Comprehensive Guide to the Miles and Misra Method (Surface Viable Count Technique)
The Miles and Misra method, also known as the surface viable count or drop plate technique, is one of the most accurate and convenient microbiological methods used to determine the number of viable microorganisms present in a given sample. This method was developed by Miles, Misra, and Irwin in 1938 and remains a valuable quantitative microbiological technique for assessing viable bacterial counts with minimal resources and media.
1. Introduction
Enumeration of viable microorganisms is a critical process in pharmaceutical microbiology, food microbiology, environmental monitoring, and clinical laboratories. The viable count refers to the number of living microorganisms capable of forming colonies on a solid medium under suitable conditions.
Among the various viable count methods such as the pour plate, spread plate, and membrane filtration, the Miles and Misra drop plate method stands out due to its simplicity, accuracy, and low sample and media requirements.
2. Principle of the Miles and Misra Method
The Miles and Misra method is based on the principle that a small, known volume (usually 10 µL) of serially diluted microbial suspension is placed as a drop on the surface of a solidified agar plate. After incubation, each drop produces discrete colonies that can be counted. The number of colonies obtained is used to calculate the viable count of microorganisms in the original sample, expressed as colony-forming units per milliliter (CFU/mL).
The assumption is that each viable microorganism develops into a single visible colony, enabling a reliable estimate of microbial concentration in the test suspension.
3. Materials and Equipment Required
- Microbial suspension or test sample
- Sterile saline or diluent (e.g., phosphate-buffered saline)
- Micropipette and sterile tips
- Sterile Petri dishes containing nutrient agar or suitable medium
- Marker pen for labeling
- Incubator (set at suitable temperature, e.g., 30–37°C)
- Colony counter
- Vortex mixer (optional)
4. Procedure of the Miles and Misra Method
Follow these steps carefully to perform the Miles and Misra surface viable count technique:
- Preparation of Serial Dilutions:
- Prepare a series of tenfold (1:10) serial dilutions of the sample in sterile diluent.
- Typically, 1 mL of the sample is transferred into 9 mL of diluent to achieve a 10⁻¹ dilution, and the process continues until the desired dilution range (e.g., 10⁻⁶) is achieved.
- Labeling of Plates:
- Divide each agar plate into six to eight equal sectors using a marker on the bottom of the plate.
- Label each sector with the respective dilution number.
- Plating the Drops:
- Using a micropipette, dispense 10 µL of each dilution onto the surface of the agar plate in its respective sector.
- Allow the drops to absorb or dry naturally (do not spread them manually).
- Incubation:
- Invert the plates and incubate them at the appropriate temperature for 24–48 hours, depending on the microorganism.
- Counting the Colonies:
- After incubation, count the number of discrete colonies formed in each drop.
- Average the colony counts from replicate drops of the same dilution.
5. Calculation
The viable count (CFU/mL) is calculated using the formula:
CFU/mL = (Average number of colonies × Dilution factor) / Volume of drop (in mL)
Example: If an average of 12 colonies is observed from a 10⁻⁴ dilution and the drop volume is 0.01 mL, then:
CFU/mL = (12 × 10⁴) / 0.01 = 1.2 × 10⁷ CFU/mL
6. Interpretation of Results
- Choose the dilution that yields 3–30 colonies per drop for the most accurate result.
- Results outside this range may be unreliable due to crowding or statistical variation.
- Always record observations and calculations in a laboratory notebook or LIMS system.
7. Advantages of the Miles and Misra Method
- Requires less medium and space compared to pour or spread plate methods.
- Allows multiple dilutions to be tested on a single agar plate.
- Reduces incubation space and cost.
- Provides accurate quantitative results.
- Less labor-intensive and time-efficient.
8. Limitations of the Method
- Not suitable for highly motile organisms as they may spread over the agar surface.
- Requires careful pipetting technique to ensure accurate drop volumes.
- May show overlapping colonies at high bacterial densities.
- Manual counting can introduce variability; automation is preferred in high-throughput labs.
9. Applications of the Miles and Misra Method
The Miles and Misra drop plate method is widely used in different microbiological and pharmaceutical settings:
- Pharmaceutical microbiology: For testing bioburden, preservative efficacy, and environmental monitoring.
- Food microbiology: Enumeration of bacteria in food samples and dairy products.
- Clinical diagnostics: Estimation of bacterial load in clinical samples.
- Research laboratories: Quantification of bacterial or yeast cultures for experiments.
- Water and environmental testing: Estimation of viable bacterial counts in environmental samples.
10. Precautions and Good Laboratory Practices (GLP)
- Use sterile equipment and aseptic techniques throughout the procedure.
- Ensure accurate pipetting to maintain consistency of 10 µL drops.
- Allow drops to dry completely before inverting the plates.
- Maintain appropriate temperature and humidity during incubation.
- Always include negative controls to check for contamination.
11. Comparison with Other Viable Count Methods
| Method | Sample Volume | Advantages | Limitations |
|---|---|---|---|
| Pour Plate | 1.0 mL | Accurate for large samples | Time-consuming, more media required |
| Spread Plate | 0.1 mL | Good for isolated colonies | Labor intensive, requires multiple plates |
| Miles and Misra (Drop Plate) | 0.01 mL | Quick, cost-effective, uses less media | Requires precision pipetting |
12. Conclusion
The Miles and Misra method remains one of the most practical and efficient techniques for determining viable microbial counts in a wide range of microbiological applications. Its minimal media usage, reproducibility, and adaptability make it an ideal choice in pharmaceutical quality control and academic research laboratories. By following proper aseptic techniques and accurate dilution procedures, reliable and consistent results can be achieved for routine microbial enumeration.
Written by: Pharmaceutical Microbiology Insights Team
Category: Pharmaceutical Microbiology & Microbial Enumeration Methods
💬 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
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