How to Calculate the Number of Bacteria in a Sample Using the Miles and Misra Method

The Miles and Misra method, also known as the drop count or surface drop plate method, is a quantitative microbiological technique for estimating the number of viable bacteria in a liquid sample. It involves dispensing replicate, calibrated drops (typically 20 µL) of serial dilutions onto the surface of a dry agar plate, allowing the drops to be absorbed, incubating the plate, and then counting colonies that grow within each drop area. The method is particularly useful when rapid enumeration is needed, when only small sample volumes are available, or when the sample contains particulate matter that would interfere with spread or pour plate techniques. The result is expressed as colony-forming units per milliliter (CFU/mL), calculated from the average colony count of replicate drops at a countable dilution.

At a Glance

Scientific Principle

The Miles and Misra method relies on the fundamental assumption that each viable bacterial cell, when deposited on a suitable solid growth medium, will divide and form a single visible colony. By dispensing a known, small volume of a diluted bacterial suspension onto the agar surface, the number of colonies that develop is directly proportional to the number of viable cells originally present in that volume.

The method was originally described by Miles and Misra in 1938 as a modification of the surface viable count technique. The key innovation was the use of replicate drops from each dilution, which allows for statistical averaging and identification of counting errors. The method exploits the Poisson distribution: when a dilute bacterial suspension is dispensed in small volumes, the probability of a drop containing zero, one, or multiple cells follows a predictable statistical pattern. By counting only drops that yield between 3 and 30 colonies, the method operates in a range where the Poisson error is acceptably low.

The surface of the agar plate must be dry enough to allow the drop to be absorbed within 15–30 minutes without spreading excessively. If the agar is too wet, drops will coalesce; if too dry, the drop may not be absorbed and may roll off. Proper drying is achieved by incubating poured agar plates with lids slightly ajar in a laminar flow hood or incubator for 30–60 minutes before use.

Materials and Instrumentation Choices

Agar Plates

The choice of agar medium depends on the target organism and the purpose of the enumeration. For routine total viable counts of non-fastidious bacteria, tryptic soy agar (TSA) or nutrient agar is appropriate. For selective enumeration, such as when enumerating a specific organism from a mixed culture, selective agar containing antibiotics, bile salts, or other inhibitors should be used. The agar should be poured to a consistent depth (typically 4–5 mm) to ensure uniform drying and absorption characteristics.

Pipettes and Tips

Accurate delivery of 20 µL drops requires a calibrated micropipette capable of dispensing 10–100 µL with an accuracy of ±1–2%. Positive displacement pipettes are recommended when working with viscous samples or when the sample contains particulate matter, as they minimize carryover and volume errors. Standard air-displacement pipettes are acceptable for clear bacterial suspensions. Tips should be sterile and, ideally, low-retention to minimize sample loss.

Dilution Tubes and Diluent

Serial dilutions are typically prepared in sterile saline (0.85% NaCl), phosphate-buffered saline (PBS), or a suitable broth medium. The diluent should be isotonic to prevent osmotic shock to the bacteria. For fastidious organisms, the diluent may be supplemented with 0.1% peptone or other protective agents. Tubes should be sterile, clearly labeled, and arranged in a logical order (e.g., 10⁻¹ through 10⁻⁷ or as needed based on expected bacterial load).

Incubator and Atmosphere

Incubation conditions must match the growth requirements of the target organism. Most routine bacteria are incubated aerobically at 35–37°C for 18–24 hours. For obligate anaerobes, plates must be incubated in an anaerobic chamber or gas-pack system. For microaerophilic organisms (e.g., Campylobacter species), a microaerophilic atmosphere (5% O₂, 10% CO₂, 85% N₂) is required. The incubation time may need to be extended for slow-growing organisms.

Colony Counter

A manual colony counter with a magnifying lens and a ruled grid is sufficient for most applications. Automated colony counters can be used but must be validated for the drop plate format, as the irregular shape of absorbed drop areas can confuse some image analysis algorithms.

Controls

Positive Control

A positive control consists of a bacterial suspension with a known viable count (e.g., a standardized suspension of Escherichia coli ATCC 25922 or another reference strain). This control is processed in parallel with test samples to verify that the method, media, and incubation conditions are functioning correctly. The positive control should yield a CFU/mL value within the expected range for that strain and dilution scheme.

Negative Control

A negative control consists of sterile diluent dispensed as drops onto the agar plate. This control confirms that the diluent, pipette tips, and agar are sterile and that no contamination has occurred during the procedure. No colonies should be observed in the negative control drops.

Dilution Blanks

For each set of serial dilutions, a "dilution blank" control should be prepared by pipetting the same volume of sterile diluent into a sterile tube and then dispensing drops from that tube onto the agar. This control detects contamination introduced during the dilution process.

Replicate Drops

The use of replicate drops (typically 3–5 per dilution) serves as an internal control. The colony counts across replicates should be consistent; large variation (e.g., coefficient of variation >30%) may indicate pipetting error, uneven mixing, or a problem with the agar surface.

Conceptual Workflow

Step 1: Sample Preparation

If the sample is a liquid culture, vortex or mix thoroughly to ensure a homogeneous suspension. If the sample is a solid (e.g., food, soil, or tissue), it must first be homogenized in a sterile diluent using a stomacher or blender. The homogenate is then allowed to settle briefly, and the supernatant is used for serial dilution.

Step 2: Serial Dilution

Prepare a series of ten-fold dilutions in sterile tubes. For example, to prepare a 10⁻¹ dilution, add 1 mL of the sample to 9 mL of sterile diluent and mix thoroughly. Continue this process to achieve the desired dilution range. The number of dilutions needed depends on the expected bacterial load. For a typical overnight bacterial culture (10⁸–10⁹ CFU/mL), dilutions from 10⁻⁵ to 10⁻⁸ are usually appropriate.

Step 3: Plate Preparation

Label the bottom of each agar plate with the sample identification, dilution factor, and date. Using a permanent marker, divide the plate into sectors (typically 4–6 sectors) by drawing lines on the bottom of the plate. Each sector will receive drops from one dilution.

Step 4: Drop Dispensing

Using a sterile micropipette set to 20 µL, carefully dispense 3–5 drops of each dilution onto the surface of the agar in the corresponding sector. Hold the pipette tip vertically, approximately 1 cm above the agar surface, and dispense the drop gently. Avoid touching the agar surface with the tip. Dispense drops in a pattern that allows them to remain separate (e.g., in a row or triangle). Work from the highest dilution (lowest concentration) to the lowest dilution to minimize carryover.

Step 5: Absorption

Allow the drops to be absorbed into the agar. This typically takes 15–30 minutes at room temperature in a laminar flow hood. Do not move or tilt the plates during this time. The drops should be fully absorbed before incubation; if they are not, extend the drying time.

Step 6: Incubation

Invert the plates and incubate at the appropriate temperature and atmosphere for the target organism. For routine aerobic bacteria, incubate at 35–37°C for 18–24 hours.

Step 7: Colony Counting

After incubation, examine the plates. Select the dilution where each drop contains between 3 and 30 colonies. Count the colonies in each drop of that dilution. If multiple dilutions fall within the countable range, use the dilution that gives the most consistent counts. Record the counts for each replicate drop.

Quality Checks

Consistency of Replicate Counts

Calculate the mean and standard deviation of the colony counts from the replicate drops. The coefficient of variation (CV = standard deviation / mean × 100%) should be less than 30%. A higher CV suggests uneven mixing, pipetting error, or a problem with the agar surface.

Absence of Spreading Colonies

If colonies have spread across the agar surface beyond the original drop area, the count may be unreliable. This can occur if the agar was too wet, if the drop was too large, or if the organism is particularly motile. In such cases, the plate should be discarded, and the procedure repeated with drier plates or a smaller drop volume.

Negative Control Verification

Confirm that no colonies are present in the negative control drops. If colonies are observed, the diluent, pipette tips, or agar may be contaminated, and the results from that batch should be considered invalid.

Positive Control Verification

The positive control should yield a CFU/mL value within the expected range. If the value is outside the expected range, investigate potential causes such as incorrect dilution, expired media, or improper incubation conditions.

Result Interpretation

Calculation of CFU/mL

The formula for calculating CFU/mL is:

CFU/mL = (Average number of colonies per drop) × (1 / drop volume in mL) × (reciprocal of dilution factor)

For example, if the average count from 20 µL drops at the 10⁻⁶ dilution is 15 colonies:

• Drop volume = 20 µL = 0.02 mL
• Reciprocal of drop volume = 1 / 0.02 = 50
• Reciprocal of dilution factor = 10⁶

CFU/mL = 15 × 50 × 10⁶ = 7.5 × 10⁸ CFU/mL

Reporting Results

Report the result as CFU/mL (or CFU/g for solid samples) with the appropriate number of significant figures. Typically, results are reported to two significant figures. For example, 7.5 × 10⁸ CFU/mL, not 750,000,000 CFU/mL.

Statistical Considerations

The Miles and Misra method is inherently less precise than the spread plate method because of the smaller volume plated. The 95% confidence interval for a count based on replicate drops can be estimated using the Poisson distribution. For a count of 15 colonies per drop with 5 replicates, the 95% confidence interval is approximately ±25% of the mean. This should be considered when interpreting results, especially for quality control or regulatory purposes.

Troubleshooting

Limitations

Precision and Accuracy

The Miles and Misra method is less precise than the spread plate method because of the smaller sample volume (20 µL vs. 100 µL for spread plate). The coefficient of variation is typically 10–30% for the drop method, compared to 5–15% for the spread plate method. This reduced precision must be considered when the method is used for applications requiring high accuracy, such as quality control release testing.

Detection Limit

The theoretical detection limit for the drop method is 50 CFU/mL (one colony in a 20 µL drop from an undiluted sample). In practice, the detection limit is higher because multiple drops are needed for reliable counting. For a sample with 50 CFU/mL, the probability of observing any colonies in 5 replicate drops is only about 99.3%, and the probability of observing 3–30 colonies (the countable range) is much lower.

Organism-Specific Issues

Some bacteria, particularly those that form chains or clumps (e.g., Streptococcus species, Staphylococcus species), may not be evenly distributed in the suspension, leading to high variability between replicate drops. Vigorous vortexing or sonication may help, but care must be taken not to kill the bacteria. Motile organisms may spread across the agar surface, making accurate counting difficult.

Matrix Effects

Samples containing particulate matter, high concentrations of salt, or inhibitory substances may interfere with the method. Particulate matter can block the pipette tip or cause uneven drop formation. High salt concentrations may inhibit bacterial growth on the agar surface. In such cases, the sample may need to be diluted further or processed using a different method.

Documentation

Essential Records

For each enumeration, the following information should be recorded:

• Sample identification and source
• Date and time of sample collection and processing
• Dilution scheme used (including dilution factors)
• Number of replicate drops per dilution
• Colony counts for each replicate drop
• Calculated CFU/mL (or CFU/g)
• Incubation conditions (temperature, atmosphere, time)
• Any deviations from the standard protocol
• Results of positive and negative controls

Data Presentation

Results should be presented in a clear, tabular format. For example:

In this example, the 10⁻⁶ dilution is selected for calculation because it falls within the countable range (3–30 colonies per drop). The 10⁻⁷ dilution yields counts below the countable range and is not used.

Biosafety Considerations

The Miles and Misra method is routinely performed at biosafety level 1 (BSL-1) for non-pathogenic organisms. However, the biosafety level must be determined by a risk assessment based on the specific organism being handled. For BSL-1 work, standard microbiological practices apply:

• Work in a clean, uncluttered area with a non-porous work surface.
• Wear a laboratory coat and gloves.
• Decontaminate work surfaces before and after use with an appropriate disinfectant (e.g., 70% ethanol or 10% bleach).
• Use a biological safety cabinet (BSC) if there is a risk of aerosol generation, such as when vortexing or pipetting concentrated bacterial suspensions.
• Dispose of all contaminated materials (pipette tips, tubes, plates) in biohazard waste containers.
• Autoclave all waste before disposal.

For BSL-2 organisms (e.g., Staphylococcus aureus, Escherichia coli O157:H7), additional precautions are required, including the use of a BSC for all manipulations and enhanced personal protective equipment. The CDC and NIH publication Biosafety in Microbiological and Biomedical Laboratories (6th edition) provides comprehensive guidance on risk assessment and containment practices [6].

Frequently Asked Questions

1. Why is the drop volume standardized at 20 µL?

The 20 µL volume represents a practical compromise between precision and practicality. Smaller volumes (e.g., 10 µL) are more prone to evaporation and pipetting error, while larger volumes (e.g., 50 µL) may not be fully absorbed by the agar and can coalesce. The 20 µL volume allows for 3–5 replicate drops per dilution on a standard 90 mm plate, enabling multiple dilutions to be tested on a single plate.

2. Can I use the Miles and Misra method for anaerobic bacteria?

Yes, but the procedure must be modified. The agar plates must be pre-reduced in an anaerobic environment before use, and the drops must be dispensed and absorbed in an anaerobic chamber. After absorption, the plates are incubated in an anaerobic atmosphere. The same calculation method applies, but the incubation time may need to be extended (48–72 hours) for slow-growing anaerobes.

3. How do I handle samples with very low bacterial counts (e.g., <100 CFU/mL)?

For samples with expected low counts, the detection limit of the Miles and Misra method (approximately 50 CFU/mL) may be insufficient. In such cases, consider using the spread plate method with a larger sample volume (100–500 µL) or the membrane filtration method, where a larger volume (e.g., 100 mL) is filtered through a membrane that is then placed on agar. Alternatively, you can increase the drop volume to 50–100 µL, but this may require longer absorption times and careful plate drying.

4. What is the difference between the Miles and Misra method and the surface drop plate method?

The terms are often used interchangeably, but the Miles and Misra method specifically refers to the use of replicate drops from each dilution, typically 3–5 drops, with the requirement that only dilutions yielding 3–30 colonies per drop are counted. The broader "surface drop plate method" may refer to any technique where drops are placed on the agar surface, including single-drop methods. The replicate drop approach of the Miles and Misra method provides better statistical reliability and allows for identification of pipetting errors.

References and Further Reading

1. Manika V, Devi PB, Majaw J, et al. Microbial production and functional assessment of γ-polyglutamic acid isolated from Bacillus sp. M-E6. 2025. PubMed ID: 41170425. Link — This study describes the isolation and characterization of a Bacillus strain from fermented food, including enumeration methods used for bacterial quantification.

2. Gilroy R, Chaloner G, Wedley A, et al. Caecal microbiome transplant inhibits transmission and intestinal colonisation of Campylobacter jejuni in broiler chickens. 2026. PubMed ID: 42367597. Link — This research on Campylobacter colonization in chickens uses quantitative bacteriology methods relevant to the Miles and Misra technique.

3. Delle Fave F, Froio M, Cisternino D, et al. Characterising the antimicrobial performance of engineered layered double hydroxide surfaces for biofilm control. 2026. PubMed ID: 42274673. Link — This study evaluates antimicrobial surfaces using CFU/mL quantification, demonstrating the application of viable count methods.

4. Falconer K, Hammond R, Parcell BJ, et al. Investigating the time to blood culture positivity: why does it take so long? 2025. PubMed ID: 39757997. Link — This investigation into blood culture detection times provides context on bacterial load and detection thresholds relevant to enumeration methods.

5. Nakagawa K, Okamoto M, Nishida M, et al. On-tissue derivatization for mass spectrometry imaging reveals the distribution of short chain fatty acids in murine digestive tract. 2025. PubMed ID: 41112572. Link — This study on bacterial metabolites in the gut uses culture-based methods for bacterial identification and enumeration.

6. CDC and NIH. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. U.S. Department of Health and Human Services, 2020. Link — Authoritative guidance on biosafety practices for microbiological laboratories, including risk assessment and containment for enumeration procedures.

7. National Institutes of Health. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. Link — Institutional framework for biosafety in research involving genetically modified organisms, relevant when enumerating recombinant strains.

8. National Center for Biotechnology Information. NCBI Bookshelf: Molecular Biology and Laboratory Methods. Link — Searchable collection of authoritative biomedical references, including methods for bacterial enumeration and quality control.

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