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