TABLE 4.
Medium for in vitro growth of G. lambliaa
| Component | Quantity/liter | Final concn |
|---|---|---|
| K2HPO4 · H2O | 1 g | 4.4 mM |
| KH2PO4 | 600 m | 4.4 mM |
| Trypticase (casein peptone) | 20 g | |
| Yeast extract | 10 g | |
| d-Glucose | 10 g | 56 mM |
| NaCl | 2 g | 34 mM |
| Cysteine-HCl | 2 g | 16.5 mM |
| Ascorbic acid | 200 mg | 1.1 mM |
| Ferric NH4-citrate (Sigma F-5879) | 22.8 mg | |
| Bovine bile (Sigma B-8381) | 40 ml of 6.5% solution | |
| NaOH | Bring pH to 7.0–7.2 (1.65 ml of 10 M solution | |
| Serum (bovine or fetal calf) | 100 ml | 10% |
Formula of TYI-S-33 (159), the most commonly used medium for growth of G. lamblia trophozoites. The ferric-NH4 citrate and bovine bile are generally kept as solutions at 4°C. All components except the serum are dissolved in water, brought to 200 ml, sterilized on a 0.22- or 0.45-μm-pore-size filter, and added to 700 ml of sterilized water. Heat-inactivated (56°C for 20 min) serum is then added. During in vitro growth, the organisms are grown in sealed glass containers nearly filled with medium. When this is impossible, such as during cloning by limiting dilution in 96-well plates (243), a sealed bag containing an anaerobic generator is used. At 4°C, the shelf life is limited to 5 to 7 days, primarily because of the degradation of the cysteine.