10 ml 10% para-formaldehyde
15 ml 1.25X fix buffer
Adjust pH to 7.3 if necessary. Add dH2O to a final volume of 25.0 ml. Final concentration
is 4% para-formaldehyde, 0.15 mM CaCl2, 4%sucrose in 0.1 M PO4 buffer.
Drain eggs add 5 ml of 0.5 mg/ml pronase for 3.5 min.
Dilute eggs with 200 ml of 8x water. Rinse 3x more with 200 ml washes of 8x water.
8x water = 12 ml stock salts per liter dH2O
Stock salts = 40 g Instant Ocean per liter dH2O
DAB is prepared by dissolving the contents of 1 g bottle of DAB in 25 ml dH2O and filtering with a disposable unit attached to a disposable syringe. Aliquots containing 1 mg DAB per 25 µl of solution are stored in 1.5 ml microfuge tubes in a -20°C freezer. Thawed aliquots may be refrozen. To minimize risk in handling this carcinogen, use disposable plastic containers and pipettes wherever possible. Treat waste with bleach.
1. Chill 200 3 d embryos after removing from chorions.
2. Rinse in 0.5% chilled bleach for 2 min and then in zero calcium Ringer for 2 min.
3. Transfer to a Dounce homogenizer with a minimum of liquid and homogenize well.
4. Resuspend in 1 ml L-15 supplemented with 0.3 mg/ml glutamine, 50 U/ml penicillin, 0.05 mg/ml streptomycin and 0.8 mM CaCl2.
5. Store at -20°C.
1. Add 10-15 grains of boiled wheat to 175 mls of dH2O.
2. Inoculate with 20 ml from an excellent existing seed culture dish or with a sample from the commercial inoculant.
3. Grow for 7 to 12 days before using.
1. Use glass finger bowls filled two-thirds full with system water.
2. To each bowl add 8-9 grains of of boiled wheat and 8 ml from a paramecia seed culture.
3. Stack the finger bowls 6 or 8 high, cover, and store at 28.5°C on well-lit shelves.
4.After 10-14 days, the paramecia are ready to feed to the fish larvae. Cultures remain useable and healthy for a month or more.
1. Fill plastic mouse cage or equivalent with 2 liters of system water.
2. Add a large pinch (about 40-50 grams) of boiled wheat, four 250 mg tablets of brewer's yeast, and 100 ml (or half the contents of a plastic seed culture dish) of paramecia culture to each mouse cage.
3. Cover and store in a warm, well-lit place. The covered mouse cages may be stacked three layers high with the highest layer closest to a warm light. It will be ready to feed first.
4. Brewer's yeast may also be added to the seed cultures (one half of a 250 mg tablet per 200 ml of seed culture). The cultures are then ready to use in four days and are depleted after two weeks.
Tricaine (3-amino benzoic acid ethyl ester also called ethyl 3-aminobenzoate)
comes in a powdered form from Sigma (Cat.# A-5040). It is also available as Finquel
(Part No. C-FINQ-UE) from Argent Chemical Laboratories, Inc. Make tricaine solution
for anesthetizing fish by combining the following in a glass bottle with a screw
400 mg tricaine powder
97.9 ml DD water
~2.1 ml 1 M Tris (pH 9).
Adjust pH to ~7. Store this solution in the freezer. (Buy the smallest amount possible because tricaine gets old.)
To use tricaine as an anesthetic combine the following in a 250 ml beaker:
4.2 ml tricaine solution
~100 ml clean tank water.
66 µl nitroblue tetrazolium (NBT) stock
33 µl 5-bromo-4-chloro-3-indolyl galactopyranoside
10 ml color development buffer
Both NBT and BCIP stocks are 50 mg/ml in dimethylformamide. NBT stock is made by suspending 50 mg NBT in 700 ml dimethylformamide. Vortex. Add 300 µl distilled water to dissolve. Store both stocks at 4°C in dark.