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were prepared for testing the sensitivity of microorganisms against plant extracts.
The disc diffusion and agar well methods were used. Mueller Hinton broth (200 ml)
and 200 ml of Mueller Hinton broth plus 4% of NaCl were prepared for making the
spread plates and for MIC purposes. 5 ml of the nutrient broth was poured in each
test tube. The test tubes and the bottles of nutrient agar were autoclaved for 15
minutes at 104 kpa. The nutrient agar and the nutrient agar plus 4% of NaCl were
aseptically poured into labeled sterile Petri dishes. The nutrient agar in the Petri
dishes was allowed to solidify and kept at 4 ºC with nutrient broths until further use.
Bacterial cultures that were going to be used for agar well diffusion, disc diffusion
and MIC were prepared by transferring one colony into a flask containing 200 ml
sterile Mueller Hinton broth and incubated to a 0.5 McFarland standard
(approximately 1x10 6 CFU/ml). The spectrophotometer was set to 640 nm for
measuring the McFarland standard. The plain nutrient broth was used as the blank
to calibrate the spectrophotometer to zero. The nutrient broth with the microorganism
was measured. The reading on the spectrophotometer had to be between 0.08 nm
and 0.1 nm, which is referred to as the McFarland standard (Eloff, 1998).
2.5.3 Antibacterial assays
2.5.3.1 Agar well diffusion assay
The activity of the plant extracts was tested using agar well diffusion assay. Agar
plates were prepared using sterile Mueller-Hinton (MH) agar. Spread plates were
made to spread the cultures (30 µl) evenly on the surface of the agar, using a sterile
glass spreader which was dipped in alcohol and flamed using a Bunsen burner to
sterilize it. The culture was allowed to absorb into the agar. Wells were made in
each plate with sterile Pasteur pipette tips. 30 µl of each plant extract (100 mg/ml)
was added to each well. 30 µl of DMSO was used as a negative control and
Neomycin (0.1 mg/ml) was used as a positive control. The agar plates were allowed
to diffuse for one hour at room temperature and then incubated at 37 ºC overnight.
(Mathabe et al., 2006).
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