User Guide
Updated March 2015
The Commandments of the Fish Facility
1)
Wear gloves and in-house shoes (or shoe covers) while working in the Fish
Facility.
2)
No fish, embryos, tank parts, or water taken from the Facility, may be
brought back into the Facility. Once something leaves the Facility, it stays out!
3)
“A fish on the floor is a dead fish.” Any fish that accidentally falls to the
floor must be euthanized by an approved method, followed by proper disposal.
4)
Use a new net each time you start working with a different tank/stock of fish
or remove sick/dead fish from its tank.
5)
Keep tanks covered with lids to prevent mixing of fish.
6)
Please clean up after yourself. Place dirty nets, tanks and cages on proper
rack shelves, tubs or sinks for dirty items to be cleaned. Clean/ethanol spray the
forceps, egg spatulas, and spoons after each use and place them in appropriate
containers with ethanol if appropriate.
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Quarantine System:
Quarantine lights come on at 8:00 am and go off at 10:00 pm. The Quarantine must
NOT be entered during Lights Off.
• All fish care (feedings, tank exchange, and tank checks) is the responsibility
of the Fish Facility Staff.
• If you perform work in the Quarantine System, do not enter the basement
Fish Facility until the next day and/or after showering/changing clothes.
Receiving Embryos or Fish from Other Labs:
• ONLY Facility Staff are allowed to transfer fish/larvae into the Quarantine
Facility.
• If embryos are received before or on 1 day post fertilization (dpf), they will
be bleached (sterilized) and dechorionated in Quarantine. The larvae can
then be transferred into the Fish Facility immediately.
• All embryos received post 1 dpf, are raised within the Quarantine. Once
grown, their offspring can be bleached and transferred to the Fish Facility.
• Adult Fish received remain in the Quarantine. They are kept off of the
system for a week to undergo fluke tablet treatment and observation by
Facility Staff. If given a clean bill of health, fish are placed in the Quarantine
system. They are bred and their offspring bleached by Fish Facility Staff and
transported into the Fish Facility.
• ONLY Facility Staff are allowed to bleach embryos and transfer bleached
embryos into the Fish Facility.
• Please notify Facility Staff with at least 24-hour notice via email of any
fish/larvae shipment, along with package tracking number.
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Embryo Care:
• Embryos need to be sorted on 0 days post fertilization (dpf) and distributed
into Petri dishes at appropriate densities (50 embryos per Petri dish).
• There should be no more than than 50 embryos per Petri dish, as this
maximizes larval health and survival.
• Petri dishes must be labeled with:
1. Genotype
2. Date of Birth
3. Density
4. Lab/Owner initials.
• Use Methylene Blue to egg water to prevent fungal growth if desired.
• Embryos should be placed within 28.5 C incubators for normal
development.
• Embryos need to be cleaned and have their water exchanged at least twice
prior to being put on the system:
1. On 1 dpf remove dead and unfertilized embryos and exchange water.
2. On 3 dpf remove dead embryos and chorions (embryo casing) and
exchange water.
• Embryos on day 0 can be discarded in “Retired Embryo” containers and
rinsed down the sink. Embryos 1 dpf and older must be euthanized using an
approved protocol (see Euthanasia section of this User Guide).
• Between 3-5 dpf, embryos to be grown up should be cleaned and new stocks
of fish are to be recorded into the Facility Stock List and assigned a stock ID
number (S#). All applicable information regarding the stocks’ pedigree and
starting quantity of larvae should be included. The Facility Stock List is
located on the Nursery computer.
• Embryos/larvae should be added to the Rotifer rack on 4 or 5 dpf. Individual
users can do this or embryos should be placed within the Walker Nursery
incubator for Fish Facility Staff to put on the system.
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Nursery (Walker) Guidelines:
Nursery lights come on at 7:00 am and go off at 11:00 pm. The Nursery must NOT
be entered during Lights Off.
• Tank labels are to be printed from the above-mentioned stocks list and
affixed to the front of the tank in a manner that does not obstruct the view of
the fish or the flow of water or the water level of the tank.
• Each Lab is assigned a color. Please attach the appropriate color flag to your
tank labels. This is required for facility organization and accurate billing.
• Larval fish are to be placed into appropriate tanks, at appropriate densities:
Ø 1-10 fish: 1.1L tank
Ø 11-40 fish: 3.5L tank
Ø 41-80 fish: 8L tank (Petri dishes are combined to reach 80
embryos, do not house 80 embryos in a single Petri dish.)
• An 800-micron BABY BAFFLE must be used for all tanks going onto the
Nursery system. Be sure not to use an Adult Baffle on embryos, as they will
flow through and be lost.
• Fill tank with about 2 inches of 2 ppt Larval Culture Water or Fish Systems
Water and place on Rotifer Rack.
• Once tanks are added to the Nursery system, they become the Fish Facility
Staff’s responsibility. No additional care is required from the user until the
tank leaves the Nursery, with the exception of checking on your fish’s health
and status.
• In general, fish will reach sexual maturity by 6-8 weeks. Once the majority
of fish in a tank have reached sexual maturity, they will be moved to a clean
tank, and placed within the users adult section in Streisinger or Chien
systems.
4
Common Nursery Requests:
Below are common Nursery requests that can be performed by Facility Staff. If
you would like any of these done, please CLEARLY label your tank with said
request.
ü Grow Fast: we can push the growth of fish to reach sexual maturity by 6
weeks. However, survivorship can be affected and sex will likely be
skewed towards females. Also, please note that the quicker fish reach
sexual maturity, the faster their fertility declines as Adults.
ü Grow Big: Tank is housed in the Nursery until all individuals have
reached sexual maturity. High productivity should be expected
immediately out of the Nursery.
ü Grow Small: Tank is removed a week or two before sexual maturation.
People do this for stocks that are not immediately needed or in hopes of
extending productivity.
ü Want Males: Sex can be skewed toward males through reduced feeding.
It takes longer for these fish to reach sexual maturity, due to reduced
feedings.
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Adult Rooms (Streisinger and Chien) Guidelines:
Adult room lights come on at 8:00 am and go off at 10:00 pm. The Adult Rooms
must NOT be entered during Lights Off.
• Fish within the Adult rooms are the responsibility of Tank Owner and Fish
Facility Staff.
• An exchange of all tanks is required every 3 months; the owner or Fish
Facility Staff can perform this for a fee.
• For optimal fish health, we suggest users spend 5 minutes per day with their
fish. During this time the following should be checked:
1. Sick and dead fish should be removed (one net per tank),
recorded in “Sick and Dead” fish log (located on book shelf in
each room), and disposed appropriately.
2. Clogged and overly dirty lids should be exchanged.
3. Excessively dirty and over-flowing tanks should be exchanged.
4. Fish health and feeding labels should be checked, for desired
results, and exchanged if needed. Food label books are located
on the bookshelf within each room. Users should consult with
Fish Facility Supervisor about appropriate feeding labels.
5. Check tank water flow.
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Euthanasia:
Euthanasia may be accomplished using one of the following approved methods:
1. Rapid cooling using an ice bath is the preferred method because it results in
more rapid and humane death, lower cost, and less chemical disposal into
the environment.
a. Prepare an ice bath using a container of appropriate size to hold all of
the fish to be euthanized.
b. Fill container with crushed ice (machine located in the Cleaning
Room).
c. Add system water to create a slurry and mix to ensure even cooling of
the water.
d. Place fish in ice bath, without adding excess additional water (e.g.
from a net). Stirring may be necessary to ensure even temperature
distribution with the ice bath.
e. Fish may be properly disposed of in a sink with a mechanical disposal
2 minutes after complete cessation of opercular movement.
2. An overdose of Tricaine methane sulfonate (a.k.a. Mesab, MS-222, Tricaine,
Finquel) solution of 4 g/L buffered to pH 7.4 by Trizma Base.
a. Place Tricaine solution in bowl or breeder tank bottom or similar
container, and fully immerse fish in solution.
b. Fish may be properly disposed of in a sink with a mechanical disposal
2 minutes after complete cessation of opercular movement.
Dispose of euthanized fish in the sinks, which have a garbage disposal. If this is
not possible (Quarantine), place carcasses in a biohazard bag located under the sink
and place in biohazard freezer (Mouse House deep freezer) or lab freezer for later
disposal.
Facility age limit of fish is 2 years; all stocks must be euthanized by this age to
maintain colony health.
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Breeding:
• Fish health and appropriate nutrition is vital in achieving high crossing
success. The image below shows the ideal size and health for actively
breeding female (top) and male (below) zebrafish:
• Fish can be too skinny, as well as obese. Both negatively affect productivity
and egg quality.
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Breeding Continued:
• Fish are most productive shortly after attaining sexual maturity. Productivity
starts to tail off after 9 months – 1 year or possibly sooner if fish were grown
fast. However, fish can be productive up to 2 years, if appropriately
managed.
• Keeping mixed sex groups in housing tanks is ideal when possible. Male
productivity in general, is not affected by isolation. Isolated females have to
be regularly set-up/managed to avoid becoming egg bound.
• Breeding of fish in the Core Fish Facility can be accomplished by either
allowing the fish to spawn naturally or by in-vitro fertilization (a.k.a. IVF,
squeezing).
• Fish that are regularly bred and set-up (trained) perform better. Infrequent or
irregular crossing reduces productivity.
• Mating crosses are set up on the “wet” mating racks in Streisinger or Chien
in the afternoon or evening. Usually with one male and one female, or 2-3
males and one female. Dividers can be used to separate males and females, if
one wishes to control the time of breeding the next day (see below for
details).
• The mating cages should be set up with a trickle or slow flow of water into
the tanks.
• A reduction of the water level the morning of crossing increases breeding
success.
• Maturation of eggs and the initiation of spawning behavior in zebrafish is
thought to be triggered at sunrise (the onset of the photoperiod at 8:00 am in
Streisinger and Chien). The closer to 8:00 am you pull your dividers, the
better breeding success you will have.
• Fish MUST be returned to their tanks on the day of breeding (i.e. they
cannot spend more than 24 hours on the mating rack).
• When finished, all dirty equipment associated with breeding fish is the
responsibility of the individual researcher to put in the designated racks and
containers in the Cleaning Room to ensure that the equipment is cleaned and
returned for use in a timely manner.
In Vitro Fertilization:
This procedure requires training by an experienced technician or researcher and
some practice to ensure success without unnecessary loss of fish life. Training is a
service provided by Facility Staff for a fee.
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Feedings:
The amount and frequency of feedings is up to the Tank Owner, provided the
desired feeding regimen is covered by animal use protocols. The facility has a
default feeding schedule and recommendations posted within each room. (See page
13 of this User Guide). Facility Users are encouraged to discuss breeding and
feeding strategies with Facility Staff. Together we can decide what plan best fits
your research needs and ensures the optimal wellbeing of fish.
Tritones (Feeding Robots):
• Tritone feeding times are posted within each room and attached (Page 13).
• The Tritone scans each tank at the start of each feed time, and then feeds the
appropriate amount.
• The indicator lights at the top end of each aisle show the Tritone’s current
status:
1. No Light = not in operation
2. Green Light = in operation
3. Yellow Light = in hold mode
4. Red Light = Error
• If a tank is moved in between the scan and feed out phase, the Tritone will
still feed that location, creating a mess.
• If the number of fish are reduced or increased within the tank, the
corresponding feeding label must be changed. Most overfed/underfed tanks
occur when the number of fish in a tank is changed without updating the
tank’s feeding label.
• There are 3-ring binders of Tritone feeding labels within Chien and
Streisinger on the bookshelf. If you cannot find the label you need, please
notify Facility Staff to print more.
• If the Tritone is in operation and you need to access the aisle, please press
the orange hold button at the start of the aisle before you walk in to pause
the Tritone. When you are finished, press the orange hold button once more
to take the Tritone back into operational mode.
• Tritones have light curtain barriers programmed to stop when encountering a
physical obstacle, e.g. a researcher. On occasion, these light curtains break
down and would not stop upon encountering a person. In such cases, a sign
will be posted. Please, be very careful around Tritones during this time.
Please try to avoid your aisle during the Rotifer Feeding (12:00-2:00pm)
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Facility Services and Resources:
The Facility offers a wide variety of services and training.
Services available include:
• Tissue collection for genotyping
• DNA isolation from tissue samples
• Genotyping of fish – PCR based assays or scoring larvae for
transgenics/phenotypes
• Cryopreservation of sperm
• Micro-injection
• Generating fish and embryos from frozen sperm by in vitro
fertilization
• Tank exchange
• Fish line maintenance
As the number of users grows, space and equipment may have limited availability.
To avoid conflict and ensure that the needed equipment is available on the day of
your experiment, we ask that you sign up for equipment use on the ZFISH Google
calendar.
Username: wustlzfish
Password: zfish2015
Equipment Available:
• Fluorescence microscopes
• Injection rigs
• I-Spawn (for mass embryo production)
• Presentation microscope
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Facility Contact List:
Stephen Canter
Facilities Supervisor
Email: [email protected]
Office Phone: (314) 362-4006
Facility Phone: (314) 362-1431
Cell Phone: (314) 960-2702
Anna Hindes
Lab Supervisor
Email: [email protected]
Contact For:
1. Cryopreservation
2. IVF
3. Facility ordering
4. Fish/Embryo shipping
John Engelhard
Research Technician II
Email: [email protected]
Contact For:
1. Fish/Embryo receiving
2. Bleaching
3. Fluke tablet treatment
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What does each Label feed?
Label
A
B
C
D
E
F
G
H
I
L
#Fish
1-5
5-10
10-15
16-25
26-35
36-45
46-55
56-65
66-75
76-85
ml of
Rotifers
5
6
7
8
10
11
12
14
16
18
J-Junior
Dry Fed x1
S-Senior
Dry Fed x2
A-Adult
Dry Fed x4
F-Feed a lot
Dry Fed x5
30 mg
30 mg
30 mg
60 mg
90 mg
150 mg
180 mg
240 mg
270 mg
300 mg
30 mg
30 mg
30 mg
60 mg
90 mg
150mg
180 mg
240 mg
270 mg
300mg
60 mg
60 mg
60 mg
60 mg
120 mg
180 mg
180 mg
240 mg
300 mg
360 mg
60 mg
60 mg
60 mg
60 mg
120 mg
180 mg
180 mg
240 mg
300 mg
360mg
*Volumes indicate amount per feeding*
Feeding Schedule:
Time
7:30am
9:30am
12:00pm
2:00pm
5:00pm
8:00pm
#Dry-Feedings
J-Junior
Dry
S-Senior
Dry
Rotifers
Rotifers
Rotifers
A-Adult
Dry
Dry
Rotifers
Dry
Dry
Dry
F-Feed a lot
Dry
Dry
Rotifers
Dry
Dry
Dry
2
4
5
1
Feeding Recommendations:
J-labels— Intended for low density <5, individuals, and split 1.1L tanks.
S-labels—Intended for infrequent breeding, storage of stocks, or slow growth of
3.5L and 8L stocks that will not be needed in the near future. Recommend for high
density (>5) and frequent breeding within 1.1L tanks.
A-labels—Intended for frequently bred fish with less hassle of dirty tanks. You
still can achieve good breeding success rates with these labels. Not recommended
for 1.1L tanks.
F-labels—These are intended for fish that are removed from the nursery early and
need additional growth, and for fish that are bred weekly (this is the feeding
regiment that used for daily WTs). With this feeding regimen, more time may be
needed to change dirty tanks or clogged baffles. Not recommended for 1.1L tanks.
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