Fish Microbiology and Quality Assurance

Unit 3 - Enumeration of microorganisms in foods

3.3.2 Enumeration of microorganisms by rapid methods
The conventional cultured based methods often fail to detect the presence of microorganisms in foods and also take long time for laboratory analysis. This may be due to their low cell numbers and loss of viability due to damage to cells. This can be overcome by applying culture independ methods involving the detection of bacterial cell components and metabolites by immunological and molecular based approaches. The commonly employed rapid tests in food microbiology are;

• Enzyme linked immunosorbant assay
• Polymerase chain reaction

1. Enzyme Linked Immunosorbent Assay (ELISA)

• ELISA or enzyme immunoassay (EIA) is used in food microbiology to identify the specific pathogens or toxins released by them. Detection of specific microorganism among the mixture of organisms is made easier by employing ELISA.
• This method based on the antigen (cell or toxin) – antibody reaction is highly specific and helps to detect when antigens are present in very low levels. Thus, ELISA involves specific reaction between the antigen, antibody and an enzyme. This reaction complex produces a colour in the presence of chromogenic substrate which is proportional to the amount of antigen present. The enzymes such as horse radish peroxidase and alkaline phosphotase are commonly used which release a dye (chromogen) when exposed to their substrate.

Procedure

• The antigen to be detected is taken in a test tube or microtitre plate and incubated with the antiserum.
• The excess antiserum is washed and the enzyme labelled with anti-immunoglobulin is added.
• After washing, the enzyme remaining in the tube or microtitre well is assayed to determine the amount of specific antibodies in the initial serum.
• The amount of peroxidase enzyme is measured by adding enzyme specific substrate and the colour developed is measured colorimetrically.

Polymerase chain reaction: (PCR)
Polymerase chain reaction (PCR) is a molecular biology technique employed in food microbiology to identify the presence of specific microorganism of interest especially when present in very low numbers by targeting the specific gene sequence. As the foods may contain pathogenic microorganisms which may be present in low numbers or injured by conditions of food processing, the recovery of such organisms by routine plating techniques is not possible. In such situations PCR based methods become very useful in identifying the target organism. The PCR for the detection of pathogens generally targets virulence associated genes such as ctx gene encoding cholera toxin for Vibrio cholerae 01 and 0139; tdh gene coding thermostable direct hemolysin for V. parahaemolyticus; hns and invE gene for Salmonella; stx1, stx2 and eae gene for enterohaemorrhagic E. coli.
Principle of PCR
The method is based on the amplification of target DNA sequences in the genomic DNA in the presence of specific primers, oligonucleotides, buffers and polymerase enzyme. Under the conditions of repeated heating and cooling cycles millions of copies of target DNA will be synthesized with in a short period of time. The amplified product is detected after performing electrophoresis on agarose gel and visualized under UV light after staining with ethydium bromide.
Advantages

• PCR is highly specific as it can amplify a target DNA fragment of pathogen of interest against DNA of other organisms.
• PCR is highly sensitive as it is possible to amplify target sequence from samples having very few microbial cells of interest.
• PCR is a rapid test since the results can be obtained with in a few hours.
• It facilitataes use of food samples directly or after enrichment. Sample lysate or enrichment lysate can be use for extracting DNA for further PCR amplification.