•DNA ANALYSIS
•NUCLEIC ACID HYBRIDIZATION
MODERN SYSTEMS OF BACTERIAL
TAXONOMY
Traditional vs Modern
Binomial taxonomy
DNA sequences
Modern systems
 The routine laboratory identification are the
microscopic observation, culturation
biochemical test and serology test
 Latest and the most modern era of
identification are molecular biological test
 Most of the methods relates to the content of
genetic materials
Serology
 Studies of serum and immune responses thru
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serum
m/o is antigenic, normally response as
antibodies
The reagent used in test kit is antiserum
Present of m/o usually causing the clotting
effects towards antiserum (+ve result)
Oppositely, the absent of m/o will be diluted
effects (-ve result)
Types of serology test
 Slide agglutination test
 ELISA (Enzyme-linked Immunosorbent
Agglutination)
 Western blotting (same approach but not
categorize as serological test)
Phage typing
 To determine which phages a bacterium
susceptible to
 Eg. Bacteriophage (the bacterial virus) only
attacking the particular strain or types of
bacteria
Fatty acid analysis
 To compare the fatty acid profiles among m/o
 Also known as FAME (fatty acid methyl ester)
 Widely used in clinical and public health
laboratories
Flow cytometry
 To identify bacteria in sample without
culturing the bacteria
 Cell and m/o go through a small portion
 Pass thru a laser and show scattering graph
determining size, shape, density and surface
 Also consist of fluorescent detection
DNA base composition
 Show the relatedness via % of (Guanine G and
cytosine C)
 G complement with C, A complement with T
 Pair s of GC resulting pairs of AT; (GC + AT =
100%)
 Eg. If there are 2 m/o, might be 1st have
40%GC but the other have 80%GC
DNA Fingerprinting
 To determine the source of hospital-acquired
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infections
Spread via finger tips
Relate to hospital personnel or patients from
the same ward
Short-time period required, instead of routine
process
Should have the control sequences
Similar sequences shows >% of relationship
Polymerase Chain Reaction
 m/o cannot be cultured through conventional
methods
 This method can be used to increase the
amount of microbial DNA to levels that can
be tested by gel electrophoresis
 Eg. From the amber of ancient period
 If there are primer of particular m/o in the
sample, there will be amplified DNA indicates
that m/o, obviously shows in the gel
5 minutes break
Nucleic acid hybridization
•Southern Blotting
•DNA chips
•Ribotyping and Ribosomal RNA sequencing
•Flourescent In Situ Hybridization (FISH)
Introduction
 Each complimentary DNA containing
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complementary bases
When heat, the hydrogen bonds separated
When cool, it reunite almost back to normal
Basic purpose is to identify unknown m/o
Can hybridize any single-stranded nucleic
acid chain; DNA-DNA, RNA – RNA, DNA-RNA
Nucleic acid hybridization
Nucleic acid hybridization
Southern Blotting
 To identify unknown m/o
 Using DNA probe for rapid identification
 Method involves breaking enzyme, then
selecting a specific fragment as a probe , then
the probe able to hybridize with the DNA of
all particular m/o strains, but not with the
DNA of closely related group m/o
DNA probe used to identify bacteria
Southern Blotting
Arrangement of papers
in Blot tray.
Southern blot
DNA chips
 Possible to quickly detect a pathogen in a
host by identifying a gene that is unique to
that pathogen
 The chips composed of DNA probe
 Unknown DNA label by fluorescent dye and
placed in a chips
 Hybridization between DNA probe and DNA
detect by fluorescent
DNA chip technology
Fluorescent In Situ
Hybridization
 Fluorescent dye-labeled RNA or DNA probe
are used to specifically m/o in place or in situ
 Cell will be treated then the probe will enter
the cell easily and react to the target
ribosome in the cell (in situ)
 To determine the identity, abundance and
relative activity of m/o in an environment
 Also to detect bacteria that have not yet been
cultured (tiny quantity)
FISH
Ribotyping and Ribosomal RNA
Sequencing
 To determine the phylogenic relationships
among organisms
3 advantages:
 All cells contain ribosomes
 RNA genes undergo <changes over time –
each sequences looks ‘same’ to each other
 Cell do not have to be cultured in laboratory
Ribosomal sequencing
Task of the week
 Compare between Western Blotting and
Southern Blotting. Differentiate in table
form.
SCORE A!!!
THE END