Bacteriological laboratory Diagnosis

Process

Bacterial Identification Revealing of the organism up to species level, it involves the use of methods to obtain pure cultures from single colonies, followed by tests designed to characterize and identify the isolate can be done by:

Culture characteristics and Colonial Morphology.Biochemical Reactions.Toxin production.Animal inoculation.Serologic Tests.Molecular Tests.

Macroscopic growth features:Shape.Margin.Elevation.Size.Texture. Appearance.Pigmentation.Opacity.

Colonial Morphology: Appearance

Colonial Morphology: Pigment productionEndopigment production

E.g. golden colonies with staphylococcus aureus

Exopigment production

Pseudomonas aerogenosa produces green pigment

Colonial Morphology: Haemolysis on Blood AgarBeta haemolysis

E.g. streptococcus pyogenes

Alpha Haemolysis

E.g. streptococcus mutans

Colonial Morphology: Indicator mediaMacConkey agar

E.g E.coli is a lactose fermenter

Mannitol Salt agar

E.g. staphyolcoccus aureus is Mannitol fermenter

he ability of the organism to attack various substrates to produce particular metabolic products. Common biochemical reactions involve:Presence or absence of single enzyme:

Catalase test:Oxidase test:Coagulase test:Urease test:DNAase testIndole test:

Presence of metabolic pathways;Sugar fermentation test.Citrate utilization.VP test.Methyl red test.

Other:H2S production

Biochemical reactions

Catalase test:

Principle:Testing the ability of bacteria to produce the enzyme which is capable to break H2O2 to produce water & oxygen

+ve catalase test appear as bubble in the medium

e.g. catalase +ve (staphylococci)

Oxidase test:Principle:The cytochrome oxidase enzyme catalyzes the transport of electrons from a donor compound to the final electron acceptor, oxygen.

using an artificial electron donor, phenylenediamine, a redox is used to reduce the cytochrome oxidase.

If the enzyme is present, the colorless dye will turn a purple to blue color.

E.g. Oxidase +ve (Pseudomonas, Neisseria)

Coagulase test

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Tubes coagulase

To detect the free coagulase

Slide coagulase

To detect the bound coagulase

+VE -VE +VE -VE

Co-agulase is the enzyme produced by staphylococcus aureus which is capable of converting fibrinogen to fibrin (to produce clot).

Two types of the test:

Urease testtests the ability of an organism to produce an enzyme urease. hydrolyzes urea to ammonia and carbon dioxide.  The broth contains the pH indicator phenol red.Phenol red turns yellow in an acidic environment and pink in an alkaline environment. urea is degraded and ammonia is produced, an alkaline environment is created, and the media turns pink.  E.g to urease positive (proteus sp and helicobacter pylori)

Indole test

oThe indole test examines the ability of a bacteria to breakdown the amino acid tryptophan.

o  The presence of   indole  is confirmed by the addition of a few drops of Kovac's or Ehlrich reagents.

o  The red ring at the top indicates a positive test.

oE.g. to indole positive E.coli.

Hydrogen sulphide (H2S) production

his test determines whether the microbe reduces sulfur-containing compounds to sulfides during the process of metabolism.agar contains peptone, sodium thiosulfate, and ferrous ammonium sulfate. 

When hydrogen sulfide gas is produced,  it combines with iron compounds to produce FeS, a black precipitate.

E.g to H2S producer (salmonella)

Sugar fermentation testTesting the ability of the bacteria to enzymatically converting sugars to acid (lactic acid, acetic acid etc.), neutral (ethyl alcohol etc.), gases (carbon dioxide, hyrogen, etc).The medium contains:

Broth.Suger tested (glucose , lactose etc).pH indicator phénol red (yellow in acidic medium) .Durham‘s tubes for gas collection.

Citrate utilization testTesting ability of the bacteria to use Citrate as carbon source.

The Simmon's Citrate Agar Slant contains: Sodium Citrate as the carbon source. Ammonium Phosphate as the nitrogen source. Bromthymol blue as the pH indicator (green at acidic blue at alkaline).

bacteria use the enzyme Citrase to degrade the citrate.

bacteria convert Ammonium Phosphate to Ammonia and Ammonium Hydroxide.

Both of these molecules will cause the agar to become alkaline and produce a blue color.e.g. Klebsiella sp

Voge’s Proskauer test

Testing the ability of the bacteria to utilize glucose in butyline glycol pathway and produces Acetyle methyle carbinol (AMC) or Aacetoin.

Contain pepton, glocose and buffer.

the end products will react with alpha naphthol and KOH (Barrit's reagents) produce red maroon color.

Methyl red testTesting the ability of the bacteria to utilize glucose in mixed acid fermentation pathway and produces stable acidic end-products (lactate, acetate).

Contain pepton, glocose and buffer.

the acids will overcome the buffers in the medium and produce an acidic environment.

methyl red is added, if acidic end products are present, the methyl red will stay red.

Biochemical reactionsAnalytical Profile Index (API):

Fast identification system combines biochemical reactions and computor database for group of bacteria.Examples API20E, API Staph

6 7 0 6 5 1 3 = Staphylococcus sp

Testing the ability of the bacteria to produce certain toxin (invitro) or observe its effect (invivo).

Toxin production

Elek's test (corynebacterium diphteriae)

Nagler’s reaction. (clostridium perfringens

Animal inoculation ( animal Pathogenicity test)Animals commonly used:

guinea pigs. rabbits. mice.

Importance of pathogenicity test:• Differentiate pathogenic and non

pathogenic bacteria.• Isolation organism in pure form.• To test ability of toxin production.• Evaluation of vaccines and

antibiotics.

Serologic testsimmunologic methods taking advantage of the specificity of antigen antibody binding to certain bacterial components:

Cell wall antigens (O Ag).Capsular antigen (K Ag).Flagellar antigen (H Ag).

Example for Serological test;Latex agglutination test.Haemagglutination.Complement fixation.Counterimmuno-electrophoresis (CIE).Enzyme linked immunosorbent assay (ELISA).Fluorescent antibody test.

highly sensitive and specific method for pathogen detection by identification of its nucleic acid in patient specimen.

detect a short nucleotide sequence of DNA or RNA (target sequence) that is unique to the pathogen.

Examples of Molecular tests used:Polymerase Chain reaction (PCR).Ligase Chain Reaction.Plasmid profile analysis.

Molecular tests

Revealing of the organism beyond to species level.It is the ability to discriminate between similar strains (population of bacteria from single cell).

Used for:investigation and control of epidemics.Identification of pathogen reservoir and source of transmission.Surveillance. Study Pathogenesis of the particular bacteria.

Two categories:Phenotypic methods.Genotypic methods.

Bacterial typing

Bacterial Typing based on expressed phenotypic characteristics.

Biotyping.

Antibiograms.

Serotyping.

Bacteriophage typing.

Phenotypic typing

Phenotypic typing: Biotyping

Based on biochemical & cultural characteristics like colonial morphology, growth requirements, carbon source utilisation and toxin production.

Example: Vibrio Cholerae biotypes.

Phenotypic typing: Antibiogram typing

Testing the clinical isolates (pathogen) for antibiotic resistance or sensitivity.

Phenotypic typing: Serotyping

Using species specific Antibodies to screen for antigens expressed on bacterial cells.Examples:

Agglutination.Immunofluorescence.

Bacteriophages are viruses that infect and lyse bacteria.

Bacteriophage typing is based on sensitivity of bacterial isolates to type-specific bacteriophages.

Example:Staphylococcus aureus bacteriophage typing.

Phenotypic typing: Bacteriophage typing

Based on analysis of genetic (DNA-based) relatedness of bacterial strains.

Plasmid analysis.Restriction Endonuclease analysis.PCR based (RAPD).Pulse-field gel electrophoresis. Ribotyping.DNA sequencing:

MLST (Multilocus Sequence Typing).SNP (Single Nucleotide Polymorphism).

Genotypic typing