Derivation of Gnotobiotic Stickleback Fish

Put on gloves

Gently squeeze eggs from females into sterile 20 or 60mm petri dishes. Place the cover on the eggs and transfer to the dissection area.

Clean the surface of the dissection area with 70% ethanol

Sterilize dissecting tools (scissors and forceps) with a brief rinse in 10% bleach followed by rinse in 70% ethanol

Euthanize the male in freshly made, filter sterilized tricaine/MS222 buffered with sodium bicarbonate

Spray the outside of the euthanized male with 70% ethanol prior to dissection, and wipe body with Kimwipe or paper towel

Dissect the testes out of the male fish by cutting from next to the anal vent to jaw, removing or pushing the intestine to the side, and using the sterile forceps to remove the testes. These are generally black but may be silver. Transfer the testes to the top of a sterile petri dish

When testes have been collected, use a clean, new razor to macerate the testes using lengthwise cuts and cross cuts

Add 5-10 drops of filter-sterilized antibiotic stickleback medium to the macerated testes with a sterile transfer pipette. Pippette up and down to fully mix the testes. The solution should be cloudy. Transfer 3-5 drops of the testes to each clutch of stickleback eggs. 1 set of testes is typically enough to fertilize up to 3 clutches of eggs.

Incubate eggs with sperm for 15 minutes at room temperature.

Add filter-sterilized antibiotic stickleback medium to the fertilized eggs to cover the eggs.

Incubate fertilized eggs at 20-23°C until eggs reach 2-8 cell stage, ~2 hours post fertilization

Transfer fertilized eggs to sterile 100mm petri dishes and rinse in ~100ml sterile antibiotic stickleback medium

Remove non-viable embryos from fertilized eggs, remove egg goo with a sterile transfer pipette, separate eggs gently with sterile tweezers or paint brushes, and rinse viable embryos with fresh antibiotic stickleback medium 2X

About 6 hours post fertilization, transfer viable embryos into sterile autoclaved 50ml beakers with foil tops, about 100 eggs per beaker

add ~40ml filter sterilized stickleback medium to the eggs in the beaker

Clean the following with 70% ethanol and transfer the following items into the hood to minimize number of times entering and exiting the hood (per 100 embryos cleaned):

• 3 sterile 50ml beakers with foil tops
• Individually wrapped, sterile transfer pipettes or sterile, individually wrapped 2 ml pipettes
• 1 L filter sterilized stickleback medium
• 0.003% bleach
• 0.2-0.4% PVP-I (some flasks may be contaminated with 0.2% PVP-I, but more fish are likely to die in the 0.4% PVP-I)
• Large beaker for collecting liquid waste

Sterilize the items above by exposing them to UV light for a minimum of 10 minutes.

Turn UV light off, then bring in embryos. Spray outside of beaker with 70% EtOH prior to bringing embryos into the hood. MAKE SURE NO ETHANOL GETS INTO THE BEAKER WITH THE EGGS

Transfer embryos to clean autoclaved, foil covered beaker

Pour off all but ~10ml stickleback medium carefully into waste container

Transfer remaining 10ml stickleback medium with eggs into clean 50 ml beaker

Add ~20ml sterile stickleback medium to old beaker to get remaining eggs

Transfer remaining eggs to beaker

Bring volume in new beaker up to 50ml with sterile stickleback medium

Rinse embryos 3X with 50ml filter sterilized stickleback medium

Immerse embryos in ~50ml 0.2-0.4% PVP-I solution for 10 minutes

Rinse embryos with sterile stickleback medium 1X

Transfer embryos to fresh beaker as in step 21-25

Rinse embryos in sterile stickleback medium additional 2X

Immerse embryos in 50ml 0.003% bleach for 10 minutes.

Rinse embryos in sterile stickleback medium 1X

Transfer embryos to fresh beaker as in steps 21-25.

Rinse embryos additional 2X in sterile stickleback medium

Add sterile stickleback medium to tissue culture flasks.

• 25cm² flasks add 19ml of stickleback medium (assumes about 1ml of medium will be used to transfer eggs)
• 75cm² flasks add 48ml of stickleback medium (assumes 2ml of medium in transfer)
• 150cm² flasks add 96ml of water (assumes about 4ml of medium in transfer)
• 300cm² flasks add 194ml of water (assumes about 6ml of medium in transfer)

Transfer embryos to flasks containing sterile stickleback medium using sterile, individually wrapped pipettes. Tightly close flasks after transfer

• For 25cm² flasks add up to 10 eggs
• 75cm² flasks add up to 20 eggs
• 150cm² flasks add up to 40 eggs
• 300cm² flasks add up to 80 eggs

Move flasks to incubator in upright position to ensure eggs do not get stuck in the neck of the flask in the transfer

Incubate embryos in light-controlled 18-20°C in incubator, laying flasks flat. Make sure eggs do not go into the neck of the flask.

Check for contamination of water by microscopy throughout the experiment. Using an inverted light microscope, examine the inside of the bottom of the flask for cocci, rods, or filamentous microbes, which may be moving or stationary. Presence of microbes indicates contamination

Daily, record the number of fish dead or not moving in response to external stimuli. Always transfer flasks in upright position and ensure no eggs are in the neck of the flask.

Fish can remain in germ free flasks until 14 days post fertilization without food. At that point they will require food.

To determine contamination of water by culture, plate 50ul to 100ul of water on TSA plates and incubate at 20°C. Presence of microbes indicates contamination.

To determine contamination of flask by microscopy, visualize bottom of flask using inverted light microscopy under a minimum of 40X magnification. Presence of microbes on inside of bottom of flask indicates contamination.

To determine contamination of flask by 16S PCR, collect water on 0.22um filter, extract DNA from filter, and perform PCR of 16S ribosomal RNA gene using standard 27F and 1492R primers. Presence of a band of about 1400 bp from the PCR product on gel electrophoresis indicates contamination.