Table 1 Description of the release treatments methods applied to L. pneumophila strains and Acanthamoeba strains in a free-living state or in co-culture

Mechanical

 21 gauge needle (21G)

Passage three times through the needle

 27 gauge needle (27G)

Passage three times through the needle

[22, 31]

 Centrifugation (Centrif.)

Centrifugation at 10,000 × g for 10 min

[21]

 Freeze in liquid nitrogen

 (N₂)

Immerse liquid N₂ for 2 min and thawed at 35 °C for 10 min

[33]

 Freezing-thawing cycles

 (Freeze Thaw.)

3 cycles of freezing-thawing consisting of 15 min at −80 °C followed by 10 min at 35 °C

[18]

Chemical

 Triton™ X-100

0.02 % Triton™ X-100 for 20 min at RT

[23]

 SDS

0.5 % SDS for 10 min at rt

[24]

 Salt treatment (KCl)

Centrifugation at 10,000 × g for 10 min, the supernatant was replaced with 1 mL of 0.038 M KCl, vortexed and incubated for 3 h at RT

[19]