M Mahfuzul Hoque
Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh.
Shemona Rattila
Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh.
M Asaduzzaman Shishir
Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh.
M L Bari
Centre for Advanced Research in Sciences, University of Dhaka,Dhaka-1000, Bangladesh.
Y Inatsu
NFRI, Tsukuba, Ibaraki, Japan.
S Kawamoto
NFRI, Tsukuba, Ibaraki, Japan.
Antibacterial activity, Ethanol extract, Essential oil, Betel leaf, Food borne pathogen
Pest Management
Plant: Betel leaves (Piper betle L., family- Piperaceae) were tested for antibacterial activity. Leaves of the betel plant were collected from local market of Dhaka Metropolitan City. Test organisms and Maintenance: Vibrio cholerae ATCC 6395, E. coli ATCC 25922, Staphylococcus aureus ATCC 25923, E. coli O157:H7 NCTC 12049, and Shigella dysenteriae-1 MJ-84, was obtained from American Type Culture Collection, National Collection of Type Culture, and the Department of Microbiology, University of Dhaka, respectively. The stock cultures of the test organisms in medium containing 20% glycerol in cryogenic vials (Apogent Company) were kept at -70oC. Working cultures were kept at 4oC on tryptic soy agar (TSA) slants (NISSUI, Tokyo, Japan) and were periodically transferred to fresh slants. A loopful of culture from the slant was transferred to Tryptic soy broth (TSB) and grown overnight at 37oC. The overnight grown culture was used for the subsequent study. Media and Chemicals: Mueller–Hinton Broth (MHB) and Agar (MHA), Trypticase Soya Broth (TSB) and Trypticase Soya Agar (TSA), ciprofloxacin (NISSUI pharmaceuticals Co. Ltd. Japan) were used for this study. Preparation of ethanol extract of betel leaves: The betel leaves were cut in small pieces; washed in distilled water; dried at 40oC for 48 hours in an oven (Barnstead/Lab-Line; USA; Model No. 121; Serial No, 0203-1541). The dried leaves were grounded using a mortar and pestle into fine powder. Twenty grams of betel leaf powder were soaked in 80ml of 95% ethanol in sterilized bottle and kept in fume hood chamber for overnight. The ethanol fraction was separated using sterilized cheesecloth and filtered through sterilized Whatman filter paper (No.3). Preparation of the McFarland standard: A 0.5 McFarland standard was prepared by mixing 0.05 ml of 1.175% barium chloride dihydrate (BaCl2.2H2O), with 9.95 ml of 1% sulfuric acid (H2SO4)24 in a test tube with constant stirring. The tube was then sealed tightly stored in the dark at room temperature to prevent loss by evaporation. Antibacterial sensitivity testing: The antibacterial activity of the ethanol extract was done according to the method of Bauer et al. (1966)25. The 8-mm diameter discs (Advantec; Toyo Roshi Kaisha, Ltd., Tokyo, Japan) were impregnated with 50 ml of ethanol extract from10mg/ml (w/Plant Betel leaves (Piper betle L., family- Piperaceae) were tested for antibacterial activity. Leaves of the betel plant were collected from local market of Dhaka Metropolitan City. Test organisms and Maintenance Vibrio cholerae ATCC 6395, E. coli ATCC 25922, Staphylococcus aureus ATCC 25923, E. coli O157:H7 NCTC 12049, and Shigella dysenteriae-1 MJ-84, was obtained from American Type Culture Collection, National Collection of Type Culture, and the Department of Microbiology, University of Dhaka, respectively. The stock cultures of the test organisms in medium containing 20% glycerol in cryogenic vials (Apogent Company) were kept at -70oC. Working cultures were kept at 4oC on tryptic soy agar (TSA) slants (NISSUI, Tokyo, Japan) and were periodically transferred to fresh slants. A loopful of culture from the slant was transferred to Tryptic soy broth (TSB) and grown overnight at 37oC. The overnight grown culture was used for the subsequent study. Media and Chemicals Mueller–Hinton Broth (MHB) and Agar (MHA), Trypticase Soya Broth (TSB) and Trypticase Soya Agar (TSA), ciprofloxacin (NISSUI pharmaceuticals Co. Ltd. Japan) were used for this study. Preparation of ethanol extract of betel leaves The betel leaves were cut in small pieces; washed in distilled water; dried at 40oC for 48 hours in an oven (Barnstead/Lab-Line; USA; Model No. 121; Serial No, 0203-1541). The dried leaves were grounded using a mortar and pestle into fine powder. Twenty grams of betel leaf powder were soaked in 80ml of 95% ethanol in sterilized bottle and kept in fume hood chamber for overnight. The ethanol fraction was separated using sterilized cheesecloth and filtered through sterilized Whatman filter paper (No.3). Preparation of the McFarland standard A 0.5 McFarland standard was prepared by mixing 0.05 ml of 1.175% barium chloride dihydrate (BaCl2.2H2O), with 9.95 ml of 1% sulfuric acid (H2SO4)24 in a test tube with constant stirring. The tube was then sealed tightly stored in the dark at room temperature to prevent loss by evaporation. Antibacterial sensitivity testing The antibacterial activity of the ethanol extract was done according to the method of Bauer et al. (1966)25. The 8-mm diameter discs (Advantec; Toyo Roshi Kaisha, Ltd., Tokyo, Japan) were impregnated with 50 ml of ethanol extract from10mg/ml (w/ v) solution of and were dried at 40oC in a dry oven (Model No.- 121, Sl. No.- 0203-1541, 1999, Burnstead Lab-Line, USA) before being placed on the inoculated agar plates. The inocula of the test organisms were prepared by transferring a loopful of culture in TSA into 9ml of sterile Mueller Hinton broth (Difco, Sparks, MD) and incubated at 37oC for 5 to 6 hours. The bacterial cultures were compared with McFarland turbidity standard (108 CFU/ml)24 and streaked evenly in three directions keeping at a 60o angle onto the surface of the Mueller Hinton agar plate (10x40mm) with sterile cotton swab. Surplus suspension was removed from the swab by rotating the swab against the side of the tube before the plate was seeded. After the inocula dried, the impregnated discs were placed on the agar using sterile forceps and were gently pressed down to ensure contact. For each plate 5 discs were placed and were placed in a way so that they were no closer than 24 mm . Plates were kept at refrigeration temperature for 30 minutes for better absorption. During this time microorganisms will not grow but absorption of extracts would take place. Negative controls were prepared using the same solvent without the ethanol extract. Reference antibiotic such as ciprofloxacin (30mg/disc) were used as positive controls. The inoculated plates containing the impregnated discs were incubated in an upright position at 37oC overnight and/or 24 to 48 hours. The results were expressed as the diameter of inhibition zone around the paper disk (8 mm).
Bangladesh J Microbiol, Volume 28, Number 2, December 2011, pp 58-63
Journal