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

64 comments:

J.A.M.M.Y.S said...

heya,
just wanna ask ya what do you mean by McFarland turbidity? can briefly explain what is that? thanks ya

michelle
tg02

ALsubs said...

hello lizzie,
why is thawed rabbit plasma used in the coagulase test? Thank u..

Shu Hui
TG02

J.A.M.M.Y.S said...

Hey

Im in bacteriology too yet i've never come across this Microgen GN-ID test.

Its quite interesting because it simplifies the work of a med tech in identifying the GNB (gram negative bacteria) and thus frees up the med tech's time to do other stuff.

Anyways I know what's indole kovac, and that VP is voges proskauer. But im just wondering what TDA is.

Thanks

Azhar

we are the XiaoBianTai-7! said...

hi michelle,

macfarland turbidity is the standard set by the NCCLS, it range from 0.5 to 5, it is depending on how turbid or concentrated you want the saline to be with the isolates...

hi shu hui,

regarding to why must use thaw rabbit plasma instead of other type of plasma, i am not very sure, i just know is a antigen-antibody reaction but i will check it out for you then tell you again.

hi azhar,

TDA - Tryptophan deaminase which convert trytophan to indolepyruvic acid giving a cheery colour when ferric acid is added. indole positive isolates may give a brown colour which is a negative result.

Lizzie

we are the XiaoBianTai-7! said...

hi lizzie,

why do we need this step in the microgen test: Overlay specific will with 1 drop of mineral oil on the black circles? and can i know the difference between uncomplicated and complicated UTI???

Thanks and regards from Gail
0503160D
TGo1

BloodBank.MedMic.Haematology said...

Hello Lizzie

If urine phase contrast show few (1+) bacteria present, is there a need to do urine culture? If patient is diagnosed with UTI, what can she do, other than taking antibiotic therapy, to reduce the number of bacteria? Thanks

Ci Liang
TG01

royal physicians said...

Hey Lizzie,

Just wanna ask about your catalase test. When you say bubbles will appear, is it seen microscopically or you can see it with the naked eye? Also, if there is more bubbles formed, does it mean there's greater catalase activity?

Thanx
Johanna
TG02

MedBankers said...

hey

what are the most common microorganism you encoutered so far?

then what are the distinct test-tube results you will see when you come across ecoli?

elaine

we are the XiaoBianTai-7! said...

hi gail,

overlay specific well with 1 drop of mineral oil is to create non-aerobic condition since most bacteria can break down energy aerobically or anerobically so if they break down the sugar anerobically they will release different chemicals.Uncomplicated UTI, they only have one type of micro-organism while complicated UTI, they have more than one type of micro-organism, this could be done from reading of morphology of the colony, retest need to be done.

hi ci liang,

there will still be a need to do a culture to see wheter the bacteria is normal flora or is UTI bacteria. Some prescription and non-prescription medications are used in the short-term relief of UTI symptoms. These medications are not antibiotics and only help with the temporary relief of symptoms (they do not cure the UTI infection itself) and drink plenty of water to clense the urinary tract bacteria.

hi johanna,

the bubbles can be see with naked eye, more bubbles may mean more catalase activity but this is a non-specific test, it is still depend on how big the colony u picked.

Hi elaine

the most common micro-organism are K. Pneunmoniae, E.coli, P. mirabilis. My lab didn't use the tube method instead they use the microgen GN- ID, the distinct change is the colour change of the substrate.

Lizzie

first6weeks said...

hihi

Why is there a need of concern regarding the turbidity? As you mention to emulsify colony into the saline till 0.5 McFarland turbidity.

Juexiu
tg02

we are the XiaoBianTai-7! said...

hi juexiu,

the higher the turbidity, the higher the concentration of the bacteria cells in the saline, this might interfere with the step that is done further and give false result. eg overgrowing of the colonies make it difficult to interpret the morpholgy in purity plate.

Lizzie

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