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:

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.

CHEMICAL STERILIZATION

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.

The Easy Bleach Method
For sterilizing seawater of average cleanliness, this protocol will yield water of acceptable sterility and quality for most microalgae (greenwater) and larval culture.

**Be sure to familiarize yourself with proper chemical handling techniques before attempting these procedures. Always wear protective goggles and gloves when handling any chemicals**

1.  Collect a known quantity of water to be sterilized.  If water is turbid, it may be necessary to prefilter for clarity.  This can be accomplished by dripping the water through a few coffee filters.

2.  Add 0.5ml unscented laundry bleach per liter of water.

3.  Seal the vessel and swirl the media so that all internal surfaces are wet.

4.  Allow the vessel to sit at room temperature for at least 6 hours, preferably 12-24.  The vessel may be stored for long period of time with the bleach inside, provided the vessel remains uncontaminated.

5) Prepare a 1M stock solution of Sodium Thiosulfate.  The most commonly available crystalline form is the pentahydrate, and should be added at 248 grams per liter.  This solution may be filter sterilized or autoclaved to ensure sterility.

6.  To de-chlorinate the water, add 0.1ml of 1M sodium thiosulfate per liter of water.

7.  Aerate at least 2 hours.

8.  Confirm that no residual chlorine exists before using the medium.  Dip tests for pools work well, as do the DPD reagent available in convenient liquid or powder pillow-packets.

Acid Sterilization

**If you are not familiar with the safety hazards associated with handling concentrated acids, DO NOT attempt this method**

1. Collect a known quantity of water to be sterilized.  If water is turbid, it may be necessary to prefilter for clarity.  This can be accomplished by dripping the water through a few coffee filters.

2.  Add sufficient hydrochloric acid or muriatic acid to reduce the pH of the solution to 3.6.  The amount needed will vary depending on the concentration of the acid stock, the initial pH of the water, and its buffering capacity, which may also be affected by residual calcium deposits in the vessel.  A good starting point for muriatic acid (about 32% concentration) is 2ml/L.

3.  Confirm pH with a pH meter or narrow range test strips.

4.  Swirl the sterilized solution so that all internal surfaces are moistened.

5.  Allowing a sufficient sterilization time (2-4 hours).

6.  Return the pH to normal with sodium bicarbonate (baking soda).  A good starting point is 1/8 tsp/L.

6.  Confirm pH is appropriate for culture with a pH meter or narrow range test strips.

GENETIC CONTROL OF BIOFILM FORMATION

Several genes and gene clusters have been identified in Vibrio cholerae that have aided in the understanding of how biofilms are formed and what environmental factors are determinants in their formation. The first gene involved is in the vps gene cluster. This gene cluster is involved in the creation of the VPS exopolysaccharide, a primary component in the biofilm matrix. The vps genes are clustered in two regions within the Vibrio cholerae genome, with vpsA-K in one region, and vpsL-q in another. Transposon inactivation mutants were screened, leading to the description of several of the genes in this cluster.

Although the function of vpsA and vpsL have not been fully described, mutations in these genes lead to a reversion from R-type colonies to L-type with low expression of VPS exopolysaccharide. . Several regulatory genes have also been described. VpsR is a positive regulator, and HapR and CytR are negative regulators. Recently, a second positive regulator has also been found, termed vpsT, it is a transcriptional activator that works in concert with vpsR to regulate each others expression, and both can also autoregulate their own expression. It is only with both of these genes activated that maximal biofilm is deposited. These regulatory genes show homology to typical two-component regulatory systems, although the sensor kinases have yet to be identified.

Utilizing a vpsL deletion mutant, it was also discovered that there are at least two pathways associated with biofilm production in Vibrio cholerae, and that they each respond to different environmental cues. These pathways were termed vps-dependent or vps-independent. The vps dependent pathway requires a nutrient rich media with the presence of pre-formed monosaccharides in the media. There is no calcium requirement for this pathway. The O-antigen is also required for vps-dependent formation. The vps independent pathway does not require the presence of monosaccharides in the media and can be expressed on minimal media, so long as milimolar amounts of calcium is present. It has been hypothesized that because Vibrio cholerae is associated with both marine and estuarine environments, different mechanisms are needed for survival in these environments.

THE VPS-DEPENDENT PATHWAY

The vps-dependent pathway is associated with nigh nutrient concentrations and is able to grow with low salinity and low calcium concentrations, as one would expect during rainfall and runoff entering a positive estuary. Nutrient concentrations are highly variable in this environment and the biofilm may even play a role in the accumulation and storage of nutrients for starvation conditions. On the other hand, the vps-independent pathway only has a requirement for calcium. This less selective pathway would be expressed in the marine environment, where vibrios are often found colonizing nutrient sources such as the chitinous exoskeletons of planktonic organisms.

An additional gene has been found that plays a role in producing the proper structures for biofilm development. It has been termed mbaA, for maintenance of biofilm architecture. In vps-dependent biofilm formation, the vps gene clusters are activated by the presence of a mannose-sensitive hemagglutinin type IV pilus (MSHA). Once initial attachment has occurred, the EPS is synthesized as the cells move via their flagella, and additional planktonic cells are recruited for settlement. The gene mbaA is not associated with these early stages of biofilm development. Instead, they appear to be regulators that control the amount of matrix EPS that is being produced in mature biofilms.

To find the mbaA gene, a mini-Tn10 mutant was isolated by researchers from a screen that produced abnormally robust biofilms. The insertion was determined to be at the 315th codon in a 2,376-bp open reading frame with no determined function. Sequence analysis indicated that this is likely a three-gene operon. Based on the identification of this mutant, an mbaA deletion mutant was created. These mutants showed extremely high levels of EPS being secreted, and none of the biofilms had the peaks, valleys, and channels associated with mature biofilms. Although the biofilms being formed were vigorous, when mbaA mutants were constructed with defects in either the MSHA or flagella genes, no biofilms were formed. This indicates that early requirements for biofilm formation are not circumvented by the mbaA mutation, and its presence occurs later in formation. Similarly, deletions in the vps gene clusters that are associated with defects in biofilm formation were not overcome by the mbaA deletion. It was also determined that the increased EPS production was not associated with either increased cell density or cell division, again supporting the hypothesis that mbaA is a regulator of EPS production.

SUMMARY

Based on these findings, it is apparent that biofilm formation is a critical factor in the environmental survival of Vibrio cholerae, and is likely a strong determinant of pathogenesis in many primarily marine pathogens, especially those associated with coral beaching, wound infections of fish, and secondary infections following parasite attack. The presence of two distinct and independently regulated pathways for the formation of these structures demonstrates their importance. With different structural types created in response to varying environmental conditions, these biofilms are able to survive starvation conditions, create ideal conditions for exchange of genetic information via horizontal gene transfer, and maximize survival when attached to the exoskeletons of planktonic crustaceans for long-term oceanic survival and dispersal. Similarly, survival within a biofilm while attached to the chitinous exoskeletons might facilitate transport and survival through the GI tract when ingested by larger organisms, allowing the possible infection of a new host.

In the next sections, we will look at how biofilms may aid in coral pathogenesis, aquaculture mortalities, and filtration systems.