Sterilization of Culture Materials

All vessels used for culture purpose should be scrubbed (abrasive brushes not appropriate for most plastics) and soaked with warm detergent (not domestic detergents, which leave a residual film on culture ware, but phosphate-free laboratory detergent), then rinsed extensively with tap water. After soaking in 10% HCl for 1 day–1 week (not routinely necessary, but particularly important for new glass and polycarbonate material), vessels should be rinsed extensively with distilled and finally bidistilled water, and left inverted to dry in a clean, dust-free place, or in an oven.

Sterilization can be defined as a process which ensures total inactivation of microbial life, and should not be confused with disinfection, which is defined as an arbitrary reduction of bacterial numbers. The primary purpose of sterilization is to prevent contamination by unwanted organisms, but it may also serve to eliminate unwanted chemicals. Sterilization can be obtained by means of several methods, the choice depending on the purpose and material used, either empty glassware/plasticware or medium-containing vessels, but also on the facilities available in a laboratory.

Several methods are available for sterilization of material, some of which can be used also for growth media:

• Gas such as ethylene oxide, EtO, finds the best application on heat sensitive equipment on which steam autoclaving (sterilization with heat) cannot be performed. EtO sterilizes by alkylation, it substitutes for hydrogen atoms on molecules such as proteins and DNA, and, by attaching to these molecules and disrupting them, EtO stops these molecules’ normal life-supporting functions. This method is widely used for the sterilization of medical devices, but it is not a routinely available technique for algology laboratories. Moreover, EtO is a potent carcinogen.
• Dry heat: some laboratories use dry heat to sterilize empty vessels, putting the material in an oven for at least 3–4 h at 160°C; however, only higher temperature (200–250°C) guarantees an effective result. Vessels are covered with aluminum foil to maintain sterility on removal from oven. This procedure is suitable only for few materials that stand high temperatures, such as glass, teflon, silicone, metal, and cotton.
• Autoclaving (moist heat) is the most widely used technique for sterilizing culture media and vessels, and is the ultimate guarantee of sterility (including the destruction of viruses). A commercial autoclave is the best, but pressure cookers of various sizes are also suitable. Sterility requires 15 min at 1–2 Bar pressure and a temperature of 121°C in the entire volume of the liquid (i.e., longer times for larger volumes of liquid; approximately 10 min for 100 ml, 20 min for 2 l, 35 min for 5 l). Flasks containing media should not be more than half full, and should be left partially open or plugged with cotton wool or covered with aluminium foil or paper, because for sterility the steam must penetrate the material. Autoclave steam may introduce chemical contaminants; empty glass and polycarbonate vessels should be autoclaved containing a small amount of bidistilled water which is poured out (thus diluting contaminants) under sterile conditions immediately prior to use. Vessels should never be closed, because of the risk of implosion, by using cotton wool bungs, or by leaving screw caps slightly open. Ensure that heating elements are covered with distilled water, and the escape valve should not be closed until a steady stream of steam is observed. After autoclaving, the pressure release valve should not be opened until the temperature has cooled to below 80°C. Autoclave steam may contaminate the media (i.e., with trace metals from the autoclave tubing). Autoclaving also produces leaching of chemicals from the medium receptacle into the medium (silica from glass bottles, toxic chemicals from plastics). Autoclaving in well-used Teflon or polycarbonate vessels reduces leaching of trace contaminants.
• Pasteurization (heating to 90–95°C for 30 min) of media in Teflon or polycarbonate bottles is a potential alternative, reducing the problems of trace metal contamination and alteration of organic molecules inherent with autoclaving. Pasteurization does not, however, completely sterilize seawater containing media; it kills all eukaryotes and most bacteria, but some bacterial spores probably survive. Heating to 90–95°C for at least 30 min and cooling, repeated on two or more successive days (“tyndallization”) may improve sterilization efficiency; it is assumed that vegetative cells are killed by heat and heat resistant spores will germinate in the following cool periods and be killed by subsequent heating.
• Ultraviolet radiation (240–280 nm) is not often used for culture materials, because very high intensities are needed to kill everything in a medium such as seawater (1200 W lamp, 2–4 h for culture media in quartz tubes). Such intense UV light necessarily alters and destroys the organic molecules in seawater and generates many long lived free radicals and other toxic reactive chemical species (Brand, 1986). Seawater exposed to intense UV light must, therefore, be stored for several days prior to use to allow the level of these highly reactive chemical species to decline.
• Sterile filtration is probably the best method of sterilizing certain media, especially seawater-based media, without altering their chemistry, as long as care is taken not to contaminate the seawater with dirty filter apparatus. Sterilization efficiency is, however, to some extent reduced compared with heat sterilization methods. Membrane filters of 0.2 µm porosity are generally considered to yield water free of bacteria, but not viruses. 0.1 µm filters can be used, but the time required for filtration of large volumes of culture media may be excessively long. The filtration unit must be sterile: for small volumes (<50 ml) pre-sterilized single use filter units for syringe filtration (e.g., Millipore Millex GS) can be used; for volumes up to 1 l reusable autoclavable self-assembly filter units (glass or polycarbonate) with 47 mm cellulose ester sterile membrane filters (e.g., Millipore HA) can be used with suction provided by a vacuum pump; for larger volumes an inline system with peristaltic pump and cartridge filters may be the best option. Filter units (particularly disposable plastic systems) and the membrane filters can also leak toxic compounds into the filtrate. The first portion of filtrate (e.g., 5% of the volume to be filtered) should be discarded to alleviate this problem.
• Most stock solutions of culture medium additions can be sterilized separately by autoclaving, although vitamin stock solutions are routinely filtered through 0.2 µm single use filter units (e.g., Millipore Millex GS), because heat sterilization will denature these organic compounds. Filter sterilization of all additions may reduce uncertainties about stability of the chemical compounds and contamination from autoclave steam, but absolute sterilization is not guaranteed. Stock solutions can be stored in ultraclean sterile glass, polycarbonate, or Teflon tubes/bottles. In order to minimize effects of any microbial contaminations, all stock solutions should be stored in a refrigerator at 4°C, except vitamin stocks which are stored frozen at –20°C and thawed immediately prior to use.