basics in identification of bacteria dr.t.v.rao md · cara umum untuk identifikasi bakteri...
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Identifikasi Bakteri
1
Oleh Irda Safni
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Cara umum untuk identifikasi bakteri
Langkah 1 : Sampling
Langkah 2 : Mengkulturkan isolat
Pada media isolasi
Langkah 3 : Melakukan
teknik identifikasi
Langkah 4 : Hasil
= memberi nama bakteri
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Cara umum untuk identifikasi bakteri
Different identification
techniques
Physical methods Based on the
characterization of proteome of the bacteria
Genetical methods Based on the
characterization of specifics genes of the bacteria
Biochemical methods Based on the
characterization of metabolic pathways of the bacteria
To identify unknown bacteria , results are compared to databases
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Q: What was that clue to help us remember the hierarchy of
biological classification?
Dr.T.V.RaoMD 2
From the Virtual Microbiology Classroom on ScienceProfOnline.com
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ClassificationFamily: a group of related genera.
Genus: a group of related species.
Species: a group of related strains.
Type: sets of strain within a species
(e.g. biotypes, serotypes).
Strain: one line or a single isolate of
a particular species.
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Methods in bacterial identification
1. Microscopic morphology - Gram Staining, Shapes, arrangements, motility
2. Macroscopic morphology – colony appearance, motility
3. Physiological / biochemical characteristics – aerobic, anaerobic, photosynthetic, growth on selective media
4. Chemical analysis – e.g. peptides and lipids in cell membranes
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Methods in bacterialidentification
1. Phage Typing – which phage infects the bacterium
2. Serological analysis – what antibodies are produced against the bacterium
3. Pathogenicity – what diseases does the bacterium cause.
4. Genetic & molecular analysis• G + C base composition
• DNA analysis using genetic probes
• Nucleic acid sequencing & rRNA
analysis Dr.T.V.RaoMD 5
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Steps in diagnostic isolation and
identification of bacteria
• Samples of body fluids are streaked onculture plates and isolated colonies ofbacteria appear after incubation.
Observation of these colonies for size, texture, color, and (if grown on blood agar) hemolysis reactions, is highly important as a first step in bacteria identification.
Whether the organism requires oxygen for growth is another important differentiating characteristic.
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Identification of Bacteria after
isolation in pure forms
• The
bacteria is
obtained in
pure culture
it has to be
identified
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Bacterial cells
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Morphology
• Depends on
number of factors
such as strain
studied nature of
the culture medium
• Temperature and
time of incubation
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Shape of thecolony
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Shape of BacteriaBacteria display three
basic shapes:
i.round- cocci, (from the
Greek kokkos - a berry),
ii.rod shaped – bacilli (from
the Latin bacillus - a stick
or rod),
iii.spiral (quelled).Dr.T.V.RaoMD 11
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Gram staina. Gram stain divides the bacteria into Gram
positive & Gram negative.
The basic procedure :i. Take a heat fixed bacterial smear.
ii. Flood the smear with CRYSTAL VIOLET or Methyl violet
for 1minute, then wash with water. [PRIMARY STAIN]iii. Flood the smear with IODINE for 1 minute, then wash with
water.
iv. Flood the smear with ETHANOL-ACETONE, quickly, then wash with water. [DECOLORI
v. Flood the smear with SAFRANIN for 1 minute, then wash with
water. [COUNTERSTAIN]
vi. Blot the smear, air dry and observe.Dr.T.V.RaoMD 12
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Crystalviolet
Gram'siodine
Decolorise withacetone
Counterstain withe.g. methyl red
Gram-positives appear purple
Gram-negatives appear pink
The Gram Stain
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Gram-positive rods
Gram-negative rods
Gram-positive cocci
Gram-negative cocci
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Coccus
Staphylococcus species
Streptococcus species
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Gram stain morphology
• Shape– cocci (round)
– bacilli (rods)
– spiral or curved (e.g.spirochetes)
• Single or multiplecells– clusters (e.g. streptococci)
– chains (e.g. streptococci)
• Gram positive ornegative
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Gram-positiveCocci
• Staphylococci
– Catalase-positive
– Gram-positive cocci
in clusters
• Staphylococcus
aureus
– coagulase-positive
• Staph. epidermidis
– and other coagulase
negative
staphylococci
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Gram-Positive Cocci
• Streptococci
– Catalase-negative
– Gram-positive cocci
in chains or pairs
• Strep. pyogenes
• Strep. pneumoniae
• Viridans-type
streps
• Enterococcus
faecalis
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Gram-Negative Cocci
• Neisseria
gonorrhoea
– The Gonococcus
• Neisseria
meningitides
– The Meningococcus
• Both Gram-negative
intracellular
diplococci
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Gram-Negative Bacilli
• Enteric Bacteria
– E. coli
– Salmonella
– Shigella
– Yersinia
– Pseudomonas
– Proteus
– Vibrio cholerae
– Klebsiella pneumoniae
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Classifying Bacteria: Gram-Negative & Gram-Positive
Gram +
•Peptidoglycan is the thick, outermost layer of the cell wall.
• About 90% of cell wall is made of peptidoglycan.
Gram -
• Cell wall is more chemically complex, thinner and less compact.
• Peptidoglycan only 5 – 20% of the cell wall.
• Peptidoglycan is not the outermost layer,but between the plasma membrane and theouter membrane.
• Not accessible to the action of antibiotics.
• Outer membrane is similar to the plasma membrane, but is less permeable and contains lipopolysaccharides (LPS).
• LPS is a harmful substance classified as an endotoxin.
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From the Virtual Microbiology Classroom on ScienceProfOnline.com
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Classification: Dichotomous Key
Simple Stain
Cocci
Gram Stain
Gram negative
cocci
Gram
positive
cocci
Mannitol Salt
yellow pink
Staphylococcus
aureusStaphylococcus
epidermis
Bacilli
Gram Stain
Gram
negative
bacilli
Gram positive bacilli
No
color
change
Salmonella
pullorum
Pink
colonies
E. coli
Enterobacter
Acid Fast stain
MacConkey’sAcid Fast
Mycobacterium
tuberculosis
Not
acid
fast
Endospore stain
Forms
endospores
Bacillus subtilus
smegmatis
Since we will be working with a limited number of bacterial species and identification techniques, we will be using a limited dichotemous key in lab.
aerogenesDr.T.V.RaoMD 24
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GRAM-POSITIVEFacultative anaerobe, cocci
Thick cell wall, ~50% of cell’s mass. (When you Gram stain it, the cells are intensely purple.)
Found in many places throughout the environment human skin, animals, water, dust, and soil.
M. luteus on human skin transforms chemicals in sweat into body odor.
Grow well even with little water or high salt concentrations. (You may find it growing on your Mannitol Salt nasal sample.)
Normal flora that can become opportunistic in immune compromised.
Bacterial Genus:
This is your lab friend Micrococcus luteus.
Dr.T.V.RaoMD Images: M. luteus colonie2s5, T.Port;M. luteus, Janice Carr, PHIL #9761
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CULTURE MEDIA
2
Culture media generally provide sources of carbon , energy
and nitrogen in the form of available carbohydrates and amino
acids
Special media provide specific requirements as inorganic
salts or particular growth factors
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Types of Culture Media
1. Basic media
2. Enrichment media
3. Selective media
4. Indicator (Differential) media
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1. BASIC MEDIA
- These are simple media used to support the growth
of microorganisms that do not have special nutritional
requirements.
- E.g. nutrient broth, nutrient agar, and peptone water.
Nutrient Broth Nutrient Agar Peptone Water
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2. ENRICHMENT MEDIA
Fluid media that contain substances which favour the growth of
wanted organisms on the expense of others.
Usually used as a preliminary step for isolation of pathogensbefore subculturing on solid selective media.
Examples are: Alkaline peptone water
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3. SELECTIVE MEDIA
Solid media that contain substances (e.g. bile salts or other
chemicals, dyes, antibiotics) which inhibit the growth of one
organism to allow the growth of another.
Used when culturing a specimen from a site having a normal
microbial flora to prevent unwanted contaminants overgrowing
a pathogen.
Example: sucrose peptone agar (Xanthomonas spp)
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4. INDICATOR (DIFFERENTIAL) MEDIA
These are media to which dyes or other substances
(Indicators) are added to differentiate microorganisms.
Indicators change colour when acid is produced following
fermentation of a specific carbohydrate e.g. MacConkey's
agar medium, Tetrazolium Chloride (TZC) medium
Isolate of Serratia marcescens on
MacConkey agar
Isolate of Ralstonia solanacearum on
Tetrazolium Chloride agar
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On solid medium the following
characters are observed
Shape: circular, irregular, radiate or rhizoid.
Size: The size of the colony can be a useful characteristic for identification. The diameter of a representative colony may be measured.
Elevation:
Margin: Entire, wavy, lobate, filiform
Surface: smooth, wavy, rough, granular, papillate, glistening etc.
Size in mm
Texture : dry, moist, mucoid, brittle, viscous, butyrous (buttery).
Color : colorless, pink, black, red, bluish-green.
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Bacillus subtilis Proteus spp.
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Metabolism of the bacteria and
biochemical bacterial
identification
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Metabolism of the bacteria
Bacteria are living cells who :
- consume nutrients (carbohydrates, proteins…)
- reject metabolic waste.
Nutrients Metabolic
waste
Enzymes
Bacteria
Biochemical techniques for
identifying bacteria are based on
the characterization of enzymes
and metabolic waste
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Metabolism of the bacteria
The main metabolic pathways :
CarbohydratesAcidic
moleculesBacteria
Proteins, aminoacids Ammonia,
CO2, amino…
Enzymes
These waste are frequently acidic
or alkaline
Possibility of detection with a colorimetric pH
indicator
For some particular produced molecules, detection
requires some special reagents
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Different packaging of identification media
Agar plate
media
Tubes media
Classics
tubes
Multi-test
miniaturized
systems
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PHENOTYPIC CHARACTERISTICS
Metabolic differences
•Biochemical tests
– Sugar fermentation
• e.g., Lactose, sucrose, glucose, etc.
• Fermentation results in acid production– pH indicator changes color
– Pink yellow
• Inverted tube (Durham tube) collects any gas produced
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Key identification
characteristics for
EnterobacteriaceaeGENUS/SPECIE
S
Fermentation of Gas MR VP Indole Citrate Urease H2
G L S M
Escherichia coli (+) (+) (+) (+) (+) (+) (-) (+) (-) (-) (-
Shiegella (+) (-) (-) (+) (-) (+) (-) (-/+) (-) (-) (-
Shiegella sonnei (+) (+) (-) (+) (-) (+) (-) (-) (-) (-) (-
Salmonella (+) (-) (-) (+) (+) (+) (-) (-) (+) (-) (+
Klebsiella
Pneumo.
(+) (+) (-) (+) (+) (-) (+) (-) (+) (+) (-
Enterobacter (+) (-) (+) (+) (-) (+) (-) (+) (-) (+) (+
Serratia (+) (+) (-) (+) (+) (-/+) (+) (-) (+) (-) (-
Proteus (+) (-) (-) (+) (-/+) (+) (-) (+) (-/+) (+) (+
morganella (+) (-) (-) (+) (+) (+) (-) (+) (-) (+) (+
Yersinia (+) (-) (-) (+) (-) (+) (-) (-/+) (-) (-/+) (-
G: Glucose, L:Lactose, S:Sucrose, M: Manitol, MR: Methyl Red, VP: Voges Proskauer
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Indole TestPrinciple:
Indole test is performed to
determine the ability of the
organism to split tryptophan
molecule into Indole.
Indole is one of the
metabolic degradation
product of the amino
acid tryptophan
Bacteria that possess the
enzyme tryptophanase are
capable of hydrolyzing and
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Indole Test
Property it tests for:
•This test is performed to help differentiate
species of the family Enterobacteriaceae.
Media and Reagents Used:
•Tryptone broth contains tryptophan.
•Kovac’s reagent—contains hydrochloric
acid, dimethylaminobenzaldehyde, and
amyl alcohol—yellow in color.
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Indole test
• Procedure:-Inoculate Tryptone broth with the
test organism and incubate for18 to 24 hrs at 37°c
-Add 15 drops of Kovac’s reagentdown the inner wall of the tube• Interpretation:-Development of bright red color
at the interface of the reagent and the broth within seconds after adding the reagent is indicative of the presence of Indole and is a positive test
Indole Positive:E.coliProteus vulgaris
Indole Negative: Salmonella spp. Klebsiella spp.
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OxidaseTest
Principle:
Oxidase test is used to determine the presence of bacterial cytochrome oxidase enzyme using the oxidization of the substrate ―tetramethyl-p-phenylenediamine dihydrochloride‖
to indophenol a dark purple colored end product.as positive test. No colour development indicates a negative test and the absence of the enzyme.
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OxidaseTest….
2. Direct plate method
The reagent acts as an artificial electron acceptor substituting the oxygen. In the reduced stage dye is colorless , but in the presence of enzyme cytochrome oxidase dye is oxidised to indophenol blue
1. Moist filter papermethod
Quality controls
Positive control- Pseudomonas spp
Negative control – E. coli
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OxidaseTest…..
Positive
• Pseudomonas spp.
• Aeromonas spp.
• Vibrio spp.
• Alcaligenes spp.
• Neisseria spp.
• Haemophilus sps
Negative
• Enterobacteriaceae
• Acenitobacterspp.
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Methyl Red/Voges- Proskauer(MR/VP)
• Properties these test for: Both tests are used to differentiate species of the family Enterobacteriaceae.
• Media and Reagents Used:– Glucose Broth– Methyl Red indicator for MR test– Voges Proskauer reagents- A: 5% Alpha-Naphthol ðanol, B: Potassium Hydroxide; (3:1 ratio) & Deionized Water.
Principle of MR test:
To test the ability of the organism to produce and maintain stable acid end products from glucose fermentation and to overcome the buffering capacity of the system
This is a qualitative test for acid production.
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MR test(contd…)
Left: negative/Right: positive
Procedure:
-Inoculate the MR/VP broth with a pure culture of the test organism
and incubate at 35° for 48 to 72 hrs.
Add 5 drops of MR reagent to the broth
Result interpretation:
-Positive result is red (indicating pH below 6)
-Negative result is yellow (indicating no acid production)
MR Positive: E. coli
MR Negative: Enterobacter aerogenes Enterobacter cloacae Klebsiella
spp.
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Nitratereduction….
• To distinguish between these two reactions, zinc dust
must be added. Zinc reduces nitrate to nitrite. If the
test organism did not reduce the nitrate to nitrite, the
zinc will change the nitrate to nitrite. The tube will turn
red because alpha- naphthylamine and sulfanilic acid
are already present in the tube
• Thus a red color after the zinc is added indicates the
negative nitrate reduction test.
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Negative
Negative
Posiitive Positive
Nitrate reducedto
NH3 or N2 gas,
nitrite absent
Nitrate reducedto
nitrite
Nitrate not
reducedNitrate not
reduced
Nitrate reduction test….
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Addition of Zndust or
Nitrate reduction test….
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MotilityTest
• Property it tests for: This test is done to help differentiate species of bacteria that are motile from non-motile.
• Media and Reagents Used: Motility media contains tryptose, sodium chloride, agar, and a color indicator.
• How to Perform Test: Stab motility media with inoculating needle.
• Reading Results: If bacteria is motile, there will be growth going out away from the stab line, and test is positive. If bacteria is not motile, there will only be growth along the stab line.
A colored indicator can be used to make the results easy to see
y
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Uji Motilitas bakteri
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Urea Hydrolysis (Ureasetest)
• Property it tests for: This test is done to determine a bacteria’s ability to hydrolyze urea to make ammonia using the enzyme urease.
• Media and Reagents Used: Stuarts Urea broth (pH 6.8) contains a yeast extract, monopotassium phosphate, disodium phosphate, urea, and phenol red indicator.
• Principle
To determine the ability of the organism to split urea
forming 2 molecules of ammonia by the action of
the enzyme Urease with resulting alkalinity
• How to Perform Test: Inoculate Urea broth with inoculating loop.
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ReadingResults:
• Urea broth is a yellow-orange
color. The enzyme urease will
be used to hydrolyze urea to
make ammonia. If ammonia is
made, the broth turns a bright
pink color, and is positive. If
test is negative, broth has no
color change and no ammonia
is made. Figure in the right shows negative
and left shows positive
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Coagulasetest
Principle:
-This test is used to differentiate Staphylococcus aureus (positive) from coagulase negative Staphylococci. S. aureus produces two forms of coagulase: bound and free.
-Bound coagulase or clumping factor, is bound to the bacterial cell wall and reacts directly with fibrinogen. When a bacterial suspension is mixed with plasma, this enzyme causes alteration in fibrinogen of the plasma to precipitate on the staphylococcal cells, causing the celss to clump.
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Coagulasetest
• Free coagulase is produced extra-cellularly by
the bacteria that causes the formation of a clot
when Staphyllococcus aureus colonies are
incubated with plasma
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CoagulaseResults
Reading Results:A. Slide test:
-Positive: Macroscopic clumping in 10 secondsor less in coagulated plasma drop and noclumping in saline or water drop.
-Negative: No clumping in either drop.
-Note: All negative slide tests must be confirmedusing the tube test.
B. Tube test:
-Positive:
-Negative:
Clot of any size
(a) No clot (b)
a b
Coagulase Positive : Staphylococcusaureus
Coagulase negative: Staphylococcus epidermidis
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Molecular Analysis
It would be ideal to compare sequences of entire
bacterial chromosomal DNA.
Alternatively, genomic similarity has been assessed
by the guanine (G)+ cytosine (C) content (% GC).
This has been replaced by two alternatives:
1.Hybridization
2.Sequencing specific genes
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DNA-DNAhomology
1. How well two strands of DNA from different
bacteria bind (hybridize) together.
This technique is employed to compare the
genetic relatedness of bacterial strains/species.
2. If the DNA from two bacterial strains display a
high degree of homology (i.e. they bind well) the
strains are considered to be members of the same
species.
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