Autoclaving Liquids: Ensure Sterility, Avoid the Burn

There are a number of concepts to be aware of when autoclaving liquid loads. We’ve covered the basics of liquid sterilization in this previous post, as well as challenges related to liquid boil-over. Many labs still struggle to achieve the correct balance between reliable sterility and damage to their products when it comes to liquid loads. Agar broth and other nutrient media can be extremely susceptible to damage if exposed to high autoclaving temperatures for too long and liquid loads are notorious for being a source of sterilization failures derailing experimental results. In this blog we’ll tackle the concept of thermal mass as it relates to sterilization, and a couple of autoclave features that can save time and money on your liquid loads.

First let’s talk about thermal mass and load lag. The goal of sterilization in an autoclave is to ensure that the load achieves a particular temperature for a particular time. Some sterilizers are equipped with a temperature probe that you put into the liquid (often called a load probe), but most control the cycle based on a temperature probe in the drain line. For many types of loads, the temperature in the drain is the coolest spot, however for high thermal mass loads (such as 1 liter+ liquid loads), the liquid will be considerably cooler than the drain temperature. This is because loads with high thermal mass take longer to come up to temperature. At time of writing, Thanksgiving is right around the corner, and this concept of thermal mass applies just as much to a turkey in an oven as it does to goods in an autoclave. A cook can’t rely on the oven temperature to know if their turkey is thoroughly cooked, they typically use a meat thermometer pushed in to the center where they know the bird will be the coolest. same thing happens in an autoclave – the autoclave might be at 250F but the liquid is still coming up to temperature.

This difference in temperature is called load lag – both in heating up and cooling down, the actual load temperature will lag behind the chamber temperature probe. If load lag is not successfully accounted for in cycle design, you may have problems with achieving full sterility of liquid loads.

This graph shows a liquid cycle where a large number of temperature probes were placed either in various points around the autoclave chamber (highlighted in green) or inside of flasks containing liquid volumes (highlighted in blue). The chamber probes quickly rise to the correct sterilization temperature, but there is a period of almost 24 minutes where the liquid hasn’t yet come up to temperature.

This brings us to our second concept, which is that sterilization and the sterility assurance level (SAL) is determined by the time that the load is at temperature – not the time that the sterilizer is at temperature. For example, take a load needs to be at 250F for 15 minutes – if this load has a high thermal mass then it will also experience appreciable load lags. If the sterilizer is only set for 15 minutes, the liquid may not even reach 250F by the time the sterilizer’s 15 minute timer expires. Therefore, the total sterilization time needs to be considerably longer than 15 minutes in order to ensure that the load experiences adequate conditions to achieve the desired SAL. If you’re running a typical liquid cycle without a load probe, then you will need to account for the load lag experimentally. Please note that the effects of load lag can be minimized by reducing the amount of liquid per container and therefore reducing the thermal mass. For example, two 1L containers with 500mL each will experience less lag than a 1.5L container with 1L of liquid – both scenarios have 1L of liquid but spreading it into smaller volumes speeds up the process. To return for a moment to the turkey analogy – multiple smaller birds placed in the same oven and spaced out could be cooked in a shorter amount of time than one large turkey would take to fully cook through.

Validation of SAL is extremely important to confirm that proper sterility has been achieved. The last thing you want to be dealing with in your lab is contamination corrupting results. Assuming you don’t have a load probe or temperature logger, you won’t know whether you have achieved a specific SAL. Rather than rolling the dice, you can establish specific SOPs regarding load types, loading practices as well as sterilization temps/times. To ensure that these combinations produce the desired results, you would want to validate the process using a self-contained biological indicator (SCBI). Recommendations regarding validation can be found in this blog. For a typical liquid load, you would want to use a BI in a sealed ampule with a population of 10^6 of G. Stearothermophilus (something similar to this: https://mesalabs.com/products/sterilization-cleaning-monitoring/magnaamp-biological-indicator). Suspend this ampule in your load, process the load, then incubate the SCBI. Successful inactivation of the SCBI would mean you can rest assured that you have achieved a sterility assurance level of 10-6SAL (10^-6 or 1 in 1,000,000 chance of spore survival) and therefore contamination would be highly unlikely. Validation should also be repeated on a regular basis to ensure that wear and tear on the equipment, changes in microbial load on the goods being sterilized or other changes to the parameters don’t reduce SAL. One last point of caution is that a successful biological indicator test only really applies to the specific load and run parameters used, so loads need to be consistently run at the same volume.

Third and finally, the concept of F0. For some types of liquid loads, excessive total heat can damage certain types of load – for example, sugars could start caramelizing or other desired nutrients of broth/agar/chemical mixture may start breaking down. Stating this another way, you don’t always want to sterilize for 2 hours just to be sure you achieve your full SAL – aside from taking a long time, using more energy/water and adding wear & tear to the equipment, you could negatively impact your load. In this type of situation, you need to carefully balance getting enough heat input to achieve the desired SAL while avoiding any negative impacts to your load. To return one last time to our turkey – leaving the turkey in the oven at full temperature until the very center of the bird is fully cooked can often leave the outer portions completely burned or dried out, so some creativity must be employed to ensure that the entire mass is cooked thoroughly.

The rule of thumb of 15 minutes at 250F is not a magic sterilization combination, you can achieve similar results with shorter times at higher temperatures or longer times at lower temperatures. Stating this a different way, there is some inactivation of microbiological contamination occurring as the load heats up.

An F0 cycle, accounts for and adds up all of this partial credit as the load is heating up using a mathematical formula, thereby reducing the overall cycle time and minimizing the heat input into the load. In many cases, this approach isn’t needed; but this principle of sterilization may be critical for certain applications. Depending on the autoclave make & model, load probe & F0 capabilities may already be present or could be retrofit. However, depending on your application, you may not need (nor want) either of these features. Another easy way to reduce cycle time and potential damage to the load for liquid cycles is to reduce the density of the load; a single 10L volume would take much much longer to sterilize than 20 spaced out 500mL flasks.

In summary, the larger the liquid volume in each container being sterilized, the larger the load lag will be and the more additional time will need to be added to the cycle to account for that. Validation of cycle design using Biological Indicators is important for all loads but especially so for high thermal mass loads or other types of critical loads. Lastly, some additional options like Load Probes and F0 cycles may be used to optimize liquid cycles.