Antimicrobial activity (agar diffusion assay)
Microorganisms were prepared as described elsewhere (Alzoreky
and Nakahara, 2003). Each pathogen was activated by
transferring a loopful from (BHI) slants into nutrient broth (10 ml)
followed by incubation at 37 ◦C for 18 h. The optical density of each
active culture was adjusted to 0.1 at 625nm using fresh broth to
give a standard inoculum of ca. 106 colony forming units (cfu) per
ml. Bacterial counts were confirmed by plating out on nutrient
agar (NA) plates (Oxoid, UK) incubated at 37 ◦C for 48 h. The agar
diffusion assay (Patil and Gaikwad, 2010) was adopted for evaluating
the antimicrobial activity of test materials. One hundred
ml of a sterilized NA tempered at 50 ◦C (water bath) was aseptically
inoculated with active cultures. The seeded agar media (ca.
105 cfu/ml) were immediately mixed and poured into Petri plates
(Ø 90 mm). Wells (6mm diameter) were punched in media using
a sterilized stainless steel borer. Each well was aseptically filled
with 100l (5.3 mg) extracts from fresh or dried spathe. The plates
were left atroomtemperature for 30 minto allow diffusion of materials
in media. Chloramphenicol (50g/ml) and blanks (solvents)
were also used under the same conditions. Plates were incubated at
37 ◦C for 12–48 h, until visible growth of test microorganisms was
evident in control plates. Inhibition zones in millimeter (including
well diameter) around wells were recorded. The test was assayed
in triplicates.