Api zym system

The phenotypic tests of Gram staining, cell morphology, colony morphology grown onto TYES agar, and oxidation/fermentation (O/F) of glucose were determined according to the procedures described in Buller [56 ]. Furthermore, several key characteristics for the description of bacterial strains belonging to the Flavobacteriaceae family [57 (link)], such as production of oxidase and catalase, hydrolysis of aesculin and gelatin, reduction of nitrate to nitrite, indole production, production of flexirubin-type pigment, growth at 25 °C on R2A (Becton-Dickinson, Sparks, MD, USA), MacConkey (Becton-Dickinson, Sparks, MD, USA), Trypticase Soy (TSA, Becton-Dickinson, Sparks, MD, USA) agar, and in Brain Heart Infusion (BHI, Becton-Dickinson, Sparks, MD, USA) broth added with 1.0, 2.0, 3.0, 4.5, and 6.5% NaCl were performed using procedures as previously described [56 ,58 ]. In addition, growth at 15, 25, 30, 37, and 42 °C in a BHI broth was assayed.
Other enzymatic activities of FP105 and FP223-J200 strains were determined using the API ZYM system (bioMérieux, Marcy-l’Etoile, France) according to the manufacturer’s guidelines. Test strips were read after 5 min as indicated by the manufacturer, and each assay was performed twice to ensure reproducibility.

In order to determine enzymatic activity of C. osculatum ES proteins, the API^® ZYM system (Biomerieux SA, Sweden) was applied. This is a semi-quantitative method testing 19 enzyme reactivities. The system consists of 20 cupules containing enzymatic substrate in the base and any enzymatic reactivities are detected through coloured reactions rated according to a table provided by the manufacturer. ES concentrate (65 μL of ES to each well) was incubated for 4 h and 40 min at 37°C whereafter reagents ZYM A and ZYM B were added and allowed to react for 5 min for colour development.

The Bacillus strains contained in the PCHS detergent (namely B. subtilis, B. pumilus and B. megaterium), previously culturally isolated, were assessed by the API-ZYM system (BioMérieux, Florence, Italy) for their ability to produce enzymes potentially useful to degrade virus components, following the manufacturer’s instructions.

The API ZYM system (bioMérieux, Marcy l’Etoile, France) was used for the assay of enzymatic activities of the ZDB strains. After subcultured twice, the cells of each ZDB strain were collected through centrifugation (8000× g for 20 min) and resuspended in API suspension medium with turbidity adjusted to 5–6 McFarland. All detection tests were performed according to the manufacturer’s instructions.

API ZYM system (BioMerieux, France) was used to evaluate the enzymatic activities of the strain P. pentosaceus MZF16 (Humble et al., 1977 (link)). The API strips were inoculated with an overnight bacterial culture grown in MRS broth, subsequently incubated at 37°C for 4 h. The appraisal of the enzymatic activity was carried out according to the intensity of coloration.

Cell morphology and gliding motility were observed using an Olympus BX50 light microscope with a drop of lactophenol blue added to enhance observational ability. A slide with thin SAP2 agar medium overlay was used to examine gliding motility. After placing a swarm colony on an overlay and allowing incubation, we observed gliding motility. Catalase activity was tested by bubble formation in 3 % H₂O₂ solution. Oxidase activity was determined by the oxidation of 1 % tetramethyl-p-phenylenediamine on filter paper. Acid production from carbohydrates was investigated using the commercial API 50 CH and API 20 E systems (BIOMERIEUX). Enzyme production was tested using the commercial API ZYM system (BIOMERIEUX), in accordance with manufacturer instructions.