Biochemical Tests

Good evening everyone. This is Jeremy (0702919B) from TG01. Today, I will be elaborating on several biochemical tests performed in the lab to aid in identifying bacteria.

Kliger’s Iron Agar (KIA)

KIA is a differential medium. It is similar to the Triple Sugar Iron (TSI) agar that we used in the school's lab. It tests for organisms’ abilities to ferment glucose and lactose to acid and acidic gas products. This media is commonly used to separate lactose fermenting members of the family Enterobacteriaceae (e.g. Escherichia coli) from members that do not ferment lactose. These lactose non-fermenting enterics are generally more pathogenic in the gastrointestinal tract.

Glucose fermentation creates acidic byproducts that turn the phenol red indicator in the media yellow. Upon depletion of glucose, organisms that can ferment lactose will continue to produce acidic byproducts and the media will remain yellow. Gaseous products produce might lift the agar off the bottom of the tube.

Non lactose fermenters metabolize amino acids and proteins in the media, creating alkaline byproducts that turn the indicator red at the slant.

Non glucose and lactose fermenters will use solely amino acids and proteins, creating a red slant and unchanged color at the butt.

Organisms that sulfer reducing enteric will produce H₂S that creates a black precipitate at the butt.

	Slant	Butt	Examples
Lactose fermenters	Yellow	Yellow	Escherichia coli
Non lactose fermenters	Red	Yellow OR Black (H2S production)	Shigella dysenteriae Proteus mirabilis (produce black ppt)
Non glucose and lactose fermenters	Red	Unchanged	Pseudomonas aeruginosa

Simmons’ Citrate Agar

Simmons’ citrate agar tests the ability of an organism to use citrate as a sole carbon source and ammonium ions as a sole nitrogen source. The medium contains citrate, ammonium ions, and also a pH indicator, bromothymol blue. Organism that can grow on Simmons’ citrate agar are capable of metabolizing citrate as the sole carbon source and ammonium ions as the sole nitrogen source. This will create alkaline byproducts which will turn bromothymol blue indicator blue.

Growth on Simmon’s citrate agar or an intense blue color formation is an indication of a positive result.

Urease Test

This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink. Proteus mirabilis is a rapid hydrolyzer of urea.

Well, that is all for now. Thank you for your attention.

Jeremy.

Immunohaematology (Blood Bank)

Firstly, I would like to discuss on how a patient's blood sample is being processed when it arrives at the blood bank. When a blood sample is first received, the Medical Technologist would check the EDTA blood tubes for any blood clots. This is being done by using 2-3 wooden sticks to "dig" the blood sample and try to "fish" out for any clots. If clots are absent, the sample would be suitable to be processed using the Ortho Autovue Innova System. If clots are present, the form accompanying the patient's sample should have "clotted" indicated on it. The blood sample with blood clots present cannot be processed by the Ortho Autovue Innova System, this the type and screen has to be carried manually.

After the blood samples in the EDTA are checked for presence of clots, the tube is loaded into the Ortho Autovue Innova System for the machine to read and screen the sample. The machine is able to carry out forward, reverse & Rh grouping of the blood samples. In addition, it is also able to detect abnormal antibodies that might be present in the blood. If the machine is malfunctioned, a manual type and screen can be carried out instead.

In type and screen which is done manually, forward group involves placing patient's blood sample into commercial sera. The 3 main groups of anti-sera are anti-A, anti-B & anti-AB. In reverse grouping, patient's serum (or plasma) is added to commercial blood. The 3 types of commericial blood used are blood group A, B & O. Lastly, to determine the presence of antigen D in patient's blood, patient's blood is added to anti-D commercial serum. This concludes the process of type and screen, and from this the patient's blood group and Rh status can be determined.

In an event that a patient requires blood transfusion, a cross-match must be carried out to check for compatiblity. In cross-matching, patient's serum is added into a sample of packet of blood (to be transfused). It is incubated at room temperature for 5 minutes, then centrifuged @ 3000rpm for 15s. The glass tube is inspected visually for any agglutination present. If there is no agglutination present, it can be concluded that the packet of blood can be safely transfused into the patient.

Regards,
Zhu Zhijie Dennis
0700847G
TG01

Oral administration of compound in animal models

Raymon

Oral administration

In a toxicity test involving oral administration of the drug/compound of interest, several methods can be considered mainly the oral gavaging and the dietary method
Gavaging is the process of force feeding whereby a tube is inserted into the mouth and down the oesophagus of the rat and the substance is subsequently emptied into the stomach via the syringe connected to the gavage tube.

Gavage VS Dietary method
The dietary method can be used if compound is suitable for mixing with the diet and that is stable under storage conditions similar to the diet. The compound must also be palatable to the animal.There are pros and cons to the dietary method. The advantage include beeing less laborious as compared to the need to gavage. The disadvantages include the exact concentration of compound for test which is mixed with food is hard to determine rather it is based on predicted food consumption and body weight. With the gavage method of dosing, a more precise amount of test compound can be delivered. However, there is a risk of punturing the oesophagus or even the lung if mishandled.

Gavage Techniques
1. Measure the distance from the tip of the nose to the last rib. This is the length of the needle that should be used. A curved needle is easiest to use and least likely to induce trauma
2. Fill syringe with appropriate amount of article to be dosed.
3. Restrain rat as mentioned in previous post
4. Place tip of the needle in rat's mouse
5. Slide ttip down back of the mouth, moving tip forward in one smooth motion
6. Needle should slide down the oesophagus easily. Resistance felt indicates that needle is not in place
7. Once everything is in place, administer the article

Fig 1: Gavaging of rat with tube

Fig 2: Measuring the tip and the anatomy of the rat

Fig 3: Gavage needles/tubes

Sexing and Gavaging of rats

Sex Determination

(extracted from http://research.uiowa.edu/animal/mp7.jpg)

Sexing is based upon anogenital distance. Male rats have a greater distance between the anus and urogenital opening. As compared to the females, males have a larger genital papilla. Well unlike the case of dogs, you cant tell the gender apart based on the testicles and phallus. Rats have open inguinal rings and can retract their phallus and testicles into their abdomen. How convenient. Thus the lack of testicles and phallus upon visual observation does not indicate that the rat is a female.

TDX analyzer

hello everybody!
can u believe it!
its going to be the 13th week of our SIP!
yay yay!

alright lets get things started.
this week i'm allocated to one of the analyzers being used in the lab called TDX analyzer.

This TDX analyzer perform tests for therapeutic drugs such as Cyclosporine, Everolimus and Methotrexate .
The therapeutic drugs mentioned are examples of immunosuppressive drugs.

The purpose of performing tests on the TDX analyzer is to quantitatively measure the amount of drug administered in the body to monitor the levels of these drugs to ensure therapeutic dosage and to minimise the levels of toxic side-effects,

im going to talk about one of the tests performed which is Cyclosporine test.
Cyclosporine is effective in fighting against tissue rejection after an organ transplantation.

The Cyclosporine Monoclonal Whole Blood assay uses FPIA (FLuorescence Polarization Immunoassay) Technology as the principle of the test.

This is when there will be a competitive protein binding technology that utilizes the specificity of immunoassay with a fluorescent tracer in order to differentiate the bound from the free analyte. The analyte, in this case, is a drug/drug derivative. During the process, the analyte is labelled. A specific Antibody against the analyte will then be prepared. The assay will cause the excitation of fluorescein tracer with polarized light and the level of fluoresent polarization of tracer is measured. The concentration of analyte is inversely proportional to the polarized light,

Methods for The Cyclosporine Monoclonal Whole Blood assay :
1. Microcentrifuge tubes are labelled accordingly for each of the patient's sample
2. The sample is mixed with gentle inversion and 150ul of it is pipetted out into the corresponding microcentrifuge tubes.
3. 300ul of Whole Blood Precipitation Reagent/ Probe Wash is pipetted accurately into each microcentrifuge tube.
4. Each tube is capped securely to avoid any spillage and vortexed for 10 seconds to make sure thorough mixing.
5. The specimens are centrifuged 10800rpm for 5 minutes to obtain a clear supernatant and a hard compact pellet of denatured proteins.
6. the supernatant is decant completely into the corresponding sample well of a sample cartridge (supernatant's minimum volume required is 150ul)
7. The Reagent Pack is mixed with gentle inversion. The formation of bubbles is avoided.
8. The reagent Pack is placed on the reagent platform.
9. The sample carousel is placed onto the analyzer.
10. Close the cover of the analyzer and press RUN.

Additional info must be noted that Cyclosporine must undergo a pretreatment step prior executing the methods above to extract the drug from the blood sample.

:)

Stella
0701059H

Influenza A & B Test Kits

Before I begin, I would like to apologize for the lateness of my entry, which was supposed to be posted a week ago.

At my workplace, I had the opportunity to use an Influenza A & B test kit to detect the presence of the virus which might be present in nasal swabs. We use a test kit to detect the presence of these two types of influenza. It should be noted that this is a qualitative and not quantitative test. If the patient shows a positive result, we would send out the test for PCR testing.

Above: The patient's nasal swab, the test kit and the extraction reagent solution vial.

Principles of test:
The test uses the principles of an immunochromatographic assay. As you can see from the picture above, the test kit has two sides to it: Testing for Influenza A, and testing for Influenza B. The picture actually shows a test kit that had already been done, which shows a negative for both A & B. The lines seen are the control lines.

If there is a positive result, another test line would appear nearer to the middle of the control line. The test area contains monoclonal anti-human influenza virus. If there is a presence of the virus in the specimen, the virus would bind to the antibody and react to form a purple-red line.

Methods of test:
A sterile swab should be inserted into the nasal cavity and rubbed on the mucosal surface for several times to collect mucous epidermis. The swab should be at a horizontal angle.
The swab is then dipped inside the extraction reagent solution vial, which contains buffer salt and detergent with 0.09% sodium azide.
The tip of the solution vial should be squeezed to contact with the swab and the swab should be rotated clockwise and anti-clockwise 5 times so that the virus (if any) would be transferred into the extraction reagent solution.
The swab is removed and the vial is gently shaken to mix the specimen.
The vial has a dropper on top. 4 drops of the solution is added to the specimen area and incubated at room temperature for 10 minutes.

We would have to write the results in a logbook for future reference. If the test shows negative for the influenzae, the swab as well as the test kit and the vial are disposed. If it's a positive we would have to put all 3 items in a ziplock bag and we would place it aside.

Hakim
0703555C