Single vs. Dual Temperature Incubation in Environmental Monitoring

Environmental monitoring (EM) is crucial in pharmaceutical microbiology and quality control, helping ensure cleanrooms and production areas are not harboring harmful levels of bacteria, mold, or fungi.

Perhaps the most contended aspect of EM is incubation temperature for growth media post-sampling. According to a 2017 PDA survey¹, 60% of respondents reported using a dual temperature incubation approach, where samples are placed in two separate incubators at two distinct temperatures for set durations.

Single temperature incubation methods are currently less commonly used. However, with the right strategy, technology, and implementation partner, any EM program could adapt to a single temperature process and unlock the efficiency, simplicity and risk reduction this approach delivers

A single temperature approach offers significant advantages, while providing comparable results to those achieved with dual temperature methods.

The two main benefits of single temperature methods are increased efficiency and reduced risk. Moving samples between incubators is a cumbersome task that creates bottlenecks and introduces opportunity for error — whether that’s contamination, missed timing, or even a dropped plate. A single temperature approach eliminates those risks, minimizing the need for time-consuming investigations and allowing staff to focus on higher-value tasks.

Additionally, since fewer incubators are needed, upfront capital investment is significantly reduced. 

The shift to a single temperature approach isn’t without hurdles. Chief among them is identifying and validating a temperature that ensures reliable microbial recovery.

Studies show that a single temperature incubation is certainly possible, though the optimal conditions rely on type of media, timeframe, target microorganisms, amongst other parameters. Research suggests that 25°C to 30°C is a viable range for a single incubation temperature, though the exact setting must be tailored to each unique situation.

Regulatory uncertainty and compliance are also common concerns for manufacturers. However, no Pharmacopeia or guideline explicitly mandates a specific method for incubation. The primary regulatory references – such as Annex 1, USP <1116>, and the FDA's aseptic processing guidance - only call for a justified and documented contamination control strategy and risk assessment.

Across the pharmaceutical industry, there's a growing shift toward simplifying incubation strategies. A 2023 poll² revealed that 70% of companies still using dual-temperature incubation are open to transitioning to a single temperature approach. This trend reflects a broader movement toward operational efficiency, digital transformation, and regulatory confidence.

One example is Selkirk Pharma, a U.S.-based contract manufacturing organization. After implementing bioMérieux’s 3P® ENTERPRISE platform, Selkirk adopted a single-temperature incubation strategy to streamline processes, reduce errors, and optimize resource allocation. As their Microbiology and EM team put it, “Our goal was to ensure we implement the solutions that can bring us efficiencies in our processes. For this reason, we decided to implement a single-temperature approach in our incubation strategy as it will help reduce potential errors, while optimizing resource allocation.”

This decision wasn't made in isolation. It was supported by the robust capabilities of 3P® ENTERPRISE— a fully integrated EM solution that combines: 

• 3P® SMART PLATES: Traceable culture media with GS1 barcodes for reliable data capture
• 3P® CONNECT SOFTWARE: Centralized software for continuous tracking across your workflow
• 3P® STATION: Automated incubation and reading for standard Petri dishes

Together these components enable a seamless transition to single temperature incubation by helping to ensure data accuracy, audit readiness, and real-time visibility, all of which are key requirements for modern pharmaceutical operations; regulatory guidance also supports this evolution. While no pharmacopeia mandates a specific incubation method, Annex 1 and other global standards emphasize the importance of a documented contamination control strategy and risk-based justification. Research shows that a single incubation range of of 25°C to 30°C is viable for most EM needs, provided it's validated for the specific media and organisms involved.

And most importantly, the bioMérieux team — scientists, implementation experts, and service professionals — are here to support your journey every step of the way.

Start your path to simplified, compliant, and efficient EM. Get in touch with our team so we can start the conversation.

References

1. 2017 PDA Aseptic Processing Survey: https://www.pda.org/bookstore/product-detail/4102-2017-pda-aseptic-processing-survey

2. Survey carried out by the European Pharmaceutical Review in November 2023 during the webinar "How to implement single temperature incubation in your environmental monitoring routine", based on 72 respondents. https://www.biomerieux.com/corp/en/education/resource-hub/webinars/pharmaceutical-qc-on-demandwebinars/How-to-implement-single-temperature-incubation-in-your-EM-routine.html