Xenopus tropicalis and Xenopus laevis environmental parameter standard operating procedure (Kroll lab)

Procedures input by VGP

link to Kroll lab Xenopus environmental procedure SOP document at Washinton University: [1]

Modified format below:

A. Room Environmental Parameters

1. Room temperature (controlled automatically by building system)

• Set point = 68°F (20.0°C) ± 3°F (± 1.7°C)

• Alarm points = <65°F, >71°F (<18.3°C; >21.7°C)

2. Light cycle and intensity (controlled automatically by automated system)

• Circadian periodicity = 14 hours on, 10 hours off (14/10)

• Lights on at 6:00 am, lights off at 20:00 (8:00 pm) Central Time. Light timer clock will be changed to coincide with local time (i.e., adjusted for Daylight Savings in spring and fall).

• Room light override for after-hours access will automatically shut off after 1 hour. However, incursions into the room during the dark cycle are discouraged, as the abrupt provision of light will startle the frogs.

• Light bulbs provide full-spectrum lighting

• Average light intensity at 1 m from floor in center of room = XX lux (ZZ foot-candles)

B. Frog Water Parameters

1. Water temperature for X. laevis (chilled by Marine Biotech system)

• Set point = 64.4°F (18°C) ± 3°F (± 1.67°C)

• Alarm points = <61.4°F; >67.4°F (<16.3°C; >19.7°C)

2. Water temperature for X. tropicalis (heated by Marine Biotech system)

• Set point = 77°F (25°C) ± 2°F (± 1.0°C)

• Alarm points = <75°F; >79°F (<24°C; >26°C)

3. Dissolved gases (oxygenation)

Not considered a critical parameter for Xenopus frogs—adult Xenopus are air breathers that must come to the water’s surface to breathe. Some gases may be exchanged through the skin, but it is possible for Xenopus frogs to drown if kept underwater for several hours.

4. Dissolved solids (conductivity measured in microSiemens)

• conductivity set point = 1600 µS ± 50 µS

• conductivity alarm points = <1500 µS; >1700 µS

• Step 1: A reverse osmosis (RO) system will remove nearly all dissolved solids from the water that is used in the frog housing system.

• Step 2: A formulated synthetic sea salt mixture (e.g., “Instant Ocean,” “Crystal Sea,” or equivalent) will be added to the RO water in defined amounts to bring the water back to an appropriate osmotic pressure for frog maintenance. The salt mixture is added via an automated dosing system. The addition of salt is controlled by sensors that assess the conductivity (or osmotic pressure) of the water.

• Frogs are very susceptible to toxicity due to metal ions in solution. All piping and components in the frog water system are plastic.

5. Nitrogenous wastes (values in parts per million—ppm)

• Nitrification of water will be controlled by 1) a biological filter (bacteria that break down ammonia and nitrites); and 2) water replacement (see following).

• Frog wastes contain urates that are converted to ammonia.

• Nitrosomonas or Nitrosococcus spp. convert ammonia to nitrite

• Nitrobacter spp. convert nitrite to nitrate.

• Nitrate (the least toxic of the nitrogenous components of frog waste breakdown) will be removed by water replacement.

• Levels of nitrogenous compounds in frog system water will be monitored. The following water quality standards will be applied.

	Ammonia (NH3)	Nitrite (NO2)	Nitrate (NO3)
Target	< 0.25 ppm	< 0.50 ppm	< 20 ppm
Acceptable range	0 to 0.8 ppm	0 to 0.75 ppm	0 to 20 ppm
Alarm point	> 1.0 ppm	> 0.80 ppm	> 30 ppm

6. Water replacement

• Water replacement of 10% per day will be accomplished automatically.

• Water replacement is the primary means of removal of nitrates in the water.

• Water replacement will commence in System A at 8:00 am. When the System A change is complete, the sequencer will initiate water replacement in System B, followed by System C.

7. Water pH

• pH set point = pH 6.8 ± 0.2

• pH alarm points = pH <6.5; pH >7.1

• An automated pH sensor and sodium bicarbonate solution doser will maintain the frog water at the targeted pH level. Frog waste products will acidify the water, and this acidification is neutralized by the addition of sodium bicarbonate solution.

8. Water circulation and disinfection

Starting at the frog cage, the flow of water within the Marine Biotech system is as follows:

• Frog cages

• Cage drainage system

• Large particulate solids removal (filtration mesh overlying reservoir tank)

• Biofilter (bacteria growing on plastic kaldness) inside reservoir

• Particulate filter—pleated cartridge filter—removes fine particulate solids

• Carbon filter—activated carbon in mesh bags inside core of particulate filter—removes dissolved organic compounds

• Ultraviolet (UV) light disinfection unit—kills most water-borne microorganisms

• Water heater (for X. tropicalis) or water chiller (for X. laevis)

• Pump to circulate water

• Automated proportioners (to adjust pH and osmotic pressure)

• Flow meter and water distribution system for cages

• Frog cages

9. General water considerations

• Frogs must ALWAYS be kept in “frog system water” to avoid shocking their systems with changes in water quality and temperature. System water for frog transport and for making special frog solutions (e.g., anesthetic immersion solutions) is readily available in the frog housing rooms.

• Frogs are not to be placed in distilled or deionized (e.g., ultra-pure RO) water.

• Do not use tap water from sink for frogs. The sink in the frog room is for hand washing only. The chlorine and chloramine present in city water are caustic to frogs’ skin.

• Once removed, frog system water must not be returned to the system. Frog system water that has been removed for frog transport or other uses (e.g., anesthetic agent diluent, egg harvest solution, etc.) must be discarded after use.

• Do not inadvertently “mix and match” frogs from different cages. Recirculated frog system water is disinfected before it is returned to frog cages. Therefore, frogs are exposed only to the microbial flora (including potential pathogens) of direct contact cagemates. Exposure to non-cagemates may result in unwanted microbial transfer.

• Do not add any chemical agents to frog water without specific instructions from DCM vet staff. The frog water composition is automatically controlled, and any alterations in osmotic pressure or pH of the water could prove harmful to frogs.

C. Feeding

1. Food item

• Frogs will be fed “frog brittle” from Nasco (or equivalent). Frog brittle is a complete, balanced diet. See diet composition at http://www.enasco.com/Static.do?page=xen_brittle

• Frogs may eat segments that are shed from their skin—this is normal.

• Natural food items (e.g., beef liver) will not be provided to frogs due to risk of bacterial contamination and hypervitaminosis A.

• Any unusual frog feeding proposals need to be reviewed and approved by the ASC.

2. Feeding frequency and quantity

• Adult X. laevis will be fed 2 times per week (Tue, Thur).

• Approximately 5 pellets of frog brittle will be provided per frog.

• The quantity of food supplied will target what frogs will eat within 1 hour following feeding. Post-feeding tank cleaning of food debris and fecal material will be handled automatically by water flow and filtration within the frog housing system. However, excessive feeding is wasteful, and food debris will tax the biological filter capacity.

3. Food intake assessment

• Frog food intake will be monitored in conjunction with feeding (Tue, Thur).

• Frogs that appear underweight may be housed 2 per cage to reduce competition for food. Single housing is to be avoided, as is has been noted that the visual stimulation of another frog feeding may be important in activating a frog’s desire to eat.

4. Pre-anesthetic food restrictions

• Frogs should be fasted 12-18 prior to anesthesia induction to reduce food regurgitation.

D. Frog Housing and Identification

1. Housing density

• Adult X. laevis

o 23 liter (large) cages: 9 frogs per cage

• Adult X. tropicalis

o 16 liter (small) cages: 16 frogs per cage

2. Housing enrichment items

• A section of 4” diameter plastic (PVC) pipe, 8-12” long, will be placed in each frog cage to provide a “shelter” where frogs can hide.

• Frog food pellets should be the only small items that are introduced into cages. A frog may ingest any item that is small enough to fit in its mouth. Ingestion of non-food items can cause serious illness or death.

3. Frog identification (ID) methods

The following ID methods are approved for Xenopus frogs:

• cage cards (required on all occupied frog cages—see general DCM requirements)

• microchip transponders or readable tags (implanted under skin)—see http://tropicalis.berkeley.edu/home/husbandry/tags/microchips.html and http://tropicalis.berkeley.edu/home/husbandry/tags/E-ANTs.html#ANT

• plastic beads sutured to skin

• injection with plastic elastomer under skin (see : http://tropicalis.berkeley.edu/home/husbandry/tags/E-ANTs.html#ANT

E. Sanitation

1. General Considerations

• Frogs are highly susceptible to contamination of their water with soaps, detergents, disinfectants, and other chemical agents.

• Unapproved chemicals and cleaners must not be stored in the frog room. Keeping unapproved chemicals out of the frog room will reduce the risk of inadvertent contamination of frog water.

2. Room—floor, walls, ceiling, shelf racks, sink area

• Routine cleaning of room surfaces will employ warm water applied by hand (via a sponge or mop).

• Activities that generate aerosols (e.g., spray misting, power washing) will not be used for general surface cleaning within the frog housing room.

• The use of soaps, detergents, disinfectants, and other chemical agents will be avoided as much as possible. Cleansing chemicals will be used only when necessary and only under the direction of authorized DCM personnel.

3. Frog cages

• Frog cages are expected to remain quite clean due to the flow of clean water within the frog housing system. Over time, there may be a build-up of algae in some cages. In many cases, the majority of the algae buildup may be removed from the cage surfaces using a plastic scraper on the cage in situ. The algae will then be removed via water flow and filtration by the automated system. When possible, cages should be scraped to remove algae when frogs have been temporarily removed for experimental procedures.

• If in situ scraping of the cage is not sufficient, the frogs will be placed in another clean cage and the dirty cage will be removed from the system. Algae will be removed by physical scrubbing action using clean paper towels and frog system water. If additional disinfection is desired, 70% alcohol may be used sparingly on the cage surfaces. When cleaning is complete, the cage must be rinsed thoroughly using frog system water. The cage is then allowed to air-dry completely before it is re-used. Air drying will kill most of the remaining vegetative microorganisms that may be present on cage surfaces.

• Do not allow dirty (e.g., algae-covered) cages to air-dry without first cleaning the surfaces. Once dried, the algae accumulation will be much more difficult to remove.

• The plastic materials used for frog cage construction have an “active surface” that tends to bind to chemicals. For example, alkaline detergents leave a tightly bound residue on the plastic that even repeated rinsing cannot remove. The slow leaching of these residues back into the frogs’ water supply could have deleterious results. Therefore, alkaline detergents or disinfectants should not be used for cleaning frog cages.

4. Water system components

• Frog system components are expected to remain quite clean due to the flow of clean water within the frog housing system. All component surfaces that come in contact with water are expected to develop a microbial biofilm. This biofilm is not visible, and the organisms that comprise the biofilm are not generally pathogenic. The bacterial agents that accomplish the de-nitrification of the system water reside in the biofilm.

• Over time, there may be a build-up of algae in some components. While the algae in itself does not generally pose a health risk to frogs, overgrowth may become undesirable in some system components. The procedures for algae removal from water system components will be the same as those outline above for frog cages.

5. Accessories (nets, individual frog containers, etc.)

• Frog nets will be color-coded for individual investigators and specific cages or rows of cages.

• All nets must be maintained in good condition, free of holes and sharp edges that could injure frogs. Damaged nets should be discarded.

• Frog nets will be maintained in dry condition between uses. Air drying will kill most of the vegetative microorganisms that may be present on net surfaces. Hooks for hanging frog nets will be available in appropriate sites.

• A frog net must be thoroughly wetted with either clean system water or water from one specific cage prior to use. Frog skin must never come in contact with dry surfaces.

• A frog net must not be used in more than one frog cage in sequence, as the net will transfer microorganisms from one cage to another. Transferred microorganisms may cause disease when they come in contact with naïve frogs.

• When net use is completed for a specific cage, the net should be rinsed thoroughly using frog system water. If net disinfection is desired, the net may first be sprayed with alcohol, taking care not to cause excessive alcohol aerosols in the room. Allow several minutes for the alcohol to exert its disinfectant effects, then rinse it well and hang it up to dry.