Culturing daphnia

From "Environmental Inquiry", http://ei.cornell.edu/toxicology/bioassays/daphnia/culture.asp

Culturing Daphnia

Using Daphnia for bioassays requires advance planning to make sure that you have a healthy, non-stressed population from which to choose your test organisms. If you order cultures through the mail, be sure to allow sufficient lead time for shipping of replacement cultures in case the original ones arrive in poor condition. After the Daphnia arrive and have stabilized at room temperature, lower the shipping jar into an aquarium or gallon jar containing unchlorinated water (see Culture Water, below).

Plan on maintaining a healthy culture for at least a week or two before using the organisms for bioassay experiments. EPA recommends not using a culture for bioassays if more than 20 percent of the Daphnia die during the two days preceding the test.

Life Cycles of Daphnia

Daphnia typically live 40 to 56 days, varying according to species and environmental conditions. Each brood typically holds 6-10 eggs, which turn into embryos and are released within a few days. Juveniles reach sexual maturity in 6 to 10 days. A healthy population of Daphnia consists mostly of females that have been produced asexually. The culture can become stressed if the population density gets too high or if there is a food shortage, poor water quality, or extreme temperatures. Under stressful conditions, Daphnia produce more male embryos and begin to reproduce sexually. The resulting resting eggs will not hatch until they have gone through a certain sequence of environmental changes, including several freeze/thaw cycles. Therefore, if you want to maintain a steady supply of Daphnia in your lab, you will need to avoid the stressful conditions that lead to sexual reproduction.

Culture Water

Daphnia are quite sensitive to the chemistry of the water in which they live. In order to provide standardized culture water, professional scientists start with distilled water and add essential minerals and nutrients (see Table 1). The problem with this approach in schools is that you may not have access to distilled water of sufficient purity. Supermarket-grade distilled or deionized water may contain trace-level contaminants at concentrations high enough to be detrimental to populations of Daphnia. An alternate possibility is to use unchlorinated water such as bottled spring water, or water from a local well, spring, stream, or lake. It also is possible to start with chlorinated tap water and either let it sit long enough for the chlorine to evaporate, or treat it with activated charcoal or one of the products sold for de-chlorination of water to be used in aquaria.

With any of these water sources, you should start by testing the sensitivity of Daphnia to the water. If more than 80% of the individuals living in the water survive for two days or longer, then that source is acceptable for your culture water. In bioassay experiments, dilutions should be made with this same type of water in order to maintain consistency between treatments.

Table 1. Recipe for synthetic culture water for Daphnia

Compound Concentration (g/L)
NaHCO3 0.192
CaSO4•2H2O 0.120
MgSO4 0.120
KCl 0.008

Optimal Culture Conditions

Although Daphnia are sensitive to dissolved oxygen, pH, and chemical contaminants, it is not difficult to maintain healthy cultures. Individual students or groups can grow Daphnia in liter flasks or quart jars, or you can maintain larger populations in aquaria or gallon jars. The larger the container, the easier it will be to maintain favorable conditions for long-term survival, but the harder it will be to harvest Daphnia with a pipette for experiments.

Start by filling the containers with culture water (see discussion above). Although you can buy special food for Daphnia, it is not necessary. Healthy cultures can be maintained using either or both of two simple foods: powdered yeast (the kind used in baking) and unicellular algae such as Selenastrum capricornutum. Simply sprinkle a pinch of yeast on the water surface every couple of days, and/or add several milliliters of concentrated algal solution. Be careful – overfeeding is probably the easiest way to cause a population crash, because excess food will cause oxygen depletion. Aquarium aerators can be used but are not necessary as long as feeding is carried out in moderation. The amount of food needed varies depending on the population density. A general guideline is to feed enough so that the water becomes slightly cloudy but clears again within a day or two. Daphnia are filter feeders, so they gradually clarify the water in which they live.

Using a test-tube sized culture of Selenastrum purchased from a biological supply company, you can create your own never-ending supply of high quality Daphnia food. Simply transfer the algal culture to a larger container such as a liter flask or quart jar, fill with water, and add houseplant fertilizer at the concentration recommended on the packaging. Place the algal culture in a well-lit location, and shake or mix it every few days. Once the solution becomes bright green, it contains billions of algal cells and is ready for use as Daphnia food. Occasionally add more water and fertilizer to replenish the algal solution after feeding your Daphnia cultures.

It is not necessary to periodically clean the Daphnia culture containers. In fact, once organic debris has begun to accumulate it is possible to develop cultures that are relatively self-sustaining because the Daphnia will feed on detritus and decomposer microorganisms. Without supplemental feeding, population growth rates will decline, but the culture may survive weeks or even months unattended.

For optimal culture growth, the following conditions are recommended:

Factor Optimal Range
pH 7-8.6
temperature 20-25°C
dissolved oxygen > 6 mg/L
hardness 160-180 mg CaCO3/L
lighting cycle 16 light/8 dark

If cultures are maintained under these optimal conditions, a 3-L vessel stocked with 30 Daphnia will produce approximately 300 young per week. You can either let the populations rise and fall in natural cycles, or periodically remove some of the individuals to prevent overcrowding and keep the culture reproducing rapidly. It is a good idea to maintain more than one culture, since even under the best of conditions Daphnia populations occasionally crash for no apparent reason.

Procedure for culturing freshwater protists

Culturing Freshwater Organisms, by Thomas Aungst. http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artoct99/cultur.html

Materials and Methods for culturing protists

To culture you need only provide four basic needs:

1) The water for the community
2) Proper temperature
3) Sufficient light
4) Food

The water would be whatever source you collect from. The proper temperature can be maintained with nothing more than a light bulb placed in a box along with a jar of water with all of those little beasties that you have collected. The light source is the same light bulb that is providing the heat. (See safety footnote).

Proper light and heat are regulated by changing the wattage of the bulb and its placement within the enclosure you are using.

Food. Most protists and microscopic life forms, which we term to be animals eat bacteria. So we must provide an ample supply of bacteria if we want our little community to bloom.

The best, and safest, way to provide plentiful amounts of bacteria is with a tea made from dry hay, straw or weed and grass clippings. Make sure that all plant material has been thoroughly dried before using it to make the tea. The reason for this is that if the tea is made with fresh chlorophyl it will foul the water and ruin the culture.

Boiling up a tea in this way provides you with a sterile food source with which to feed and maintain a rich bacteria bloom which in turn will feed a growing population of protists, which will feed a growing population of multi-celled animals, and so on and so on.

The tea can be placed in a sterilized canning jar and sealed with a tight fitting lid, and kept in the fridge.

If you start with a liter container half full of, lets say pond water with some bottom sediment in it you can add an ounce of tea as needed. Give the culture at least 24 hours and the check a drop of water, taken from the surface, under the microscope for the presence of bacteria.

If you do not have a lively bacteria population within 48 hours add another ounce of the tea and check again and repeat if needed. Add more tea 1 ounce at a time as needed to maintain a flourishing community of protists and algae.

DO NOT add too much tea or the culture will go foul. If the jar gets full siphon off about 1/3rd of the water and keep going.

Safety footnote.

Be especially careful when using mains powered lights near water. Ensure the wiring is properly insulated and metal objects are correctly earthed and keep well away from the water. Also ensure that the container will not overheat and catch fire.