Geniii microplates

Biochemical traits and enzymatic activities were determined using GEN III Microplates (Biolog, USA) and API 20NE tests (bioMérieux, France) according to the manufacturer’s protocols, in two independent experiments. The urease activity of the tested strains was also verified, in two biological replicates, using the urease-indole diagnostic broth (BioMaxima, Poland). The results were collected after 24 and 48 hours of incubation at 28°C. The Columbia blood agar (BTL, Poland) was used to assess hemolytic activity. The MacConkey agar from BTL (Poland) was used to determine the growth on this medium. Catalase activity was determined by assessing the generation of oxygen bubbles in the presence of 3% (v/v) hydrogen peroxide. Oxidase activity was tested using plastic strips with a paper zone saturated with N,N-dimethyl-p-phenylenediamine oxolate and α-naphthol (Sigma-Aldrich, USA). LB agar and Tryptic Soy Agar TSA (Thermo Fisher Scientific, USA) were used for determination of colony morphology. Motility was assessed in glass tubes filled with 5 mL of the M9 minimal medium [64 ] containing 0.3% agar supplemented with 0.4% glycerol or 0.4% glucose, and with or without the addition of 0.5% casamino acids. Media in tubes were stab inoculated and incubated at 28°C for 4 days.

The putative Bacillus isolate was tested for the utilization of 95 carbon sources using the BIOLOG program (Jeon et al., 2003 ). Briefly, bacterial cells cultured on TSA medium for 24 h at 28°C were suspended in an inoculating fluid (i.e., 0.4% NaCl, 0.03% Pluronic F-68, and 0.01% gellan gum). They were inoculated onto GENIII MicroPlates (BIOLOG Inc., BiOLOG GP MicroPlateTM, Hayward, CA, United States), and incubated at 28°C. After 24 h of incubation, turbidity in the wells was measured using a MicroLogTM 3-Automated Microstation system (BIOLOG, Hayward, CA, United States). Bacteria were identified using the MicroLog Gram-positive database (ver. 4.0; BIOLOG).

Carbon source utilization abilities of Acidovorax sp. A1169 were assessed using GEN III Microplates (Biolog Inc., Hayward, CA, USA) as described in Gawor et al. (2016 (link)). After incubation in R3A broth on a rotary shaker (WL-972, JWElectronics) for 3 days in 15 °C the cells were harvested by centrifugation (9000 rpm for 3 min), washed twice, suspended in sterile 0.9% saline and added to a vial of MicroPlate IF C inoculation fluid until transmittance reached 90%. Biolog GEN III Microplates (Biolog Inc., Hayward, CA, USA) were inoculated according to the manufacturer’s instructions. The plates were incubated in darkness at 15 °C, the color development was read at 590 nm (A590) in a Varioskan plate reader (Thermo Fisher Scientific, Waltham, MA, USA), and cellular respiration was measured kinetically by determining the colorimetric reduction of tetrazolium dye. Data were collected twice a week over a 15-day period.

Carbon source utilization abilities of Acidovorax sp. A1169 were assessed using GEN III Microplates (Biolog Inc., Hayward, CA, USA) as described in Gawor et al. (2016 (link)). After incubation in R3A broth on a rotary shaker (WL-972, JWElectronics) for 3 days in 15 °C the cells were harvested by centrifugation (9000 rpm for 3 min), washed twice, suspended in sterile 0.9% saline and added to a vial of MicroPlate IF C inoculation fluid until transmittance reached 90%. Biolog GEN III Microplates (Biolog Inc., Hayward, CA, USA) were inoculated according to the manufacturer’s instructions. The plates were incubated in darkness at 15 °C, the color development was read at 590 nm (A590) in a Varioskan plate reader (Thermo Fisher Scientific, Waltham, MA, USA), and cellular respiration was measured kinetically by determining the colorimetric reduction of tetrazolium dye. Data were collected twice a week over a 15-day period.

Strain ncl2^T and N. vaccinii DSM 43285^T were screened for a broad range of phenotypic properties, including their ability to metabolize diverse sole carbon and nitrogen sources, to grow in the presence of several concentrations of sodium chloride, at a range of pH values and in the presence of antibiotics using GENIII microplates and an OMNILOG device (Biolog Inc., Hayward, CA, USA), as described previously [8 ]. The resultant data were analysed using version 1.3.36 of the OPM package [62 , 63 ]. They were also tested for their ability to produce niacin, arylsulfatase after 3 days [64 ], and to reduce tellurite [65 ]. All of these tests were carried out in duplicate using a standard inoculum. Enzymatic and additional metabolic properties of the strains were determined using API-ZYM kits and the protocol provided by the manufacturer (Biomerieux, France).

The biochemical metabolite profile of VI4T1 and VI4.1 was determined using Gen-III MicroPlates (Biolog, Hayward, USA). Bacteria were grown in 869 rich medium overnight, subsequently resuspended in sterile 10 mM sodium phosphate buffer (pH 7.2) to an Optical Density (OD) of one checked at 600 nm. Next day, 100 μL of each strain was inoculated into each well of the Gen-III microplate and incubated at 30 °C for five days. Each microplate allows to carry out 94 phenotypic tests: 71 carbon source utilization assays and 23 chemical sensitivity assays. Each well contains a redox tetrazolium dye that changes colour as a result of cellular respiration providing a reliable metabolic fingerprint, which can be evaluated by measuring absorbance at 595 nm.
In addition, a GC Analysis of total fatty acid methyl esters (FAMEs) was performed at EMSL Analytical (Cinnaminson, NJ, USA) to identify the strains based on the FAME fingerprint and using the Sherlock Microbial Identification system (MIS).

The chemotaxonomic analysis sample was prepared from biomass produced in M.65 medium (L-1) containing 4.0 g glucose, 4.0 g yeast extract and 10.0 g malt extract (pH 7.2). Whole-cell sugars and isomers of diaminopimelic acid were diagnosed with standard samples by TLC on cellulose plates according to the method of Staneck and Roberts [34 (link)]. Polar lipids were extracted from freeze-dried material in chloroform:methanol:0.3 % aqueous NaCl, separated by 2D TLC and detected according to the study by Tindall and colleagues [35 ]. Cellular fatty acids were extracted, methylated and analysed using minor modifications of the methods of Miller [36 (link)] and Kuykendall et al. [37 ]. The fatty-acid methyl esters were separated by GC and identified using the Sherlock Microbial Identification System (MIDI). Menaquinones were extracted in hexane, purified by silica-based solid phase extraction and analysed by reverse phase HPLC-DAD-MS. Phenotypic characterization was done in BioLog GENIII MicroPlates (71 carbon source utilization assays, 23 chemical sensitivity assays) with further biochemical tests done with API 20E strips. All analyses were performed by DSMZ services, Leibniz-Institute DSMZ, Braunschweig, Germany.