Susceptibility testing of Listeria monocytogenes to extracts

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Preparation of crude plant extracts

One fairly polar solvent ethyl acetate and another solvent of medium polarity (chloroform) were selected. Due to finance constraints water and ethanol solvents were not included (This would had increased the number of samples to be analysed). Ethyl acetate and chloroform extracts of each plant sample were prepared. To obtain these extracts one hundred grams (100 g) of fresh plant material was homogenised and extracted with ethyl acetate or chloroform. The extract was filtered and concentrated. The residue was later dissolved in 10% DMSO to a final stock concentration of 50 mg/ml.

Bacterial strain and inoculum preparation 

The pathogenic strain of Listeria monocytogenes (LMG 21263) used in this study was obtained from the Department of Pharmaceutical Sciences at Tshwane University of Technology. It was activated by transferring a loopful from the Brain Heart Infusion (Merck) slants into Tryptone Soya Broth (Merck), followed by incubation at 37°C for 24 hours. The bacterial counts of the standardized culture were confirmed by plating out on TSA (Merck) plates and incubating at 37°C for 24 hours. Stock cultures were maintained at -70 °C (Alzoreky & Nakahara, 2003).

Antimicrobial bioassay 

The disc diffusion method as described by Alzoreky & Nakahara (2003) was used for testing the susceptibility of L. monocytogenes to the plant extracts. Two hundred microlitres of prepared culture (106 CFU/ml) was spread on surfaces of Mueller–Hinton agar. Sterile filter paper discs (10 mm in diameter) were impregnated with 50 microlitre (50 mg/ml) of the extracts.
Erythromycin (150 μg/ml) was used as a positive drug control. The DMSO (2.5%) was used as control to test the inhibition of the bacteria. Spread plates were then kept at ambient temperature for 30 minutes to allow diffusion of the extracts prior to incubation at 37 °C for 24 hours. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)
Extracts (ethyl acetate or chloroform extracts of Acacia karroo, Eucomis autumnalis, Drimia altissima, Aloe arborescens, Plectranthus ecklonii and Senecio inonartus) which showed antilisterial activity in the initial screening using the disc diffusion method were further evaluated to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using 96-well microtitre plates. The micro dilution method as described by Eloff (1998) was used. Briefly, the extracts were first dissolved in 10 % DMSO and then added to Tryptone Soya Broth (TSB) to obtain a final concentration of 25 mg/ml in the first well. The DMSO (2.5%) was used as control to test the inhibition of the bacteria. A serial double dilution was performed to obtain a concentration range of 25- 0.01 mg/ml for the extracts. For compounds the antibiotic erythromycin (Merck) at concentrations ranging from 150 – 0.29 μg/ ml served as a positive drug control. Hundred microlitres of bacterial inoculum 106 CFU/ml of L. monocytogenes was added to the wells thereafter, the plates were then incubated at 37 °C for 24 hours. After 24 hours incubation microbial growth was tested by adding 40 μl of (0.2 mg/ml) piodonitrotetrazoilium violet (INT) (Sigma-Aldrich, South Africa) to the micro titre wells and reincubated at 37 °C for 1 hour. Change in colour to orange red indicated that the cells were still viable. The MIC was defined as the lowest concentration of the extract that caused no colour change. To determine the minimum bactericidal concentration (MBC) against L. monocytogenes, fifty microlitres of the sample (from the wells which did not show bacterial growth during MIC determination) were transferred into 150 microlitres of fresh TSB on the new plates. The plates were then re-incubated for another 24 hours and the MBC (lowest dilution of extracts with no growth after 24 hours incubation at 37 °C) was determined according to Reimer et al. (1981).

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CHAPTER 1 INTRODUCTION
1.1 Background
1.2 Listeriosis
1.3 Scope of the thesis
1.4 References
CHAPTER 2 BACKGROUND INFORMATION ON THE SELECTED PLANTS
Abstract
2.1 Introduction
2.2 Plants selected for the present study
2.3 References
CHAPTER 3 Susceptibility testing of Listeria monocytogenes to extracts of selected South African medicinal plants
Abstract
3.1 Introduction
3.2 Materials and methods
3.3 Results and discussion
3.4 References
CHAPTER 4 Bioactivities of Plectranthus ecklonii constituents
Abstract
4.1 Introduction
4.2 Experimental: Materials and Methods
4.3 Results and Discussion
4.3 References
CHAPTER 5 GENERAL DISCUSSION AND CONCLUSION
5.1 Motivation of this study
5.2 Discussion and conclusion
5.7 References
CHAPTER 6
ACKNOWLEDGEMENTS

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