A critical factor in the success of a plankton culture system is the proper sterilization of both culture vessels and any solutions that go into them. Such sterilization prevents the overgrowth of the target species with microbial contaminates. Such contaminates may include undesirable bacterial, fungal, and protozoan species. Microbial contaminates may exert deleterious effects on the target species via predation, release of toxins, or through secretion of harmful metabolic byproducts and competition for nutrients and space. Contamination of rotifer and microalgae cultures by certain species of dinoflagellates has been shown to be a major factor in the mortality of clownfish larvae in hatchery settings. At the very least, maintaining a clean and sterile culture system will go a long way in producing reliable production levels of planktonic or larval organisms and will help speed the troubleshooting process when things go wrong.
Below are descriptions of various methods used in aquaculture hatcheries, as well as an explanation of their typical applications, limitations, and in some cases, links to protocols for implementation.
STERILIZATION BY MEMBRANE FILTRATION:
Primarily utilized for small culture volumes, membrane filtration provides a high level of sterility while being extremely gentle to water chemistry. Sterilization is accomplished by forcing liquid through a filter that has a defined pore size, typically either 0.45 or 0.22 microns. This allows for the elimination of bacteria and fungi, which are too large to fit through the pores, without modification of the chemical constituents of the culture media. Effective for bacterial, fungal, and protozoan species, these filters are not generally effective against viruses as these are small enough to pass easily through the pores.
Membrane filters typically come in two flavors:
Syringe filters are handy for sterilizing very small volumes of liquid, less than 100ml. These filters come in convenient disposable cassettes that attach to the tip of syringes. Liquid is forced from the syringe through the filter and the exiting solution is sterilized.
Vacuum filtration setups are effective for larger volumes, up to a liter. These allow the culture media to be pulled through a larger filter membrane via a vacuum pump. The sterilized culture media is collected in a receiver vessel, often a side-arm Erlenmeyer flask. It is important to note that for either of these methods, all downstream vessels and apparatus that contact the sterilized media must be sterile themselves. Thus, it is common to use these methods only for making small stocks of solutions such as f/2 that are sensitive to other forms of sterilization, and may be stored in pre-sterilized, disposable containers.
Heat sterilization, when properly performed, can be among the best methods of sterilization. However, many desirable constituents of culture media may be temperature sensitive and can be destroyed by heat. Most notable are vitamins, fertilizers, and antibiotic solutions, which are typically filter sterilized and added to heat sterilized media after it cools. In addition to being damaging to additives, high temperatures can cause undesirable precipitation of a variety of constituents of seawater, especially as temperatures approach boiling. Such precipitation may or may not have adverse effects on the culture, depending on the species and conditions that are utilized.
Autoclaving is the most common and effective method for sterilizing moderate amounts of material, especially in a laboratory setting. Requiring specialized equipment, material is heated under pressure in the presence of steam. Given an adequately long exposure time, this is an effective method of destroying bacteria, fungi, spores, and viruses. The most common exposure conditions are 121 degrees C at 15psi. Similar levels of sterility can be attained in a pressure cooker without the expense of an autoclave.
Boiling is a moderately effective method of sterilization. It does a good job of killing most bacteria, viruses, and fungi. However, it often is not successful at destroying the environmentally resistant spores produced by some species. To ensure complete killing of spores, it may be necessary to boil the medium on 2 or 3 consecutive days, allowing the medium to cool between treatments.
Pasteurization is not quite as assured a method for complete destruction of microorganisms as autoclaving, but properly executed can reach nearly the same kill rates without the problems of precipitation that may occur with boiling or autoclaving. Pasteurization can be accomplished by heating the solution to 80 degrees Celsius, allowing the solution to cool naturally, then heating again, generally 24 hours later. This may be repeated a third time for extra safety.
The most economical and convenient method for sterilizing moderate to large volumes of water is chemical sterilization. This is most often accomplished through the addition of strong oxidizing agents such as chlorine, or by dropping the pH below 4 through the use of hydrochloric or muriatic acid. Highly effective, these methods are affected by factors such as temperature, contact time, dissolved organics etc., therefore, sterilization parameters will need to be adjusted in response to these conditions.
It is important to return the pH to normal before using the media if acid-sterilization was employed, and chlorine solutions must be neutralized, generally through additions of sodium thiosulfate. Chemical sterilization may destroy additives such as vitamins, antibiotics, and fertilizers, so sterile stocks of these should be added only after neutralization of the chlorine or acid.