Notes

The antimicrobial activity of citrus extract was determined by agar well diffusion method [29]. Each bacterial isolate preserved in 15% glycerol broth was subcultured on freshly prepared MacConkey agar (Oxoid Ltd., Basingstoke, Hampshire, UK) plate and incubated at 37˚C for 24 hours. A bacterial colony was obtained with a sterile wire loop and emulsified in 5 ml Mueller Hinton broth (Oxoid Ltd., Basingstoke, Hampshire, UK). The turbidity of the resulting mixtures was adjusted with sterile saline solution to 0.5 Mcfarland standard (1.5 × 108 cells∙mL−1). Fresh Mueller Hinton agar plates were labeled and seeded with the test bacterial suspension using a sterile swab steak. These plates were allowed to stand for 15 minutes and agar punched out with sterile cork borer to create wells of 5 mm diameter. The essential oils were diluted to 8 µg/ml, 40 µg/ml and 80 µg/ml with methanol, ethanol or Tween 80 (0.5% v/v). 100 µl dilute essential oils of the different concentrations were dispensed into each agar well in a plate; plates were allowed to stand for 1hour and then incubated at 37˚C for 24 hours. A suspension of 0.5% w/v ciprofloxacin (Fidson Healthcare Ltd., Nigeria) was used as positive control while the solvent only was used as negative control. This experiment was performed in triplicate plates for each isolate per dissolution solvent. The diameter (mm) of zone of inhibition was recorded for the different concentrations of oil samples.