Vacuum-sealed anaerobic plate culture enhances growth and confluence of Giardia intestinalis trophozoites

Giardia intestinalis trophozoites, the replicative stage responsible for human giardiasis, require anaerobic settings for microtiter plate culture. However, residual ambient oxygen (AAO₂) within plates and enclosed compartments could hinder consistent confluence. This study evaluated a vacuum-sealed bag (VS) combined with an anaerobic gas generator sachet (AS) (VS + AS) as an alternative culture method to minimize residual AAO₂. The conventional airtight compartment plus AS (AT + AS) served as the reference, while VS without AS (VS–AS) functioned as the negative control. Two-fold serially diluted trophozoites were seeded in 96-well plates, and confluence was scored at 48-, 96-, and 144-h post-seeding using a 5-point scale (0: 0–20 % to 4+: 80–100 %). MTT assays were used to validate the association between seeded cell number and parasite viability, as well as inter-well variation among fully confluent wells. VS + AS cultures achieved ≥3+ confluence within 48 h and maintained this through 96 h, whereas AT + AS required 96–144 h to reach comparable levels. VS–AS consistently showed poor growth. Optimal confluence in the VS + AS system was observed with seeding densities of 6 × 10⁴ to 5 × 10⁵ trophozoites per well. MTT assays confirmed a strong linear correlation between seeded cell number (10^3.9–10^5.7) and OD₅₄₀ readings (r² = 0.98). The inter-well variation among fully confluence wells was below 10 %. In conclusion, the VS + AS system accelerates trophozoite growth and improves reproducibility compared with conventional methods. This approach provides a reliable platform for studying Giardia trophozoite biology and supports the discovery of novel anti-giardial compounds.