11 Identification

Fanny Hesse

chromogen is a colourless chemical compound that becomes "coloured" by chemical reactions

Bacteria are characterised by enzyme systems that metabolise the substrates to release the chromogen

Typical appearance of microorganisms E. coli → dark pink to reddish

Enterococcus → turquoise blue

Klebsiella, Enterobacter, Citrobacter → metallic blue

Proteus → brown halo

Pseudomonas → cream, translucent

S. aureus → golden, opaque, small

Staphlococcus saprophyticus → pink, opaque, small

Vibrio cholerae

Helicobacter pylori

Campylobacter jejuni

Difficult to Gram stain due to their morphology

• Borrelia burgdorferi

• Treponema pallidum

Acid-fast organisms contain large amounts of lipid substances within their cell walls (mycolic acids )

Used to identify acid-fast microbes

• Mycobacterium tuberculosis (mycolic acids)

• Nocardia spp

• A quick, cheap and simple test for the presence of the enzyme catalase

• Present in aerobes and some facultative bacteria

• Principle use is to differentiate between Staphylococcus (positive) and Streptococcus (negative)

2 H2O2 —> 2 H2O + O2

• If a Gram-positive coccus is catalase positive it is probably a pathogenic Staphylococcus spp.

• Coagulase acts by a thrombinase-like action

• A coagulase-positive strain of Staphylococcus will cause fibrin formation, i.e., will clot plasma

• Performed only on Gram-negative bacteria

• Detects presence of cytochrome oxidase

• When reagent N,N-dimethyl-p phenylenediamine is oxidized by oxidase, a purple colour develops

• Oxidase-negative

  • Escherichia coli, Salmonella, Shigella

• Oxidase-positive

  • Neisseria, Pseudomonas, Vibrio, Helicobacter, Campylobacter

Standardised, miniaturized phenotypic tests for a range of micro-organisms

• higher sensitivity and accuracy

• ability to monitor DNA amplification in real-time through fluorescence intensity → negating need for any post-PCR detection techniques

• Uses universal random primers as alternatives to gene specific molecular marker identification (i.e., 16S rRNA)

• Useful when analysing a large number of samples of diverse species and without any prior genetic information

• BUT RAPD suffers from poor reproducibility between laboratories largely because of the requirement of consistent PCR amplification conditions

  • complex patterns of RAPD means there are challenges in consistent scoring of images even in single-source samples

Method for identifying bacterial strains using unique fingerprints which relies on the presence of variations (polymorphisms) in homologous DNA sequences

BUT method slow with poor reproducibility

Method of separating large fragments of DNA

• particularly useful for characterising and typing bacteria for epidemiological studies

• PFGE used to be the current “gold standard” fingerprinting method

• BUT now transitioning toward using whole genome sequencing

Reactions where the Ag-Ab reaction can be directly observed can be used for bacterial identification purposes

Antigen reacts with its corresponding antibody → visible clumping of bacterial cells

Agglutination tests are frequently used for initial confirmation of specific pathogens

• e.g., Salmonella and Vibrio cholerae

• Analytical technique to detect the presence of an antigen or antibody in a given sample •

• Virtually all microbial species have unique antigen(s), and such type of antigen(s) can be exploited as specific molecules of detection by ELISA

• Variations in ELISA allows detection of either antigen or antibody

  • identify different bacteria (strains) at a time

  • also characterisation of epitope distribution on the microbial surface

1. Direct ELISA-use in the detection of antigen

2. Indirect ELISA-use in the detection of antibody

3. Sandwich ELISA-use in identification of different epitopes at a time

4. Competitive ELISA-use in quantifying the antigen/antibody

5. Multiplex ELISA-use in identification of multiple antigens/antibodies at a time

Covid tests

• Also known as an immunochromatographic assay

Two main types of lateral flow immunoassay used in microbiological testing

• Double antibody sandwich assays

• Competitive assays

• gas chromatography (GC)

• matrix-assisted laser desorption ionization time-of-flight mode (MALDI-TOF)

• electromigration techniques

• electrospray ionization (ESI)

• Latest next generation tool being used for the rapid identification and classification of microorganisms

• Based on the ionization of microbial cells with short laser pulses and then accelerating the particles in a vacuum system using an electric field

• After ionization, a molecular fingerprint in the form of a spectra profile is obtained → specific for each microorganism

• Molecular fingerprint in the form of a spectra profile. Spectrum compared to an existing database, resulting in its identification by an automated program

• Rapid and robust method for accurate microbial identification

• Can be used to identify closely related bacterial species

• MALDI-TOF-MS based method to discriminate between methicillin-sensitive and methicillin-resistant S. aureus

• Beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based approaches