basics in identification of bacteria dr.t.v.rao md · cara umum untuk identifikasi bakteri...

Identifikasi Bakteri

1

Oleh Irda Safni

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

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

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

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.

Dr.T.V.RaoMD 3

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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

Dr.T.V.RaoMD

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

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.

Dr.T.V.RaoMD 6

Identification of Bacteria after

isolation in pure forms

• The

bacteria is

obtained in

pure culture

it has to be

identified

Dr.T.V.RaoMD 7

Bacterial cells

Morphology

• Depends on

number of factors

such as strain

studied nature of

the culture medium

• Temperature and

time of incubation

Dr.T.V.Rao MD

9

Shape of thecolony

Dr.T.V.RaoMD 10

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

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

Crystalviolet

Gram'siodine

Decolorise withacetone

Counterstain withe.g. methyl red

Gram-positives appear purple

Gram-negatives appear pink

The Gram Stain

Dr.T.V.RaoMD 14

Gram-positive rods

Gram-negative rods

Gram-positive cocci

Gram-negative cocci

Coccus

Staphylococcus species

Streptococcus species

Dr.T.V.RaoMD 16

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

Dr.T.V.RaoMD 17

Gram-positiveCocci

• Staphylococci

– Catalase-positive

– Gram-positive cocci

in clusters

• Staphylococcus

aureus

– coagulase-positive

• Staph. epidermidis

– and other coagulase

negative

staphylococci

Gram-Positive Cocci

• Streptococci

– Catalase-negative

– Gram-positive cocci

in chains or pairs

• Strep. pyogenes

• Strep. pneumoniae

• Viridans-type

streps

• Enterococcus

faecalis

Gram-Negative Cocci

• Neisseria

gonorrhoea

– The Gonococcus

• Neisseria

meningitides

– The Meningococcus

• Both Gram-negative

intracellular

diplococci

Gram-Negative Bacilli

• Enteric Bacteria

– E. coli

– Salmonella

– Shigella

– Yersinia

– Pseudomonas

– Proteus

– Vibrio cholerae

– Klebsiella pneumoniae

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.

Dr.T.V.RaoMD 22

From the Virtual Microbiology Classroom on ScienceProfOnline.com

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

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

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

Types of Culture Media

1. Basic media

2. Enrichment media

3. Selective media

4. Indicator (Differential) media

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

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

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)

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

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.

Bacillus subtilis Proteus spp.

Metabolism of the bacteria and

biochemical bacterial

identification

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

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

Different packaging of identification media

Agar plate

media

Tubes media

Classics

tubes

Multi-test

miniaturized

systems

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

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

Dr.T.V.RaoMD 39

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

Dr.T.V.RaoMD 40

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.

Dr.T.V.RaoMD 41

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.

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.

Dr.T.V.RaoMD 43

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

OxidaseTest…..

Positive

• Pseudomonas spp.

• Aeromonas spp.

• Vibrio spp.

• Alcaligenes spp.

• Neisseria spp.

• Haemophilus sps

Negative

• Enterobacteriaceae

• Acenitobacterspp.

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 &ethanol, 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.

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.

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.

Negative

Negative

Posiitive Positive

Nitrate reducedto

NH3 or N2 gas,

nitrite absent

Nitrate reducedto

nitrite

Nitrate not

reducedNitrate not

reduced

Nitrate reduction test….

Dr.T.V.RaoMD 50

Addition of Zndust or

Nitrate reduction test….

Dr.T.V.RaoMD 51

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

Uji Motilitas bakteri

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.

Dr.T.V.RaoMD 54

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

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.

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

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

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

Dr.T.V.RaoMD 59

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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