A little system for zebrafish.

A few years ago, when we moved to Western Michigan University, getting our fish system going was running into the snags common to the wonderful hierarchy typical of most public universities. We needed a fish system to work with fish, we needed it fast, and it had to be in the laboratory… oh, and it had to be smallish, we just had a little corner between a freezer and a bench.

So we made a twenty-five tank system which worked great…and then ended up using it for more than two years, long after our main fish facility was on line. After disassembling it, I realized that this system could be used for many little projects, from a quarantine closet to an ambitious High School laboratory (or a mouse lab interested in doing experiments on a creature that you didn’t have to write new IACUC protocols for every week.)

Basic Design.

One rack zebrafish system.

The system is a plastic shelf system lined with cafeteria trays that hold overflowing type fish tanks; the water movement is powered by two Little Giant submersible water pumps; it also had a fluidized bed sand filter and a homemade particle filter. The temperature and humidity was controlled with a shower curtain.  Light cycle? Nothing but natural window light…and, yes, the fish produced eggs every morning, summer (16h/8h) and winter (8h/16h)....So much as for the Streissinger 14h/10h light cycle.

This system is a small version identical to our big system in terms of growing & feeding fish, producing eggs, raising babies, and controlling water quality. Each tank holds up to 40 fish without crowding, and wiith snails doing the housework, a family of fish will live for two years in the same tank.

The system works by pumping water through a particle filter to overflow type fish tanks at the rate of about 5 water changes per hour; the water flows to trays, and back to the reservoir; then the cycle begins again. Overflow water goes to a pressure stand tube, which maintains constant water pressure in the system. On a separate flow circle, water is pumped through a fluidized bed of sand and it returns to the reservoir.

Air exchange occurs in the reservoir and trays; pH control is maintained by adding coral to the fluidized bed; biological scouring of water occurs in the fluidized bed  (and to some degree in in the particle filter); salinity is checked and adjusted after filter back flushing, which is done about once per month. Cleaning is done by snails. Evaporation of the water supply was replenished by pouring a bucket of RO water into the reservoir once or twice a week. The heat from the water pumps keep the system at about 5°C over ambient, so no heaters are necessary. Best of all, UV filters and routine water changes apparently are unnecessary.

The only disadvantage of this system that I am aware of it that it is essential that the water remain flowing to each tank or the fish will suffocate, necessitating a tank check each evening.

Preparing the floor.

Floor preparation is important if you are not in the basement and you need to worry about the computer lab beneath you. Whatever. We have always been in places where if we wanted the fish in the lab, which is pretty nice, we had to control the water spillage. There two parts to water control, sealing the floor and draining the water.

A full sized facility normally needs a drain, and it has to be in the mist of the fish system and not isolated. Overflow drains on the reservoirs and back flush from the filters need to go to directly to drains without crossing an aisle. However for our little system we did not worry about drains, since we were back flushing into a bucket and filling the system manually.

But we did seal the floor, or, more accurately, the floor edges, using clear bathroom silicone caulking to seal around all the baseboards and cabinets. (In most laboratories, the walls and cabinets receive electric, water and other services from holes that open directly into the ceiling of the room below.)

Shelf System.

Three cafeteria trays glued together

The shelf system was an inexpensive plastic shelf systems, the kind sold at Lowes or Home Depot (like “Enviro Elements 72"H x 36"W x 24"D Plastic Freestanding Shelving Unit”)  for less than $50. We bought two of these and shortened or combined posts to put one shelf directly on the floor (no supporting posts), a gap of 36” to the first fish shelf, and then 12.5” spacing for the next 4 shelves (fish shelves 2 though 5) and then 14” to a “roof” shelf. For the shelf set we used, 1¼“ pipe was the correct diameter to substitute for the posts that come with the shelf set; they were the long legs dropped to the floor tray; we also inserted ¾” pipe thru the posts from the top for extra strength to help keep the structure square.

To capture water from the overflowing fish tanks, each shelf was covered with a home made water tray; these were made from cafeteria trays that were cut up and reassembled to a somewhat larger size and then glued together with heat glue.

For the main reservoir, the bottom shelf (on the floor) had a big 80 or 90 gallon Nalgene tank (30x24x30 inches), the type used for soaking lab glassware before washing. You can buy lids for these to control evaporation, but what with all the water on the shelves, we never thought it would matter.

Plumbing the Shelves/Trays.

Nipples and tubing, on our main system.

Each shelf needs a water supply and drainage. Supply is from a horizontal manifold made from 1” PVC piping with ¼” nipples tapped into holes drilled along the length of the pipe. This manifold is attached by strap ties to the bottom of shelf above. To reach tanks, to each nipple is attached an 8 inch length of 5/16th  inch tubing with an adjustable clip to regulate the water flow. The manifold for each shelf screws into a larger vertical manifold that supplies water to all the shelves.

One inch NPS couplings used to make drains. Note plastic shim.

Tapping of pipes in this way is not “normal” plumbing practice, but it works if the water pressure is not too high and if one is a little careful. If you have a real plumber helping, don’t be surprised if they are more than skeptical.  We drill a  3/8 hole with a rather steeply stepped drill bit (otherwise it will go through the other wall of the pipe!). When tapping the ¼” NPA hole, we only drive the bit about halfway in. In any case, some practice on scrap pipe is a good idea. If you make an occasional hole too big, it will leak, but the leak will be on the tray and will not signify. Or, a little t-tape can be added.

The drains were made from 1” threaded pipe coupling after drilling  ½” holes in the top of the male coupling so that water drains directly from the plastic of the tray. Because the couplings don’t close tightly enough to clamp on to the trays, we usually had to use plastic spacers to get a good fit, and pipe compound to help stop drips. Besides cost, two advantages of this drain over a more conventional bulkhead fitting is that the drain clears water flush to the surface of the plastic and that these drains are hard to plug: water keeps flowing even if you inadvertently place a fish tank on the top of the drain! One other advantage is that the drain from a shelf above can be fed directly to the drain in the shelf below.

Particle Filter.

Home made particle filter.

The particle filter is based on the BBF-XS2000 or BBF-1 bubble bead filter used in our main system. It is just smaller and made out of stuff you can buy at Home Depot or Lowes. These filters work using floating beads to physically retain particles that as water moves upwards through the filter. The beads act as a depth filter that trap a large amount of material in the beads before clogging.

The beauty of these filters is that when back flushed, the beads break up and the load is released to go down the drain as brown sludge. The “bubble” feature is that when the filters are under negative pressure (when they are being drained with the top valve closed) a check valve opens allowing air into the throat of the filter, “bubbling” the beads as they come down through. It seems to work; after 10 years we see no difference in the effectiveness of the filters, and a lot of dirt comes out of them when they are flushed.

Slotted 1" pipe glued into wrong end of a 1¼” male connector.

The filter is made of two sections of 4” pipe connected by a throat of 2” piping. At either end are threaded 1¼”  connectors, and in the middle, near the throat, is a  1¼”” threaded opening for the bubble value. To retain the beads, short sections of 1” slotted pipe are capped and forced and glued into the threaded 1¼” male connectors that fit into the three 1¼”  female connectors in the filter.  The slotted pipe was made by hand, there are 3 columns of about 30 1x10mm slots, which gives a square sectional area about 50% larger than the square section of the 1” pipe feeding the filter.

The Fluidized Bed (Sand Filter).

The sand filter is constructed from an study cylindrical plastic garbage container. A 1” tube is runs to the bottom of the container and a 2” Bulkhead Fitting was used to make a stand tube drain. The sand is the stuff they use for kids sandboxes.

Getting these things going after they stop can be a challenge. One thing that helps is to put a check valve between the pump and the discharge so that when the motor stops water doesn't siphon back though the pump. When this happens, sand fills the discharge tube and the sand is impossible to clear. (If that happens, you need to dissemble the down tube and clear the sand.)

Fish Tanks.

The tanks are mouse cages that have 1/8” holes drilled on the front for drainage; we constructed tops cut from 3/8 inch polycarbonate sheeting.

The tanks are  made of polycarbonate and they measure 11-1/2" x 5-3/4" x 6" deep. They were purchased from Lab Products, Inc. in cases of 20 for $212.00; the product number is #10017. These tanks are rugged; they have survived multiple drops on the floor. Also,after 15 years of use in our main system, they have remained clear. We still just pull the tanks out of the system to sex the fish for crosses directly from their tanks.

Tanks, main system.

To drill the fronts, we used hole guides and a stepped drill bit; it is important that the holes are only 1/8”; teenage fish swim through ¼” holes. Some tanks have holes on the sides, to let high flowing water out without spilling off the tray. These are higher than the front holes because when doing water checks, it’s important to see water flowing down the front of the tank.

The tops are cut from sheets of polycarbonate plastic, dimensions are 12" x 5-7/8". It is important to use polycarbonate for these and not cut costs by using polystyrene. Polystyrene absorbs water and warps, and, after about 2 weeks, they do not fit tightly against the tanks. To feed the fish, we have a 1" hole drilled towards the front of the lid. This hole should be back a bit, ½”, from the front of the tank so that fish that jump along the front of the tank don't make it out of the hole. Also, for feeding purposes, it is really nice if all of the holes are in the same place as measured from the front of the lid. The smaller holes, ½”, are for inserting in the inflow tubing. They are spaced at roughly 2" intervals to allow for variation in the placement of the water lines/ length of the tubing in our system. The lids have two 1/8" holes in the back; these align with similar holes in the backs of the tanks, and the lids are attached with small strap ties.

General plan for water flow.

The 1st figure shows general flow of water, including the location of control valves and pumps; 2nd two figures (side and top views) sketch plumbing details for the pressure stand tube at the top of the tank rack.

The system uses two pumps in the main reservoir, one for providing filtered water to the fish shelves and one for the sand filter. The fish shelf pump pumps water into the bottom of the filter; after the filter the water is spilt between the pressure stand tube and the vertical shelf manifold. The water to the shelves ultimately goes into and out of the tanks, onto the shelf, down the drain, and back to the main reservoir. The excess water goes to the pressure stand tube and overflows at about the 12 foot level; this maintains a constant pressure in the system. When the water flow drops enough so that water is no longer overflowing the pressure stand tube, it’s probably time to back flush the filter.

There is some excess plumbing beneath the pressure stand tube, forming a large "H"; it is to help support and stabilize the stand tube, which sits some 5 or 6 feet above the rack.

The sand filter pump is only used to float the sand by pumping water to the bottom of the sand filter; the water returns to the reservoir.

The system uses 5 valves, all of which are used while back flushing the filter. The valve to the shelves can be turned off while flushing the system so not to introduce dirt into the shelf manifold. The bypass valve allows water to get to the top of the system when the filter is shut down. The top and bottom valves on the filter control which direction water goes to the filter, and the drain valve allows back flush to go down the drain (or into a bucket).

Water calculations:

Water volumes:
            tanks: 25x3500ml = 80 to 90 liters
            shelves: 5x1000ml = 5 liters
            main reservoir: 200 liters (about half full)
            sand filter: 80 liters
            filter: 15 liters
            pipes: 10 liters
Water overage on motor shut down:
            25x400ml (from tanks) +5x5 liters (from shelves) +10 liters (from pipes)
                        = about 25 liters (only raises the water a few inches in the reservoir.)

Flow rates:
            Tanks: 25x25 liters/h = 625liters/h
            Little giant flow at 7psi back pressure: about 1500 liter/h


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