Molecular Microbiology:

Microbe Identification
BC34M
(Lecture notes 2006/7)
Prepared and presented by
Dr. Marcia E. Roye
Office: Biotechnology Centre, ground floor
Tel: 927-0304 (Office)
977-1828 (Centre)
ext. 2518-2520 (from campus)
Email: marcia.roye@uwimona.edu.jm
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 BC34M Course Website:
www.uwimona.edu.jm/biochem/courses/bc34m

Getting
notes
from
Web

2
 Lecture Outline
• Microbe Identification
• Definitions, importance of microbes, overview of microbe identification
methods, unculturables, specimen collection, 5 “I”s.
• Phenotypic methods: media (enriched, selective, differential,
characteristic), direct microscopic examination, biochemical tests, rapid
tests, 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)
• *Brock’s Biology of Microorganisms. Madigan, M., Martinko, J. & Parker, J.
Pearson Education 9,10 or 11th Ed (or other advanced microbiology text)
• *Jawetz, Melnick and Adelberg’s 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 of
lectures is to examine the
phenotypic, immunological and
genotypic methods commonly
used in microbe identification.
• Why is microbe identification
important?
4
 Objectives of this Lecture
• Definitions
• The importance of
microorganisms
• An overview of microbe
identification methods

5
 What is Micro- and Molecular-
biology?
• Microbiology is a specialized area of
biology that deals with living things
ordinarily 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 the
macromolecules of life (particularly DNA, RNA
and proteins) and their interactions at the
molecular (molecule) level.
6
 What is Molecular
Microbiology?
• What is molecular microbiology?

• Deals with the interaction of microorganisms

with 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 7
The Importance of Microorganisms

8
 The
Importance
of
Microbes

9
 Microorganisms and Earth
• Microorganisms have profound effects
on the earth and its residents.
• Microorganisms are critical for the flow
of energy and food thru the
ecosystem.
• Microorganisms (including algae)
account for more than 50% of the
earth’s photosynthesis that contribute
the majority of O2 to the atmosphere.
10
 Microorganisms and Earth
• Microorganisms is crucial for the
decomposition and nutrient recycle which
help to keep the earth in balance.
• What molecules are cycled by microorganisms?

• In the long term microorganisms are the main

force that drive the structure and content
of the soil, water and atmosphere e.g.:
• Greenhouse gases CO2 and CH4 create an
insulation layer in the atmosphere that help
retain heat.
11
Carbon Cycle

12
Nitrogen
Cycle

13
Sulphur Cycle

14
 Microorganisms and Earth
• Bacteria and fungi live in complex associations
with plants and assist the plant in obtaining
nutrients, water and protect against diseases.
• E.g.???

• Microorganisms form similar associations in the

stomach of ruminants to help in the digestion of
complex carbohydrates.
• E.g. of ruminants?

• Recent estimates propose that up to 50% of all

microorganisms exist beneath the earth’s crust
in rocks etc. and they significantly influence
weather, mineral extraction and soil formation.15
Biochemical
Rxns in the
Rumen

16
Soil Profile

17
 Human and Useful Microorganisms
• What are some of the microorganisms and their products that are useful to us?

• Yeast- bread, alcoholic beverages

• Cheese making
• Antibiotics
• Genetic engineering
• Bacteria produce natural pesticides
• Yeast and E. coli - insulin
• Bioremediation involves the use of
microorganisms to restore the environment of
cleanup pollution e.g. oil spills, pesticides,
degrading garbage esp. plastic and paper. 18
Products from Microbes

19
 Human and Diseases
• Despite all their beneficial effects, microorganisms
also 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 new
infections each year.
• Worldwide death toll from infection is about 13
M/year.
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 Figure of
infections

21
 Microbes and Infection

• The hardest hit countries are the ones with

inadequate medical care.
• One third of the world population live on less
than $1 USD/day, are malnourished and
not fully immunized.
• We are also seeing increases in the # of
new 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 microorganisms
e.g. gastric ulcers caused by Heliobacter.
• A connection has also been established between
certain cancers and viruses, diabetics and
Coxsackie virus, schizophrenia and borna agent
(virus).
• ‘Microorganisms: we have to learn to
live with them because we cannot live
without them’.
23
 Identification of Microorganisms
• For many students and professionals the most
pressing topic in microbiology is how to identify
unknown specimens.
• Why is this important?
• Labs can grow, isolate and identify most routinely
encountered bacteria within 48 hrs of sampling.
• The methods microbiologist use fall into three
categories:
♣ Phenotypic- morphology (micro and
macroscopic)
♣ Immunological- serological analysis
♣ Genotypic- genetic techniques
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 Identification of Microorganisms
• For many students and professionals the most
pressing topic in microbiology is how to identify
unknown specimens.
• Why is this important?
• Labs can grow, isolate and identify most routinely
encountered bacteria within 48 hrs of sampling.
• The methods microbiologist use fall into three
categories:
♣ Phenotypic- morphology (micro and
macroscopic)
♣ Immunological- serological analysis
♣ Genotypic- genetic techniques
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 Microbe Identification
• The successful identification of microbe
depends on:
♥ Using the proper aseptic 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.
26
 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 clinical
samples it is used in susceptibility tests to
find which method of control is most
effective.
27
 Phenotypic Methods
• ‘Old fashioned’ methods via biochemical,
serological and morphological are still used
to identify many microorganisms.
• Phenotypic Methods
• Microscopic Morphology include a
combination of cell shape, size, Gram stain,
acid fast rxn, special structures e.g.
endospores, granule and capsule can be
used to give an initial putative
identification.
28
 Phenotypic Methods
• Macroscopic morphology are traits that can be
accessed with the naked eye e.g. appearance of
colony 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, capacity
to digest or metabolize complex polymers and
sensitivity to drugs can be used in identification.
29
 Immunological Methods
• Immunological methods involve the
interaction of a microbial antigen with an
antibody (produced by the host immune
system).
• Testing for microbial antigen or the
production of antibodies is often easier than
test for the microbe itself.
• Lab kits based on this technique is available
for the identification of many
microorganisms. 30
 Genotypic Methods
• Genotypic methods involve examining the
genetic material of the organisms and has
revolutionized bacterial identification and
classification.
• Genotypic methods include PCR (RT-PCR,
RAPD-PCR),use of nucleic acid probes, RFLP
and plasmid fingerprinting.
• Increasingly genotypic techniques are
becoming the sole means of identifying
many microorganisms because of its speed
and accuracy. 31
 Microbe
Identification

32
 Specimen Collection
• Successful identification depends on how the
specimen is collected, handled and stored.
• It is important that general aseptic procedures be
used including sterile sample containers and
sampling methods to prevent contamination of
the specimen.
• E.g. Throat and nasopharyngeal swabs should not
touch the cheek, tongue or salvia.
• What other precautions must be taken when collecting specimens?
• After collection the specimen must be taken
promptly to the lab and stored appropriately
(e.g. refrigeration).
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Specimen
Collection

34
Lab safety

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Phenotypic Methods of Identification
• Microbiologists use 5 basic techniques to grow,
examine and characterize microorganisms in
the lab.
• They are called the 5 ‘I’s: inoculation,
incubation, isolation, inspection and
identification.
• Inoculation: to culture microorganisms a tiny
sample (inoculum) is introduced into medium
(inoculation).
• Isolation involves the separating one species
from another.
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Phenotypic methods of Identification

• Incubation: once the media is inoculated it

is incubated which means putting the culture
in a controlled environment (incubation) to
allow for multiplication.
• After incubation the organisms are
inspected and identified phenotypically,
immunologically or genetically.

37
Specimen collection
isolation

5 “I” s
inoculation
inspection

identification
incubation
38
 Media
• A microbiologist can fine tune a media for almost
any purpose.
• General purpose media are designed to grow a
board spectrum of microorganisms e.g. nutrient
agar/broth, brain and heart infusion, trypticase soy
agar (TSA)-casein, soy digest and NaCl.
• Enriched media has complex organic substrates
such as blood serum and special growth factors
(vitamins, amino acids).
• A bacteria that requires requires complex growth
factors is termed fastidious.
• Enrichment media facilitates the extensive growth
of particular organism. 39
 Enrichment Media
• The addition of blood, serum extract or tryptic
soy agar will support the growth of many fastidious
microorganisms.
• Blood agar has the addition of citrated blood to
tryptic soy agar to make possible variable hemolysis
which permit the differentiation of some bacteria.
• α hemolysis: greenish brown halo around the colony ( e.g.
Streptococcus gordonii or S. pneumoniae)
• β hemolysis: complete lysis of blood cells resulting in a
clearing effect around the colony (e.g. Staphylococcus
aureus, Streptococcus pyogenes)
• Non hemolytic: no change in media (Staphylococcus
epidermidis, Staphylococcus saprophyticus
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 Enriched Media
Chocolate agar, a
medium that gets brown
from heated blood. Used
for isolation of N.
gonorrhoeae.

Blood agar plate with

bacteria from human
throat. This media
differentiates among
different colonies by
appearance 41
 Selective/Differential Media
• A selective media contains agents that inhibit the
growth of certain microorganisms and select for
the growth of others.
• A selective media is important as a primary
isolation of specific organisms.
• E.g mannitol salt agar (MSA) has a high NaCl
concentration (7.5%) which will inhibit the growth of
most microbes but will select for the growth of
Staphylococcus.
• MacConkey agar which contains bile salts as a
selective agent.
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 Selective/Differential Media
• Bile salts is a component of feces and inhibit the
growth of Gram positive bacteria and encourage
the growth of Gram negative rods.
• Other selective agents:
• Methylene blue and crystal violet inhibit Gram
positives
• Selenite and brilliant green are used in media to
isolate Salmonella from feces.
• Sodium azide is used to isolate enterococci from
water and food.
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General vs Selective Media

44
Differential Media
• Differential media
grow several types of
organisms and
display visible
differences among
organisms.
• Differences may show
up as colony size,
media colour, gas
bubble formation and
precipitate formation.45
Selective/Differential Media

MacConkey agar differentiates

Mannitol salt agar is between lactose-fermenting bacteria
used to isolate and (pink-red centre) and lactose-
negative bacteria ( no pink
members of the genus
colouration). 46
Staphylococcus
♦ Triple sugar iron agar (TSIA).
Differential
♦ This media contain
fermentable carbohydrates Media
♦ 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 the
bottom only
♦ Acid production in the
bottom and H2S gas
formation (black) 47
Chromagar orientation Differential
uses colour-formation to Media
distinguish at least 7
common urinary
pathogens.
This allow for rapid
identification and
treatment.
 In this example, the
bacteria were streaked
as to spell their names. 48
 Characteristic Media
• Characteristic media are used to test
bacteria for particular activity, product or
requirement.
• E.g. urea broth used to detect for urease.

49
Direct Microscopic Examination
• Direct microscopic examination of a stained
specimen is often the most rapid method for the
identification of characteristics.
• Stains include Gram, acid fast, direct
fluorescent antibody test (DFA).
• DFA can be used to highlight the presences of
microorganisms in a specimen.
• DFA test are available for Staphylococcus
aureus, Streptococcus pyogenes, Neisseria
gonorhoeae and Haemophilus influenza.
50
 Direct Examination

Micrococcus luteus

E. coli (white), Micrococcus luteus

(yellow), Serratia marcescens (red)
Serratia marcescens 51
Direct Microscopic Examination

52
 Direct
Fluorescent
Antibody Test

53
 Biochemical Tests
• The microbe is cultured in a media with a special
substrate and tested for an end product.
• Prominent biochemical tests include
carbohydrate fermentation, acid or gas
production and the hydrolysis of gelatin or
starch.
• Many of these test in rapid miniaturized system
that can detect for 23 characteristics in small cups
called Rapid test.
• The info from the rapid test are input into a
computer to help in identification of the organisms.
54
Carbohydrate  . This medium show
fermentation (acid
Fermentation production) and gas
formation.
 The small Durham tube
for collecting gas bubbles.
 Left- right:
 Uninoculated negative
control
 Centre, positive for
acid (yellow) and gas
(open space).
 Growth but no gas or
acid.
55
 Methyl Red Test
• This is a qualitative
test for acid
production.
• The bacteria is grown
in MR-VP broth.
• After addition of
several drops of
methyl red solution
a bright red colour is
positive and yellow-
orange negative.
56
Nitrate Reduction ♫ After 24-48 hrs of
incubation, nitrate
reagents are added.
♫ Left to right:
♫ Gas formation
(positive for nitrate
reduction).
♫ positive for nitrate
reduction to nitrite
( red colour).
♫ Negative control
57
 Starch Hydrolysis
• After incubation on
starch agar, plates
are flooded with
iodine solution.
• Positive test indicated
by colourless area
around growth.
• Negative test
indicated below.
58
 Catalase Test
• Place a drop of H2O2
on the culture.
• A positive reaction
show gas bubbles.
• Often used to
differentiate
Streptococcus
from
Staphylococcus.
59
 Biochemical Tests
• Other biochemical tests of interest include:
H2S production
Indole test
Oxidase test
Oxidation fermentation
Phenylalanine deaminase test
Antibiotic susceptibility tests
• Principle, procedure, most common use.
60
 Rapid Tests
• Rapid test: a biochemical system for the
identification of Enterobacteriacae and other
Gram –ve bacteria.
• It consist of plastic strips with 20 μl of
dehydrated biochemical substrates used to
detect biochemical characteristics.
• The biochemical substrates are inoculated with
pure cultures and suspended in physiological
saline.
• After 5 hrs-overnight the 20 tests are converted to
7-9 digital profile.
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 Rapid Tests

positive

negative

ONPG (β galactosidase); ADH (arginine dihydrolase); LDC (lysine decarboxylase);

ODC (ornithine decarboxylase); CIT (citrate utilization); H2S (hydrogen disulphide
production); URE (urease); TDA ( tryptophan deaminase); IND (indole production);
VP (Voges Proskauer test for acetoin); GEL ( gelatin liquefaction); the fermentation
of glucose (GLU), mannitol (MAN), inositol (INO), sorbitol (SOR), rhamnose (RHA),
sucrose (SAC); Melibiose (MEL), amygdalin (AMY), and arabinose (ARA); and OXI
62
(oxidase).
Rapid Test GLU--4

Results
OXI--0 +
- GEL-0 4
ARA-2 2 -
+ VP--0
AMY-0
-
IND--4
+
MEL--4 TDA-0 4
+ -
SAC-2 7 URE-0
+ -
RHA-1
+
H2S--0
-
SOR--4 CIT-0 1
+ -
INO-0 5 ODC-1
- +
MAN-1
+
LDC--4
+
normal 7 digit code ADH-0 5
-
5 144 572 = E. coli ONPG-1
+
63
 Bacteriophage Typing
• What are bacteriophages?
• Bacteriophage typing is based on the specificity
of phage surface receptor for the cell surface
receptor.
• Only those phages that can attach to the
surface receptors can cause lysis.
• The procedure involves:
• A plate is heavily inoculated so that there is no
uninoculated areas.
• The plate is marked off in squares (15-20 mm)
and each square inoculated with a drop of
suspension for different phages.
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Heavily Inoculated Plate

65
 Bacteriophage Typing
• The plate is incubated for 24 hrs then
observed for plaques.
• The phage type is reported as a specific
genus and species followed by the types
that can infect the bacterium.
• E.g. 10/16/24 means that the bacteria is
sensitive to phages 10, 16 and 24.
• Phage tying remain a tool for research and
reference labs.
66
 Unculturable Organisms

• Environmental researchers estimate

that < 1% of microorganisms are
culturable and therefore it is not
possible to use phenotypic methods of
identification.
• These microorganisms are called
viable nonculturable (VNC).
67
 Flow Cytometry
• Classical techniques are not successful in
identification of those microorganisms that cannot
be cultured.
• What do is the name of microorganisms that cannot be cultured?
• Flow cytometry allows single or multiple
microorganisms detection an easy, reliable and fast
way.
• Inflow cytometry microorganisms are identified on
the basis of the cytometry parameters or by
means of certain dyes called fluorochromes that
can be used independently or bound to specific
antibodies.
68
 Flow Cytometry
• The cytometer forces a suspension of
cells through a laser beam and
measures the light they scatter or the
fluorescence the cell emits as they
pass through the beam.
• The cytometer also can measure the
cell’s shape, size and the content of
the DNA or RNA.
69
 Immunological Methods
• The study of antibody (Ab)- antigen (Ag) rxns in
vitro is called serology.
• Serological rxns are the basic of immunological
identification and diagnostic methods.
• The usefulness of serological test is dependent on
its sensitivity and specificity.
• Sensitivity is the ability to detect minute amounts
of Ab or Ag.
• Specificity is the ability to detect a single Ag or
Ab.
70
 False Negatives/Positives
• High sensitivity prevents false
negatives.
• False negatives occurs when there is
not reaction when the Ag or Ab is
present.
• High specificity prevents false positives.
• False positives occurs when there is
cross reaction with another molecule.
71
 Precipitation Reactions
• Precipitation (ppt) is the interaction of a soluble Ag
with 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 optimal
proportions 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.
72
Precipitation Reactions

73
 Agglutination Reactions
• Agglutination (Aggn) is the visible
clumping of an Ag when mixed with a
specific Ab.
• Aggn tests are widely used because they are
simple to perform, highly specific,
inexpensive and rapid.
• Standardized tests are available for the
determination of blood groups and
identification of pathogens and their
products. 74
Agglutination Reactions

75
 Direct/Indirect Aggn
• Direct aggn occurs when a soluble Ab results in
clumping by interaction with an Ag which is part
of a surface of a cell.
• E.g. Blood typing and detection of mycoplasma
pneumonia.
• Indirect (passive) aggn. Ab/Ag is adsorbed or
chemically coupled to the cell, latex beads or
charcoal particles which serves as an inert carrier.
• The latex beads can be used to detect for surface
Ag.
76
 Use of Latex Agglutination Tests
• Commercial suspension of latex beads are
available for the detection of Staphylococcus
aureus, Streptococcus pyogenes,
Haemophilus influenza and Camplyobacter
spp.
• A similar technology is used for urine pregnancy
tests.
• What is this the basis of the urine pregnancy test?

• Passive aggn tests are simple, specific and

inexpensive which make them suitable for large
scale screening purposes. 77
Agglutination
Reaction

78
 Fluorescent Antibodies
• Abs can be chemically modified with
fluorescent dyes such as rhodamine B,
fluorescent red, fluorescien isothiocynate
and fluoresces yellow or green.
• Cells with bound fluorescent Ab emit a
bright red, orange, yellow or green light
depending on the dye used.
• There are two distinct fluorescent Ab
procedure: direct and indirect.
79
 Fluorescent Antibodies
• In the direct method the fluorescent Ab is directed
to surface Ag of the organism.
• In the indirect method a non-fluorescent Ab
reacts with the organism's Ag and a fluorescent Ab
reacts with the non-fluorescent Ag.
• Fluorescent Ab can be used to detect
microorganisms directly in tissue, long before a
primary isolation technique yield the suspected
pathogen.
• Fluorescent Ab has been used for the detection of
Bacillus anthracis and HIV virus.
80
Direct Fluorescent Antibodies

81
Indirect Fluorescent Antibodies

82
 ELISA
• During the ELISA test enzyme labeled Ab are
used 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 ß galactosidase.
• During indirect ELISA the Ag is trapped between
two Ab molecules (sandwich ELISA).
• Suggest that happens during direct ELISA.

• The specimen is added to a well with attached Ab.

83
 ELISA
• If the Ag (microbe) is present it will attached to the
Ab.
• 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 of
many organisms including Staphylococcus
aureus, E. coli and Salmonella.
84
ELISA Movie

85
 Immunoblot/Western Blot
• Immunoblot detects for a specific protein
associated 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) and
identification of the protein with a specific Ab.
• The method is sensitive for detecting proteins in
complex mixtures.
• Immunoblot is laborious, time consuming and less
sensitive than ELISA. 86
Immunoblot

87
 Immuno/Western Blot
• Immunoblot is used as a confirmatory test for HIV.
• Although it is less sensitive than HIV ELISA???.
• The ELISA test for HIV often yields false positive
and 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 and
inactivate the virus.
• The proteins (at least 7) are resolved by
polyacrylamide gel electrophoresis and the
proteins are blotted unto a membrane and
incubated with the test serum.
88
 HIV Immunoblot Test
• The test is considered positive if bands occur at, 2
locations e.g. gp160 and gp 120 or p24 and gp 41-45.

89
 HIV Immunoblot
Test
• Test was done at 6
different times (after the
suspected exposure).
• Test is positive if bands
occur at two locations
e.g. gp160 or gp 120
and g31 or g24.
• Test is negative if no
bands are present for
any HIV antigen.
• SRC is positive control
90
 Genotypic methods
• Genotypic methods of microbe
identification include the use of :
Nucleic acid probes
PCR (RT-PCR, RAPD-PCR)
Nucleic acid sequence analysis
rRNA analysis
RFLP
Plasmid fingerprinting.

91
 Nucleic acid probes
• Nucleic acid hybridization is one of the most
powerful tools available for microbe
identification.
• Hybridization detects for a specific DNA
sequence 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. 92
 Nucleic Acid Probes
• A single stranded probe is added and if there is
sequence complementality between the target
and the probe a positive hybridization signal will be
detected.
• Hybridization is detected by a reporter molecule
(radioactive, fluorescent, chemiluminescent) which
is attached to the probe.
• Nucleic acid probes have been marketed for the
identification of many pathogens such as N.
gonorrhoeae.
93
 Two Component Probes
• Molecular probes are also finding wide spread use
in 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 a
capture probe which are attached to each other).
• Following hybridization the dipstick with the capture
probe (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.
94
Two Component Probe

95
 Advantages of Nucleic Acid Probes

• Nucleic acid probes has many advantages over

immunological methods.
• Nucleic acid are more stable at high temperature,
pH, and in the presence of organic solvents and
other chemicals.
• This means that the specimen can be treated
very harshly to destroy interfering materials.
• Nucleic acid probes can be used to identify
microorganisms which are no longer alive.
• Furthermore nucleic acid probes are more
specific than antibodies.
96
 Polymerase Chain Reaction (PCR)
• PCR is widely used for the identification of
microorganisms.
• Sequence specific primers are used with PCR in
the amplification of DNA or RNA of specific
pathogens.
• PCR allows for the detection even if only a few
cells are present and can also be used on viable
nonculturables (see sensitivity table).
• The presence of the appropriate amplified PCR
product confirms the presence of the organisms.
• Primers are available for the identification of Niesseria
gonorrhoeae, and to monitor food for the presence of
Salmonella and Staphylococcus. 97
 Sensitivity of Microbe Detection Tests

Methods Toxin or organism Sensitivity

Flow Cytometry S. Typhimurium in milk 103/ml in 40 min
Fluorescent Salmonella 106/ml
antibody
Latex agglutination E. coli enterotoxin 32 ng/ml

ELISA C. perfringens type A 1 ng/ml

toxin
PCR E. coli 1-5 cells/100 ml
of H2O
98
 Real Time PCR and RT-PCR
• Currently many PCR tests employ real time PCR.
• This involves the use of fluorescent primers.
• The PCR machine monitors the incorporation of the
primers 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 trancriptase
PCR).
• During RT-PCR an RNA template is used to generate
cDNA and from this dsDNA is generated.
• The enzyme used is reverse transciptase.
• RT-PCR is used to detect for HIV and to monitor the
progress of the disease.
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RT-PCR

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 RAPD-PCR
• Random amplified polymorphic DNA PCR uses a
random primer (10-mer) to generate a DNA
profile.
• What are random primers?
• The primer anneals to several places on the DNA
template and generate a DNA profile which is used
for 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 of
Listeria monocytogenes from milk.
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 PCR vs
RAPD-PCR

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RAPD Profile

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 DNA sequencing
• The determination of a small amount of DNA
sequence can be used for microbial identification.
• The most common sequence used for microbe
identification is DNA sequence of the 16S rRNA
gene.
• PCR is used to amplify the 16S rRNA gene and the
sequence determined.
• rRNA is a major component for ribosome and
ribosome have the same function in protein
synthesis in all cells.
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 DNA Sequencing
• Computer analysis of 16S rRNA sequence has
revealed the presence of signature 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 between
Mycobacterium and Legionella.
• 16s rRNA sequence can also be used to identify
microorganisms from a microbial community.
• Review evolutionary chronometers (Brock)
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 Restriction Fragment Length
Polymorphism
• RFLP involves digestion of the genomic
DNA of the organism with restriction
enzymes.
• The restricted fragments are separated by
agarose gel electrophoresis.
• The DNA fragments are transferred to a
membrane and probed with probes specific
for the desired organisms.
• A DNA profile emerges which can be used
for microbe identification.
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RFLP of M.
tuberculosis
from 17
patients

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 Plasmid fingerprinting
• What is a plasmid?
• Plasmid fingerprinting identifies microbial
species or similar strains as related strains
often contain the same number of plasmids
with the same molecular weight.
• Plasmid of many strains and species of E.
coli, Salmonella, Camylobacter and
Psseudomonas has demonstrated that this
methods is more accurate than phenotypic
methods such as biotyping, antibiotic
resistance patterns , phage typing and
serotyping. 108
 Plasmid fingerprinting
• The procedure involves:
• The bacterial strains are
grown, the cells lysed and
harvested.
• The plasmids are
separated by agarose gel
electrophoresis
• The gels are stained with
EtBr and the plasmids
located and compared.
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 Computer and Bacteria
Identification
• Computers improve the efficiency of
the lab operations and increase the
speed and clarity with which results
can be reported.
• Computers are also important for the
result entry, analysis and preparation.

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