Media fill testing: Ensuring Aseptic Processing for Client Projects and Licence Applications. Blog Post by Natasha Tal

August 22, 2025

Running a lab doesn’t just mean performing experiments and getting results. One of the many important aspects of being a properly functioning lab includes verifying the performance of lab procedures, and staying up-to-date with regulatory affairs. One of the many ways SteriLabs ensures the effectiveness of their testing procedures is with media fill testing.

 

What is media fill testing?

Media fill testing, also known as aseptic process simulation, is a technique used to assess the effectiveness of aseptic techniques used by staff performing microbiological processes. It is required by various standards and licenses, such as a Drug Establishment License (DEL), to give evidence of good manufacturing practices (GMP). Health Canada issued various guiding documents for GMP, including the details for media fill testing found under Annex 1 to the Good manufacturing practices guide - Manufacture of sterile drugs (GUI-0119) (Health Canada, 2024). This document is referenced in the Drug GMP and DEL Enforcement Policy (POL-0004), used to guide companies when applying for a DEL (Health Canada, 2025).

 

In general, media fill requires testing some of the procedures that a lab uses, and analyzing if the staff is properly following aseptic techniques for that procedure. For example, at SteriLabs, direct inoculation of medical devices and membrane filtration are the procedures most commonly used for sterility testing, as per USP <71>, so those are the techniques that are simulated for media fill testing.

 

What are the steps to performing media fill testing?

Media fill requires performing a technique such as direct inoculation or membrane filtration for sterility testing, omitting the use of samples, and instead using sterile media and controls. The staff member being tested will prepare sterile media, such as tryptic soy broth (TSB) or tryptic soy agar (TSA), and any other necessary sterile reagents, such as Fluid A, a rinsing reagent used for membrane filtration. The process is started by transferring any necessary tools or reagents to a biological safety cabinet (BSC).

 

To perform the test for direct inoculation, sterile bags are filled with sterile TSB (as if part of a sterility test), following aseptic procedures. Membrane filtration is performed in a similar manner, using sterile Fluid A as a rinsing agent to be pumped through a membrane filter by a vacuum pump, instead of using a sample. This membrane filter can then be plated onto TSA (see Figure 1), or placed into a tube containing TSB.

 

In addition, the inclusion of positive and negative controls are necessary to validate the results, and ensure that the experiment and materials are functioning properly. All bags are incubated at 32-35˚C for 14 days, before observing any results.

 

What do the results mean?

The target is to observe no microbial growth in the samples, resulting in a passed test. If growth is observed (turbidity in liquid media or colony formation on solid media), this indicates that the sample has been contaminated during the procedure simulation, and aseptic techniques are not being followed. If a test fails, there must be an investigation into the cause of the contamination, appropriate actions to correct any errors in processing and the test must be successfully repeated a minimum of three times before continuing any related procedures.

 

Media fill testing is one of many ways to give evidence of good manufacturing practices in a laboratory. Additional tests such as glove print testing or environmental monitoring may also be performed to demonstrate the verification of aseptic processing in a laboratory space. When applying for a DEL, the test results may be included to demonstrate that a lab complies with GMP and validated aseptic techniques. Ultimately, passing a media fill test demonstrates that the lab staff are effectively following aseptic techniques, which allows clients to place trust in their work. It shows their commitment to product sterility, regulatory compliance, and ultimately, patient safety.

 

Figure 1. Media fill test performed through membrane filtration showing no growth after the incubation period.

References:

Health Canada. (2024, May 21). Annex 1 to the Good manufacturing practices guide - Manufacture of sterile drugs (GUI-0119). https://publications.gc.ca/collections/collection_2024/sc-hc/H139-49-2024-eng.pdf

Health Canada. (2025, January 21). Good manufacturing practices guide for drug products (GUI-0001). Canada.ca. https://www.canada.ca/en/health-canada/services/drugs-health-products/compliance-enforcement/good-manufacturing-practices/guidance-documents/gmp-guidelines-0001/document.html#appc02