Detection Methods

Selective Enrichment

• Principle: A multi-step process that uses specific culture media to first recover injured cells and then selectively encourage the growth of a target microorganism while inhibiting others.

• Microbes: Used for specific pathogens or groups, like Salmonella, Listeria, etc.

• Steps:

  1. Pre-enrichment: Uses a non-selective broth to heal injured cells.

  2. Selective Enrichment: Uses a selective broth to grow only the microbe of interest.

  3. Isolation: Streaking onto a selective/differential agar to obtain pure colonies.

  4. Confirmation: Using biochemical tests or PCR to confirm the identity.

Biochemical tests (API strips)

• Principle: Detects microorganisms based on their unique enzymatic activities and metabolic capabilities (e.g., carbohydrate fermentation, hydrolysis) which cause visible changes, like color change.

• Microbes: Used for identifying a wide range of bacteria (e.g., differentiating Enterobacteriaceae).

• Steps:

  1. Inoculate a test strip (like an API strip) with a bacterial suspension.

  2. Incubate the strip for 18-48 hours.

  3. Observe color changes in the microtubules as substrates are utilized.

  4. Interpret the profile to identify the bacterium.

PCR

• Principle: A molecular technique that amplifies a specific, target DNA sequence to detectable levels.

• Microbes: Can detect any microorganism with DNA (bacteria, fungi, parasites). 16S rDNA for bacteria, 18S rDNA for yeast/mould.

• Steps:

  1. Sample Prep: Harvest cells, lyse them, and extract DNA.

  2. Amplification: In a thermal cycler, cycles of denaturation, annealing, and elongation exponentially copy the target gene.

  3. Detection: Run the amplified DNA (amplicon) on a gel electrophoresis; a visible band confirms the microbe's presence.

RT-PCR

• Principle: A variation of PCR used to detect RNA by first converting it into complementary DNA (cDNA) using the enzyme reverse transcriptase.

• Microbes: Used for RNA viruses (e.g., Norovirus, Hepatitis A) and for gene expression studies.

• Steps:

  1. Extract RNA from the sample.

  2. Use reverse transcriptase to synthesize cDNA from the RNA template.

  3. Proceed with standard PCR to amplify the cDNA.

  4. Detect the amplicon via gel electrophoresis.

Microscopy

• Principle: Directly visualizes and enumerates microorganisms using magnification. Stains (like Gram stain or Live/Dead stains) enhance contrast and provide information.

• Microbes: Bacteria, yeast, moulds, and parasites via light microscopy. Viruses require electron microscopy.

• Steps:

  1. Prepare a sample smear on a slide.

  2. Apply appropriate stains (optional, but common).

  3. Observe under the microscope for the presence, shape, and arrangement of cells.

  4. For Live/Dead: Live cells fluoresce green (SYTO-9), dead cells fluoresce red (Propidium Iodide).

ELISA

• Principle: A colorimetric or fluorescent assay that uses antibodies to detect and quantify specific antigens (or antibodies).

• Microbes: Used for viruses, bacteria, fungi, parasites, and microbial toxins (e.g., botulinum toxin).

• Steps (Sandwich Method):

  1. Coat a well plate with capture antibodies.

  2. Add sample; target antigen binds to the antibodies.

  3. Add an enzyme-linked detection antibody that binds to the captured antigen.

  4. Add a substrate; the enzyme converts it to a colored/fluorescent product, indicating a positive result.