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Autoclaving is an important step to ensure your culture media is sterile and ready to culture your target organisms. But autoclaves are complicated machines and there are many ways they can fail, due to problems with the autoclaves themselves or to user error. When these failures happen, the consequences can range from unsterilized media and unexpected visitors in your cultures to loss of entire media batches. Checking for and preventing the common problems listed below can help to ensure that your culture media is sterilized and ready for use, and that valuable resources aren’t being wasted.
The autoclave fails to reach the correct pressure/temperature
To properly sterilize culture media, the autoclave needs to generate a temperature of 121°C and a pressure of 14-16 PSI. If these conditions aren’t reached, contaminants can survive to grow and ruin your media. Reasons that the autoclave may not reach the correct temperature or pressure include:
The gasket is no longer creating a seal, allowing steam, pressure and heat to escape. As gaskets can last from a few months to a few years, this can be a common cause of failure. To prevent this problem, perform visual inspections of the gasket and clean the gasket groove and the sealing surfaces of the door on a regular basis. Lubricate the gasket when it appears to be dry with a lubricant designed for high pressures and temperatures. Keep a spare gasket on hand and replace the gasket on the schedule recommended by the manufacturer.
The steam generator isn’t working properly. The steam generator could be turned off if it’s on a separate power source, or there could be damage that will need to be repaired. Ensure that the steam generator is turned on before starting a run, and contact a maintenance professional if needed.
There is a clog in the chamber drain. Clogs can prevent air from escaping the chamber and steam from reaching the ideal temperature and pressure. To prevent clogs, always check the drain for objects that may be blocking it before starting a run. Follow proper procedures to prevent containers from exploding or overflowing in the autoclave (see below), and clean up any spills as soon as it is safe to do so, to prevent agar from hardening in the drain.
There is not enough water in the autoclave. The autoclave needs water to generate steam for sterilization. Without it, the correct temperature and pressure can’t be reached. Always check to make sure that the water is filled to the proper level before starting a cycle.
After starting a cycle, you should always wait until the autoclave has reached the correct temperature and pressure before leaving it to complete the run. Make sure to check on any alarms that may sound to indicate a problem with the cycle. Autoclave tape, which changes colors at 121°C, should be put on each item in your batch to help visually confirm that the temperature was reached during the cycle.
Regular maintenance and calibration of the autoclave, along with thorough training for all personnel involved in media preparation, are essential to prevent lapses in sterilization.
The media doesn’t reach the correct temperature
Even if your autoclave reaches 121°C during the cycle, this doesn’t necessarily mean that all your media have also reached this temperature. There are several reasons that the media might not get hot enough to kill off contaminants.
The autoclave is too full. Overfilling the autoclave can inhibit airflow and block steam from reaching the center of the batch. Larger batches may need a longer autoclave time to thoroughly sterilize the entire batch. Always follow the manufacturer’s guidelines when loading the autoclave to ensure that all of your media is reaching the correct temperature.
The autoclave time is incorrect for the media volume and type. Larger volumes of media heat more slowly than smaller ones, and the center of a large volume may not reach temperature in a typical 20 minute run. Media with high salt content and more viscous liquids such as agar will also take longer to heat thoroughly. It is important to select the correct time to autoclave your media, as autoclaving too long can overcook and damage it. Consult your manual or protocol for the correct time to use.
Using the wrong tray/not adding water to the tray. Optimizing the heat transfer during the run can help to improve media heating. Plastic autoclave trays conduct heat more slowly than metal ones. Additionally, not all types of plastic can be autoclaved and some can melt during the cycle. Metal trays are recommended for optimal heating. If you use a plastic tray, always make sure that it is autoclave safe. Adding 1-2 cm of water to the bottom of the tray will improve the heat distribution around your bottles or flasks. These steps will help your media reach temperature more quickly and ensure proper sterilization.
Validation samples should be run in the autoclave at least once per month to test whether the media is being properly heated and sterilized. The samples should be autoclaved under the same conditions as your regular batches – that means in the same size batches, in the same media volumes, and in the same tray type as usual. The validation sample should be placed in the middle of the batch in the autoclave. The validation samples can then be tested for growth of the known organism to see if your media is truly sterile.
Overflows and explosions
Improper handling of bottles and flasks can cause your media to overflow or even cause the bottle to explode, wasting media and potentially clogging the autoclave drain. Causes of overflows and explosions include:
Using the gravity or dry cycle for media. The gravity cycle releases steam rapidly during the exhaust phase, which can cause media to overflow. The liquid cycle releases the steam more slowly, preventing overflows and keeping your media in the container.
Overtightening bottle caps. Bottles that are capped too tightly will build up pressure when their contents heat and expand, causing overflows or explosions. Bottle caps should be tightened no more than halfway to allow pressure to escape. Flasks should be covered with foil rather than stoppered for the same reason.
Overfilling containers. As explained above, media expands when it is autoclaved. Overfull bottles or flasks are likely to overflow. Containers should never be more that ½ full when autoclaving media to prevent overflows.
Using an autoclave tray as described above will not only help your media heat more quickly and evenly, but will also catch any overflows that may happen. If you do have a bottle explode or overflow, carefully clean the autoclave to prevent further problems.
Learn more
By following these steps to prevent autoclave failures, you can help ensure your media is optimal for culturing, achieve accurate results, and reduce wasted money and time from lost batches and rework. Your lab may choose to skip the hassle of autoclaving altogether by choosing prepared culture media that is ready to use for microbial culture.
Read our Media Preparation Guide for more tips on autoclaving media and other advice to help you:
Prepare the best quality media every time
Avoid failed media batches
Avoid atypical colonial morphology or reduced shelf-life
Maintain efficiency in your test processing
