Powder SOP CA-01-02
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Effective Date: 1-23-18
Title: Daily Care of Zebrafish (Danio rerio)
Facility: Aquatic Animal Research Laboratory (AARL) Room 3
Author(s):
Date: 1-23-18
Kara E. Powder, Principle Investigator
AARL Facility
Manager:
John A. Smink
Date:
Attending
Veterinarian:
Dr. John Parrish
Date:
1.0 OBJECTIVE
This SOP describes daily care of zebrafish (Danio rerio). This SOP is based on standard
aquarium trade procedures and care protocols from The Zebrafish Book: A Guide for the
Laboratory Use of Zebrafish (Danio rerio), by Monte Westerfield.
2.0 HEALTH AND SAFETY
All personnel will be enrolled in the Clemson University Medical Surveillance Program. Attire
appropriate to the task must be worn at all times.
3.0 PERSONNEL/TRAINING/RESPONSIBILITIES
All personnel must have completed the required Animal Care and Use Committee on-line
training. Thereafter, any Clemson employee familiar with the techniques and trained in this and
referenced SOPs may perform this procedure. Prior to being assigned full responsibility for
performing this procedure, personnel must have demonstrated proficiency in the use of the
technique in a closely supervised environment. Documentation of training should be kept for
each person performing the procedure.
4.0 REQUIRED AND RECOMMENDED MATERIALS
4.1 Required Materials
Aquaneering zebrafish system, GM300 adult food, larval food (Ziegler brand <100-micron),
zebrafish tanks and baffles, tape for labeling, permanent markers, transfer pipette, API brand (or
comparable) water test strips, API brand (or comparable) ammonium test kit, handheld monitor
for conductivity, pH and temperature
5.0 GUIDELINES
The following procedures were developed to meet or exceed the National Institute of Health
(NIH) Guide for the Care and Use of Laboratory Animals.
Westerfield, M, 2007, The Zebrafish Book, 5th edition. Eugene: University of Oregon Press.
http://zfin.org/zf_info/zfbook/zfbk.hmtl.
6.0 PROCEDURE
6.1 Zebrafish System
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Effective Date: 1-23-18
Zebrafish are maintained on a purpose-built recirculating system purchased from a leading
manufacturer, Aquaneering. This system includes all necessary mechanical and biological
filtration, UV sterilization, RO water preparation, automated timed water exchange (10% per
day), automated chemical dosing, and automated monitoring of pH, temperature, conductivity,
and water level. Necessary components are on a backup generator in case of power failure. Per
standard zebrafish protocols described in Westerfield 2007, all zebrafish will be maintained on a
14 hour light/10 hour dark cycle, managed by built in timer systems.
6.2 Animal acquisition and identification
Zebrafish will be obtained from established commercial facilities like the Zebrafish International
Resource Center (ZIRC), donated from other academic institutions, or produced via breeding
within AARL Room #3. All additions to the system will be noted in the Zebrafish Inventory and
Mortality Log.
6.3 General Housing
Animals are kepts in tanks between 1.8 and 6L with a density of 10 adults per liter of system
water. Different transgenic strains are housed in separate tanks on the same water system. All
tanks should be labeled using tape and permanent marker with the strain name and birth date.
6.4 General Care of Fish
The below points will be addressed daily, with information recorded on the Daily Care Log and
Checklist. If any of the listed parameters are outside the acceptable range, the care taker should
take immediate action to bring the value to an acceptable value (or tell the PI or facility manager
if you don’t know what to do) and record the action taken in the comments section of the Daily
Care or Water Quality Logs. A monitoring system provides text and email alerts to relevant lab
members, the PI, and John Smink, Aquatics Facility manager, if there are significant deviations
for water temperature, water level, pH, and conductivity. In the event of an alert, the alert and
action taken will be recorded in the comments section of the Daily Care or Water Quality Logs.
A. Room maintenance
As needed, clean floors, put away equipment, and clean zebrafish tanks from accumulated food
or algae through siphoning. As needed, tanks will be manually scrubbed and put on the rack to
dry. Tanks will be disinfected periodically using a bleach solution (1/4 cup or 60mL sodium
hypochlorite in 10 gallons of water). Tanks should be rinse VERY well after bleaching and
allowed to dry completely before use.
B. Water temperature
Optimal temperature is 28.5˚C. Zebrafish can be can be grown +/- 5 ˚C (23-33˚C) without any
side effects – as would be expected from animals that live in rivers in the wild that are subject to
changes in the weather and temperature fluctuations. However, acceptable range for the system
will be considered +/- 2.5 ˚C, or 26-31˚C. Water temperature readings from the automated
system will be recorded daily in the log. Room temperature should be between 70-80˚F (2127˚C). As needed, room temperature will be adjusted using a space heater. Room and water
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Effective Date: 1-23-18
temperature will be periodically verified independent of the system using a thermometer or
handheld device and recorded in Water Quality Log.
C. Water conditions
The system is automated to adjust pH and salt levels. Conductivity should be between 3001500 microSiemens (S) (500-700S optimal) and pH should be 7.0-8.0. Readings from the
automated system will be recorded daily in the log. Other deviations (e.g. an increase in water
cloudiness) should be recorded in the Water Quality Log and reported immediately to the PI.
Water conditions will be verified independent of the system by the PI through occasional
checking by test strips and kits (e.g. API 5 in 1 aquarium water test strip and API Ammonium
test kit) to measure nitrate, nitrite, alkalinity, general hardness, and ammonium levels.
Conductivity and pH will be verified independent of the system with a handheld device or test
strips. Results of all independent verifications will be recorded in the Water Quality Log. All
water quality checks and verifications independent of the automated system will be conducted
monthly, or as needed.
Acceptable water quality parameters are in below table and on Daily Care and Water Quality
Logs. Values are based on industry standards, as reported in Westerfield’s The Zebrafish Book
5th edition (2007) and Lawrence’s The Laboratory Zebrafish: A Volume in The Laboratory
Animal Pocket Reference Series (2012).
Parameter
Optimal
Measured by*
Acceptable
Range
Water temperature
28.5 ˚C
26-31˚C
Daily readings from automated
system, periodically verified
independently by second
thermometer or handheld device
pH
7.0-8.0
Daily readings from automated
system with meter calibrated every
2weeks-1 month, periodically
verified independently by
handheld device or test strips
Conductivity
Daily readings from automated
500-700S 300-1500 S
system with meter calibrated every
2weeks-1 month, periodically
verified independently by
handheld device or test strips
Nitrate
<200 ppm
Test strips
Nitrite
<0.5 ppm
Test strips
60-150 ppm
Test strips or titration
Alkalinity (Carbonate
hardness)
General hardness
75-200 ppm
Test strips or titration
Ammonium
<0.5 ppm
Test kit
*All measurements not obtained daily from the automated system will be collected monthly, or
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Effective Date: 1-23-18
as needed.
D. Feeding
Adult and larval zebrafish food will be stored in the dedicated fridge in AARL Room #17,
labeled with PI name, purchase date, and date opened. Zebrafish must be fed once a day per the
following ratios:
 15 adults receive roughly 1/3 of a “drop” scoop of GM300 feed, using the
provided measuring spoons. This is equivalent to roughly 5% of their body mass.
 Larval food is prepared by mixing 1/3 of a “drop” scoop of larval (<100micron)
food per 50mL system water. 50 larvae receive 2 fills of a transfer pipette
(~5mL) of this prepared solution.
E. Monitoring of fish and water flow
Each day, every tank containing animals should be observed. Check that water is actively flowing
into the tank; use the valve to adjust as needed. Fish are monitored daily for health through
observation of normal swimming and feeding behaviour, correct orientation, normal interaction
within the school, smooth skin, and normal coloration. See section 6.4.A for further instructions of
sick or dead animals.
F. Cleaning screen
The filtration screen should be rinsed daily and replaced.
G. Inventory
Any changes to fish number will be noted daily on the Inventory and Mortality Log.
H. Sanitation
 Tanks which can be pulled off line will be disinfected periodically using a bleach solution
(1/4 cup or 60mL sodium hypochlorite in 10 gallons of water). Tanks should be rinse
VERY well after bleaching and allowed to dry completely before being returned to the
system for use.
 Dirty nets will be placed into the appropriately marked container. Nets are sanitized by
soaking in a 5% bleach (sodium hypochlorite) solution for 24 hours in the dirty net
container. Nets should be rinsed VERY well after bleaching and allowed to dry
completely before being placed with other clean nets in the appropriately marked area.
 Petri dishes will be manually scrubbed and sprayed with 70% ethanol prior to reuse.
6.5 Specialized Animal Care
A. Sick animals
Animals that look sick should be transferred to a separate tank to be assessed by the PI. The tank
should contain system water, but not be on the system to prevent spread of sickness. The AARL
Facility manager and the Attending Veterinarian will be notified immediately. Sick and injured
animals may be treated with antibiotics if this is deemed necessary. If animals are too distressed or
deemed by the PI or the AV to be unable to recover, they will be euthanized.
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B. Euthanasia
Animals are sacrificed as described in Powder SOP EU-01-01 and Powder SOP EU-02-01.
C. Dead animals
If any dead animals are noticed during daily inspection, it will be noted in the Zebrafish
Inventory log. Carcasses are disposed of as described in Powder SOP EU-03-01.
6.6 Specialized System Care
The following system maintenance will be done by the PI or a senior member of the
lab. If there are any alarms or monitors going off for these, notify the PI or
senior lab members immediately. All system maintenance will be recorded under
the comments column on the daily log or in the Water Quality Log. If you have any
doubts about any of this, ask the PI before touching anything!
A. Preparation of stock solutions
As needed, the stock solution of bicarbonate and salt will be added to the automated dosing
system reservoirs and noted on the comments column. THIS SHOULD HAPPEN EVERY FEW
WEEKS AT MOST—IF THIS IS HAPPENING MORE FREQUENTLY, SAY SOMETHING
(the probe may be broken or in need of calibration). The solution should be prepared at roughly
¼ lb (113g) sodium bicarbonate per 3 gallons, and ½ lb (226 g) Instant Ocean salt per 3 gallons
of system water. Before adding, double check that you are adding the salt solution to the salt
container and the bicarbonate solution to the bicarbonate container.
B. UV bulb
A UV bulb is used to kill control biological contaminants such as bacteria. This will be replaced
by the PI roughly every year, or as necessary. Crystal sleeve will be cleaned roughly yearly, and
replaced roughly every 2 years, or as necessary.
C. Carbon filtration
Carbon filtration sleeves and activated carbon will be replaced roughly every 3 months or as
necessary, per manufacturer’s instructions. Per manufacturer’s instructions, replacement of
carbon will be alternated between the two filters (i.e. change A after 3 months, then B after
another 3 months, then A again after another 3 months).
D. RO system filters
The carbon and sediment filtration units on the RO water maker will be replaced roughly every
6-12 months or as necessary, per manufacturer’s instructions.
E. Cleaning and calibration of probes
The pH and conductivity probes will be cleaned and calibrated roughly every 2wks-1 month or
as necessary, per manufacturer’s instructions.
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Effective Date: 1-23-18
F. Bead biofilter
As necessary, glass beads will be added to the biofilter.
7.0 QUALITY CONTROL CHECKS AND ACCEPTANCE CRITERIA
All procedures are subject to review by the Attending Veterinarian(s) and the Institutional
Animal Care and Use Committee.
8.0 LITERATURE CITED
1. Westerfield, M, 2007, The Zebrafish Book, 5th edition. Eugene: University of Oregon Press.
http://zfin.org/zf_info/zfbook/zfbk.hmtl.
2. Aquaneering system manual.
3. Lawrence, C. (2007) ‘The husbandry of zebrafish (Danio rerio): A review’ Aquaculture 269,
p1-20.
4. Harper, C and C. Lawrence, 2012, The Laboratory Zebrafish: A Volume in The Laboratory
Animal Pocket Reference Series. CRC Press.
9.0
APPENDICES
Zebrafish Daily Care Log and Checklist
Zebrafish (Danio rerio) Water Quality Log
Zebrafish (Danio rerio) Inventory and Mortality Log