Techniques for Total Aerobic Microbial Count (TAMC) and Pathogen Testing

1.TAMC (Total Aerobic Microbial Count)

TAMC determines the total count of aerobic (oxygen-requiring) bacteria and fungi in a sample. This helps assess product quality and ensure compliance with regulatory standards (e.g., USP, ISO).

Techniques for TAMC:

1.Serial Dilution:

Objective: Reduce the concentration of microbes to countable levels.

Process:

A sample (liquid or dissolved solid) is diluted stepwise in sterile saline or peptone water (e.g., 10^-1, 10^-2, 10^-3).

Each dilution is tested to ensure countable colonies (30–300 colonies) appear.

2.Pour Plate Method:

Procedure:

1 mL of the diluted sample is transferred into a sterile Petri dish.

Molten agar (45–50°C) is poured over the sample and mixed gently.

After solidification, plates are incubated at 30–35°C for 48–72 hours.

Advantages: Suitable for high microbial counts.

Limitations: Some microbes may be sensitive to heat or oxygen availability in the agar.

3.Spread Plate Method:

Procedure:

0.1 mL of the diluted sample is pipetted onto the solidified agar surface.

A sterile spreader evenly distributes the sample.

Incubate at 30–35°C for 48–72 hours.

Advantages: Better for heat-sensitive microbes and quantitative analysis.

Limitations: Inefficient for high microbial loads.

4.Membrane Filtration (for liquid samples):

Procedure:

Sample (e.g., 100 mL) is passed through a 0.45 µm membrane filter.

The filter is transferred to SCDA or nutrient agar and incubated.

Advantages: Suitable for samples with low microbial loads (e.g., water testing).

5.Colony Counting and Reporting:

Colonies are counted, and results are expressed as CFU/mL (Colony Forming Units per mL).

Calculation:

CFU/ml = Number of colonies × Dilution factor/Volume of sample plated(ml)

2.Pathogen Testing:

Pathogen testing focuses on detecting harmful microorganisms such as E.coli, Salmonella, Pseudomonas, and Staphylococcus aureus.

Techniques for Pathogen Testing:

1. Enrichment Culture:

Objective: Enhance the growth of target pathogens while suppressing others.

Process:

The sample is inoculated into selective enrichment broths like:

Lactose Broth (for E. coli).

Selenite Cystine Broth (for Salmonella).

Broth is incubated at specific temperatures for 24–48 hours.

2. Streak Plate Method:

After enrichment, a loopful is streaked on selective media:

E. coli: MacConkey Agar – Pink colonies due to lactose fermentation.

Salmonella: XLD Agar – Black-centered colonies.

S. aureus: Mannitol Salt Agar – Yellow colonies due to mannitol fermentation.

Incubation: Plates are incubated at 37°C for 24–48 hours.

3.Biochemical Identification:

IMViC Test (for E. coli): Differentiates between coliforms.

Coagulase Test (for S. aureus): Detects coagulase enzyme production.

Oxidase Test (for Pseudomonas): Identifies oxidase-positive bacteria.

4.Molecular Methods (PCR):

PCR detects pathogen-specific DNA sequences.

Process:

DNA is extracted from the sample.

Specific primers amplify target genes, providing precise identification.

Advantages: Highly specific, rapid, and reliable.

5.Immunological Tests (EL

ISA):

Procedure:

Uses antigen-antibody interactions to detect pathogens.

Advantages: Suitable for large-scale testing and rapid screening.