Environmental Monitoring: How to Choose the Incubation Temperatures for Your Culture Media?

This article is brought to us by a SuperMicrobiologist, Ségolène Charrat, who is a contamination control consultant (among other roles) at Cophaclean. If you would like to write an article for the blog, contact us.

The goal of environmental monitoring is to ensure that cleanrooms and air treatment equipment continue to provide a suitable quality environment in line with design and regulatory requirements (§9 Eudralex Volume 4 EU Annex 1, August 25, 2022).

Environmental monitoring is a tool used to control the quality of production environments for medicines, medical devices, cosmetics, and biotech products, which must be produced in clean and controlled environments.

Like any tool, environmental monitoring is only useful if the parameters are well thought out to highlight the factors being studied.

One essential parameter of environmental monitoring is defining the incubation program. This program affects the ability to detect certain microorganisms and, in some cases, may include or exclude the growth of specific microbes.

Let’s now see how we can define our incubation temperatures.

Sommaire :

• What is the current regulatory context on the subject?
• What do different standards and guidelines recommend?
• What is the difference between these incubation ranges, and what are their benefits?
  • The limits of 32.5°C followed by 22.5°C
  • The limits of 22.5°C followed by 32.5°C
• What are the most common programs used today, and why?
• The possibility of a single incubation temperature
• Conclusion: Which program should you choose and why?

What is the current regulatory context?

Environmental monitoring is governed by various regulatory texts, standards, and guidelines, which emphasize its importance but also its risks.

In terms of regulation, GMP for the pharmaceutical industry, ISO 22716 for cosmetics, and ISO 13485 (2016) for medical devices provide requirements for using environmental monitoring data.

However, these texts do not specify the details of an environmental monitoring program, especially incubation temperatures for agar plates.

Annex 1 of GMP (August 2022) and the NF EN 17141 standard (2020) offer more guidance on environmental monitoring programs and incubation temperatures.

The NF EN 17141 standard applies to cleanrooms and controlled environments and replaces ISO 14698-1 and 2 in France. It provides important details on incubation temperatures for environmental monitoring programs. However, this standard is voluntary and not yet harmonized internationally (ISO).

There are also guidelines like the PDA’s Technical Report No. 13, the USP 1116 (Aseptic Processing Environments) from the FDA, and the WHO guide for environmental monitoring in vaccine production facilities, which give additional insights.

What do different standards and guidelines recommend?

The most detailed text on this subject is the NF EN 17141 (2020) standard, which applies to any health industry working in cleanrooms or controlled environments.

This standard recommends incubation at 32.5 ± 2.5°C for at least 3 days, followed by 22.5 ± 2.5°C for at least 4 days. If a company uses this, the incubation between 20 and 35°C with a tolerance of ± 2.5°C applies.

For USP 1116, which is for aseptic processing environments, the same range of 20-35°C is recommended for a minimum of 3 days.

The WHO guidelines for vaccine production suggest double incubation: 3-5 days at 22.5 ± 2.5°C, followed by 2-3 additional days at 32.5 ± 2.5°C.

What is the difference between these incubation ranges, and what are their benefits?

The two most commonly used temperature ranges for environmental monitoring in cleanrooms are 22.5 ± 2.5°C and 32.5 ± 2.5°C.

The range of 22.5 ± 2.5°C promotes the growth of most yeasts and molds, as well as some bacteria that survive in cleanrooms. This temperature is close to the ambient conditions of production areas, which are often around 20°C.

The 32.5 ± 2.5°C range is generally accepted to cultivate aerobic bacteria. However, some microorganisms may struggle to grow if they are incubated directly at this temperature after being collected from a low-temperature environment like a cleanroom.

While each range helps grow certain microbes, using both ranges together has more benefits than just using a single temperature.

The limits of 32.5°C followed by 22.5°C

The incubation program with 32.5 ± 2.5°C for at least 2-3 days followed by 22.5 ± 2.5°C for 3-4 days theoretically allows the growth of total aerobic bacteria and then fungi (yeasts and molds).

However, there are limitations:

• Physiological stress: Going from around 20°C to 32.5°C could stress already weakened organisms, affecting their ability to grow or even survive.
  
• Underestimating fungi populations: Some fungi might be killed off by the sudden temperature increase, preventing them from growing before the plates move to the lower temperature.

The limits of 22.5°C followed by 32.5°C

An alternative is to start with 3-4 days at 22.5 ± 2.5°C and then incubate for 2-3 days at 32.5 ± 2.5°C. Once again, this program could allow the cultivation of both fungal flora and total aerobic bacteria.

This may reduce the limitations of the previous method, but there are still issues:

• Some human-origin bacteria, which grow best at 30-35°C or even 37°C, might be harmed by the lower temperature of 22.5°C ± 2.5°C.

• The initial cultivation of fungal flora can result in the invasion of the agar by certain molds, making it impossible for bacteria to grow or to read the agar and properly count the bacterial colonies that may have developed during the first and second phases of incubation

What are the most common programs used today, and why?

According to a customer survey conducted in 2017 by BioMérieux in France, as well as field observations I have made on this subject, a majority of manufacturers use double temperature ranges for incubations.

Still according to the BioMérieux survey, nearly 60% of them have chosen this type of program, which includes double incubation. Among them, 37.5% implemented the program 20-25°C followed by 30-35°C, while 21.4% opted to incubate first at 30-35°C, then 20-25°C.

Less than 40% of French manufacturers perform single incubations or use other types of incubation programs.

A similar study conducted by the PDA (Parenteral Drug Association) indicates that 61% of the manufacturers surveyed perform their incubations at 20-25°C followed by 30-35°C. Around 12% opted to incubate at 30-35°C followed by 20-25°C, meaning more than three-quarters of the manufacturers have chosen a combined temperature incubation program.

The reason why incubation programs with double temperatures seem so popular is that they allow the growth of a greater variety of microorganisms, particularly enabling the detection of most microorganisms of interest that may be found in a controlled clean environment.

However, the lack of a consensus on this subject is due to the fact that the primary requirement for such a program is to allow the detection of so-called microorganisms of interest.

Microorganisms of interest are defined by the standard NF EN 17141 (2020) as “microbiological contamination identified as dangerous for the product, the process, or the user in a controlled clean environment.”

Since each process and each product may present specificities, it is logical that microorganisms of interest vary from one production site to another and, consequently, that environmental monitoring programs to detect them differ as well.

The possibility of a single incubation temperature

Despite significant advantages in detecting microorganisms of interest, incubation programs involving two temperature ranges can present technical drawbacks, as previously described, but also a major economic and quality disadvantage: time.

Indeed, applying a dual temperature range for incubation extends the total incubation time compared to using a single temperature range.

However, it has been noted that, in most cases, single temperature ranges may present the technical drawback of not allowing the growth of as many microorganisms as a synergy of two temperature ranges.

In this context, a study was initiated by two experts from Sanofi Vaccines and bioMérieux to investigate the effectiveness of a single temperature range described in the Japanese Pharmacopoeia: incubation at 25-30°C for at least 5 days.

The results of this study were presented during the microbiology days in 2022. The study, conducted at a temperature range of 25-30°C for 7 days, showed that a significant portion of the microorganisms studied (reference strains and wild site strains) achieved a satisfactory recovery rate (between 50% and 200%) compared to the target value of the study, which was performed with incubation at 22.5 ± 2.5°C (5 days) followed by 32.5 ± 2.5°C (2 days).

However, the study also highlighted that this single incubation program might be less effective for certain types of microorganisms, such as Cladosporium spp., which do not grow well at this temperature. Given that this fungal genus is among the top three molds most commonly found in industrial sites, it would be unwise to set an incubation program with this temperature when the current regulations require ensuring the detection of microorganisms that can grow in production environments.

Based on these observations, additional protocols were conducted to test single temperature ranges of 25°C and 22.5°C. Microorganisms with optimal growth temperature ranges extending up to 30-37°C were studied.

At these lower single temperatures, the growth of Cladosporium spp. is optimal, with satisfactory recovery. However, the growth of Corynebacterium striatum is challenging and does not yield satisfactory recovery under the study’s parameters.

The study concludes that a single incubation temperature is possible for an environmental monitoring program. However, none of the temperatures tested allows optimal growth for all the microorganisms studied in this context.

Conclusion: Which program should you choose and why?

There is no universal “best” program for environmental monitoring. Each program must be adapted to the production process, product, and potential risks from microorganisms in the environment.

After identifying the microorganisms of interest, the incubation program should be designed to detect them effectively. The chosen program should be based on a risk assessment and be justified, ensuring it can reveal the presence of these microorganisms.

The only requirements during audits are that the program demonstrates its ability to detect the targeted microorganisms and that it is validated.

The key to an effective environmental monitoring program is choosing the right incubation temperatures and duration to gather valuable data on the production environment.

After reading this article, if you have any objections or additional questions, feel free to join the discussion by leaving a comment below.