Columbia agar with 5 sheep blood

The Helicobacter hepaticus strain Hh-2 (ATCC 51448)^{59 (link)} was purchased from the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures (DSM No.22909) and cultivated at the Max von Pettenkofer-Institute, LMU Munich. Bacteria from cryo stock were resuspended in Brain Heart Infusion (BHI) medium and put on blood agar plates (Columbia agar with 5% sheep blood, BD, Cat: 4354005). Plates were incubated in a chamber with anaerobic conditions (83% N₂, 10% CO₂, 7% H₂) for 4 days at 37°C. A subculture was cultivated further on in BHI medium with 3% sheep serum in a culture flask in the chamber with anaerobic conditions for additional 4 days at 37°C.
For Hh lysate (HhL) preparation, bacterial cells were harvested and washed 2–3 times with PBS. Cell pellets were resuspended in PBS and lyzed by sonification with the Sonifier 150 Cell Disruptor (Branson) 6 times for 3 min at level 3 on ice. Lyzed cells were centrifuged at 20,000 x g for 30 min at 4°C and the supernatant was mixed with a protease inhibitor (cOmplete ULTRA Tablets, Roche, Sigma-Aldrich, Cat: 05892953001). Protein concentration was determined using the Qubit Protein Assay Kit and Fluorometer (Invitrogen), according to the manufacturer’s instructions and the lysate was stored at −20°C until use for immunoblot or ELISA.

Characterisation and comparison of the alternative method were carried out with routine samples according to DIN EN ISO 13843:2018 [39 ], investigating more than 20 different water samples from natural sources in a parallel study. After incubation on CCA and CCA including a CV supplement, colonies were counted and morphologically described by colour. After that, colonies were inoculated on Tergitol 7-lactose TTC agar (Oxoid^® Deutschland GmbH, Wesel, Germany) for determination of lactose fermentation. Inoculation on non-selective media Columbia Agar with 5% Sheep Blood (BD^® Austria GmbH, Schwechat, Austria) was carried out for further MALDI-TOF MS analysis. If no classification of the target colony was possible, Gram-staining was performed to obtain more information about the selected bacteria. Cytochrome oxidase testing was performed according to ÖNORM EN ISO 9308-1:2017 [25 ] for all pink and red colonies.

American Type Culture Collection (ATCC) strains (Table 1) were purchased from Microbiologics (St. Cloud, MN, USA) and supplied as quantitative lyophilized pellets. Stock suspensions of all microorganisms with a theoretical concentration of 10 ≤ CFU/mL <100 and subsequent four dilutions were prepared in peptone water (Becton Dickinson [BD], Franklin Lakes, NJ, USA). Before and during the validation phases, viability, purity, and titer determination were assessed by plating 100 μL of each diluted suspension in triplicate on TSA (for aerobic suspensions), Columbia agar with 5% sheep blood (for anaerobic suspensions), and Sabouraud dextrose agar (SDA; for fungal suspensions) plates (all from BD). Plates were incubated at 32.5°C ± 2.5°C for bacteria and 22.5°C ± 2.5°C for fungi. Before incubation, agar blood plates with the anaerobic microorganisms were placed inside resealable GasPak EZ anaerobe pouch systems (BD). The CFU were quantified after 2–3 days for bacteria and 4–5 days for yeast and mold. For each experiment, the theoretical titer of each diluted suspension was confirmed by calculating the arithmetic average of the CFU values obtained. Purity and identity were determined as described elsewhere.³³ (link)