TITLE: Derivation of Gnotobiotic Stickleback Fish
AUTHORS: Kat Milligan-McClellan

[DESCRIPTION]
This protocol is used to generate gnotobiotic stickleback fish. Based on the zebrafish protocols, this was optimized for stickleback. See the paper https://doi.org/10.1242/dmm.021881 for the first description of the use of this protocol. This was created by Kat Milligan-McClellan with extensive help from Erika Mittge at the University of Oregon.            

                

[BEFORE_START]
Tools for dissections should be rinsed in 10% bleach and soaked in 70% ethanol prior to use
Gloves should be worn throughout the protocol to prevent contamination from skin microbiome
Any person handling fish should be trained on proper fish care BEFORE starting this protocol and should receive IACUC or other training (see ethics statement below)
25cm2 flasks can hold about 10 eggs; 75cm2 flasks can hold about 20 eggs and 50ml of water; 150cm2 flasks can hold about 40 eggs and 100ml of water; 300cm2 flasks can hold about 80 eggs and 200ml of water

[GUIDELINES]
Use a biosafety cabinet class II to ensure sterility of fish. If this protocol is performed without a biosafety cabinet, sterility drops considerably. 

[STEPS]
SECTION: Fertilize eggs
1. Put on gloves
SECTION: Fertilize eggs
2. Gently squeeze eggs from females into sterile 20 or 60mm petri dishes. Place the cover on the eggs and transfer to the dissection area.
SECTION: Fertilize eggs
3. Clean the surface of the dissection area with 70% ethanol
SECTION: Fertilize eggs
5. Euthanize the male in freshly made, filter sterilized tricaine/MS222 buffered with sodium bicarbonate


SECTION: Fertilize eggs
6. Spray the outside of the euthanized male with 70% ethanol prior to dissection, and wipe body with Kimwipe or paper towel
SECTION: Fertilize eggs
7. 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
SECTION: Fertilize eggs
8. When testes have been collected, use a clean, new razor to macerate the testes using lengthwise cuts and cross cuts
SECTION: Fertilize eggs
9. 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.
SECTION: Prepare eggs for cleaning
12. Incubate fertilized eggs at 20-23°C until eggs reach 2-8 cell stage, ~2 hours post fertilization
SECTION: Prepare eggs for cleaning
14. 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
SECTION: Prepare eggs for cleaning
15. About 6 hours post fertilization, transfer viable embryos into sterile autoclaved 50ml beakers with foil tops, about 100 eggs per beaker
SECTION: Prepare eggs for cleaning
16. add ~40ml filter sterilized stickleback medium to the eggs in the beaker
SECTION: Prepare the hood
17. 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
SECTION: Prepare the hood
19. 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
SECTION: Clean surface of chorions
20. Transfer embryos to clean autoclaved, foil covered beaker
SECTION: Clean surface of chorions
21. Pour off all but ~10ml stickleback medium carefully into waste container
SECTION: Clean surface of chorions
22. Transfer remaining 10ml stickleback medium with eggs into clean 50 ml beaker
SECTION: Clean surface of chorions
23. Add ~20ml sterile stickleback medium to old beaker to get remaining eggs
SECTION: Clean surface of chorions
24. Transfer remaining eggs to beaker
SECTION: Clean surface of chorions
25. Bring volume in new beaker up to 50ml with sterile stickleback medium
SECTION: Clean surface of chorions
26. Rinse embryos 3X with 50ml filter sterilized stickleback medium
SECTION: Clean surface of chorions
27. Immerse embryos in ~50ml 0.2-0.4% PVP-I solution for 10 minutes
SECTION: Clean surface of chorions
28. Rinse embryos with sterile stickleback medium 1X
SECTION: Clean surface of chorions
29. Transfer embryos to fresh beaker as in step 21-25
SECTION: Clean surface of chorions
30. Rinse embryos in sterile stickleback medium additional 2X
SECTION: Clean surface of chorions
31. Immerse embryos in 50ml 0.003% bleach for 10 minutes.
SECTION: Clean surface of chorions
32. Rinse embryos in sterile stickleback medium 1X
SECTION: Clean surface of chorions
33. Transfer embryos to fresh beaker as in steps 21-25.
SECTION: Clean surface of chorions
34. Rinse embryos additional 2X in sterile stickleback medium 
36. 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 
SECTION: Post-cleaning fish husbandry
38. 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.
SECTION: Post-cleaning fish husbandry
41. Fish can remain in germ free flasks until 14 days post fertilization without food. At that point they will require food. 
SECTION: Post-cleaning fish husbandry
40. 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.
SECTION: Post-cleaning fish husbandry
42. 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.
SECTION: Post-cleaning fish husbandry
43. 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.
SECTION: Post-cleaning fish husbandry
44. 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.
SECTION: Prepare the hood
18. Sterilize the items above by exposing them to UV light for a minimum of 10 minutes.
SECTION: Fertilize eggs
4. Sterilize dissecting tools (scissors and forceps) with a brief rinse in 10% bleach followed by rinse in 70% ethanol
SECTION: Fertilize eggs
10. Incubate eggs with sperm for 15 minutes at room temperature.
SECTION: Fertilize eggs
11. Add filter-sterilized antibiotic stickleback medium to the fertilized eggs to cover the eggs. 
SECTION: Prepare eggs for cleaning
13. Transfer fertilized eggs to sterile 100mm petri dishes and rinse in ~100ml sterile antibiotic stickleback medium
SECTION: Clean surface of chorions
35. 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)
SECTION: Post-cleaning fish husbandry
37. Move flasks to incubator in upright position to ensure eggs do not get stuck in the neck of the flask in the transfer
SECTION: Post-cleaning fish husbandry
39. 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