Sterilization

Autoclaving

We use autoclaving as the prime method for sterilizing media and particularly for making media for axenic cultures. Many of our media recipes refer to fully autoclaved media, where the last stage in media preparation is for individual culture flasks to be autoclaved so that the flasks remain totally unopened until culture transfer. However where maintaining axenic cultures is not as critical then filter sterilizing particular components of the media and adding them aseptically to presterilized seawater (or freshwater) can be used. Concentrated nutrient solutions can be prepared in this way so that phosphate stocks do not precipitate and vitamin activity is not impaired.

While autoclaving at 121 ⁰C for 15 minutes appears to be a common mantra, the required duration of autoclaving can cause some confusion. It should be noted that the autoclave time refers to the duration the goods or total volume of liquid is maintained at the set temperature, and not simply the time in the autoclave or the time after 121 ⁰C is reached. Autoclaves come with different chamber sizes and with different capacities for pressurization, air removal, vacuum generation, steam generation and air exhaust, and all these factors influence the rate of temperature increase, holding capacity and decrease. Therefore it is difficult to give predefined autoclave times for anything other than hard goods such as pipettes and empty glassware or containers with up to 100mL of media where the typical 121 ⁰C for 15 minutes is probably appropriate. Note, however that a carboy with 10 L of media may require in excess of 60 minutes once the autoclave reaches the set temperature of 121 ⁰C. The only reliable means of monitoring temperature in bulk media is to have a thermistor within a reference container (e.g. PT100) and this would only have to be done periodically to “calibrate” the system. Relatively small benchtop pressure cookers may be the sole autoclave in many aquaculture facilities or used as backup autoclaves in laboratories. In this case, where for example the largest container that will fit may be a 1 L culture bottle, it is rare for more than 800 mL to be autoclaved and in this instance a time of 25-30 minutes may be appropriate. Some confusion also exists around pressure and steam, with a perception that these factors are important in lethality. This is not true, temperature above 100 ⁰C is the lethal factor and pressure and steam are simply the means of delivering that temperature to the entire contents of the autoclavable goods. Without steam, air pockets and voids can exist in both glassware and within autoclave bags and these can persist at lower temperatures than the chamber temperature but steam penetration into these voids ensures the the required temperature is met.

Autoclaving drives off carbon dioxide, needed for algal photosynthesis, and raises the pH to undesirable levels.  Leaving the media for at least 1-2 days before use will allow sufficient time for CO₂ equilibration.

Autoclaving is also a mandatory method for disposing of our microalgal stock cultures under our Approved Arrangement (provided by the Department of Agriculture, Water and the Environment, DAWE). Minimum draft regulations set in June 2004 are 121 ⁰C at 15psi for 30mins. Note these guidelines are more strict than those for preparing media. Other countries may have other regulations. This section does not consider alternative methods, e.g. chemical, for culture waste disposal as these may vary with regulatory compliance standards imposed in different countries. In Australia each State and Territory has waste disposal operators certified to remove laboratory biological waste. In ANACC these operators would only be called upon if there was a lengthy delay in the maintenance or replacement of the laboratory autoclave.

Filter sterilization

While autoclaving is the preferred methodology for ensuring zero viability of cultures pre disposal, it is not necessarily the optimum method for preparing media in situations where sterility is secondary to attaining acceptable algal growth. Convention suggests that 0.2 μm filter sterilization will exclude all culturable bacteria in downstream media without vitamin denaturing and other unwanted chemical reactions that may occur during autoclaving. If algal strains are showing poor growth in autoclaved media then test using filter sterile media before necessarily changing media type. Various 0.2 μm filter systems from syringe filters to cartridge formats, in pre-sterile disposable units to reusable autoclavable units are available to filter several mL’s to many litres of the base water or fully formed media. Bacteriophage and viruses are not effectively excluded by 0.2 μm filtration. While filters certainly exist with smaller nominal pore sizes, the achievable flow rates diminish and generally the cost per unit increases. The type of filter format and its composition (out of scope here) has a large bearing on the efficiency of particle retention, the filter flow rate and volume that can be passed. Filters may work via positive pressure or a vacuum source and ideal flow rates will be achieved within specified pressure levels. These properties directly influence filter failure. Filtering (i) excessive volumes or (ii) too high a pressure (ii often occurs due to i) will increase the potential for filter failure which is not easy to detect routinely (e.g. via the bubble point test). At laboratory culture scale, doubt surrounding filter integrity in the absence of routine testing is one reason why autoclaving may be preferential, while at larger production scales (e.g. aquaculture) filter sterilization may be the only logistically viable means of sterile media production.