Microbe Identification 3 Farook

8/9/2019 Microbe Identification 3 Farook

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Molecular Microbiology:Microbe Identification

BC34M(Lecture notes 2006/7)

Prepared and presented by

Dr. Marcia E. RoyeOffice: Biotechnology Centre, ground floor

Tel: 927-0304 (Office)977-1828 (Centre)

ext. 2518-2520 (from campus)Email: [email protected]


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Gettingnotesfrom

Web

www.uwimona.edu.jm/biochem/courses/bc34m

BC34M Course Website:


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Lecture Outline Microbe Identification Definitions, importance of microbes, overview of microbe identification

methods, unculturables, specimen collection, 5 Is. Phenotypic methods: media (enriched, selective, differential,

characteristic), direct microscopic examination, biochemical tests, rapidtests, bacteriophage typing, flow cytometry.

Immunological methods: precipitation, agglutination reactions,fluorescent antibodies, ELISA, immunoblot/western blot. Genotypic methods: nucleic acid probes, PCR, nucleic acid

sequencing, RFLP, plasmids fingerprinting. Textbooks* (limited copies available from book rental scheme) *Brocks Biology of Microorganisms. Madigan, M., Martinko, J. & Parker,

J. Pearson Education 9,10 or 11th Ed (or other advanced microbiologytext) *Jawetz, Melnick and Adelbergs Medical Microbiology. Brooks, G.F. et

al. 22nd edn. Appleton and Lange. 2001. *Medical Microbiology and Immunology. Leveinson and Jawetz, latest

edn.

Bacterial Pathogenesis: A Molecular Approach. Salyers, A.A. & Whitt,D.D.


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Overall Lecture Objectives

The objectives of this group oflectures is to examine the

phenotypic, immunological andgenotypic methods commonlyused in microbe identification.

Why is microbe identificationimportant?


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Objectives of this Lecture

Definitions

The importance ofmicroorganisms

An overview of microbe

identification methods


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What is Micro- and Molecular-

biology? Microbiology is a specialized area ofbiology that deals with living thingsordinarily too small to be seen with the

naked eyes (microorganisms). Microorganisms include .

These organisms include bacteria, fungi,viruses, protozoa and some parasitic worms.

Molecular biology is the study of themacromolecules of life (particularly DNA, RNAand proteins) and their interactions at themolecular (molecule) level.


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What is Molecular Microbiology?

What is molecular microbiology?

Deals with the interaction of microorganismswith their environment at the molecular

level. Microbiology deals with all aspects of

microorganisms including their:

Genetics

Physiology

Disease causing

Interactions with humans

Uses in industry and agriculture


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The Importance of Microorganisms


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TheImportance

ofMicrobes


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Microorganisms and Earth

Microorganisms have profound effectson the earth and its residents.

Microorganisms are critical for the flowof energy and food thru theecosystem.

Microorganisms (including algae)account for more than 50% of theearths photosynthesis that contributethe majority of O2 to the atmosphere.


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Microorganisms is crucial for thedecomposition and nutrient recycle whichhelp to keep the earth in balance.

What molecules are cycled by microorganisms?

In the long term microorganisms are themain force that drive the structure andcontent of the soil, water and atmosphere

e.g.: Greenhouse gases CO2 and CH4 create an

insulation layerin the atmosphere that help

retain heat.


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Carbon Cycle


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NitrogenCycle


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Sulphur Cycle


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Bacteria and fungi live in complex associationswith plants and assist the plant in obtainingnutrients, water and protect against diseases.

E.g.???

Microorganisms form similarassociations in thestomach of ruminants to help in the digestion ofcomplex carbohydrates.

E.g. of ruminants?

Recent estimates propose that up to 50% of allmicroorganisms exist beneath the earths crust

in rocks etc. and they significantly influence

weather, mineral extraction and soil formation.


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BiochemicalRxns in theRumen


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Soil Profile


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Products from Microbes


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Human and Diseases

Despite all their beneficial effects, microorganismsalso contribute to our misery as pathogens.

Most microorganisms cause no damage and is

an important part of human ecology (normal flora). Nearly 2,000 different microorganisms can cause

disease e.g.???

WHO indicates that there are about 10 B newinfections each year.

Worldwide death toll from infection is about 13M/year.


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Figure ofinfections

es ir tor infections

2

1i rr e l ise ses

1

u erculosis

12

l ri

1

e sles

et nus

r sitic ise ses

iscell neous

2


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Microbes and Infection

The hardest hit countries are the ones withinadequate medical care.

One third of the world population live on lessthan $1 USD/day, are malnourished andnot fully immunized.

We are also seeing increases in the # ofnew diseases such as SARS, AIDS,hepatitis C and viral encephalitis.


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Noninfectious Diseases??

In recent years many diseases considered non-infectious probably do involve microorganismse.g. gastric ulcers caused by Heliobacter.

A connection has also been established betweencertain cancers and viruses, diabetics andCoxsackie virus, schizophrenia and borna agent(virus).

Microorganisms: we have to learn tolive with them because we cannot live

without them.


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Identification of Microorganisms

For many students and professionals the mostpressing topic in microbiology is how to identifyunknown specimens.

Why is this important?

Labs can grow, isolate and identify most routinelyencountered bacteria within 48 hrs of sampling.

The methods microbiologist use fall into threecategories:

Phenotypic- morphology (micro andmacroscopic) Immunological- serological analysis Genotypic- genetic techniques


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Microbe Identification

The successful identification of microbedepends on:

Using the properaseptic techniques.

Correctly obtaining the specimen.

Correctly handling the specimen

Quickly transporting the specimen to the

lab.Once the specimen reaches the lab it is

cultured and identified.


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Microbe Identification

Identification measures include: Microscopy (staining)

growth on enrichment, selective, differential or

characteristic media specimen biochemical test (rapid test methods)

immunological techniques

molecular (genotypic) methods.

After the microbe is identified for clinicalsamples it is used in susceptibility tests tofind which method of control is mosteffective.


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Phenotypic Methods

Old fashioned methods via biochemical,serological and morphological are still usedto identify many microorganisms.

Phenotypic Methods

Microscopic Morphology include acombination of cell shape, size, Gram stain,

acid fast rxn, special structures e.g.endospores, granule and capsule can beused to give an initial putativeidentification.


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Phenotypic Methods

Macroscopic morphology are traits that can beaccessed with the naked eye e.g. appearance ofcolony including texture, shape, pigment, speed of

growth and growth pattern in broth. Physiology/Biochemical characteristic are

traditional mainstay of bacterial identification.

These include enzymes (catalase, oxidase,

decarboxylase), fermentation of sugars, capacityto digest or metabolize complex polymers andsensitivity to drugs can be used in identification.


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Immunological Methods

Immunological methods involve theinteraction of a microbial antigen with anantibody (produced by the host immunesystem).

Testing for microbial antigen or theproduction of antibodies is often easierthan

test for the microbe itself. Lab kits based on this technique is available

for the identification of manymicroorganisms.


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Genotypic Methods

Genotypic methods involve examining thegenetic material of the organisms and hasrevolutionized bacterial identification and

classification. Genotypic methods include PCR (RT-PCR,

RAPD-PCR),use of nucleic acid probes,RFLP and plasmid fingerprinting.

Increasingly genotypic techniques arebecoming the sole means of identifyingmany microorganisms because of its speed

and accuracy.


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Microbe

Identification


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Specimen Collection

Successful identification depends on how thespecimen is collected, handled and stored.

It is important that general aseptic procedures beused including sterile sample containers andsampling methods to prevent contamination ofthe specimen.

E.g. Throat and nasopharyngeal swabs should nottouch the cheek, tongue or salvia.

What other precautions must be taken when collecting specimens?

After collection the specimen must be takenpromptly to the lab and stored appropriately(e.g. refrigeration).


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SpecimenCollection


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Lab safety


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Phenotypic Methods of Identification

Microbiologists use 5 basic techniques to grow,examine and characterize microorganisms inthe lab.

They are called the 5 Is: inoculation,incubation, isolation, inspection andidentification.

Inoculation: to culture microorganisms a tiny

sample (inoculum) is introduced into medium(inoculation).

Isolation involves the separating one speciesfrom another.


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Phenotypic methods of Identification

Incubation: once the media is inoculated itis incubated which means putting the culture

in a controlled environment (incubation) toallow for multiplication.

After incubation the organisms are

inspected and identified phenotypically,immunologically or genetically.


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isolation

inoculation

incubation

Specimen collection

inspection

identification

5 I s


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Media

A microbiologist can fine tune a media for almostany purpose.

General purpose media are designed to grow aboard spectrum of microorganisms e.g. nutrient

agar/broth, brain and heart infusion, trypticase soyagar (TSA)-casein, soy digest and NaCl.

Enriched media has complex organic substratessuch as blood serum and special growth factors(vitamins, amino acids).

A bacteria that requires requires complex growthfactors is termed fastidious.

Enrichment media facilitates the extensivegrowth of particular organism.


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Enrichment Media

The addition ofblood, serum extract or trypticsoy agarwill support the growth of manyfastidious microorganisms.

Blood agarhas the addition of citrated blood to

tryptic soy agar to make possible variablehemolysis which permit the differentiation of somebacteria.

hemolysis: greenish brown halo around the colony ( e.g.Streptococcus gordoniiorS. pneumoniae)

hemolysis: complete lysis of blood cells resulting in aclearing effect around the colony (e.g. Staphylococcusaureus, Streptococcus pyogenes)

Non hemolytic: no change in media (Staphylococcus

epidermidis, Staphylococcus saprophyticus


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Enriched Media

Blood agar plate with

bacteria from humanthroat. This mediadifferentiates amongdifferent colonies by

appearance

Chocolate agar, amedium that gets brownfrom heated blood. Used

for isolation ofN.gonorrhoeae.


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Selective/Differential Media

A selective media contains agents that inhibit thegrowth of certain microorganisms and select forthe growth of others.

A selective media is important as a primaryisolation of specific organisms.

E.g mannitol salt agar(MSA) has a high NaClconcentration (7.5%) which will inhibit the growth of

most microbes but will select for the growth ofStaphylococcus.

MacConkey agarwhich contains bile salts as aselective agent.


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Bile salts is a component of feces and inhibit thegrowth ofGram positive bacteria and encouragethe growth ofGram negative rods.

Other selective agents: Methylene blue and crystal violet inhibit Gram

positives

Selenite and brilliant green are used in media toisolate Salmonella from feces.

Sodium azide is used to isolate enterococci fromwater and food.


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General vs Selective Media


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Differential Media

Differential mediagrow several types oforganisms and

display visibledifferences amongorganisms.

Differences may show

up as colony size,media colour, gasbubble formation andprecipitate formation.


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Mannitol salt agar isused to isolatemembers of the genus

Staphylococcus

MacConkey agar differentiatesbetween lactose-fermenting bacteriaand (pink-red centre) and lactose-negative bacteria ( no pinkcolouration).


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DifferentialMedia

Triple sugar iron agar (TSIA).

This media contain

fermentable carbohydrates Red phenol to indicate pH

change

Iron that indicate H2S gas

production. Rxns (left to right) are:

No growth

Growth with no acid

Acid production in thebottom only

Acid production in thebottom and H2S gas

formation (black)


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DifferentialMedia

Chromagar orientationuses colour-formation todistinguish at least 7common urinarypathogens.

This allow for rapididentification andtreatment.

In this example, thebacteria were streakedas to spell their names.


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Characteristic Media

Characteristic media are used to testbacteria for particular activity, product orrequirement.

E.g. urea broth used to detect for urease.


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Direct Microscopic Examination

Direct microscopic examination of a stainedspecimen is often the most rapid method for theidentification of characteristics.

Stains include Gram, acid fast, directfluorescent antibody test (DFA).

DFA can be used to highlight the presences ofmicroorganisms in a specimen.

DFA test are available forStaphylococcusaureus, Streptococcus pyogenes, Neisseria

gonorhoeaeand Haemophilus influenza.


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Direct Examination

E. coli(white), Micrococcus luteus(yellow), Serratia marcescens (red)

Micrococcus luteus

Serratia marcescens


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Direct Microscopic Examination


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DirectFluorescentAntibody Test


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Biochemical Tests

The microbe is cultured in a media with a specialsubstrate and tested for an end product.

Prominent biochemical tests include

carbohydrate fermentation, acid or gasproduction and the hydrolysis of gelatin orstarch.

Many of these test in rapid miniaturized system

that can detect for 23 characteristics in small cupscalled Rapid test.

The info from the rapid test are input into acomputer to help in identification of the organisms.


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CarbohydrateFermentation

. This medium showfermentation (acid

production) and gasformation.

The small Durham tubefor collecting gasbubbles.

Left- right:

Uninoculated negativecontrol

Centre, positive foracid (yellow) and gas(open space).

Growth but no gas oracid.


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Methyl Red Test

This is a qualitativetest foracidproduction.

The bacteria is grownin MR-VP broth.

After addition ofseveral drops ofmethyl red solutiona bright red colour ispositive and yellow-orange negative.


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Nitrate Reduction After 24-48 hrs of

incubation, nitratereagents are added.

Left to right:

Gas formation(positive for nitratereduction).

positive for nitrate

reduction to nitrite (red colour).

Negative control


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Starch Hydrolysis

After incubation onstarch agar, platesare flooded withiodine solution.

Positive test indicatedby colourless area

around growth. Negative test

indicated below.


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Catalase Test

Place a drop ofH2O2on the culture.

A positive reactionshow gas bubbles.

Often used todifferentiate

Streptococcusfrom

Staphylococcus.


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Biochemical Tests

Other biochemical tests of interest include:

H2S production

Indole testOxidase test

Oxidation fermentation

Phenylalanine deaminase testAntibiotic susceptibility tests

Principle, procedure, most common use.


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Rapid Tests

Rapid test: a biochemical system for theidentification ofEnterobacteriacae and otherGram ve bacteria.

It consist ofplastic strips with 20 l ofdehydrated biochemical substrates used todetect biochemical characteristics.

The biochemical substrates are inoculated withpure cultures and suspended in physiologicalsaline.

After 5 hrs-overnight the 20 tests are converted to7-9 digital profile.


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Rapid Tests

ONPG ( galactosidase); ADH (arginine dihydrolase); LDC (lysine decarboxylase);ODC (ornithine decarboxylase); CIT (citrate utilization); H2S (hydrogen disulphideproduction); URE (urease); TDA ( tryptophan deaminase); IND (indole production);VP (Voges Proskauer test for acetoin); GEL ( gelatin liquefaction); the fermentationof glucose (GLU), mannitol (MAN), inositol (INO), sorbitol (SOR), rhamnose (RHA),sucrose (SAC); Melibiose (MEL), amygdalin (AMY), and arabinose (ARA); and OXI

(oxidase).

positive

negative


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Rapid Test

ResultsOXI--0

-ARA-2 2+AMY-0-

MEL--4+

SAC-2 7+RHA-1+

SOR--4+INO-0 5-MAN-1+

GLU--4+

GEL-0 4-VP--0

IND--4+TDA-0 4-

URE-0-

H2S--0-CIT-0 1-ODC-1+

LDC--4+ADH-0 5-ONPG-1+

normal 7 digit code5 144 572 = E. coli


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Bacteriophage Typing

What are bacteriophages?

Bacteriophage typing is based on the specificityof phage surface receptorfor the cell surfacereceptor.

Only those phages that can attach to thesurface receptors can cause lysis. The procedure involves: A plate is heavily inoculated so that there is no

uninoculated areas. The plate is marked offin squares (15-20 mm)and each square inoculated with a drop ofsuspension fordifferent phages.


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Heavily Inoculated Plate


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Bacteriophage Typing

The plate is incubated for 24 hrs thenobserved for plaques.

The phage type is reported as a specific

genus and species followed by the typesthat can infect the bacterium.

E.g. 10/16/24 means that the bacteria issensitive to phages 10, 16 and 24.

Phage tying remain a tool for research andreference labs.


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Unculturable Organisms

Environmental researchers estimatethat < 1% of microorganisms areculturable and therefore it is notpossible to use phenotypic methods ofidentification.

These microorganisms are calledviable nonculturable (VNC).


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Flow Cytometry

Classical techniques are not successful inidentification of those microorganisms that cannotbe cultured.

What do is the name of microorganisms that cannot be cultured?

Flow cytometry allows single or multiplemicroorganisms detection an easy, reliable and fastway.

Inflow cytometry microorganisms are identified on

the basis of the cytometry parameters or bymeans of certain dyes called fluorochromes thatcan be used independently or bound to specificantibodies.


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Flow Cytometry

The cytometer forces a suspension ofcells through a laser beam and

measures the light they scatter or thefluorescence the cell emits as theypass through the beam.

The cytometer also can measure thecells shape, size and the content ofthe DNA or RNA.


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Immunological Methods

The study of antibody (Ab)- antigen (Ag) rxns invitro is called serology.

Serological rxns are the basic ofimmunological

identification and diagnostic methods. The usefulness of serological test is dependent on

its sensitivity and specificity.

Sensitivity is the ability to detect minute amountsof Ab or Ag.

Specificity is the ability to detect a single Ag orAb.


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False Negatives/Positives

High sensitivity prevents falsenegatives.

F

alse negatives occurs when there isnot reaction when the Ag or Ab ispresent.

High specificity prevents falsepositives.

False positives occurs when there iscross reaction with another molecule.


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Precipitation Reactions

Precipitation (ppt) is the interaction of a soluble Agwith an soluble Ab to for an insoluble complex.

The complex formed is an aggregate of Ag and Ab.

Ppt rxns occurs maximally only when the optimalproportions of Ag and Ag are present.

Ppt can also be done in agar referred to as

immunodiffusion. Ppt test uses antibodies to detect for streptococcal

group antigens.


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Precipitation Reactions


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Agglutination Reactions

Agglutination (Aggn) is the visibleclumping of an Ag when mixed with aspecific Ab.

Aggn tests are widely used because theyare simple to perform, highly specific,inexpensive and rapid.

Standardized tests are available for thedetermination ofblood groups andidentification of pathogens and their

products.


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Agglutination Reactions


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Direct/Indirect Aggn

Direct aggn occurs when a soluble Ab results inclumping by interaction with an Ag which is partof a surface of a cell.

E.g. Blood typing and detection ofmycoplasmapneumonia.

Indirect (passive) aggn. Ab/Ag is adsorbed orchemically coupled to the cell, latex beads or

charcoal particles which serves as an inert carrier.

The latex beads can be used to detect for surfaceAg.


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Use of Latex Agglutination Tests

Commercial suspension of latex beads areavailable for the detection of Staphylococcusaureus, Streptococcus pyogenes,

Haemophilus influenza and Camplyobacterspp.

A similar technology is used forurine pregnancytests.

What is this the basis of the urine pregnancy test?

Passive aggn tests are simple, specific andinexpensive which make them suitable forlargescale screening purposes.


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AgglutinationReaction


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Fluorescent Antibodies

Abs can be chemically modified withfluorescent dyes such as rhodamine B,fluorescent red, fluorescien isothiocynateand fluoresces yellow or green.

Cells with bound fluorescent Ab emit abright red, orange, yellow or green light

depending on the dye used. There are two distinct fluorescent Ab

procedure: direct and indirect.


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Fluorescent Antibodies

In the direct method the fluorescent Ab is directedto surface Ag of the organism.

In the indirect method a non-fluorescent Ab

reacts with the organism's Ag and a fluorescent Abreacts with the non-fluorescent Ag.

Fluorescent Ab can be used to detectmicroorganisms directly in tissue, long before aprimary isolation technique yield the suspectedpathogen.

Fluorescent Ab has been used for the detection ofBacillus anthracis and HIV virus.


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Direct Fluorescent Antibodies


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Indirect Fluorescent Antibodies


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ELISA

During the ELISA test enzyme labeled Ab areused to detect Ag.

This reduces the amount of Ab necessary and

increases the detection limit of the Ag/Ab rxn. Enzymes used in ELISA include alkaline

phosphate, peroxidase and galactosidase.

During indirect ELISA

the Ag is trapped betweentwo Ab molecules (sandwich ELISA). Suggest that happens during direct ELISA.

The specimen is added to a well with attached Ab.


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ELISA

If the Ag (microbe) is present it will attached to theAb.

After washing away unbound material, a second

Ab with a conjugated enzyme is added. The second Ab is specific for the Ag.

A substrate is added which reacts with the

enzyme to give a coloured rxn. ELISA tests are available for the detection ofmany organisms including Staphylococcusaureus, E. coliand Salmonella.


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ELISA Movie


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Immunoblot/Western Blot

Immunoblot detects for a specific proteinassociated with specific organism.

The procedure involves:

Separation of the proteins on polyacrylamide gel. Transfer (blotting) of proteins from the gel to a

membrane (nitrocellulose or nylon) andidentification of the protein with a specific Ab.

The method is sensitive for detecting proteins incomplex mixtures.

Immunoblot is laborious, time consuming and lesssensitive than ELISA.


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Immunoblot


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Immuno/Western Blot

Immunoblot is used as a confirmatory test forHIV. Although it is less sensitive than HIV ELISA???.

The ELISA test for HIV often yields false positiveand the immunoblot test is used to confirm a

positive ELISA results. To perform the HIV immunoblot purified HIV is

treated with SDS to solubilize the proteins andinactivate the virus.

The proteins (at least 7) are resolved bypolyacrylamide gel electrophoresis and theproteins are blotted unto a membrane andincubated with the test serum.


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HIV Immunoblot Test

The test is considered positive if bands occur at, 2locations e.g. gp160 and gp 120 or p24 and gp 41-45.

HIV Immunoblot


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HIV ImmunoblotTest

Test was done at 6different times (after thesuspected exposure).

Test is positive if bandsoccur at two locationse.g. gp160 or gp 120and g31 or g24.

Test is negative ifnobands are present forany HIV antigen.

SRC is positive control


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Genotypic methods

Genotypic methods of microbeidentification include the use of :

Nucleic acid probes

PCR (RT-PCR, RAPD-PCR)

Nucleic acid sequence analysis

rRNA analysis

RFLPPlasmid fingerprinting.


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Nucleic acid probes

Nucleic acid hybridization is one of themost powerful tools available for microbeidentification.

Hybridization detects for a specific DNAsequence associated with an organism.

The process uses a nucleic acid probe

which is specific for that particular organism. The target DNA (from the organism) is

attached to a solid matrix such as a nylon

or nitrocellulose membrane.


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Nucleic Acid Probes

A single stranded probe is added and if there issequence complementality between the targetand the probe a positive hybridization signal will be

detected. Hybridization is detected by a reporter molecule

(radioactive, fluorescent, chemiluminescent) whichis attached to the probe.

Nucleic acid probes have been marketed for theidentification of many pathogens such as N.gonorrhoeae.


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Two Component Probes

Molecular probes are also finding wide spread usein the food industry and food regulatory agencies.

The pathogen DNA is attached to a dipstick to

hybridize to the pathogen DNA from the food. A two component probe is used (reporter and acapture probe which are attached to each other).

Following hybridization the dipstick with the captureprobe (usually poly dT to capture poly dA on the

probe) is inserted into the hybridization solution. It is traps the hybridized DNA for removal and

measurement.


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Two Component Probe


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Advantages of Nucleic Acid Probes

Nucleic acid probes has many advantages overimmunological methods.

Nucleic acid are more stable at high temperature,

pH, and in the presence of organic solvents andother chemicals.

This means that the specimen can be treatedvery harshly to destroy interfering materials.

Nucleic acid probes can be used to identifymicroorganisms which are no longer alive.

Furthermore nucleic acid probes are morespecific than antibodies.


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Polymerase Chain Reaction (PCR)

PCR is widely used for the identification ofmicroorganisms.

Sequence specific primers are used with PCR inthe amplification of DNA or RNA of specific

pathogens. PCR allows for the detection even ifonly a few

cells are present and can also be used on viablenonculturables (see sensitivity table).

The presence of the appropriate amplified PCRproduct confirms the presence of the organisms.

Primers are available for the identification ofNiesseriagonorrhoeae, and to monitor food for the presence ofSalmonella and Staphylococcus.


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Sensitivity of Microbe Detection Tests

Methods Toxin or organism Sensitivity

Flow Cytometry S. Typhimurium in milk 103/ml in 40 min

Fluorescent

antibody

Salmonella 106/ml

Latex agglutination E. coli enterotoxin 32 ng/ml

ELISA C. perfringens type A

toxin

1 ng/ml

PCR E. coli 1-5 cells/100 mlof H2O


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Real Time PCR and RT-PCR

Currently many PCR tests employ real time PCR.

This involves the use offluorescent primers.

The PCR machine monitors the incorporation of theprimers and display an amplification plot which can be

viewed continuously thru the PCR cycle. Real time PCR yields immediate results.

Another application of PCR is RT-PCR (reverse trancriptasePCR).

During RT-PCR an RNA template is used to generatecDNA and from this dsDNA is generated.

The enzyme used is reverse transciptase.

RT-PCR is used to detect for HIV and to monitor theprogress of the disease.


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RT-PCR

C


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RAPD-PCR

Random amplified polymorphic DNA PCR uses arandom primer(10-mer) to generate a DNAprofile.

What are random primers?

The primeranneals to several places on the DNAtemplate and generate a DNA profile which is usedfor microbe identification.

RAPD has many advantages:Pure DNA is not needed

Less labour intensive than RFLP.There is not need for prior DNA sequence data.

RAPD has been used to fingerprint the outbreak ofListeria monocytogenes from milk.


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PCR vs

RAPD-PCR

RAPD P fil


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RAPD Profile

DNA i


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DNA sequencing

The determination of a small amount of DNAsequence can be used for microbial identification.

The most common sequence used for microbeidentification is DNA sequence of the 16S rRNAgene.

PCR is used to amplify the 16S rRNA gene and thesequence determined.

rRNA is a major component for ribosome andribosome have the same function in proteinsynthesis in all cells.

DNA S i


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DNA Sequencing

Computer analysis of 16S rRNA sequence hasrevealed the presence ofsignature sequences,short oligonucleotides unique to certain groups of

organisms and useful in their identification. rRNA sequence can be used to fine tune identity at

the species level e.g differentiating betweenMycobacterium and Legionella.

16s rRNA sequence can also be used to identifymicroorganisms from a microbial community.

Review evolutionary chronometers (Brock)

Restriction Fragment Length


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Restriction Fragment LengthPolymorphism

RFLP involves digestion of the genomicDNA of the organism with restrictionenzymes.

The restricted fragments are separated byagarose gel electrophoresis.

The DNA fragments are transferred to a

membrane and probed with probes specificfor the desired organisms. A DNA profile emerges which can be used

for microbe identification.


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RFLP ofM.tuberculosis

from 17patients

Pl id fi i ti


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Plasmid fingerprinting

What is a plasmid?

Plasmid fingerprinting identifies microbialspecies or similar strains as related strainsoften contain the same number of plasmids

with the same molecular weight. Plasmid of many strains and species ofE.

coli, Salmonella, CamylobacterandPsseudomonas has demonstrated that this

methods is more accurate than phenotypicmethods such as biotyping, antibioticresistance patterns , phage typing andserotyping.

Pl id fi i ti


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Plasmid fingerprinting

The procedure involves:

The bacterial strains aregrown, the cells lysed and

harvested. The plasmids are

separated by agarose gelelectrophoresis

The gels are stained withEtBrand the plasmidslocated and compared.

Computer and Bacteria


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Co pute a d acte aIdentification

Computers improve the efficiency ofthe lab operations and increase thespeed and clarity with which resultscan be reported.

Computers are also important for the

result entry, analysis and preparation.

Documents

Microbe Identification 3 Farook