Eosin methylene blue emb

In this study, midstream urine samples were taken from 420 patients with community-acquired (CA) UTI referred to Ibn Balady hospital, Baghdad during two years 2018 and 2019. One hundred and fifty of these samples yielded UPEC. Among patients, 90 were females aged between 4 months and 78 years (median, 42 years) and 60 patients were males aged between 1 year and 70 years (median, 51 years). The microscopic observation of urine (presence of more than three pus cells in each microscopic field) and Gram staining was considered as an early sign of UTI infection. Urine samples were cultured separately on nutrient agar, blood agar, and eosin methylene blue (EMB) (Merck, German) medium and plates with a growth of more than 10⁵ colony forming units (CFU) per ml were considered as positive samples for UTI. An uninoculated culture medium was used as a negative control in all sample processing. Presumptive identification of UPEC strains were done Analytical Profile Index (API 20E) kit (bioMerieux, France) and confirmed using PCR (16S ribosomal RNA gene). The identified isolates were cultured in Lysogeny broth (LB) (Merck, German) and after incubation for 18–24 hours, kept in 20% glycerol at −70°C until they were tested.

The test bacterial suspension which has been standardized with McFarland 0.5 was diluted using 0.85% physiological NaCl. S. aureus or E. coli was grown in nutrient broth/NB (Merck) and then added 10 μL of yeast indigenous for 72 h at 30°C. 1 ml of bacterial suspension was platted in eosin methylene blue/EMB (Merck) for E. coli and mannitol salt agar/MSA (Merck) for S. aureus and then incubated at 37°C for 24 h. For positive (+) control treatment, 200 μL of bacterial culture of 1 × 104 CFU/mL + 2 μL amoxicillin 100 mg/mL were used, while 200 μL bacterial culture of 1 × 104 CFU/mL used as negative (-) control treatment. The population of E. coli and S. aureus were count every 24 h; all the treatments were replicated three times.