Columbia sheep blood agar

Fifty microliters of synovial fluid was subjected to Gram staining via a PolyStainer (IUL Instruments). Fifty microliters of synovial fluid was spread onto Columbia sheep blood agar, chocolate agar, and Sabouraud agar for the detection of aerobic bacteria and yeasts and on Schaedler anaerobic agar (all from Oxoid, Basingstoke, UK) for cultivation of anaerobic bacteria. Incubation was carried out at 37°C and 5% CO₂ for up to 14 days or under anaerobic conditions for 48 h. Plates were evaluated for growth after 24 h, 48 h, 7 days, and 14 days. In addition to solid media, 2 mL of thioglycolate broth was inoculated with up to 500 μL of the synovial fluid. All microorganisms obtained from cultures were subjected to pathogen identification using MALDI-TOF (Microflex; Bruker Daltonics, Bremen, Germany).
Isolates were subjected to susceptibility testing on a Vitek2 instrument (bioMérieux, Marcy l’Étoile, France) using either the Vitek2 AST-N223 (Enterobacterales) or the Vitek AST-P611 card (staphylococci and enterococci). Agar diffusion was employed to test fastidious organisms (e.g., streptococci) according to EUCAST protocols. Additionally, anaerobic bacteria were tested using brucella agar, McFarland 1.0, and anaerobic culturing conditions. Oxacillin resistance in S. aureus was confirmed using an immunochromatographic assay (Abbott, Scarborough, ME, USA).

Swabs were inoculated into Columbia sheep blood agar (Oxoid, USA) and Streptococcus selective agar (colistin-bacitracin supplemented agar-COBA) and incubated at 37 °C for 24–48 h, depending on the bacterial growth. Bacterial colonies exhibiting alpha hemolysis were selected for subculture to obtain pure colonies. Pure colonies were then stained with Gram staining and subjected to catalase test as a pre-screening test for S. suis to identify Gram-positive and catalase-negative colonies.

All Bavarian S. Agona strains were cultured on Columbia sheep blood agar (Oxoid, Wesel, Germany) and identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectroscopy (MALDI-TOF MS; Bruker, Bremen, Germany). Somatic (O) and flagellar (H) antigens were identified by using slide agglutination (antisera provided by Sifin, Berlin, Germany) according to the White−Kauffmann−Le Minor scheme [25 ].

The specimens, here nasal swabs, bronchoalveolar lavage fluid, or lung tissue samples, were analyzed in the ISO 17025 accredited laboratory at the Bavarian Health and Food Safety Authority. Samples were initially inoculated on Columbia sheep blood agar (Oxoid, Wesel, Germany) and incubated at 37 °C for 24 to 48 h under aerobic conditions as well as under a microaerophilic atmosphere, at 10% CO₂. To isolate pure suspicious colonies of P. multocida, M. haemolytica, B. trehalosi or T. pyogenes, fresh subcultures were incubated under the above-described conditions. Identification of bacterial species was carried out using MALDI-TOF MS (Bruker, Bremen, Germany).
Regarding the isolation of Mycoplasma species, animal samples were inoculated in specific Thermo Scientific™ Mycoplasma/Ureaplasma Broth that inhibits the growth of most gram-negative, gram-positive bacteria, as well as yeasts (Thermo Scientific, Schwerte, Germany), and incubated microaerophilic for 120 h at 37 °C with 10% CO₂.

A confirmed isolate of C. jejuni from chicken feces was recovered from stocks kept at −80°C by plating on Columbia sheep blood agar (Oxoid, Basinstoke, Hampshire, UK) for 48 h at 37°C under microaerobic conditions (AnaeroGen system, Oxoid). The bacteria were harvested from plate and resuspended in fresh cell culture media. The optical density (OD₆₀₀) was adjusted to 1 to achieve 10⁸ CFU/ml for their straight inoculation into INT-407 and IPEC-1 cells at a multiplicity of infection (MOI) of 100/1. All in vitro cell infections with bacteria were performed in triplicate as previously described (Aguilar et al., 2014 (link)) using two points in the study time course: 3 h (early time, T3h) and 24 h (late time, T24h). The same time course was used with the uninfected controls.

Members of the genus Streptococcus referred to the British Columbia Centre for Disease Control Public Health Laboratory (BCCDC PHL) were selected for study. These isolates, though identified as belonging to the mitis group streptococci by partial 16S rRNA gene sequencing, could not be classified definitively as S. pneumoniae, S. mitis or S. pseudopnuemoniae. All isolates of members of the genus Streptococcus were grown on 5 % Columbia Sheep Blood Agar (Oxoid) at 37 °C in a CO₂ incubator for 18–24 h. Fifty strains of members of the genus Streptococcus isolated from various sample types were selected; three of these isolates were identified as S. pneumoniae, one as Streptococcus gordonii and one as Streptococcus australis. The remaining 44 (plus one repeated sample) isolates belong to the mitis group streptococci, but after 16S rRNA sequencing had uncertain laboratory identification beyond the viridans grouping. ATCC strains S. mitis (ATCC 49456^T), Streptococcus oralis (ATCC 9811), S. pneumoniae (ATCC 49619) and S. pseudopneumoniae (ATCC BAA-960^T) were included as controls for the real-time PCR.

Briefly, as described in our study protocol [10] , an aliquot of S. pneumoniae serotype 6B BHN418 was thawed, centrifuged and washed before being resuspended in 0.9% normal saline at the pre-specified concentration (20,000 CFU/100 µl or 80,000 CFU/100 µl). The prepared inoculum was taken immediately (less than 5 min transit) to the clinical area where 100 µl was instilled into each nostril of the participant [3] (link).
Nasal wash was collected as described elsewhere [3] (link). Briefly, 5 ml of 0.9% saline was instilled into each naris; this was repeated twice (10 ml per naris, 20 ml total). Samples were transported to the laboratory and centrifuged at 3400 g for 10 min. Following centrifugation, 1 ml aliquots of the supernatant were stored at −80 °C. The nasal wash bacterial pellet was resuspended in 100 µl of skim milk, tryptone, glucose, and glycerine (STGG). Samples were retained for bacterial culture and DNA extraction followed by lytA PCR.
To quantify colonization density by culture, serial dilutions of the pellet, in 100 µl STGG, were plated on Columbia sheep Blood Agar (Oxoid, UK) containing 4 mg/ml gentamicin (CBG). Plates were incubated at 37 °C with 5% CO₂ for 18–24 h and inspected to identify pneumococcal phenotype. Serotype (23-valent kit) was confirmed by latex agglutination using Immulex Pneumotest reagents (Statens serum institute).