The Zebrafish Science Monitor, Vol 3 (6)
AN ECONOMICAL ZEBRAFISH GENETICS FACILITY
By E.G. Gestl and K.C. Cheng; Division of Experimental Pathology and Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, PA 17033 USA.
General Description
Our goal was to set up a zebrafish facility at minimum cost that would support a mutant hunt, maintain healthy fish with minimum effort, and offer modular flexibility and the greatest efficiency with regard to feeding, water changes, and system maintenance. We have just finished construction of a facility with a capacity of 51 ten gallon tanks and 144 five gallon tanks at a cost of about $36,000. The water temperature is regulated by a mixer valve and by room temperature, and is usually set at 28C. A light/dark cycle of 14h/10h is achieved using electronic timers. System water recirculates at a rate of up to 5 water changes per hour with standard use being 3-4 changes per hour. The system was designed to fit in an existing room in the Animal Facility at the Penn State College of Medicine, Hershey Medical Center, and was slowly brought to 50% capacity over about 6 months. Our fish appear to be very healthy.
Tanks and Stands
The tanks were made by All-Glass Aquarium to be 1" shorter than the standard 10- and 5-gallon tanks (11" and 9", respectively) to allow more working space above the tanks. The tank walls are approximately 1/8" thick with a 1" hole drilled in an upper corner (centered at 1" down from the plastic lip and 1" in from the side) into which a " bulkhead (Aquatic Eco-Systems, TFK1) is siliconed in place. The lids consist of 2 overlapping glass pieces sliding on a plastic rail purchased from Tropical Isle. Each glass lid has a corner cut off, the rear lid for a water inlet into the tank and the front lid for liquid feeding such as brine shrimp.
The stands are constructed of grade 304 #4 polished stainless steel (Staco). Other labs would use local metal companies to minimize shipping costs. The structural supports for the stands are made of 1" square tubing and the shelves from 18 gauge stainless steel sheet metal. The shelves have a " lip and a " hole in one corner so that in the case of an overflowing tank the water is contained and exits through the hole into the drain. Each leg also has a 2" square footplate to distribute the weight evenly. The stands for the five gallon tanks have 4 rows of tanks with 3, 4, or 5 tanks in a row, while the ten gallon tank stands have 3 rows of tanks with either 3 or 4 tanks per row.
Description of the Recirculation System
Water leaving the tanks exits through the bulkhead by gravity and enters an angled 1" common drainage pipe through open tees. These lead to vertical 1" drainage pipes which in turn exit into 90 gallon fiberglass sump tanks (Aquanetics, 18" D x 16" W x 75"L). Before entering the sumps the water passes through DSL pad filters (Aquanetics) to eliminate large particles. Two of the sumps are interconnected via a 2" PVC pipe which is below the water line. To make efficient use of the available space, tanks had to be placed on either side of a walkway. The sumps on each side are connected by a siphon system (3" PVC pipe) which transports 25 gpm or 40% of the system's water back to the side of the room containing the pump. This inverted "U" also has a small peristaltic pump which pumps out the air that accumulates at the top of the "U." This is necessary for maintaining the siphon.
Operating only one of two M1000-H pumps (Aquanetics), the water then goes through two sets of two filters (Aquanetics, 420) each set containing a 150 m and 25 m bag filter (Aquanetics, 400-150 and 400-5). The water is then sterilized using 2 ultraviolet (UV) sterilizing units placed in series (Aquanetics, Q480IL and Q240IL). The sterilizing power of the large unit at the maximum flow rate is 45,000 wsec/cm2, 3-fold the killing power needed for viruses and bacteria (15,000 wsec/cm2) so that many fungi, protozoa, and spores are also destroyed (Aquacultural Engineering, Fredrick Wheaton, 1985). The UV units are installed in series so that when bulbs burn out, water will still be sterilized. Therefore, the small unit has an output of at least 15,000 wsec/cm2 and is placed in series with the first to ensure that the water is sterilized at a minimal level. The water leaving the UV system enters a network of PVC piping whose pressure is equalized by circular loops throughout the system. The water flow into the individual tanks is controlled by no kink style globe valves (Aquatic Eco-systems, VK-2), and the cycle begins again.
The aeration system uses a 1/6 H.P. turbo-blower (Aquanetics, 104P) with silencer and filter. Air hose leads to every tank, and is plumbed in circular loops to yield equal pressure. The air exits the PVC pipe from plated brass valves (Aquatic Ecosystems, VN2), goes through 1/8" silicone tubing (McMaster- Carr, 51135K16), and enters the water through Jungle glass-beaded airstones (Jungle Laboratories, NJ297). The usual rate of aeration per tank is 0.7 L/min while 4L/min is its maximum rate.
Water Changes
To ensure that ammonia and other harmful toxins do not accumulate, the system is designed for continuous automatic water changes; we exchange about 10% of the system volume per day. Waste water exits the system through a valve which is located between the filtration and UV systems. The temperature of the replacement water is regulated using a mixing valve. This water then passes through a pressure reducing valve and two carbon filters (Aquanetics, model 220) in series before entering the sump. The flow rate is regulated by two parallel float valves (AREA, FLV550-050) in one sump.
In our second fish room, we wished to have automatic water changes in tanks in which babies are raised. Because babies are fed powdered food and paramecia, we did not want small food to plug the particulate filters. Therefore, we raise babies in the flow-through portion of our system. To keep things simple, we decided to use "conditioned" water from the recirculating system in the flow-through system. We also use flow-through tanks for quarantine purposes when new fish are brought into the system. The water leaving the tanks in the flow-through system goes to waste drains. In our basement system, 28 five-gallon tanks are used as flow through tanks. Another 44 five-gallon and 33 ten-gallon tanks will have this capability in the future. The flow through of these tanks represents from 0 to 100% of the water exchange in the system, with the remainder draining from the recirculating portion of the water flow. System water pressure is adjusted using two lines which bring water directly back to the sump either after the filter bags or after the UV system.
In order to maintain the maximum number of fish per volume of water, the dissolved oxygen level of greater than 96% saturation is obtained using a bioreactor trickle filter (Aquanetics, B126) which draws about 20% of the system flow following the bag filters, and returns aerated water to the sump.
Individual Adult Tank System
In the recirculation section of our separate fish room, we recently added 160 one liter tanks, of polystyrene. These tanks (model T49F) were purchased from Alpack Inc. for $1.25/tank. The unhinged lids were drilled with 2 holes, one 3/8" in diameter in a back corner for a water inlet line and the other 1 3/8" in diameter in the front center for feeding (we would recommend using a 1' hole next time, since fish occasionally jump out of the 1 3/8" holes). A 1/8" wide, 1" deep slit was cut in the front center of each tank for water to exit. The shelf frame was constructed of materials previously mentioned and has a pitch of " over the length of 51" to aid in drainage. The shelves are constructed of " thick acrylic with 1" high by " thick acrylic sides. Each shelf contains 2 rows of 10 tanks with access from either side of the rack.
UV-sterilized water is pumped through 1" PVC pipe to the individual adult tank system where it is regulated by a ball valve for each shelf. The water exits the common water inlet by a 1/4" threaded adapter with a 1/8" barbed end (Aquatic Eco-Systems, 62001). A pipette tip attached to 1/8" tubing transports the water to the tank. Water flows onto the acrylic shelf through the tank slits, where gravity takes the water to one end of the shelf. It then exits through a pair of 1/2" bulkheads, enters a common drainage pipe, and empties into a sump which is connected to the recirculation system.
Other Features of the System
An important advantage of the system is its flexibility. First, back-up systems are important for flood prevention and to ensure continuous running. The room is divided into 4 zones with the ability to isolate and stop water or air flow to that zone when the need arises. The water pump, bag filtration, and UV systems all have bypass routes which can be activated without stopping water flow when damage occurs or routine maintenance is required. For example, when the siphon action in the inverted "U" siphon is broken, there is a back-up pump (Aquanetics, TE-6-MO-SC) activated by a seesaw switch (AREA, LP19), which returns water to the other side of the room. In addition, when the water level of the sumps becomes abnormally high for any reason, two parallel condensate pumps (McMaster-Carr, 9907K11), each capable of pumping 100 gph, are activated. The sterilization power of the UV system was designed to be higher than normally used, with two UV sterilizing power units in series. This decreases the possibility of cross-contamination when bulbs inevitably burn out.
Monitoring of the system is eased by plumbing T's and valves so that water can be collected and chemically analyzed at critical points. Valves between and after the carbon filters allow us to test their efficiency. At other locations, branches have been added to allow measurement of water exchange and bioreactor trickle flow rates. True unions and true union ball valves were used in many areas to create easy access to many parts of the system. The low cost of this system must be balanced against the time needed for design and construction. However, the knowledge gained from setup has already helped us to make changes and correct problems as required.
Businesses/Companies
All-Glass Aquarium Co., Inc.
9675 South 60th Street
Franklin, WI 53132
(414) 421-9670
(414) 421-9682 - FAX
Alpack, Inc.
7 Overhill Rd.
Natick, MA 01760
(508) 653-9131
(508) 650-3696 - FAX
Aquaculture Research/Environmental Associates, Inc. (AREA)
P.O. Box 1303
Homestead, FL 33090
(305) 248-4205
(305) 248-1756 - FAX
Aquanetics Systems
5252 Lovelock Street
San Diego, CA 92110
(619) 291-8444
(619) 291-8335 - FAX
Aquatic Eco-Systems, Inc.
2056 Apopka Blvd.
Apopka, FL 32703
(407) 886-3939
(800) 422-3939 - Order
(407) 886-6787 - FAX
Jungle Laboratories Corporation
Box 630
Cibolo, TX 78108-0630
(210) 658-3505
(800) 245-1446 - Order
(210) 658-8413 - FAX
McMaster-Carr Supply Co.
P.O. Box 440
New Brunswick, NJ 08903-0440
(908) 329-3200
(908) 329-3772 - FAX
Staco
P.O. Box 216
Route 501 North
Schaefferstown, PA 17088
(717) 949-2630
(717) 949-3103 - FAX
Tropical Isle
4 Pierce Street
Framingham, MA 01701
(508) 875-5303
(508) 872-1916 - FAX
The Zebrafish Science Monitor, Vol 3 (6)
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