Inoculums Preparation and Standardization

The bacteria were selected based on availability and considering the likely bacterial strains that can cause diarrhea for which L.ocymifolia is indicated traditionally. Nutrient agar was set by using the manufacturer’s procedure. After cooling of the culture medium at 45°C, it was poured into a prelabeled sterile petri dish and given time for congealing of the agar. The test bacteria were then inoculated and spread on the prepared agar with an inoculating wire loop following aseptic condition and incubated for 24 hours at 37°C.

The bacterial turbidity of every bacterium was set and standardized as described by Chikezie.²¹ The bacterial suspension in a broth was set by the growth method as follows. After preparing nutrient broth in distilled water, 5 mL of the broth was added to test tubes and sterilized. Isolated colonies of similar morphology of every bacterium from three-to-five wells were picked up by wire loop from fresh agar plates of bacterial culture and aseptically transferred into pre-labeled test tubes containing the sterile nutrient broth and incubated for about 6 hours. The inoculum tube was adjusted visually by either adding bacterial colonies or by adding sterile normal saline solution to that of the already prepared 0.5 McFarland standard which is assumed to contain a bacterial concentration of 1×10⁸ colony forming unit (CFU)/mL. The adjustment and comparison of turbidity of inoculum tube and that of 0.5 McFarland standard was performed by visually observing them with the naked eye against a 0.5 McFarland turbidity equivalence standard card with white background and contrasting black lines in the presence of adequate light.