Membrane Heterotrophic Plate Count Agar (mHPCA): Principle, Composition, Preparation, and Applications

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Membrane Heterotrophic Plate Count Agar (mHPCA): Principle, Composition, Preparation, and Applications

Membrane Heterotrophic Plate Count Agar (mHPCA) is a nutrient-rich solid culture medium used for the enumeration of heterotrophic bacteria in water, pharmaceutical products, and purified water systems. It is a modified version of the standard Heterotrophic Plate Count (HPC) medium, specifically optimized for use with the membrane filtration method.

🧫 Principle of mHPCA

The mHPCA medium supports the growth of a wide variety of heterotrophic microorganisms that utilize organic compounds as their energy source. In the membrane filtration technique, a measured volume of water sample is passed through a sterile membrane filter (0.45 μm pore size), which traps bacteria. The membrane is then placed onto the surface of the mHPCA agar plate, allowing nutrients to diffuse through the membrane and support bacterial growth during incubation.

The number of bacterial colonies appearing on the membrane surface corresponds to the heterotrophic bacterial count in the sample.

🧪 Composition of Membrane Heterotrophic Plate Count Agar (mHPCA)

IngredientsQuantity (g/L)Function
Casein Enzymic Hydrolysate5.0Source of amino acids and nitrogen
Yeast Extract2.5Provides vitamins and growth factors
Glucose1.0Energy source for bacterial growth
Agar15.0Solidifying agent
Final pH (at 25°C)7.0 ± 0.2

*Composition may slightly vary depending on the manufacturer (HiMedia, Merck, or Difco).

⚗️ Preparation Procedure

  1. Weigh 23.5 grams of mHPCA medium powder and suspend it in 1 liter of distilled water.
  2. Heat gently while stirring to dissolve the medium completely.
  3. Distribute into appropriate containers and sterilize by autoclaving at 121°C for 15 minutes.
  4. Cool to 45–50°C, mix well, and pour into sterile Petri plates (approximately 20–25 mL per plate).
  5. Allow the agar to solidify and store at 2–8°C until use.

🧫 Test Procedure (Membrane Filtration Method)

  1. Filter a measured volume of the water sample (usually 100 mL) through a sterile 0.45 μm membrane filter.
  2. Using sterile forceps, place the membrane filter onto the surface of a freshly prepared mHPCA agar plate.
  3. Ensure no air bubbles are trapped beneath the membrane.
  4. Incubate plates at 35 ± 2°C for 48 ± 2 hours.
  5. After incubation, count the number of colonies that appear on the membrane surface.

📊 Result Interpretation

  • Each visible colony represents one viable heterotrophic bacterial cell present in the original sample.
  • Results are expressed as Colony Forming Units (CFU) per mL or per 100 mL of sample.
  • Typical colonies are small, round, and may be colorless or slightly pigmented depending on the species.

🧫 Quality Control Organisms

The performance of mHPCA medium can be checked using the following standard ATCC strains:

Test OrganismATCC No.Expected Growth
Escherichia coli25922Good, small circular colonies
Pseudomonas aeruginosa27853Good, creamy colonies
Staphylococcus aureus25923Good growth
Bacillus subtilis6633Moderate growth

🧴 Applications of mHPCA

  • Used for enumeration of heterotrophic bacteria in potable water, purified water, and pharmaceutical-grade water.
  • Essential in Microbial Limit Tests (MLT) and Environmental Monitoring.
  • Useful in the validation of water systems in pharmaceutical manufacturing.
  • Applied in food and beverage industries for water quality control.

💡 Precautions

  • Avoid overheating or prolonged autoclaving — it can cause degradation of nutrients.
  • Ensure membrane filters are handled aseptically to avoid contamination.
  • Plates should not be used if signs of dehydration or contamination appear.

📚 References

  • Indian Pharmacopoeia (IP)
  • United States Pharmacopeia (USP)
  • European Pharmacopoeia (EP)
  • HiMedia Laboratories Product Data Sheet

Conclusion: The Membrane Heterotrophic Plate Count Agar (mHPCA) is a crucial culture medium for assessing microbial quality of water systems in pharmaceutical, food, and environmental laboratories. Its optimized composition and compatibility with membrane filtration make it a reliable medium for detecting low levels of heterotrophic microorganisms.

💬 About the Author

Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.

📧 Contact: siva17092@gmail.com
Mobile: 09505626106

📱 Disclaimer: This article is for educational purposes and does not replace your laboratory’s SOPs or regulatory guidance. Always follow validated methods and manufacturer instructions.

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