Wednesday, October 31, 2007

18th week of SIP - Lab Techniques (Research)

Hey! Last 2nd week of SIP now aye? Soon, it'll be over! =D

Anyway, I'll be touching on this assay called Rat/Mouse Insulin ELISA Assay

Rat/Mouse Insulin ELISA Assay

Insulin is a hormone made by the beta cells in the islets of Langerhans that regulates the level of glucose in the blood.

When blood glucose level increases, insulin will be produced and released into the blood circulation.

This picture gives an overview on how insulin regulates blood glucose level.

Taken from: here.

This assay is performed in vitro.

Principle:
The basis of this assay is through sandwich ELISA -

1. Binding of insulin molecules to the wells of a multi-well plate by a pre-titered amount of monoclonal mouse anti-rat antibodies, and bindin of biotinylated polyclonal antibodies to the captured insulin molecules.

2. Unbound materials are washed away.

3. Binding of horseradish peroxidase to the bound biotinylated polyclonal antibodies.

4. Unbound materials are washed away.

5. The antibody-enzyme conjugates are quantified by monitoring the activities of horseradish peroxidase in the presence of the substrate, 3,3',5,5'-tetramethylbenzidine.

6. The enzyme activity will be measured spectrophotometrically at 450nm. An increase concentration of insulin will lead to an increase formation of the blue color.



The more insulin molecules captured by the antibodies, the higher the absorbance.
Thus an increase in absorbance is directly proportional to the amount of captured insulin.

-------------------------------------------

Message from Mr. Poh:
Poster format -
1. Go to MS Powerpoint
2. Under "Page Setup", key in the height and width of A1 size
Height: 33.07"
Width: 23.38"
3. Type everything that is to be in the poster on just this ONE slide.

Price for printing A1 size poster in the school : $18. *faints*

That's all!
Charmaine Tan
TG01

Saturday, October 27, 2007

17th week of SIP - Laboratory Management

(This entry will get drier as it goes.....)


Since the school semester ended, our job as a TSO revolves around laboratory management and cleanliness! It's the best time to carry out 'spring cleaning" and organising paperwork! And it's maintenance of both MCT and MBIO lab! There're 2 more lab in MCT lab for those who took MCT before should know.. '4' labs down to me, charmaine and my poor supervisor to clean up.. (T_T) Chemicals such as media prepared by students were discarded. Contaminated media were autoclaved befor disposal. Equipment were cleaned with 70% alcohol or detergent. We had cleaned the incubators, water baths, 4 degrees fridge which we kept our DMEM for MCT practical (due to its size, we took one whole day to clean it and it looks exactly like a brand new fridge!) And the most shocking thing about the fridge was that the exhaust fan cover was transparent in colour! the amount of dust accumulated deserved 10 times "oh my god"! Latest news - all the labs had undergone big renovation! the lecturer's bench disappeared.. It's a pity we're not using MCT or MBIO lab anymore.. We know all the passwords and keys to everything! haha..

Other than spring cleaning, we had done tonnes of admin work! I guess we spent at least a month on the paperwork! Each lab have to maintain 4 types of documents. For File#1, it consists of general records like list of chemical, accessories and equipment (abbreviated LOC, LOA, LOE).. For the LOC we had to check if all the chemicals in the list are atill available.. For your information, there are 2 safety cabinet, 1 flammable cabinet, 4 4degree fridge, 4 -20degree fridge and 1 -80degree fridge! after checking every chemicals, our hands will be so freezing numb! And the checking was not only done once.. I suppose we can memorise where all the chemicals are... become walking chemical inventory list! Equipment were checked against the LOE.. As the LOA was updated quite recently, therefore we can leave it aside.. *phew*

For File#2, it contains all the MSDS! For this particular file, we spent almost 7 days on sorting the MSDS for two labs! Imagine 7 days 9-6 only MSDS! MSDS-phobia... As our supervisor just took over these 2 labs therefore the workload is heavier.. Lab file #3 basically contains all the maintenance records and service contracts for equipment. Lab file #4 is on the laboratory equipment SOPs and the inventory list. During LMQA, Ms Chew said before that our school has spent much money and alot of paperwork to get ISO 9001 & ISO 14001 acreditation.. Now I really know how much paperwork was required! It's an eye opener! So next time, we see any TSOs.. we must really appreciate them! on top of preparing reagents for us, they still have to do all the paperwork and maintaining a clean and organised lab!

Joan
TG01

Sunday, October 21, 2007

16 week of SIP- Urinary Stone Analysis


I.
Introduction













If you've ever passed a kidney stone, you're not likely to forget the experience — it can be excruciatingly painful. Kidney stones (renal lithiasis) are an ancient affliction dating back to the age of the Egyptian pyramids, yet they are still a common disorder today. The incidence of kidney stones has been increasing in recent decades. Although the reasons for this are still unclear, many experts believe that diet choices and lack of fluids are important factors that have contributed to this increase.

Your kidneys are two bean-shaped organs, each about the size of your fist. They're located in back of your abdomen on each side of your spine, and their main function is to remove excess fluid, unneeded electrolytes and waste from your blood in the form of urine. The ureters carry urine from your kidneys to your bladder, where it's stored until you eliminate it from your body.

Kidney stones usually form when your urine becomes too concentrated. This causes minerals and other substances in urine to form crystals on the inner surfaces of your kidneys. Over time, these crystals may combine to form a small, hard mass, or stone.



What a rock! Yup, its a kidney stone.







II. Signs and symptoms

A patient is not likely to have signs and symptoms unless a kidney stone is large, causes a blockage, is associated with an infection or is being passed. Then the most common symptom is an intense, colicky pain that may fluctuate in intensity over periods of five to 15 minutes. The pain usually starts in the patient’s back or side just under or below the edge of his ribs. As the stone moves down the ureter toward the patient’s bladder, the pain may radiate to his lower abdomen, groin and genital structures on that side. If the stone stops moving, the pain may stop too. Other signs and symptoms may include:

  • Bloody, cloudy or foul-smelling urine
  • Nausea and vomiting
  • Persistent urge to urinate
  • Fever and chills if an infection is present



The patient must try to catch the stone in a strainer during urination.













III. Causes

The crystals that lead to kidney stones are likely to form when the patient’s urine contains a high concentration of certain substances — especially calcium, oxalate, uric acid and rarely, cystine — or low levels of substances that help prevent crystal formation, such as citrate and magnesium. Crystals also may form if the patient’s urine becomes too concentrated or is too acidic or too alkaline.

A number of factors can cause changes in the patient’s urine, including the effects of heredity, diet, drugs, climate, lifestyle factors and certain medical conditions. Each of the four main types of kidney stones has a different cause:

  • Calcium stones

Roughly four out of five kidney stones are calcium stones. These stones are usually a combination of calcium and oxalate. Oxalate is a compound that occurs naturally in some fruits and vegetables. A number of factors can cause high concentrations of these substances in urine. Excess calcium, for instance, may result from ingesting large amounts of vitamin D, from treatment with thyroid hormones or certain diuretics, and from some cancers and kidney conditions. You may also have high levels of calcium if your parathyroid glands, which regulate calcium metabolism, are overactive (hyperparathyroidism). On the other hand, certain genetic factors, intestinal bypass surgery and a diet high in oxalic acid may cause excess amounts of oxalate in your body.

  • Struvite stones

Found more often in women than in men, struvite stones are almost always the result of chronic urinary tract infections caused by bacteria that produce specific enzymes. These enzymes increase the amount of ammonia in the urine, which is incorporated in the crystals of struvite stones. These stones are often large, may have a characteristic stag's-horn shape and can seriously damage the patient’s kidneys.

  • Uric acid stones

These stones are formed of uric acid, a byproduct of protein metabolism. A patient is more likely to develop uric acid stones if he has undergone chemotherapy, consumes a high-protein diet or has certain genetic factors that predispose him to the condition.

  • Cystine stones

These stones represent only a small percentage of kidney stones. They form in people with a hereditary disorder that causes the kidneys to excrete excessive amounts of certain amino acids (cystinuria).


Kidney stones vary in size and shape.


Golf-ball sized and round




Small and smooth








Jagged and yellow






IV. Material & Methods

Sample Preparation

Dissolve a sample as homogenously as possible of the urinary calculus to be analysed. From this solution the various components of the calculus are determined semi-quantitatively, the titrimetric method being used for calcium and a colorimetric method (ie. visual colour comparison) being used for oxalate, phosphate, magnesium, ammonium, uric acid and cysteine. The composition of the urinary calculus is obtained form the results of these determinations with the help of the test kit’s calculation aid.

1. 1. Finely triturate the calculus to be analysed in a motar.

2. 2. Mix the resultant powder thoroughly and using a spatula, transfer a tipful to a plastic boat.

3. 3. Add 5 drops of Sulfuric acid. Stir with spatula to ensure complete dissociation.

4. (Evolution of gas during dissolution indicates carbonate.)

5. 4. Transfer the solution into a 100ml graduate filed to one-third with distilled water.

6. 5. Make up to the 50ml mark with distilled water and mix well with the plastic boat.

7. 6. Transfer 5ml of the sample solution into each of several test tubes for other calculi composition testing, with the exception of Magnesium.

8. 7. For Magnesium testing, transfer 1 ml of the sample solution into another test tube with 4 ml DI water.

Individual calculi composition analysis

  1. Calcium

Reagents

Reagent 2: Sodium hydroxide solution 27%

Reagent 3: Calconcarboxylic acid tituration

Reagent 4: Titriplex III solution

Procedure

To the sample solution, add 2 drops of reagent 2 and one spatulaful of Reagent 3 and shake.

Continue shaking, and while doing so add reagent 4 drop by drop until the colour of the solution changes from red to blue. Count the drops required for the colour change to occur.

The number of drops required multiplied by 5 gives the percentage calcium content of the calculus.


  1. Oxalate

Reagents

Reagent 5: Borate buffer solution

Reagent 6: Iron (III) chloride solution

Reagent 7: Sulfosalicylic acid solution

Procedure

To the sample solution, add subsequently while shaking

2 drops of Reagent 5,

2 drops of Reagent 6,

3 drops of Reagent 7; allow to stand for 2 minutes.

Compare the colour of the solution with the appropriate colour scale and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process.

Read off the percentage oxalate content of the calculus.






  1. Ammonium

Reagents

Reagent 8: Potassium tetraiodomercurate (II)

Reagent 2: Sodium hydroxide solution 27%

Procedure

Add subsequently to the sample solution, while shaking,

3 drops of Reagent 8 and

3 drops of Reagent 2.

Compare the colour of the solution with the appropriate colour scale and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process.

Read off the percentage ammonium content of the calculus.









  1. Phosphate

Reagents

Reagent 9: Ammonium molybdate solution

Reagent 10: Reducing solution 4: methylaminophenol sulfayte soldium disulfite)

Procedure

Add subsequently to the sample solution, while shaking

5 drops of Reagent 9 and

5 drops of Reagent 10; Allow to stand for 5 minutes

Compare the colour of the solution with the appropriate colour scale and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process.

Read off the percentage phosphate content of the calculus.






  1. Magnesium

Reagents

Reagent 11: Buffer solution (borate buffer)

Reagent 12: Colour reagent (1-azo-2-hydroxy-3-(2, 4-dimethyl-carboxanilido)-napthalene-1’-2-hydroxylbenzene-5-sodium sulfonate) solution

Procedure

Pipette 1 ml of sample solution into a reaction vessel and make up to the calibration mark with distilled water. Add 10 drops of Reagent 11 and 10 drops of Reagent 12 while shaking.

After 1 minute, compare the colour of the solution with the appropriate colour scale and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process. Read off the percentage magnesium content of the calculus.








  1. Uric Acid

Reagents

Reagent 13: Molybdatophosphoric acid solution

Reagent 5 Borate buffer solution

Procedure

Add 3 drops of Reagent 13 to the sample solution, shake, and allow to stand for 2 minutes. Then add 2 drops of Reagent 5 and shake. Immediately compare the colour of the solution with the appropriate colour scale and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process. The colour comparison should be performed within 10 seconds after the addition of Reagent 5 to the sample solution because the colour is not stable and liable to change to blue.

Read off the percentage uric acid content of the calculus.








  1. Cystine

Reagents

Reagent 14: Ammonia solution

Reagent 15: Reducing agent (sodium sulphite)

Reagent 16: Sodium nitroprusside titruation

Procedure

To the sample solution, add 10 drops of Reagent 14 and a red dosing spoonful of Reagent 15, and swirl until dissolution is obtained. 1 minute after the addition of Reagent 15, add a black dosing-spoonful of Reagent 16, and again shake until dissolution is obtained. Compare the colour of the solution in the reaction vessel with the appropriate colour scale 30 seconds after the addition of reagent 16, and determine which of the reference colours most closely matches the colour of the solution, looking through the solution from the above process.

Read off the percentage cystine content of the calculus.









Result Reporting

Record down the percentage values for each of the calculi components on a result slip.


V. Conclusion


Urinary calculi analysis may sound tedious, but its actually very fun to perform....if there isn't much samples. Its like O'Level Chemistry practical..Qualitative Analysis. Hope it rings a bell.

I'm sure u got lotsa questions to ask. Just show some mercy ok? This topic is really vast. This message goes out especially to those from my workplace. . =P

Will upload more exciting photos soon. Stay tuned!


Kent Lieow
TG 01
0503261J


Sunday, October 14, 2007

15th Week of SIP - Medical Microbiology

Hi everyone,

Sorry for the late posting, it's me again! I am currently attached to the microbiology department! For the last week in the Department, my section head give me a mini test. Sound scary but fun...

Identification of Bacteria Species in Urine Culture

A urine culture may be ordered when symptoms indicate the possibility of a urinary tract infection, such as pain and burning when urinating and frequent urge to urinate. Antibiotic therapy may be prescribed without requiring a urine culture for symptomatic young women, who have an uncomplicated lower urinary tract infection. If there is suspicion of a complicated infection, or symptoms do not respond to initial therapy, then a culture of the urine is recommended.

Step 1: Gram Stain

Principle: A method of differentiating Gram Positive and Gram Negative bacteria based on the properties of the cell wall. Gram Positive bacteria's cell wall has a stronger attraction for crystal violet. Gram iodine is known as a MORDANT which will form a complex with crystal violet and will retain the crystal violet stain after washing with alcohol. Gram negative bacteria will become colorless after washing with alcohol, counterstaining with safranin will make them pink.

Procedure:
  1. Emulsify a colony of the bacteria (from the culture plate) into a drop of saline on a clean microscope slide.
  2. Heat fix the slide.
  3. Flood the slide with crystal violet for 1 min. Wash with running tap water.
  4. Flood with Gram's iodine for 1 min. Wash with running tap water.
  5. Carefully decolourize with 95% ethanol.
  6. Flood with safranin 1 min. Wash with running tap water.
  7. Look at the slide under microscope.
Result: Gram Negative - Pink, Gram Positive – Purple

For Gram Positive bacteria,

Step 2: Catalase Test

Principle: To identify which organism produces catalase enzyme that convert hydrogen peroxide to water and oxygen gas. Catalase enzyme help to protect bacterial cell wall against hydrogen peroxide which is a highly reactive compound. It is useful in differentiating Staphylococci and Streptococci. Catalase positive will produce bubble which is an indication of presence of Staphylococci.

Procedure: Simply place a loopful of cells from isolated colonies into a drop of 3% hydrogen peroxide on a clean microscope.

Result: Immediate generation of bubbles - Positive (Staphylococci), no bubbles - Negative (Streptococci).

Step 3: Coagulase test

Principle: This test is to identify the presence of bound coagulase or clumping factor which will attach to the cell wall of the bacteria. Bound coagulase reacts with fibrinogen in plasma causing the fibrinogen to agglutinate which will give a positive result. This test is useful in differentiating S. aureus from Catalase positive Staphylococci.

Procedure: Simply add a colony into a drop of thawed rabbit plasma and mix them thoroughly.

Result: Agglutination – Positive (S. aureus), No agglutination – Negative (Staphylococci)

Step 4: Sensitivity Plate and Purity Plate

Principle: Sensitivity Plate is to determine the effectiveness of antibiotic against micro-organism that has been isolate from the culture. Purity Plate is to check for the morphology of the bacteria growth on the plate to tally with the result found.

Procedure:
Sensitivity Plate:
  1. Emulsify half a colony from the culture plate into the saline till 0.5 McFarland turbidity.
  2. Lawn the whole MH agar plate and ¼ of 5% NaCl agar plate with the micro-organism.
  3. Place the antibiotic disks: VA, E, SXT, TE, FT, P, AM, CIP, AMC, FOS, FT on MH agar plate and OX on 5% NaCl agar plate.
Purity Plate:Streak the plate.

Result: For Sensitivity Plate, sensitive – antibiotic is effective, micro-organism grow far away from it, intermediate – antibiotic may or may not be effective and resistant – the antibiotic is ineffective, micro-organism grow around it.

For Gram Negative Bacteria

Step 2: Oxidase test

Principle: To identify organism that produce enzyme cytochrome oxidase. Cytochrome oxidase participates in the electron transport chain by transferring electron from donor molecule to oxygen. The oxidase test reagent contain reducing agent which is a compound that change colour when become oxidized.

Procedure: Simple smear an isolated colony onto the oxidase card

Result: Change of colour – positive, no change of colour - negative

Step 3: Microgen GN-ID

Principle: To identify the complete range of Enterobacteriaceae and other non-fastidious, oxidase positive and negative, gram negative bacilli. Microgen GN-ID comprises of 2 separate, 12 substrates, microwell strips, GN-A and GN-B. GN-A can be used alone and identifies oxidase negative, non-fastidious gram negative bacilli while GN-B needs to be used with GN-A, identifies the complete set of Enterobacteriaceae and other non-fastidious, oxidase positive and negative, gram negative bacilli. The wells contain dehydrated biochemical substrates that are reconstitute with a saline suspension of the organism to be identified. If the substrate is metabolized by the organism, colour change will occur during incubation or after adding addition of reagents.

Procedure:
  1. Perform oxidase test on the isolate prior to strip inoculation.
  2. Emulsify a single colony from the culture in sterile saline.
  3. Mix thoroughly.
  4. Add 3-4 drops of suspension to each well of the strip.
  5. Overlay specific will with 1 drop of mineral oil on the black circles.
  6. Seal the top of the microwell strip with adhesive strip and incubate at 37oC for 18-24 hours.
  7. Add a drop Indole Kovas, VP I and II and TDA to the respective well.
Result: Compare the colour shown on the microwell with the colour chart provides by the supplier and type in the result into the software provided by the supplier to identify the organism.

Step 4: Sensitivity Plate and Purity Plate

Principle: Sensitivity Plate is to determine the effectiveness of antibiotic against micro-organism that has been isolate from the culture. Purity Plate is to check for the morphology of the bacteria growth on the plate to tally with the result found.
Procedure:
Sensitivity Plate:
  1. Emulsify half a colony from the culture plate into the saline till 0.5 McFarland turbidity.
  2. Lawn the whole MH agar plate and ¼ of 5% NaCl agar plate with the micro-organism.
  3. Place the antibiotic disks: CRO, CXM, AM, SAM, GM, CIP, FT, SXT, FOS, AMC on blood agar plate.
Purity Plate: Streak the plate.

Result: For Sensitivity Plate, sensitive – antibiotic is effective, micro-organism grow far away from it, intermediate – antibiotic may or may not be effective and resistant – the antibiotic is ineffective, micro-organism grow around it.

Clinical Significance:

It is used to diagnose Urinary Tract Infection (UTI). It is done to determine the type of bacteria in the urine and the appropriate antibiotic for treatment. A UTI is an infection of one or more components of the urinary tract. UTI occurs at any age but women are much more likely than men to have them.

Hope that you all have learnt something after reading my blog. Feel free to ask questions.

Lizzie Chew (0503194C) TG01