General Microbiology
Lecture Twelve
Identification of Bacteria
• Enzyme and Growth characteristics
Identification of bacteria involves testing
the pure culture in as many ways as
possible. Incubating is many relevant
indicating media and collecting all the data
regarding characteristics including: growth
characteristics in solid and broth media,
enzymic reactions, morphology and its
physical, chemical and biological
requirements for growth.
Serology
• Serology In addition to enzyme reactions
and growth features a bacterium can be
tested to see what anti-gens it carries on
its cell and extra-cellular material. This is
done by testing the cells with a range of
anti-bodies to produce a sero-type
Pharge typing
• Pharge typing Additional information can
be determined by testing which
bacteriophage (bacterial viruses) will
attack the bacteria. This produces a
phage type
Antibiogram
• Bacteria can develop resistance to many antibiotics. The
range and degree of resistance to antibiotics can be
measured using antibiotic sensitive test, which involve
placing small antibiotic impregnated disks on a gar plate
inoculated with the bacteria.
• The bacteria will not grow near the disk if it is sensitive to
that antibiotic. A zone of inhibition will exist around each
effective antibiotic the diameter of which is an indication
of the degree of sensitivity the bacteria has for the
antibiotic.
• A histogram can be draw showing the range and
sensitivities. This is called an antibiogram and will be an
indicator is an organism is the same as some suspected
strain.
Phenotypes
• All this information is what can be observed and
is referred to as phenotypic characteristics.
When at hand an identification can be
determined using a dichotomous key.
• The problem with this approach is that not
bacteria exhibit the characteristics they could.
• For a characteristic to be manifested a
bacterium must stimulate a gene to express it
self. All the characteristics are the result of gene
expressions.
• But in some strains of a bacterium some genes
can be dormant
Miniaturized Kits
Biochemical reactions:
API 20E Enterobacteriaceae +
18-24 h
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PAGE
• There are many proteins coded for by the genes in a
bacterium that does not produce visible results. The total
of all the proteins expressed by genes can be detected
by isolating chemically all the protein and separating
them by electrophoresis using polyacrilamide gel.
(PAGE).
• When the polyacrilamide is stained with a dye specific
for protein a pattern of protein bands are revealed. That
is highly specific for a particular variety of a bacterial
species.
PSAGE
Interpretation
• All these phenotypic characteristics need
to be interpreted in a method that allows
for the probability of certain features not
occurring.
• Computer programs are available to
calculate these probabilities.
• Identification can be expressed as a
certain % probability of being one species
and another % of being something else.
Genotype
• It is possible that not all genes are
expressed and two different bacteria can
still appear the same with all the above
phenotypic information.
• An examination of the genetic material is
possible were the DNA is examined
directly
Southern Blot Technique
• One method is to extract chemically the DNA
then chop it into fragments with specific
enzymes (endonuceases).
• These fragments can be separated using
electrophoresis similar to PAGE except that
agarose gel is used as a support medium
instead of polyacrilamide.
• The separated DNA fragments have to be
transferred on to an acetate paper for dyeing. By
a blotting process.
• This is called a Southern Blot technique.
Northern Blot
Specific Single Tests
• All the above testing takes a lot of time and is requires a lot of
apparatus.
• If one were looking to see if there is a particular species or strain
present in food, it would be much more preferable to have a single
test for a particular organism.
• There is a lot of money to be made by identifying a particular antigen
or gene that is always present in a particular organism. Using
monoclonal antibodies that target a single entity can look for these
unique features.
• The binding of a monoclonal antibody to a bacteria can be detected
by having the antibody attached to an enzyme the will trigger a
colour change.
• Theses are called Enzyme Linked Immuno Assays or ELISA tests.
• To save the time needed for incubation it is possible to look for
pieces of DNA by multiplying a minute amount of bacterial DNA
using a PCR (polymer Chain Reaction) technique.
• A specific piece of DNA can be detected by an ELISA test attached
to a gene probe.
ELISA
Latex Agglutination – E. coli O157