General MicrobiologyLecture 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

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Biochemical reactions:

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