of the samples that are taken to detect for the presence of MRSA are nasal swab. Screening for MRSA requires the use of BHIB (Brain Heart Infusion Broth) and MRSA agar which is a selective agar that only allows the growth and differentiate MRSA from other bacteria. BHIB contain a type of antibiotic called oxicilin and this antibiotic will kill most of the bacteria in the nasal swab except for MRSA as MRSA can withstand this antibiotic and is an enriched broth for staphylococcus species.

MRSA selective agar BHIB

Procedures
1. Incubate the cotton bud that contain the nasal swab into BHIB and incubate for 24 hours to give enough time for the antibiotic to kill off most of the other types of bacteria and allow MRSA to grow, in an oxygen incubator.
2. After 24 hours incubation, sub the cotton bud which was incubated in the BHIB on MRSA selective agar and do normal agar streak.
3. Incubate the agar in an incubator for 2days for the bacteria to grow.

Results
If MRSA is present and grow on the agar, the positive result is shown a purple colony growing on the agar which is shown on the diagram below.

Purple colonies growing on MRSA agar

Lower Respiratory Tract Infection

Sputum Culture
Usually, sputum samples are obtained from patients to detect for the presence of lower respiratory Tract Infection. The appearance of sputum can be watery, clear or mucoid. Before doing sputum culture, we have to do a gram stain for the sputum first as we need to identify whether the sputum sample is a good sample to do the culture or not.

After viewing the gram stain of sputum under the microscope, it can be determine whether the remaining sputum sample can be carried on to do the culture or should it be rejected.
A good sputum sample should have absence of epithelial cells and presence of PMN (Polymorph nuclear leukocyte) as presence of high number of epithelial cells shows that the sample obtained could be from saliva as saliva sometimes does contain epithelial cells from our cheeks. High number of PMN shows that the sample is as good sample as it shows that there is inflammation by the infections caused by pathogens.

Other types of samples such as CSF (Cerebrospinal Fluid) and ETTA (Endotracheal Tube Aspirate) also requires gram staining and cultures. The only difference is that these two types of samples do not require doing gram stain first before culture can be done. Both the culture and gram stain can be done at the same time.

Below is a tabulated table showing which agar or broth to use for each of the type of samples.

(Staphylococcus streak is done on all blood plates after normal streaking for any type of samples as staphylococcus provide a particular essential nutrient for the bacteria on the blood agar to grow.)

hm_med()

Procedures (For sputum)
1. The appearance of the sputum was checked. (Such as whether the sputum is mucoid, watery, clear, or purulent.)
2. Using a labeled slide, a small circle was drawn on the under side of the slide.
3. The surface of the slide was swab by alcohol swab.
4. Using a cotton bud, the sputum was stirred and a small amount of sputum was obtained.
5. A small amount of sputum was applied onto the slide between the circles that was drawn on the underside.
6. The sputum smear was fixed by putting the slide on the heater until smear was dried.
7. Gram stain was performed.
8. The slide was viewed under the microscope.

If the sample is a good sample, carry on with sputum culture. If the sample is not good sample, reject it.
For a good specimen of sputum, there should be no or minimal epithelial cells seen and PMN (Poly morphs nuclear). If too many epithelial cells are seen, it could mean that the specimen is the saliva of patient not the sputum as saliva may contain epithelial cells that are actually the cheek cells. Presence of PMNs show that the is inflammation of the patient’s lower respiratory tract as pathogens are present and cause the activation of PMNs.
Cont’
9. The remaining sputum was subbed onto Mackonkey and Blood agar.
10. Normal streaking was done.
11. Staphylococcus streak was done on blood agar.


References:
MRSA Screening: The Test. Retrieved from 12 July 2009 from Lab Test Online: http://www.labtestsonline.org/understanding/analytes/mrsa/test.html

Medical Microbiology

Hi, this is Muna. For the past 4 weeks, including this week, Shameema, Jennifer and I have been posted to the Central Processing Area(CPA) in the Department of Diagnostic Bacteriology. Each week, we rotate to a different testing bench. We receive patient samples from the wards and clinics for testing.

I shall start by posting what I did last week - Urine Culture.

The purpose of urine culture is to isolate and identify potential pathogenic organisms from urine. This helps in the diagnosis of renal or urinary tract infections (UTI). Since urine is mostly sterile, any presence of microorganisms is usually clinically significant, unless it is contaminated by normal flora.

The samples we receive are usually mid-stream urine (MSU) for aerobic culture and dipslides. Mid-stream urine, as its name implies is collected in the middle part of the urination flow. The initial part is not collected as it usually contains the flushed out normal flora and cellular debris from the urethra and urinary tract. This may cause a false positive reading.

The samples are inoculated onto a split plate containing CLED agar and blood agar (BAP). CLED stands for Cystine Lactose Electrolyte Deficient agar, which can differentiate between lactose fermenters and lactose non-fermenters. Lactose fermenters produce a lower pH in the media, thus causing the indicator (bromothymol blue) to change from the original greenish yellow to yellow. It also enhances the growth of dwarf-colony coliforms.

Blood agar plates (BAP) supports growth of fastidious bacteria. It is also able to detect the pattern of bacterial haemolytic activity, which can determine the pathogenicity.

CLED and BAP split plate.


The streaking of plates for urine culture is unique as it does not follow the ocnventional way of streaking plates. I was taught that this method is used so that the pattern of growth can be obseved and the calculation of colony forming units per ml(cfu/ml) of urine can be counted easily. Also, instead of a 10 ul inoculating loop, a 1ul loop is used since bacterial presence in urine should be minimal or none in normal persons.

Urine culture streaking technique.

Normally, the BAP is streaked first, followed by the CLED plate so that any residual inhibitory substances picked up from the cLED plate in streaking does not affect the growth on the BAP.


NOTE: I shall continue where I left off soon. Need to clarify some doubts before continuing.

Clincal Chemistry/ Laboratory Managment and Quality Assurance

Been posted to biochemistry from 22/06 to 11/07, meaning 3 weeks in biochemistry.

In biochemistry, its more like clincal chemistry, doing tests like wat we learn, example liver function test, glucose tolerance tests(GTT) etc. But most of the tests are done automated. xD

What i learnt in the first week were mainly HbA1c test, G6PD deficiency test, drug 5/drug 7 tests, GTT, daily QC and weekly maintainance. I will be talking about what i learn in biochem in these 3 weeks in a post so its easier to read. xD

HbA1c/total hemoglobin ratio

HbA1c is actually glycosylated Hb, whereby the glucose bind/attach to the Hb. Therefore, increase in blood glucose will results in increase in HbA1c. For diabetic patients, if their HbA1c is within the acceptable range, this shows that they are controlling their disease well or they are responding to the treatment given to them and vice versa.

It is to monitor glucose of the patient over a period of 3 months. Its unit is in % and the normal amount is less than 8% (if I'm not wrong). For values more than 8%, dilution has to be done, most of the time, in 1:41 (>8-12%), 1:51 (>12-15%), 1:81 (>15-20%) and 1:101 (>20%).

However, HbA1c can be increased due to high amount of glucose, so meaning that if the glucose level is higher than HbA1c, the patient is still normal, meaning that if the HbA1C value is >8% but less than 15%, I have to wait for the blood glucose level results to see if I need to do a dilution. If the glucose level is higher than the HbA1c%, the results can be accepted and key into the LIS. This is because more glucose will attach to the Hb, so will results in higher HbA1c.

If the glucose level is lower, 1:41 or 1:51 dilution has to be done accordingly. However, for HbA1c% that is more than 15%, dilution must be done immediately as it can be quite dangerous.

When the results is > 8%, dilution don't need to be done if the glucose level is higher than HbA1C, or there is previous history of the patient.

The test requires whole blood (EDTA tube) and also before testing, bubbles have to be removed because of the probe will end up taking air if there is bubbles in the blood. I was having alot of problems removing bubbles in the first few days as I have to tilt the tube to almost horizontal, then I'm so afraid to spill the blood.

The machines used by the company are ADIVA 1650 and ADIVA 1800. ADIVA 1650 is an automated machine where it can do up to 1650 tests in an hour and for ADIVA 1800, it can do up to 1800 tests per hour.

The tests are ordered manually, meaning that I have to key in the barcode no. manually, then order as BAYER, where the machine will process to add in Hb denaturant and other reagents. Then the test tube has to be inverted a few times to mix it well, after that removes the bubbles before loading into the machine. It is very important to place the barcode facing outside as it is where the machine will scan the barcode to make sure the no. is what I had keyed. The test takes around 30 mins or smth (I'm not too sure, never take note of the timing). It takes quite long as the test is run twice. After the test is completed, the results has to be checked to see if there is any need for dilution, if not, it can be key into the LIS or they call it the ultra which I dunno why. >.>

So how's the dilution done? for 1:41 dilution, it is to add 10ul of blood sample to 400ul of denaturant then incubate for 5-10 mins in room temp before putting the sample into the machine to run. Same goes for the other dilutions but the denaturant volume increase to 500ul for 1:51, 800ul for 1:81 and 1000ul for 1:101 dilution.

The principle of the test is that the concentration of HbA1c and total Hb is measured separated then the ratio is reported as HbA1c%.

G6PD deficiency

The G6PD test done is a qualitative test, meaning that it can only test for the presence of G6PD enzmye and unable to find out the amount enzyme present in the patient.

The test is fairly easy. Add 5ul of blood sample (in EDTA tube) to 100ul of G6PDS screening reagent then incubate for 10mins before adding 5ul of the solution on a filter paper (Gurthie test paper) and incubate again for 10 mins. After 10mins, read the filter paper under UV to see if there is any fluorescence of the blood sample. If there is fluorescence, it means there is presence of G6PD. No fluorescence showing G6PD deficient.

Repeat testing has to be done if there is deficient in G6PD.

However, before the test can be, the G6PD screening reagent has to be prepared beforehand. It comes in a bottle which contains freeze dried powder which is all the reagent. Then the contents are dissolved in elution/dilution buffer provided. Then the bottle is allowed to stand for 10 mins in room temp before putting on the rotator for 20 mins. After that, 100ul of the G6PDS reagent is aliquoted into test tubes. When finished aliquoting, the test tubes are capped and labelled as " G6PDS reagent, prepared by: (name of staff) and the date". The final step is to put in the -20C freezer. Then everyday around the evening, a few racks of the G6PDS reagents are taken out to thaw and then the G6PD tests can be done~! xD

The G6PDS reagent contains glucose-6-phosphate and NADP+ and when react with G6PD will produce gluconate-6-P and NADPH and H+. NADPH will fluorescence under long wave UV. So, when there is G6PD deficient, no NAPDH will be produced, and therefore there will be no fluorescent.

I had taken some pictures which shows the results of tests under UV, but blogger now having problem in uploading pics, so I will upload when I can. Pictures are of course taken with permission from my supervisor/HOD. xD

Patients's sample in EDTA tube and G6PDS reagent in the test tube.

Gurthie filter paper

10 minutes after 5ul of blood with G6PDS reagent are pipetted onto the filter paper.

When the filter is under UV light. All are G6PD present as all blood spots are fluroscence. It should be green actually, but think when taking the picture the colour will change. The UV light is blue.

All are G6PD present. Some are different colour as the fluorescence is dimmer. As long as there is fluorescence, it means G6PD present.

G6PD deficient. There should be no fluorescence for G6PD deficient. In here is looks quite similar with those blood spot that are dimmer, but in actual fact, there is really a difference.

Drug 5/Drug 7

The test is to find out if there is any of certain drugs taken by the patient.

Drugs 5 tests for the presence of amphetamines, cocaine, marijuana, opiate and phencyclidine or drugs related to them.

It requires a urine sample from the patient and a drug device. 3 drops of urine are dropped onto the drug device whereby the urine will move up due to capillary action. If there is no presence of drug or drug present below the cut off concentration, a coloured line will appear, showing negative results. No coloured line will show that the drug present in the urine is above the cut off concentration. The coloured line is due to the Ab present in the device. Its is very similar to the pregnancy test. Their principles are the same. 3 drops of urine specimen is added into each sample well of the test device.

The princple of drug 5/7 is based on competitive binding. When corresponding drug(s) is present in urine specimen, the drug(s) will competitve against their respective drug conjugates for binding sites on the specific Ab.

When the urine samples are added into the sample wells, the urine will move upwards by capillary action. Drugs present below cut off concentration will not saturate the binding sites of their specific Ab, these allow the Ab to react with the drug-protein conjugate and resulting in a coloured line. When presence of drugs is above the cut off point, the dtugs will saturate all the Ab binding sites and thus no coloured line will be produced. When a very faint coloured line is produced, the results is still negative. This is because the drug concentration is still below the cut off point. The test is counted positive if only there is no coloured line at all.

For drug 7, additional of 2 more drugs are tests. (i will go find out what are the 7 drugs) They are barbiturates and benzodiazepines.

The reagents in the drug tests are mouse monoclonal Ab-coupled particles and the corresponding drug-protein conjugates and for the control contains goat anti-rabbit IgG polyclonal Ab and rabbit IgG.

Pictures of the test devices will be uploaded soon.

Drug 5 pack.

Drug 5 test device.

Drug 7 test devices. Benzodiazepines and barbiturates are in individual pack where as amphetamines, cocaine, marijuana, opiate and phencyclidine are in one test device.

After when 3 drops of urine are dropped into each sample well, the urine moves upwards by capillary action.

Around 5 mins later, coloured lines will appear if the drug concentration are below the cut off point. If there are no drugs above the cut off concentration, there will be total of 8 lines whereby 3 lines will belong to control. Control line must appear or else the test has to be repeated.

Test results for drug 7.

The coloured lines in the first test strip is very fade, but the results is still negative as there are still presence of coloured lines. Positive test will be absolutely no colored line at all. As long as there is some coloured line appear, it will be marked as negative.

GTT

GTT uses dipstix (glucose + ketones only) for fasting urine, 1 hour after intake of glucose and 2 hrs after intake of glucose. So same as what we had done during our cchem pract on urinanalysis, the dipstix was dip into the urine sample and excess urine is drained by the c-folded towel. Then after 2 mins, compare the colour of the dipstix with the reference on the bottle. Prescence of glucose or ketones are noted down.

Quality Control

-to be updated soon-
QC are run twice daily, one in the morning at 7.30am (and yes, i have to reach at 7.30am everday for the first week to observe the QC) and another on the in the late afternoon or evening, around 5.30 to 6pm.

So everday in the morning, (the staffs tend to reach earlier than 7.30am so they have more time to do the QC as they have to finish the QC by 9.30am) the wash solutions are checked to see if there is sufficient, if they are not enough, they will be top up.

Next, the probes will be clean using the alcohol pad, then after that the staffs will top up the reagents that are low in amount. They will then barcode scan the reagents so to update the machine the reagents in the machine and the number of tests can be done.

After that they will initalise the machine and also buffer prime (sort of washing) ISE 5 - 10 times. ISE contains electrolytes like Na, K and Cl.

After the maintainance, they will start to run QC using blank, bio-rad calbrator, bio-rad control 1 and 2, immunological control 1 and 2 (3 for certain machines), CO2 calibrator, bio-rad urine control 1 and 2 and HDL/LDL calibrator. ISE calibation involves using of serum and urine high and low. They also run QC for HbA1c where they use normal and adnormal control. The HbA1c has to do 1:41 dilution and incubate for minimum of 5 minutes before able to run the QC.

So after the QC has finished running, the QC will check if they are within the acceptable range. If it is not in the acceptable range, they have to run again until it is within the range, if not, the results for the day would not be accurate. For ISE QC, the Na, K and Cl slope will be observe for the high and low of the serum and urine. Also, if the slode is not in range, it has to be run again.

Aside of the daily QC, they also have weekly QC.

In weekly QC, they will clean the outside of the machine, and clean the ISE where they will replace Na, K and Cl with a dummy and allow the ISE to be washed. Also, the lamp of the machine is check once a week to see if the lamp is in working condition. The lamp is very important part of the machine as most tests run in the machine requires lamp to read the absorbance of the reaction. If the lamp is not in working condition, all the results can be greatly affected.

And on certain day, additional QC has to be done for ethanol tests and Apo A and B as they are run once a week as the samples are not received daily.

And for evening QC, it is more simple as they will just run the QCs of the reagents and ISE.

-the end-

and so this is almost about what i learnt in biochem. any updates will be edited in this post again.


yaNLing xD


-updated on 7 july 2009.
-updated on 12 july 2009.

Next post will be about serology~! Look forward for it okay? xD