Cracked Agar Plates and Shrinkage – The Causes and How to Address Them

Agar plates are prone to dehydration. The combination of warm, dry air and limited moisture content to achieve a solid finish leads to inevitable drying out and desiccation. Cracks in the media, a loss of volume, or shrinkage away from the sides of the Petri dish are evidence of dehydration, which greatly reduces the capability of the media to support the growth of organisms. The reduction in growth support capability is particularly so if the microorganisms deposited on the surface of the media are stressed.

Why desiccation is an issue

Microorganisms have three basic requirements for growth — a food source, ideal growth temperature, and moisture. Reducing the moisture content may inactivate and/or inhibit growth. The purpose of monitoring for microbes using solid media is to capture microorganisms from the manufacturing environment and create favourable conditions to support their optimum growth, thus allowing them to replicate and form visible colonies for inspection. Dehydration of media is considered a significant quality issue as it can lead to the inhibition of growth and/or cell death, leading to inconclusive data for a critical sampling point. 

Cleanroom environments are, by their very nature, high-airflow facilities, which is a primary reason for the loss of moisture from agar plates.

How does desiccation occur?

1. Aggressive airflow at exposure – High airflow and a lower humidity increases the evaporative effect from the surface of the media.
2. Hot and dry air – Exposure to hot (warm) and dry air increases the rate of evaporation.
3. Aggressive fan units in incubators – Plates should be inspected for desiccation post-exposure and before incubation, as well as post-incubation when reading takes place during qualification.
4. Additives – The addition of neutralisers forms chemical reactions within the media, leading to reduced strength and eventually shearing of the media and crack formation.
5. Storage time and conditions.
6. Broad temperature range changes – Rapid and extreme temperature changes in storage and in-use can lead to excess water loss from media. 
7. The concentration of agar in the plate.
8. Consistency and variability of the supplier's manufacturing process.
9. Particle bounce – Occurs in higher particulate environments and, as such, is not considered a problem for low bioburden cleanroom facility or aseptic manufacturing site.
10. High doses of irradiation – High doses of irradiation can lead to chemical alteration (oxidation) of the media.
11. Failure to validate exposure times – Not all media are created equal or will be suitable for use on your site.

How to avoid desiccation?

If you are facing issues of desiccation, below are several suggested remedies to be implemented to ensure no impact to the monitoring for microbiological control.

Short term solutions

• Primarily, assess whether any of your alternate approved suppliers, on-site supplies, or media types can be used.
• Double up on a duplicate plate at your monitoring locations when possible.
• Decrease the exposure time of the media and change your media out more frequently.
• Beware of media stored up to their expiry date, as they are potentially at higher risk of desiccation.

Medium term solutions

• Investigate the layout of your incubator shelves.
• Get a review of the fan speed settings on your incubators to determine possible optimization. 

Longer term solutions

• Approach your supplier and ask them to support your efforts to solve the desiccation issues.
• The implementation of humidity changes can improve the conditions during incubation, but bring with them their own set of challenges and contamination risks.
• Consider changes to your local manufacturing environment where requalification or validation of a new facility may have led to airflow and environmental condition changes. 
• An alternate media supply may be the only way to address the desiccation issues. 

Conclusion

In this review, we have identified various contributory factors that may be a cause for the desiccation of solid culture media. To summarize, the inherent nature of the cleanroom environment and the associated need to expose media to aggressive airflows, coupled with formulation sensitivities in the manufacturing of the media, can result in a product not suitable for use at your site. However, with this knowledge, when assessing media for use on-site, better care can be taken in ensuring suitability for the unique environment you are managing.

PMM, PharmaMedia Dr. Müller GmbH, addresses these challenges by manufacturing under the strictest controlled conditions. Both agar strength and pouring temperature are carefully regulated within a Class A cleanroom environment. The specialized packaging used by PMM provides a physical barrier against airflow and temperature fluctuations, ensuring optimal product stability.

View our assortment of PMM plates for cleanroom environments and detection of aerobic and anaerobic micro-organisms.

For more information and support, please contact Peggy Roth at peggy.roth@nordicbiolabs.se.