Bhi agar medium

During endoscopy, two biopsies were collected from the antrum of the stomach and cultured on brain heart infusion (BHI) agar medium (Oxoid, Stoke, Basin, United Kingdom) containing 5% defibrinated sheep blood, 5 mg/L trimethoprim, 10 mg/L vancomycin, 20 U/L polymyxin B, and 10 mg/L nalidixic acid under microaerophilic conditions (85% N₂, 10% CO₂, and 5% O₂) at 37°C. The strains were confirmed according to Gram-negative, positive urease, oxidase, and catalase reaction, and its morphology was spiral or curved. The strains were collected in BHI broth with glycerol at 4°C and stored at −80°C. Before the susceptibility test, bacteria were resuscitated and subcultured on BHI agar medium (Oxoid, Stoke, Basin, United Kingdom). ATCC43504 was used as the quality control with MIC from 64 to 256 mg/L for metronidazole, from 0.016 to 0.125 mg/L for clarithromycin, and from 0.032 to 0.125 mg/L for levofloxacin.

After cytokine treatment and Dectin-1 receptor blockage, neutrophils and monocytes were challenged with Pb265 for 4 h. The coculture supernatants were collected and the wells were washed many times with sterile distilled cold water to remove and lyse the cells and consequently release the phagocyted fungi. The solution obtained was considered to be experimental cultures. Cultures containing only the fungus were used as a control. Each experimental and control culture well washing resulted in a final volume of 2 mL, and 100 μL was added (in duplicate) on petri dishes containing the brain–heart infusion (BHI) agar medium (Oxoid Ltd. England) supplemented with 4% horse serum, 50 μg/mL of gentamicin, and 5% P. brasiliensis strain 192 culture filtrate (v/v), which constituted the source of the growth-promoting factor [22 (link)]. Experimental and control plates were incubated at 37°C, and after 10 days, the number of colony-forming units (CFU) per plate was counted. The fungal recovery percentage was determined by the following formula:
$\begin{array}{c}\%\text{\hspace{0.17em}fungal recovery}=\left[\frac{\text{experimental culture CFU mean}}{\text{control culture CFU mean}}\right]\times 100\end{array}$