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When sterilizing liquid media with steam, the concerns of users are approached in various ways. Monitoring the performance of the cycles with biological indicators (BIs) and applying various limitations and cycle modifications can lead to a false positive or a failed cycle

Image Credit: Duraline BioSystems, Inc.

Below, you can find a selection of the cycle modifications carried out or limitations applied by users of the steam sterilization of liquid media process:

A number of diverse questions come up when working with liquid loads that typically never arise when the cycle is for wrapped or hard goods.

Most of these items would not be temperature sensitive, so there would be no need to worry about boil-over or questioning of BI placement. However, when working with liquid loads, there are no concerns about pre-vacuum-air-removal or steam penetration as found with a (solid) porous load.

So, why are there so many issues that crop up when approaching liquid media sterilization and BI lethality? Some of the methods employed to tackle the above concerns can, in fact, contribute to a failed cycle or a cycle where the BI is positive.

This generally happens as a result of the fact that most of the liquid load/media cycle configurations employed in most clinics, universities or hospitals were not verified.

Verification of a load configuration cycle offers a “documented procedure for obtaining, recording and interpreting the results required to establish that a process will consistently yield product complying with predetermined specifications” (ISO/TS 11139:2006, Definitions 2, 55 2)

When sterilizing various media, the intended outcome is producing media with a specific pH range, growth promotion ability, sterility, etc.

When conducting validation of a specific exposure time/temperature liquid media cycle to be used, it needs to be established how long from the start of the cycle it takes for the media to reach 121 °C.

This would be noted as the necessary ‘come-up’ time for that particular cycle. If this was performed using 1 L flasks of media, an additional validation would be needed for cycles comprised of larger volumes (2 L flasks, for example).

To establish the ‘come-up’ time for a load of two 1 L flasks of TSB, a temperature recorder could be introduced into each flask of media to record the media’s temperature throughout the entire cycle duration.

It would be necessary to repeat this activity for at least two more cycles, with each containing freshly prepared flasks of media. Consequently, the ‘come-up’ times of all three cycles should show as being similar.

If, for instance, the ‘come-up’ times for the three cycles (to hit a temperature of 121 °C) were 16 minutes, 12 minutes and 14 minutes respectively, this data could be used for a contrast analysis, and it could then be established that the worst-case come-up time would be 16 minutes.

There is now supportive data that confirms, with a specific number of 1 L flasks of a particular media, to reach the media temperature of 121 °C will take around 16 minutes from the start of the cycle.

The number of flasks and the volume in each and detailing their position/placement within the autoclave chamber should be recorded. They must stay the same for future cycles unless a continued validation is conducted using other placement areas or fewer flasks.

It is crucial to validate a worst-case load to predetermine that all other loads should reach a temperature of 121 °C within the specified 16-minute ‘come-up’ time.

A simple thermistor or data logger for recording the media’s temperature throughout a sterilization cycle can be readily placed inside the actual flask of media. By doing so, it is possible to determine the actual temperature of what is being sterilized.

Merely placing the data logger on a shelf inside the autoclave chamber would not show what is happening inside the media flask. The logger should be placed directly inside the media flask.

It is not likely that running media in a longer cycle will adversely affect the media and growth promotion abilities. Running a 35-minute cycle with a 20-minute come-up time would result in a 15-minute cycle at 121 °C.

If there are issues surrounding cycle length, run the cycle and then test the media and validate as necessary to confirm that it performs as expected and that growth promotion has not been jeopardized.

Running various volumes per flask or different numbers of flasks in a cycle are scenarios where additional validation work would be necessary. A 35-minute cycle may be suitable for running 1 L flasks, but for several 2 L flasks, more time may be needed for come-up, and therefore, a 45-minute cycle or more may be required.

By using a data logger, the different exposure times needed to get varying volumes of media up to 121 °C can be determined easily.

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Last updated: May 20, 2022 at 8:40 AM

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