Various Cultures

Urine Culture
(Culture on Blood Agar and MacConkey agar)

This test is performed when a patient is suspected to have UTI ( Urinary tract infection )

Basically, when there is growth of pure culture in high colony counts and the urinalysis resulted in large amount of bacteria, we will consider it as positive result for urine culture. 

Methodology:

1. Use a smaller loop to obtain the urine sample
2. Draw a vertical line at first in order to distribute the sample evenly.
3. Then streak the plate starting with close distance to far distance between each horizontal line.

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Blood Culture(Culture on Blood Agar and MacConkey agar)
This test is performed to determine and identify the presence of fungi, bacteria or virus in the sample. It is ordered when the patient shows the symptom of sepsis.

*Normally, the FBC( Full Blood Count ) test will also be ordered along to determine whether the patient have increased WBC (White blood cell ) that indicating a potential infection.*

 Methodology:
1.

Scan the bar code.

2.

Insert the culture bottle into the appropriate position.

3.

Wait 2 days for the analyzer to detect whether there is any bacteria growth inside the culture bottle.

*Basically, there should be no bacteria inside the human blood as it should be sterile inside the body.*

4. If there is bacteria inside the culture bottle, a syringe will used to withdraw the patient's blood from the rubber sealed bottle and one drop of blood is used for culture.

Lancefield Grouping test :

-- a test that used to identify the species of detected Streptococcus.

Reagent used.

There are total 6 types of Streptococus : A, B, C, D, F, G .
The agglutination indicate that which the detected species belongs to. 

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Swab Culture
(Culture on Blood Agar and MacConkey agar)

1. If the culture consists of bacteria growth followed by beta-haemolysis, the microorganism is suspected to be the Streptococcus aureus.

   

   β-hemolysis is the lysis of red blood cell in the blood agar around the colonies due to the streptolysin which produced by the bacteria.

   
   
2. Catalase test will be conducted.
   
   Small amount of the suspected organism will mix with a drop of catalase which placed on a slide. Bubble formation indicating positive result.
3. Then, Coagulase test will be carry out.
   
   Small amount of the suspected organism will mix with the plasma (FFP) within a test tube.
   If there is coagulation of the plasma indicating positive result.
4. Gram staining and sensitivity test will take place afterward.

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Stool Culture(Culture on  MacConkey agar and XLD agar)

1. Small amount of the patient's stool is added into a test tube containing Selenite F solution and incubate for 1 day.
2. While in the same first day, the stool is also used to culture on MacConkey agar.
3. In the second day, the incubated Selenite-F solution is cultured on XLD agar plate.  

Positive result: Black colonies ( Salmonella ) present on XLD agar.
* The microbe will change the color of the agar*

Culture the "Black colonies" on TSI agar ( Triple Sugar Iron agar ).

 

(Left) Agar turned black indicating positive result with the presence of Salmonella.

(Right) Agar remained yellow in color indicating negative result.

Procedure of culturing on TSI:

1. Stab vertically into the slant agar.
2. Then, drag the loop out and streak a vertical line on the agar surface.
3. Lastly, do the horizontal streaking.

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Other culture

Upper respiratory sample( Sputum , throat swab...） ，Eye swab , high vagina swab...  use Blood agar, MacConkey agar and Chocolate agar.

Chocolate agar is placed in a sealed tin with lighted candle while incubating in order to create an anaerobic environment.

Other than Blood agar and MacConkey agar, urethral swab is also cultured on the Sabaraud agar to find out any yeast presented.

AFB testing

AFB testing ( Acid-Fast Bacillus Smear and Culture and Sensitivity )

• It is a test that used to diagnose tuberculosis ( caused be Mycobacterium tuberculosis) in the patient and help in monitoring the effectiveness of treatment.
• AFB test may also be used to detect any infection caused by the Mycobacterium species (Acid fast bacilli).

The pink rod-shaped microorganism is Mycobacterium tuberculosis

Procedure:

1. Prepare a smear and fix.

   

   
   

2. Flood the smear with ZN-carbol fuchsin stain for 5 minutes and gently heat it.
   
3. Wash gently with running tap water.
   
4. Decolorize  with TB-decoloriser for 2~3minutes
   
5. Wash gently with running tap water.
   
6. Counter stain with methylene blue for 30~60 seconds.
   
7. Wash gently with running tap water.
   
8. Dry over gentle heat.
   
9. Examine under 100x with immersion oil.

Important notes during streak plate.

Important Notes

1. Don't touch the wall using loop while streaking because the side wall of the agar plate is consider not sterile. ( except for Mueller Histon agar )
   
2. 
   
   
   
3. Blood Agar (BA) should be streaked first before MacConkey (MAC) agar to avoid contamination by the bile salt and crystal violet in MAC which used to inhibit the growth of Gram-positive organism.
   (Every microorganism can grow on BA)

   

   (Left) MAC agar  ; (Right) BA 

4. Function of every types of used agar should be studied in order to determine which types of organism can be cultured on it.
   
5. Sometimes when Urine FEME showed 3+ positive in the amount of bacteria, however there is no growth on the agar culture. This is because the bacteria observed under the microscope might be already dead or it could also be possible that there is mistake in the technique used to culture the plate.
   
6. Gram-positive and gram-negative organism should not present together in certain part of the body. If the culture shows this type of result, we must always correlate with the FBC ( Full Blood Count ) result to see whether there is any infection.  
   

C & S test

Culture and Sensitivity test

• The specimen is cultured on agar plate to identify the microorganism that present in the sample based on the culture's morphology, biochemistry profile and staining.
• The specimen is cultured on a specific antibiotic-containing agar plate to discover the microorganism based on its susceptibility.
  Also, it helps in determining what type of antibiotic can be given to the patient for infection treatment.
• This test can be done with different types of body fluids and excretion, such as urine, blood, pus, swab, saliva, mucus, stool and so on.

Procedure:

Culture and species determination :

 

1. Insert 4 full amount of normal saline into the test tube ( "Culture tube" )
2. Take little amount of bacteria from the pure culture and insert into the tube until the saline looks slightly turbid.
   
   
3. Incubate at 37°C for 2 hours.
4. Insert 100µl of the culture into each well of the Microgen kit ( contains certain addictive and reagent for growing ) to determine the species of the microorganism.
5. Add 2 drops of mineral solution.
6. Seal the kit and incubate for 1 day.
7. Add appropriate drops of reagent in to specific well.
8. Compare the resulted color with the identification system chart, calculate the number and key in to the computer software to determine the species automatically.
   
   

   

   Microgen kit which added with sample and reagent in each well.

Identification system for Streptococci and Enterococci .

Identification system for all Enterobacteriaceae and excessive range of oxidase-positive Gram-negative bacilli.

Fill in the blank and calculate the number.

Key in details into the computer software.

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Antibiotic Susceptibility Test:

1. Add 0.5ml of peptone water into a microcentrifuge tube ("Sensitivity tube" )
   
   
2. Take little amount of bacteria from the pure culture and insert into the tube until the peptone water looks slightly turbid.
   
   
3. Incubate at 37°C for 2 hours.
4. Dip a clean cotton swab into the sensitivity tube and fully spread the solution on the agar plate ( Müeller-Hinton agar ).
5. Add on the antibiotic chips with the appropriate distance ( about 2.4 cm) based on the species.
   For example, Escherichia coli

   

   For E.coli, there are 12 antibiotic chips to be tested.

   
   
6. Incubate the agar plate for 1 day.

The microbes are resistant to the AMS 20 while sensitive to the other antibiotics.

Result Interpretation:

*Often correlate with other relevant test result before the final result is reported*

For example,
FBC result :

• High lymphocytes indicate viral infection, thus no bacteria will grow on the culture plate. 
• High neutrophils indicate bacteria infection, thus there should be bacteria growth on the agar.

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Reference:

1. http://antranik.org/culture-and-sensitivity-test-the-clinical-requisition/
2. https://labtestsonline.org/understanding/analytes/urine-culture/tab/test/

WWF

Equipment:

6 types of reagent .

Procedure:

1. Add 8000μl of saline in a container.
2. Withdraw 100μl of the saline and replace it with the same amount of patient's serum. 
3. Add 1000μl of the diluted sample into 6 tests tubes which containing 6 different types of  reagents in it. (1 drop in each)
4. Incubate in water bath for 5 hours.

Result Interpretation:

• Positive result will shown in the sedimentation of the colored reagent.

Gram staining.

• Gram stain is used to differentiate the microorganism whether it is gram-positive or gram negative.
  
  
  
• Gram positive -Purple
  Gram negative- Pink
  
  
• It is conducted when the urine or swab culture have mixed growth of bacteria.
  
  
• For urine or stool culture, it is normal to have both gram-positive and gram-negative bacteria present in the culture.
  
  
• Except for stool, if there are more than 10 colonies appeared in other types of culture on blood agar, gram staining will be carry out for species identification.

Procedure:

Add caption

1. Prepare a smear and fix it by passing through gentle heating.
2. Flood the smear with crystal violet for 1 minute.
3. Rinse with running tap water.
4. Flood the slide with iodine for 1 minute.
5. Rinse with running tap water.
6. Flood with decolorizer for 30 seconds.
7. Rinse with running tap water.
8. Counter stain the slide with safranin for 1 minute.
9. Rinse with tap water and dry the slide.
10. Observe under 100x with immersion oil.

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Reference:

1. http://www.medicinehack.com/2012/02/gram-staining-procedure-mechanism.html
2. Labtestsonline.com

Immunology department

Abbott i 1000 SR

Monitor Screen: " Patient order" page

Sample: Patient's blood serum.

Reagent reloading...

Checklist:

• Daily maintenance:
1. Check if any sample is running, if no, change the analyzer to "ready mode" by pausing it.
2. Press the "system" button and select "maintenance".
3. Fill the particular container with distill water and add 4 drops of saline.
4. Fit the container on a rack and place them to "position 12"
5. Click perform, OK and proceed 2 times.
• Reagent checking:
• All the reagents which have been calibrated can be load into the analyzer when the samples are running.
• However, only trigger and pre-trigger reagents must be reloaded during ready mode.
• Only when the analyzer is in running mode, the sample can gone through the test.
• Running the test:
1. Record and key in patient's detail with the requested tests according to the requisition form.
   Steps: Select "patient order" → C: " code of the rack"  P:"position"→ Select "patient details"

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Tests that performed:

FT4, FT3 and TSH are Thyroid hormones.

• When FT3 ↓ FT3 ↓, while TSH ↑    =  Hypothyroidism
• When FT3 ↑ FT3 ↑, while TSH ↓    =  Hyperthyroidism

AFP (Alpha-fetoprotein) is Liver Tumor Marker.

• Protein that produced by fetal liver.
• The concentration of AFP elevate when a baby is born and then decline rapidly until there is liver damage or certain cancer.
• Aids in detecting liver, testes and ovarian cancer.
• Monitor the patient with chronic liver diseases or monitor the patient's treatment against hepatocellular carcinoma.

CEA (Carcinoembryonic Antigen) is common Tumor Marker.

• Monitor the treatment against colon cancer
• Determine prognosis ( the likelihood a disease to be healed )
• To stage cancer.
• Determine the spreading of cancer into body cavity.

Ca125 is Ovarian Tumor Marker.

PSA (Prostate specific antigen) is Prostate Tumor Marker

• PSA is a type of protein which produced by the prostate cells and being released into the semen, however small amount of the PSA will also being released into the blood.
• There are two forms of PSA exist in the blood:  Free PSA (not bound) & Complexed PSA (cPSA) (bound to other proteins).
• We can measure either Free PSA or Total PSA (both bound and unbound) through lab tests.
• The range for PSA rises according to the man age.

HIV Ag/Ab

Anti-Hbs (HbsAb)

HBsAg - Hepatitis antigen

Anti-HCV

External test : 

Ca199 is the Tumor Marker for Pancreatic cancer.

Ca153 is the Tumor Marker for Breast Cancer.

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Reference:

https://labtestsonline.org/understanding/

Dengue IgM/IgG and NS1

• There are total 4 distinct serotypes of dengue virus (DV) namely, DV-1,-2,-3 and -4, which are mainly transmitted by the Aedes mosquitos.
  
  
• NS1 (nonstructural protein 1) is a highly-conserved glycoprotein which present at high concentration in the serum during early stage of the disease infection.
  
• NS1 antigen is detectable within the first to nine days following the onset of symptoms in the primary or secondary dengue infected patient.
  
• IgM will only be determined after 5 to 6 days of the onset of illness for primary infection while 4 to 5 days after the illness onset for secondary infection.
  
• IgG will only appear after 14 days in primary infection while for secondary infection, it rise within one or two days and will induce the release of IgM after 20 days of infection.

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There are two types of test devices we are using in this lab to identify the dengue virus infection, Dengue NS1 and Dengue IgG/IgM.

The former is the solid phase immunochromatographic assay for the detection of dengue antigen (NS1) while the latter aids in the detection and differentiation of antibodies (IgG/IgM) against dengue virus.

Both use patient's serum/plasma or whole blood as testing sample.

Dengue NS1 test device.

Procedure:

Result Interpretation: 

Dengue IgG/IgM test device.

Procedure:

 Result Interpretation:

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Reference:

1. http://www.biogatelab.com/uploads/3/1/1/5/3115507/dengue_ns1__cassette.pdf

Serology department.

In Serology department, the equipment which is commonly used in all of the tests is the timer.

There are some tests which will be performed on a background card placed on the rotater while the other tests are performed using the reagent containing test kits.

The rotater in 100 revs/min with 4 types of background cards and timer on top of it.

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Background card.

* VDRL test uses a white background while ASOT and ANF test use dark background*

*Monospot test uses transparent background*

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About the Tests:

ASOT (Antistreptolysin O (ASO) titer)
It is a blood test which used to determine whether the patient has been infected by the Streptococcus bacteria. The test is performed by measuring the antibodies (Antistreptolysin O) against the exotoxin (known as "Streptolysin O") which is produced by the group A Streptococcus bacteria.



Procedure:

1. 1 drop of sample and 1 drop of reagent are mixed evenly on a dark background card.
2. Placed the card on the rotator and wait for 3 minutes.

Interpretation of result:
Positive result will shown in agglutination of the sample with the reagent.

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ANF Anti-nuclear factor
Also known as ANA (Antinuclear antibody) test , is used to evaluate whether a patient is having any autoimmune disorder. 

ANA are a group of autoantibodies which produced by a person's immune system when it is unable to differentiate between own cells and external antigen.


Both ANF and ASOT tests are usually carry out  when the patient is having joint pain/arthritis.



Procedure:

1. 1 drop of sample and 1 drop of reagent are mixed evenly on a dark background card.
2. Placed the card on the rotater and wait for 3 minutes.

Interpretation of result:

Positive result will shown in agglutination of the sample with the reagent.

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VDRL (Venereal Disease Research Laboratory)
Or known as RPR (Rapid Plasma Reagin) test, is a serological screening test that use RPR Carbon antigen for the qualitative and semi-quantitative detection of syphilis in patient's serum or plasma.

This VDRL antigen test is a non-Treponemal test which means that the antibodies detected in the patient's sample are not specific to the Treponema pallidium. Therefore, when the test shown positive result, the doctor might ask us to do further testing on that sample with TPHA test which is more specific.

Treponema pallidium is a spirochaete microorganism that cause the venereal disease, Syphilis.


The VDRL test is functioned by measuring the antibodies which is produced in response to the lipoidal material released from damaged host cells and lipoprotein like substance that released by the bacteria.

In addition to screening, the RPR test can also be used to monitor the treatment of syphilis. In this case, the level(titer) of the antibodies presented will measured through dilution.


*Titer: A unit of measurement in the clinical laboratory, which is the lowest dilution of a substance for the particular reaction to take place. *



Procedure:

1. Place one drop (50μl) of the patient's sample on the white test card and allow one drop of the VDRL Carbon Particle Antigen to fall on it.
2. Spread the solution on the RPR test card to cover the test circle.
3. Rotate the test card for 8 minutes at 100 revolution/minutes.

Interpretation of result:

Positive result will shown in agglutination of the sample with the reagent.

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Monospot / Mononucleuosis test /Mono test

This test is used to determine whether the patient has infectious mononucleosis.

• use transparent test card

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Various test devices: