Hydra Culture Methods

Included here is a description of the methods used in the Steele lab at UC Irvine for culturing Hydra. While the methods used for Hydra culture are generally the same in all labs, the details vary. For a thorough discussion of Hydra culture, one should refer to Hydra: Research Methods (edited by H.M. Lenhoff). This book was published in 1983 by Plenum Press, but it is, unfortunately, now out of print.

Hydra culture medium:

The recipe we use for Hydra medium (HM) is the recipe used by Hans Bode's lab at UC Irvine. We make up HM in 20 liter batches in 5.5 gallon polycarbonate Nalgene carboys (Nalgene catalog number 2322-0050). We add Milli-Q water to close to the 20 liter mark on the carboy (which we establish by filling the new carboy with 20 liters of water when we first get it). We then add 20 ml aliquots of the 1000x medium stock solutions (see below) using a 50 ml graduated conical tube to measure the aliquots. We then add Milli-Q water to the 20 liter mark and mix the contents of the carboy thoroughly. The pH of the medium does not have to be adjusted; the sodium bicarbonate will buffer it sufficiently. Because the carboys we use are transparent, it is easily to visually monitor the medium's condition. We have not found it necessary to rinse or clean the carboys between fillings.

Recipes for 1000x Stock Solutions for Hydra Medium

Compound

Fisher Catalog Number

Formula Weight

Concentration of Stock Solution

Grams to use for 600 ml of Stock Solution

CaCl2.H2O

C79-500

147.02

1.0 M

88.21 gm

MgCl2.6H2O

M33-500

203.31

0.1 M

12.20 gm

KNO3

P263-500

101.11

0.03 M

1.82 gm

NaHCO3

S233-500

84.01

0.5 M

25.20 gm

MgSO4

M65-500

120.37

0.08 M

5.78 gm

We make these stock solutions up in 600 ml batches in Corning Costar 162 cm2 cell culture flasks (Corning catalog number 3150) and store them at 4°C. The carboys containing HM are kept at room temperature.

Containers for culturing Hydra:

Large cultures of Hydra are generally maintained in some sort of glass or plastic tray. We prefer plastic trays since they are light weight and therefore much less clumsy to handle than heavy glass trays (it's also less dangerous if they are dropped). Our trays are made of polystyrene and have hinged lids attached to them. Their dimensions are approximately 21 cm wide x 32 cm long x 5 cm high. We buy them at The Container Store. We use a heated awl to make three holes near the top of each end of the tray to allow air to circulate more easily and we permanently label them with Tech Pens from Bel- Art Scienceware; to easily distinguish the various strains and species we culture, we use a different color pen for each strain or species.

Before using a particular kind of tray, one should set up a test culture in it to determine if it is ok. This is essential since some plastics can leach compounds which are toxic to Hydra. If you use trays without lids, you will need to used some sort of cover to retard evaporation and cross-contamination between cultures of different strains and species. Pieces of plexiglass cut to the appropriate size can be used as covers. Avoid using sheets of plastic wrap (e.g. Saran Wrap) as covers. The water which condenses on the sheets leaches compounds from them which are toxic to Hydra. As the condensed water drips back into the culture it will kill the Hydra.

Counting Hydra in a tray:

To estimate the number of Hydra in a tray, we place a piece of graph paper under the tray and use a magnifying glass to count the number of polyps in about 20 of the 1 cm squares of the graph paper. Using the average number of polyps per square cm, we can then calculate the total number of polyps in the tray. A dense culture of the Zurich strain of Hydra vulgaris in the trays we use contains about 5000 polyps.

Artemia hatching:

Hydra are fed freshly hatched Artemia (brine shrimp) naupili. A variety of methods have been described for hatching small or large amounts of Artemia (see the Lenhoff book). We use the following methods.

We purchase Artemia cysts from Sanders Brine Shrimp Company in Ogden, Utah. We purchase their grade B cysts from the Great Salt Lake. While Sanders is a bit inconvenient to deal with (they much prefer that purchases be made with a credit card; orders made with an institutional purchase order will take forever to arrive), their shrimp cysts are of high quality and hatch reliably. Prior to use, we aliquot the cysts into 50 ml conical tubes and store them at 4°C. Aliquot only a single can at a time, since the cysts deteriorate upon removal from the dry vacuum of the can.

We hatch our Artemia in an Aquabreed 1000, a hatcher produced by Aqualine, a German manufacturer of aquarium equipment. In the United States, Aqualine products are handled by the Aqua Medic Sales Office USA. The Aquabreed comes with a fragile plastic support for attaching it to the side of an aquarium. Instead we use chain clamps (Fisher catalog number 05-745) and an aluminum lab frame (VWR catalog number 60075-008). This lab frame can hold four Aquabreed 1000 units.

As a hatching medium, we use 30 grams of synthetic seawater salts (Reef Crystals® from Aquarium Systems) per 1 liter of deionized water. We make this up ahead of time and store it at 4°C. To start a batch of shrimp, we add 1 liter of seawater to the Aquabreed (the water doesn't need to be warmed up before use) and then 2 grams of Artemia cysts (be careful not to let the small amounts of undissolved seawater salt crystals get into the Aquabreed, as they can plug the outlet when you try to harvest the shrimp). We mix the cysts briefly by stirring with a pipette and then turn on the aquarium air pump (we use a Rena Air 200 pump) connected to the Aquabreed. Because the check valve in the Aquabreed spigot does not seal perfectly against backflow into the air pump line, we install a check valve (available at aquarium supply dealers) in the line to the pump and keep the pump above the top of the Aquabreed. Some labs add antibiotics to the shrimp culture to retard bacterial growth; we have not found this to be necessary. We culture our shrimp at room temperature. Under our conditions, we harvest the shrimp on the third day after starting the culture (i.e. a culture started on Tuesday is ready to harvest on Friday). We feed our Hydra stock cultures on Monday, Wednesday, and Friday; so we start shrimp cultures on Friday, Sunday, and Tuesday.

When the shrimp are ready for harvesting, we turn off the air pump and let the culture sit for about 10 minutes. During this time the empty cysts from the hatched shrimp float to the top and the shrimp congregate at the bottom of the Aquabreed (this can be helped by shining a light at the bottom of the Aquabreed). Unhatched cysts will also settle to the bottom (below the shrimp layer), but since we don't get very many of these under our hatching conditions, we don't try to drain them off before the shrimp. Once the shrimp and empty cysts have separated, we open the valve of the Aquabreed and collect the hatched shrimp in a beaker. The shrimp are then poured into an Artemia net (such nets in various sizes can be purchased from suppliers of aquarium supplies) and the medium is allowed to drain completely from the net. The remaining seawater is then removed by rinsing the shrimp thoroughly with deionized water (from the house tap is fine). Once the water has completely drained from the net, the net is inverted into a beaker containing about 100 ml of HM to remove the shrimp. The shrimp are now ready to use for feeding. The shrimp from one hatching are sufficient to feed about 15 trays of Hydra. When we are maintaining more trays, we use additional Aquabreed units. We keep the Artemia nets in a container of isopropanol between uses to prevent bacterial and fungal growth (rinse the isopropanol off thoroughly before using the net, and rinse any shrimp from the net with hot tap water before returning the net to the isopropanol).

If you need to culture huge amounts of Hydra, Aquaculture Supply USA sells a shrimp hatcher with a 20 liter capacity.

Feeding Hydra:

The Hydra are fed with the washed shrimp by squirting about three Pasteur pipette volumes into each tray. The tray is then rocked to evenly distribute the shrimp.

Washing Hydra after feeding:

About 1 hour after feeding, we rinse the Hydra with HM to remove uneaten shrimp. To do this we dump off the medium, add fresh medium to just cover the bottom of the tray, swish the medium around, dump it off, and add fresh medium (600 ml for our trays). A few hours later, the trays need to be rinsed again to remove the undigested material which the Hydra regurgitate. We also rinse the trays on the days when the animals are not fed (Tuesday, Thursday, and either Saturday or Sunday) to keep the medium as clean as possible. For most species/strains the animals adhere well enough to the bottom of the tray that only a few animals are lost during washing. However, some species/strains do not adhere well. For these annoying animals, one will have to collect the floating animals using either a nylon mesh or in a large bowl from which they can be collected by "swirling" (see Lenhoff book for how to do this).

Cleaning the trays:

After prolonged use (every couple of months or so under our conditions) the trays need to be cleaned of the "slime" that accumulates on the bottom (this is presumably a combination of bacteria and mucous from the feet of the Hydra). If the animals need to be retained during cleaning, they can be recovered from the tray by rubbing them off with your finger and collected in a bowl. The tray is then scrubbed with hot tap water and rinsed with deionized water (from the house tap is fine). The trays can either be left to dry on the bench or they can be dried with paper towels. Any retained animals can then be returned to the trays. In cultures kept in glass trays in closed incubators with fluorescent lighting, algae will begin to grow over time. Washing will also remove these. We have not seen any algal growth in our cultures kept at room temperature under normal room lighting (even after many weeks).