microbe identification 3 farook
TRANSCRIPT
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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 and Earth
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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Microorganisms and Earth
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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Selective/Differential Media
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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Selective/Differential Media
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.
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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.