If you have a different media protocol, add it as another header here.
Phosphate-buffered Giardia Growth Medium
5X Basic Medium (500ml)
in about 400ml milliQ water add:
- 25 g D-glucose
- 5g NaCl
- 25 g yeast extract
- 50 g casein peptone digest (SIGMA P6838, Lot#27H1113)
- Stir on stirplate for about 30 minutes.
- Bring to 500ml volume with more milliQ water.
- Prefilter with a 0.22µm filter. This keeps >4 months at 4°C.
Complete Medium (500ml)
Thaw ABS (adult bovine serum), FAC (ferric ammonium citrate) and PSF (PenStrepFungizone) in 37C water bath.
Then in a 500ml beaker, add:
- 350 ml milliQ water
- 1 g cysteine (SIGMA C-7880)
- 0.05 g ascorbic acid
- Stir to mix, and pH to 7.0-7.2 with 10M NaOH Transfer to 500ml purple cap media bottle.
- In laminar flow hood, add the following to the cysteine solution from above:
- 10 ml 50X Phosphate Buffer Solution (50X= (15g KH2PO4/32.5g K2HPO4x3H2O in 500ml)
- 100 ml semi-sterile basic medium (see above) ***make sure to warm semi-sterile basic medium to 37 degrees C for 30 min, or else it will clog the filter***
- 4 ml sterile 6.5% bovine bile (stored at 4°C)
- 1.5 ml sterile FAC (ferric ammonium citrate) (2.28mg/ml stock)
- 5 ml pen/strep+fungizone (Gibco)
- 50 ml Adult Bovine Serum (Invitrogen, lot #)
- Shake to mix in bottle, then filter with 0.22µm filter. Medium should be clear and amber-colored. The medium only lasts about 7-10 days, so it is best make it fresh every week. Cysteine precipitates out of solution easily over time (looks like crystals in microscope or flakes in bottle). If this happens, change medium immediately.
- Store at 4C.