2.6. Determination selleck chemicals Lapatinib of the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)The MICs of the extract against the susceptible Listeria isolates were determined using the broth microdilution assay method of EUCAST [21] and carried out in sterile disposable flat-bottomed 96-well microtiter plates. Twofold serial dilutions using sterile distilled water were carried out from 10mg/mL of the stock plant extract to make 9 test concentrations ranging from 0.039 to 10mg/mL. Double strength Mueller-Hinton broth (100��L) was introduced into all the 96 wells and 50��L of the varying test concentrations of the extracts were added in decreasing order along with 50��L of the test organism suspension.

Column 1 was used as the sterility wells containing 100��L of sterile distilled water in addition to the 100��L of Mueller-Hinton broth, column 2 was used as the positive control wells containing 100��L of the broth, 50��L of ciprofloxacin, and 50��L of the test organism, column 3 was used as the negative control wells containing 100��L of the broth, 50��L sterile distilled water, and 50��L of the test organism whilst columns 4 to 12 were used as test wells containing 100��L of the broth, 50��L of the test extract concentration, and 50��L of the test organism. The plates were then incubated at 37��C for 18�C24h. Results were read visually by adding 40��L of 0.2mg/mL of ��-iodonitrotetrazolium violet (INT) dissolved in sterile distilled water into each well [4]. A pinkish coloration is indicative of microbial growth because of their ability to convert INT to red formazan [23].

The MIC was recorded as the lowest concentration of the extract that prevented the appearance of visible growth of the organism after 24h of incubation [21].The method of Sudjana et al. [24] was used to determine the MBC from the MIC broth microdilution assays through subculturing 10��L volumes from each well that did not exhibit growth after 24h of incubation and spot inoculating it onto fresh Mueller-Hinton agar plates. The plates were incubated for 48h after which the numbers of viable colonies were counted. The MBC was defined as the lowest concentration killing more than or equal to 99.9% of the inoculum compared with initial viable counts [24].2.7. Rate of Kill AssayThe time-kill assay was done according to the method of Odenholt et al. [25] following the descriptions of GSK-3 Akinpelu et al. [26]. The selected test Listeria isolates, namely, L. ivanovii (LEL 18), L. grayi (LAL 15), L. monocytogenes (LAL 8), and L.