Lausannevirus was purified as described previously by Thomas et al. (19 (link)). Briefly, Lausannevirus was cocultured with A. castellanii ATCC 30010 in 30 ml of peptone-yeast extract-glucose (PYG) medium (33 (link)) within 75-cm2-surface cell culture flasks (Becton Dickinson). The flasks were kept at 32°C until complete amoebal lysis was observed (usually between 24 and 48 h); then, cocultures were harvested and centrifuged at 5,000 × g for 15 min. The supernatant was collected and passed through 5-μm filters to remove residual amoebal cells; then, the filtrate was centrifuged at 35,000 × g for 1 h, and the pellet was resuspended in 1 ml of DNA-free water. Lausannevirus DNA was further isolated from 50-μl viral suspensions according to the instructions for the Wizard SV genomic DNA purification system (Promega), using a 2-h incubation time for digestion and 50 μl of nuclease-free water for elution.
75 cm2 surface cell culture flasks
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The 75-cm2-surface cell culture flasks are a lab equipment product used for the cultivation of cells. They provide a standardized surface area for cell growth and proliferation in controlled laboratory environments.
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5 protocols using 75 cm2 surface cell culture flasks
1
Lausannevirus Purification and DNA Isolation
2
Cultivation of Acanthamoeba castellanii for ARB Analysis
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Acanthamoeba castellanii ATCC 30010 was used to cultivate ARB. A. castellanii was grown in the rich peptone yeast-extract glucose (PYG) medium [22] (link), [23] (link), at 28°C without CO2, in 75 cm2 surface cell culture flasks (Becton Dickinson, Allschwil, Switzerland). Amoebae were collected by centrifugation (1500 × g, 10 minutes) and washed with phosphate-buffered saline and finally resuspended in poor medium Page amoeba saline (PAS) [22] (link), [23] (link) to avoid extracellular overgrowth of bacteria. Amoebae were seeded in a 24-well culture microplate (Milian, Wohlen, Switzerland) at 5 × 105 amoebal cells/mL. An aliquot (100 μL) of biofilm or concentrated water sample was then inoculated, and tenfold serial dilutions were performed. The microplates were immediately centrifuged at 1790 × g for 15 minutes, and the cells were incubated for 1 hour at 28°C. Cells were gently washed once with PAS and incubated at 32°C in a humidified atmosphere without CO2. Amoebae were observed daily for amoebal lysis, and the co-cultures were reseeded on fresh confluent amoebae in PAS after 7 and 14 days [24] . At day 7 and day 14, 100 μL of each amoebae-containing well was collected and stored at −20°C until DNA extraction.
3
Cultivation of Acanthamoeba castellanii
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Acanthamoeba castellanii ATCC 30010, were cultured in peptone yeast extract glucose (PYG) medium, as previously reported (Greub and Raoult 2004 (link); Greub et al. 2004 ; Jacquier et al. 2013 ) in 75 cm2 surface cell culture flasks (Becton Dickinson, Allschwil, Switzerland) at 25 °C.
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Acanthamoeba castellanii ATCC 30010, were cultured in peptone yeast extract glucose (PYG) medium, as previously reported (Greub and Raoult 2004 (link); Greub et al. 2004 ; Jacquier et al. 2013 ) in 75 cm2 surface cell culture flasks (Becton Dickinson, Allschwil, Switzerland) at 25 °C.
4
Purification of Estrella lausannensis
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Estrella lausannensis strain CRIB-30 was co-cultured in Acanthamoeba castellanii ATCC 30010 at 32°C in 75-cm2-surface cell culture flasks (Becton Dickinson, Allschwil, Switzerland) with 30 ml of PYG medium as described previously (Greub and Raoult, 2002 (link)). Co-cultures were harvested when a complete lysis of the amoebae was observed. Then, Estrella elementary bodies were purified using successive sucrose and gastrografin gradients, as described previously (Bertelli et al., 2010 (link)). These steps were not performed by the course students.
5
Lausannevirus and E. lausannensis Purification
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Lausannevirus was co-cultured with A. castellanii ATCC 30010 at 32 °C, in 75 cm2 surface cell culture flasks (Becton Dickinson), with 30 ml of PYG medium. When complete amoebal lysis was observed, co-cultures were harvested. The same procedure was used for E.lausannensis strain CRIB30. Both bacteria and virus were purified with Wizard Genomic DNA purification kit (Promega Corporation, Madison, USA; Thomas et al. 2011 (link)).
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