The Unique Autoclave Load: Sterilizing Liquid Media

When it comes to the steam sterilization of liquid media, there are a number of user concerns that are handled in various ways. Monitoring these cycles with biological indicators (BIs) and the various restrictions and cycle modifications that are done can lead to a false positive or failed cycle. Here are only a few of the cycle modifications done or restrictions applied by users for the steam sterilization of liquid media:

In case of boil-over, I want to keep the media flask in a container while being sterilized so the boil-over does not get all over my autoclave.

According to the manufacturers instructions, Im to sterilize the media at 121 degrees C for 15 minutes. I cant find a BI that will die in this short cycle.

  • I cant run a longer cycle time since the media is heat sensitive and may not promote growth if a longer cycle time is used.
  • Our protocol states to run only in a 15 minute, 121 degrees C cycle.
  • We have determined a cycle exposure time for a 1-liter flask of TSB.

Now, what time should I use for a 2-liter flask, double the time?

As one can see, many unique questions arise when working with liquid loads that would never come up if the cycle was for hard goods or wrapped goods. Most of the items would not be temperature sensitive; we need not worry about boil-over or question BI placement.

Yet with liquid loads we do not have to be concerned about pre-vac air-removal or steam penetration as into a porous load. So, why do so many problems occur with liquid media sterilization and BI lethality?

Some of the methods used to address the concerns expressed above actually can contribute to a failed cycle or a cycle where the BI is positive. This largely happens due to the fact that most of the liquid load/media cycles used in many clinics, universities or hospitals did not actually validate their load configurations or media cycles. Validating a load configuration cycle provides 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 11139:2001). With the sterilization of liquid media we want to produce a specific media with certain qualities such as pH range, growth promotion ability and sterility.

Performing a validation of a selected exposure time/temperature liquid media cycle to be used, one would determine how long it takes from the start of the cycle to get the media up to 121degrees C. This would be recorded as our come-up time required for that particular cycle. If this was done with 1-liter flasks of media and we wanted to know the time required if the chamber was for 2-liter flasks, a separate validation would be needed for cycles to include larger volume flasks.

To determine come-up time for a load of two 1-liter flasks of TSB, one could use a temperature recorder that could actually be put into the flask of media and record the medias temperature during the entire cycle duration. Knowing what time the cycle was started and what time the media actually reached a temp of 121 degrees C would tell us how long we can expect our come-up time to be. We would then repeat this activity for at least two more cycles with each containing newly prepared flasks of media. The come-up times of all three cycles should be very similar. If, for example, the come-up times for the three cycles to hit a temperature of 121 degrees C were 16 minutes, 12 minutes, and 14 minutes respectively, we could compare this recorded data and determine that a worst-case come-up time would be 16 minutes.

We now have data that we can use to establish that with a certain number of 1-liter flasks of a particular media we can expect that the temperature of the media in the flask will reach a minimum temperature of 121 degrees C within 16 minutes of the start of the cycle. The number of flasks and the volume in each along with their position placement within the autoclave chamber should be documented and must remain the same for additional cycles used in the future unless a continued validation is performed using other placement areas or fewer flasks. We want to validate a worst-case load so we will be able to predetermine that all other loads will also hit our 121 degrees C temperature within the specified 16-minute come-up time.

A very easy-to-use thermistor or data logger for recording the medias temperature during a sterilization cycle such as the one pictured (figure 1) can easily be placed inside the actual flask of media. By doing so one can determine the actual temperature of what one is trying to sterilize. Placing the data logger on a shelf inside the autoclave chamber would not tell you what is actually going on inside the media flask. The logger must actually be placed inside the media flask. To demonstrate the very large temperature difference between chamber and media come-up time, a data logger was just placed inside the autoclave chamber while another was placed inside the media flask.

The cycle was set for 20 minutes and both temperatures were recorded simultaneously during the exposure.

One can see in figure 2 that the chamber reached the temperature of 121 degrees C within 4 minutes while the temperature of the media flask did not reach a temperature of 121 degrees C until 19 minutes into the cycle. If this 20-minute cycle were to be used for the media sterilization, the media would only have been at the sterilization temperature of 121 degrees C for approximately 1 minute. If a BI ampoule would have been placed inside the media flask during the cycle, it would not have been killed. Thus if a 15-minute cycle had been used, the media would have only reached 117 degrees C by the cycle end. Obviously this is not a suitable cycle for this load.

USP clearly states that with media sterilization of 121 degrees C for 15 minutes, the 15-minute exposure time is for the media temperature once it hits 121 degrees C. Therefore, if it takes 20 minutes for the flask of media to reach 121 degrees C, it must now remain there for a full 15 minutes. This makes a total cycle time of 20 minutes for comeup and 15 minutes for exposure or a full 35 minutes for that cycle. Anything less and you are not following the manufacturers instructions for media sterilization.

During a liquid-loads cycle, upon steam charge, the steam supply enters the autoclave chamber usually from an inlet located in the upper rear of the chamber. The steam is to push forward and down to gradually push all the air out of the chamber and down the drain. The replacement of air with steam is necessary so that a high steam quality environment exists and the transfer of heat will be much more efficient with steam being the conveyor of energy transfer. Air is an extremely poor conductor for heat transfer, especially when compared to steam transfer. In this situation, if air is still present in the chamber, it will act as an insulator and actually reduce and slow the transfer of heat energy. If the media flask is placed into a container to catch boiloff from the flask, air can be trapped in the container and insulate the lower portion of the flask from efficient heat energy transfer. Steam is trying to replace the air but cannot push the air out of the container and may actually push the air down and compact it so it remains in the container protecting the flask from getting efficient heat transfer. Thus the flask will take much longer to heat up due to being placed into a container and may result in a failed cycle.

If boil-over is a real concern, place a low height pan (like a cookie sheet) under the lower rack of the autoclave chamber. The pan will still catch boil-off and will also be under the media flask without protecting it. Better yet, if it is possible to reset some of your cycle set points, slow down the post exposure exhaust ramp so that pressure is released at a slower rate and boil-over will be eliminated.

Running your media in a longer cycle will likely not adversely affect your media and growth promotion abilities. After all, running a 35-minute cycle where it takes a 20-minute come-up time would actually be a 15-minute cycle at 121 degrees C. If one is in doubt, run this cycle and then test your media and verify that the media still performs as expected and that growth promotion has not been compromised.

Running different volumes per flask or different numbers of flasks in a cycle are situations where additional validation work would need to be done. Running 1-liter flasks may need a 35-minute cycle where several 2-liter flasks may need additional time for come-up and one may need a 45-minute cycle. By using a data logger, the various exposure times needed to get differing volumes of media up to 121 degrees C can easily be determined.

Russ Nyberg is director of technical support for the Omaha, Neb.-based biological indicator manufacturing facility of Raven Labs, a division of Mesa Laboratories, Inc. He can be contacted at (800) 728-5702 or via email at russ@ravenlabs.com

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