Specimens of Ae. albopictus were anaesthetised with ether and surface-disinfected this website as previously described [12], then crushed individually in 150 μl of sterile 0.8% NaCl with sterile piston pellets. After a brief vortexing, the homogenate was used in different isolation procedures using various media, from generalist to selective. All solid media were FRAX597 ic50 supplemented with 2.5 μg ml-1 amphotericin B to prevent the growth of fungi. An aliquot of the homogenate (10 μl) was streaked onto a modified rich solid Luria-Bertani medium (LBm, LB with 5 mg ml-1 NaCl) and incubated

at 28°C for 24 to 48 h. Another aliquot (20 μl) was inoculated into 1 ml of selective enrichment medium I (0.2% KNO3, 0.02% MgSO4.7H2O, 0.2% sodium acetate, 0.04 M KH2PO4, pH 6), a medium which is suitable for the isolation of Acinetobacter species [29]. Cultures were incubated at 30°C for 24 to 48 h with shaking. When microbial

growth occurred, an aliquot (10 μl) of the culture was streaked onto Herellea agar plates (Biolife, Italy), a medium suitable for the isolation of Gram-negative bacteria especially members of the Acinetobacter genus and the Enterobacteriaceae family [30]. These cultures were further incubated at 37°C for 24 to 48 h. In parallel, 1 ml of pre-enrichment liquid medium (pH 3.5), which is suitable for the isolation of acetic acid bacteria [31], was inoculated with an aliquot of homogenate (20 μl). These cultures were incubated with shaking at 30°C for 3 days. When microbial growth occurred, an aliquot (10 μl) was streaked onto CaCO3 agar plates see more (pH 6.8), a medium suitable for the isolation of members of the genus Asaia, and the plate was incubated at 30°C for 3 days as previously described [32]. Colonies were selected according to various characteristics including colour,

shape, or size. Individual colonies were then re-inoculated onto fresh agar plates of the appropriate isolation Ureohydrolase medium. Newly formed colonies were streaked again to check for purity and stored in 25% glycerol at -20°C for two weeks before they were transported to the laboratory in Lyon, France. Isolates were re-streaked and new glycerol stocks were made and stored at -80°C. Brief morphological descriptions of colony size, shape and colour were recorded for each isolate. PCR and amplified ribosomal DNA restriction analysis (ARDRA) For PCR, a sterile toothpick was used to transfer bacteria from a single colony freshly grown on appropriate medium into 20 μl sterile water in a 0.5 ml Eppendorf tube. The homogenate was placed on a heating block at 95°C for 2 min followed by 2 min on ice. This step was repeated and the tube was centrifuged at 16,000 g for 5 min. The supernatant (2 μl) was used as template in a 50-μl PCR reaction.