How to Calculate the Number of Bacteria Using the Petrifilm Method

The Petrifilm method is a ready-to-use, thin-film culture plate system designed for the enumeration of aerobic bacteria, coliforms, and other microbial groups in food, water, and environmental samples. This method replaces traditional agar pour plates with a dehydrated, cold-water-soluble gelling agent and a tetrazolium indicator dye that stains colonies red for easy counting. The calculation of colony-forming units per milliliter (CFU/mL) or per gram (CFU/g) follows the same fundamental principle as conventional plate counts: count colonies on plates containing 25–250 colonies, multiply by the reciprocal of the dilution factor, and adjust for the volume plated. The Petrifilm method is particularly useful for laboratories processing high sample volumes, field testing, and facilities with limited autoclave capacity, as it eliminates the need for media preparation and reduces waste. This article provides a comprehensive, step-by-step guide to calculating bacterial counts from Petrifilm plates, including grid-based counting, dilution factor adjustments, quality control measures, and troubleshooting common issues.

At a Glance

Scientific Principle of the Petrifilm Method

The Petrifilm system operates on a dehydrated culture medium principle. Each plate consists of a thin, flexible film coated with a water-soluble gelling agent, nutrients, and a tetrazolium indicator dye (typically 2,3,5-triphenyltetrazolium chloride, TTC). When a 1 mL sample is added, the gelling agent rehydrates and forms a semi-solid matrix that immobilizes bacteria. During incubation, viable bacteria metabolize the nutrients and reduce the colorless TTC to a red formazan precipitate, causing colonies to appear red or pink against the clear or slightly opaque background. This color change facilitates visual counting without the need for specialized equipment.

The Petrifilm Aerobic Count (AC) plate is the most common variant, designed to enumerate total aerobic mesophilic bacteria. Other variants include coliform/E. coli, Enterobacteriaceae, yeast and mold, and Staphylococcus aureus plates, each with selective or differential components. The fundamental calculation method remains consistent across all variants, though specific counting rules may differ (e.g., counting blue colonies for E. coli versus red colonies for total coliforms).

The method's reliability has been validated against traditional pour plate methods in numerous studies. For example, Mortazavi et al. (2024) used Petrifilm AC plates to monitor aerobic bacterial counts in beef steaks under different packaging conditions, demonstrating the method's utility in food quality assessment [1]. Similarly, Williams et al. (2023) employed Petrifilm to evaluate bacterial recovery from copper antimicrobial surfaces, noting that direct application of Petrifilm to surfaces could revive stressed bacteria, leading to higher counts compared to indirect sampling methods [2]. This highlights the importance of understanding the method's behavior with different sample types.

Materials and Instrumentation Choices

Petrifilm Plate Selection

The choice of Petrifilm plate depends on the target microorganism and the regulatory standard being followed. For routine aerobic plate counts, the Petrifilm Aerobic Count (AC) plate is appropriate. For coliform enumeration, use Petrifilm Coliform Count (CC) plates, which contain selective agents that inhibit Gram-positive bacteria. For simultaneous E. coli and coliform detection, Petrifilm E. coli/Coliform Count (EC) plates incorporate a differential indicator that turns E. coli colonies blue while other coliforms appear red.

Sample Preparation Equipment

• Sterile diluent: Butterfield's phosphate-buffered dilution water, 0.1% peptone water, or 0.85% saline. The choice depends on the sample matrix; for high-fat samples, peptone water is preferred to improve dispersion.
• Homogenizer or stomacher: For solid samples, a stomacher with sterile filter bags is standard. For liquid samples, vortex mixing or shaking is sufficient.
• Pipettes and tips: Sterile, calibrated pipettes capable of delivering 1 mL accurately. Positive displacement pipettes are recommended for viscous samples.
• Dilution tubes or bottles: Sterile containers for serial dilutions, typically containing 9 mL of diluent for 1:10 dilutions.
• Incubator: Capable of maintaining 35°C ± 1°C for aerobic counts, or 30°C for psychrotrophic organisms. Humidity control is important to prevent plate dehydration.
• Colony counter: Manual or automated. Manual counting uses a magnifying glass and tally counter. Automated counters use image analysis but require validation against manual counts for each sample type.
• Petrifilm spreader: A flat, sterile device to spread the sample evenly over the growth area. The Petrifilm system includes a plastic spreader that fits the plate's grid.

Quality Control Materials

• Positive control: A known bacterial suspension (e.g., E. coli ATCC 25922 or Staphylococcus aureus ATCC 25923) to verify medium performance.
• Negative control: Sterile diluent plated on Petrifilm to confirm sterility of materials and technique.
• Reference cultures: Certified reference materials or in-house cultures with known counts for accuracy checks.

Controls and Their Importance

Positive Controls

A positive control confirms that the Petrifilm plate supports growth of target organisms. Prepare a suspension of a known reference strain at a concentration expected to yield 30–300 CFU per plate. Plate 1 mL and incubate under standard conditions. The expected count should fall within established ranges for that strain. If counts are significantly lower than expected, the plate batch may be defective, the incubator temperature may be incorrect, or the sample may contain inhibitory substances.

Negative Controls

A negative control (sterile diluent only) verifies that the diluent, pipette tips, and Petrifilm plates are free of contaminating bacteria. Plate 1 mL of sterile diluent and incubate. Any colony growth indicates contamination of materials or technique. If contamination occurs, discard all materials from that batch and investigate the source.

Duplicate Plates

For each dilution, plate duplicate Petrifilm plates. This allows calculation of the mean count and assessment of precision. If duplicate counts differ by more than 20% of the mean, the results are suspect and should be repeated. This is especially important when counts are near the upper or lower limits of the countable range.

Spiked Samples

For complex matrices (e.g., ground meat, cheese, soil), spike a portion of the sample with a known concentration of a reference organism. Compare the recovered count to the expected count. Recovery rates below 70% or above 130% indicate matrix interference, such as inhibitory substances or poor bacterial dispersion.

Conceptual Workflow for Petrifilm Counting and Calculation

Step 1: Sample Preparation and Dilution

For solid samples, aseptically weigh 10 g into a sterile stomacher bag, add 90 mL of sterile diluent, and homogenize for 1–2 minutes. This yields a 1:10 dilution (10⁻¹). For liquid samples, directly pipette 1 mL into 9 mL of diluent for a 1:10 dilution.

Perform serial ten-fold dilutions as needed. For most food samples, dilutions from 10⁻² to 10⁻⁶ are typical. The goal is to obtain at least one dilution that yields 25–250 colonies per plate. For samples expected to have low bacterial loads (e.g., pasteurized milk), plate the undiluted sample (10⁰) and the 10⁻¹ dilution.

Step 2: Inoculation and Incubation

1. Place the Petrifilm plate on a flat, level surface.
2. Lift the top film and dispense 1 mL of the sample or dilution onto the center of the bottom film.
3. Gently lower the top film to avoid trapping air bubbles.
4. Using the flat side of the Petrifilm spreader, apply gentle pressure to distribute the sample evenly over the circular growth area (approximately 20 cm²). Do not twist the spreader.
5. Allow the gel to solidify for 1–2 minutes.
6. Incubate plates in a horizontal position, stacked no more than 10 high, with the clear side up. Incubate at 35°C ± 1°C for 48 hours for aerobic plate counts. For psychrotrophic counts, incubate at 30°C for 72 hours or 25°C for 5 days, depending on the standard.

Step 3: Colony Counting

After incubation, count all red colonies on the plate. The Petrifilm grid (approximately 1 cm² squares) aids in counting. Count colonies in several representative squares, calculate the average per square, and multiply by the total number of squares (approximately 20) to estimate the total count. For precise counts, count all colonies on the plate.

Counting rules:

• Count only red colonies (for AC plates). For EC plates, count blue colonies as E. coli and red colonies as other coliforms.
• Count colonies on the surface and within the gel. Pinpoint colonies are included.
• If colonies are too numerous to count (TNTC, >250), record as "TNTC" and use the next higher dilution.
• If fewer than 25 colonies are present, record the actual count but note that the result is an estimate.

Step 4: Calculation of CFU/mL or CFU/g

The fundamental formula is:

[ \text{CFU/mL or CFU/g} = \frac{\text{Number of colonies}}{\text{Volume plated (mL)}} \times \text{Dilution factor} ]

Where:

• Number of colonies = average count from duplicate plates (or count from a single plate if only one is countable)
• Volume plated = 1 mL (standard for Petrifilm)
• Dilution factor = reciprocal of the dilution plated (e.g., for 10⁻³ dilution, dilution factor = 10³)

Example calculation:

• Sample: Ground beef
• Dilution plated: 10⁻⁴
• Colonies counted: 87 and 93 on duplicate plates
• Average count: (87 + 93) / 2 = 90
• Volume plated: 1 mL
• CFU/g = 90 × 10⁴ = 900,000 CFU/g (or 9.0 × 10⁵ CFU/g)

Reporting rules:

• Report counts as two significant figures (e.g., 9.0 × 10⁵ CFU/g, not 900,000).
• For counts between 25 and 250, report the calculated value.
• For counts below 25, report as "Estimated count: X CFU/g" and note the limitation.
• For TNTC plates, report as ">250 CFU per plate at dilution X" or calculate an upper limit using the highest countable dilution.

Step 5: Adjusting for Sample Weight or Volume

If the sample is not exactly 10 g or 1 mL, adjust the calculation accordingly. For example, if 5 g of sample is used with 45 mL of diluent (1:10 dilution), the calculation remains the same because the dilution factor accounts for the ratio. However, if an unusual sample weight is used, calculate the effective dilution factor:

[ \text{Effective dilution factor} = \frac{\text{Total volume of homogenate (mL)}}{\text{Sample weight (g)}} ]

Then multiply by subsequent dilution factors.

Quality Checks and Validation

Precision Check

Calculate the relative percent difference (RPD) between duplicate plates:

[ \text{RPD} = \frac{|\text{Count}_1 - \text{Count}_2|}{(\text{Count}_1 + \text{Count}_2)/2} \times 100 ]

An RPD below 20% indicates acceptable precision. Higher values suggest uneven sample distribution, pipetting errors, or plate defects.

Linearity Check

When multiple dilutions yield countable plates, the counts should be proportional to the dilution factor. For example, if the 10⁻⁴ dilution yields 90 CFU and the 10⁻⁵ dilution yields 9 CFU, the ratio is 10:1, confirming linearity. If the ratio deviates significantly (e.g., 10⁻⁴ gives 90 CFU and 10⁻⁵ gives 2 CFU), suspect a counting error or inhibitory carryover.

Control Plate Acceptance

• Positive control: Count within expected range (e.g., 50–150 CFU for a standardized suspension).
• Negative control: Zero colonies.
• If controls fail, reject all results from that batch and repeat with new plates and diluents.

Inter-Laboratory Validation

For critical samples, consider sending duplicate samples to a reference laboratory. Results should agree within 0.5 log₁₀ for routine samples. Discrepancies >1 log₁₀ indicate systematic errors in one laboratory.

Result Interpretation

Normal Counts

For most food products, aerobic plate counts (APC) below 10⁴ CFU/g indicate good microbiological quality. Counts between 10⁴ and 10⁶ CFU/g suggest marginal quality, and counts above 10⁶ CFU/g indicate potential spoilage or poor hygiene. However, these thresholds vary by product type and regulatory standards. For example, pasteurized milk should have APC <20,000 CFU/mL in many jurisdictions.

Elevated Counts

Elevated counts may result from:

• Improper sample storage (temperature abuse)
• Inadequate cleaning and sanitation
• Raw material contamination
• Equipment biofilm formation

Compare results to historical data for the same product to identify trends. A sudden increase may indicate a process failure.

Low or Zero Counts

Zero counts on the lowest dilution (10⁰) suggest either a sterile product or the presence of inhibitory substances (e.g., preservatives, antibiotics, sanitizer residues). If inhibitory carryover is suspected, repeat the test with a higher dilution or use a neutralizing diluent (e.g., Dey-Engley neutralizing broth).

Troubleshooting

Limitations of the Petrifilm Method

Inability to Distinguish Viable from Injured Cells

Petrifilm plates may recover stressed or injured bacteria that would not grow on selective media, leading to higher counts compared to traditional methods. Williams et al. (2023) demonstrated that direct Petrifilm application to copper surfaces revived stressed bacteria, resulting in counts that did not reflect true viability [2]. This is advantageous for total viable counts but may overestimate risk in disinfection studies.

Matrix Interference

High-fat, high-salt, or acidic samples may inhibit bacterial growth or cause poor gel formation. For such samples, dilution in appropriate buffers or use of neutralizing agents is essential. Choi et al. (2026) used Petrifilm to enumerate lactic acid bacteria in frozen kimchi, a high-acid, high-salt matrix, and noted that sample pretreatment (centrifugal dehydration) affected recovery [3]. This underscores the need for matrix-specific validation.

Limited Differentiation

Standard Petrifilm AC plates do not differentiate between bacterial species. For identification, colonies must be isolated and characterized using biochemical or molecular methods. The Petrifilm system offers selective plates for specific groups, but these are not a substitute for full identification.

Counting Subjectivity

Manual counting of Petrifilm plates can be subjective, especially when colonies are small, overlapping, or located near the edge. Automated counters reduce variability but require calibration for each sample type. Nasser et al. (2026) used hemocytometer counts alongside Petrifilm to assess bacterial adhesion on membranes, highlighting that different enumeration methods can yield different results [4].

Incubation Conditions

Petrifilm plates are sensitive to dehydration during incubation. If the incubator has low humidity, plates may dry out, inhibiting growth. Use a humidified incubator or place plates in a sealed plastic bag with a damp paper towel.

Documentation and Record Keeping

Essential Records

• Sample identification (type, source, date collected)
• Sample preparation details (weight, diluent, homogenization time)
• Dilution series and plates plated
• Incubation conditions (temperature, time, humidity)
• Colony counts for each plate (raw data)
• Calculated CFU/mL or CFU/g
• Control results (positive, negative, duplicates)
• Any deviations from standard procedure

Data Reporting Format

Report results as:

• "Aerobic Plate Count: 4.5 × 10⁵ CFU/g"
• "Estimated Aerobic Plate Count: 18 CFU/g (below countable range)"
• "Aerobic Plate Count: >2.5 × 10⁶ CFU/g (TNTC at 10⁻³ dilution)"

Include the method used (e.g., "Petrifilm AC, 35°C, 48 h") and any relevant notes (e.g., "Sample contained visible fat globules").

Archiving

Store Petrifilm plates after counting for at least 30 days in a cool, dry place for verification. Some regulatory standards require longer retention. Label plates with sample ID, date, and count.

Biosafety Considerations

Routine BSL-1 Practices

For non-pathogenic environmental and food isolates, standard BSL-1 practices apply [5]. These include:

• Hand washing after handling samples and before leaving the laboratory
• Decontamination of work surfaces before and after use
• Use of lab coats and gloves
• No eating, drinking, or applying cosmetics in the laboratory
• Proper disposal of Petrifilm plates as biohazardous waste

Sample Handling

Even for food samples, assume that unknown bacteria may include opportunistic pathogens. Open Petrifilm plates only in a biosafety cabinet if the sample is known to contain pathogens. For routine food testing, a clean, uncluttered bench is acceptable, provided that spills are immediately disinfected.

Waste Disposal

Petrifilm plates containing viable bacteria must be autoclaved before disposal. Alternatively, they can be placed in biohazard bags and incinerated. Do not discard plates in regular trash.

Recombinant Organisms

If the Petrifilm method is used to enumerate recombinant or synthetic nucleic acid-containing organisms, follow the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules [6]. This may require BSL-2 containment and additional documentation.

Frequently Asked Questions

1. Can I use Petrifilm plates for water testing?

Yes, Petrifilm plates are suitable for water testing, including drinking water, recreational water, and wastewater. For drinking water, plate 1 mL of undiluted sample directly. For wastewater, serial dilutions are typically needed. The method is approved by the U.S. Environmental Protection Agency (EPA) for coliform and E. coli detection in water under certain conditions. However, for regulatory compliance, check the specific standard method required by your jurisdiction.

2. How do I count colonies when they are very small or overlapping?

For small colonies, use a magnifying glass (2–3×) and good lighting. Count colonies in the grid squares systematically. If colonies overlap, count each distinct colony center. For overlapping colonies that form a continuous red zone, estimate the number by counting the number of distinct colony centers visible at the edges of the zone. If overlapping is extensive, the plate is likely TNTC, and you should use a higher dilution.

3. What should I do if my sample contains particles that look like colonies?

Food particles, fat globules, or air bubbles can be mistaken for colonies. True colonies are typically red or pink (on AC plates) and have a defined, circular shape. Particles are often irregular, colorless, or have a different hue. To confirm, gently press the top film with a spreader; colonies will not move, while air bubbles may shift. If uncertainty persists, streak a colony onto a traditional agar plate to confirm growth.

4. Can I incubate Petrifilm plates for longer than 48 hours?

Extended incubation (up to 72 hours) may be necessary for slow-growing organisms or psychrotrophic bacteria. However, prolonged incubation can cause colonies to spread or merge, making counting difficult. If extended incubation is needed, check plates at 24-hour intervals and record the count at the time when colonies are most discrete. For standard aerobic plate counts, 48 hours is recommended; deviations should be documented and validated.

References and Further Reading

1. Mortazavi SMH, Kaur M, Farahnaky A, Torley PJ, Osborn AM. Microbial and Quality Attributes of Beef Steaks under High-CO₂ Packaging: Emitter Pads versus Gas Flushing. 2024. PubMed — Demonstrates Petrifilm use for aerobic counts in meat quality studies.

2. Williams TC, Woznow T, Velapatino B, Asselin E, Nakhaie D, Bryce EA, Charles M. In vitro comparison of methods for sampling copper-based antimicrobial surfaces. 2023. PubMed — Evaluates Petrifilm recovery from antimicrobial surfaces, highlighting revival of stressed bacteria.

3. Choi YJ, Kim HE, Lee MJ, Kim M, Park SJ, Choi JY, Kang M, Park SH, Lee MA. Process optimization of centrifugal dehydration-hydrocolloid pretreatments for quality preservation of frozen kimchi. 2026. PubMed — Uses Petrifilm for lactic acid bacteria enumeration in fermented food.

4. Nasser N, Hassouna MSE, Salem N, Amer R, Kandil SH, Nady N. Evaluation of innovative dual-layer modified polyethersulfone membranes in the control of biofouling. 2026. PubMed — Compares Petrifilm with hemocytometer counts for bacterial adhesion studies.

5. CDC and NIH. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. U.S. Department of Health and Human Services, 2020. CDC — Authoritative biosafety guidelines for laboratory practice.

6. National Institutes of Health. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. NIH Office of Science Policy — Framework for recombinant organism work.

7. National Center for Biotechnology Information. NCBI Bookshelf: Molecular Biology and Laboratory Methods. NCBI Bookshelf — Searchable collection of biomedical methods references.

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