Mueller hinton nutrient broth

A. hydrophila (MTCC No.1739) was grown in Mueller Hinton nutrient broth (Hi Media) by placing in shaking water bath for 12 h at 20°C and centrifuged at 9,000xg for 20 min at 4°C. The supernatant was discarded and the bacterial pellet was washed thrice with PBS (pH 7.2) and adjusted by dilution to get a concentration of 2.5x10 6 cfu/ml using Neubauerhemocytometer

The antibacterial activity of crude extract of S. urens roots was checked against bacterial strains Escherichia coli, Bacillus subtilis, and Klebsiella pneumoniae by employing agar well diffusion assay [27] (link). Obtained bacterial cultures were maintained at 37 °C in Mueller Hinton nutrient broth (Himedia, Mumbai, India). The bacterial cell suspension culture was prepared in sterile saline water, and it was optimized to 0.5 McFarland turbidity standards with the help of a spectrophotometer to achieve the desired cell density (1.5 × 108 cfu/ml). Each bacterial culture uniformly streaked on Mueller Hinton agar plates, and 100 μl hydro-methanolic root extract was filled in the wells with 200 and 300 μg/ml concentration. Bacterial plates were kept at temperature 37 °C and incubated for about 24 h. Later, antimicrobial activity was reckoned by measuring the inhibition zone.