Myanmar Health Sciences Research Journal

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MATERIALS AND METHODS

 

This study was a laboratory-based experi-mental study carried out during 2016 and 2017 at Department of Medical Research (Pyin Oo Lwin Branch), Mandalay District.

Collection of plant material

Coriandrum sativum L. was collected from Mandalay Region.

Identification of plant

Identification of plant was performed at Department of Botany, University of Mandalay by using The Flora of Ceylon.10

Phytochemical analysis

Phytochemical analysis of whole plant was also done by phytochemical techniques.11

Extraction of plant

Coriandrum sativum L. were collected and thoroughly washed with water and then air-dried for about two weeks. The whole plant was powdered and extracted by percolation method.12 The plant sample 100 g was percolated with 1000 ml of 70% ethanol for a week in a percolator. The liquid extract containing plant constituents was filtered and evaporated on the water bath until to get constant weight and stored in desiccators. Methanolic extract of C. sativum L. was obtained by the same procedure.

Determination of antibacterial activity of different extracts of the whole plant of Coriandrum sativum L.

Antibacterial activity of different extracts of Coriandrum sativum L. was determined by agar disc diffusion technique according to modified Kirby and Bauer method.13

Preparation of medium

Muller-Hinton Agar was prepared according to the procedure of the manufacturers’ recommendation and sterilized by moist heat at 121°C for 15 minutes. After autoclaving, 25 ml of the media was poured into 9 cm diameter petridishes and allowed to set at room temperature. It was prepared freshly before use. When the agar had solidified, the plates were dried at 50°C in the upright position in the oven with the lids tilted. The plates were then labeled.

Preparation of bacterial suspension

A few colonies of organisms from the sub-culture to be tested were picked with a wire loop and introduced into test tube containing peptone solution. These tubes were incubated at 37°C for 3-4 hours to produce the growth turbidity.

Preparation of impregnated disc of different plant extracts

The sterile discs, 6 mm in diameter, were spread out separately in petridishes, so that each disc was not less than 2 mm from its neighbours. They were sterilized by dry heat at 160°C for 1 hour. Different doses of ethanolic extracts of C. sativum L. (40 mg, 80 mg, 120 mg, 160 mg, 200 mg etc.,) were dissolved in 1 ml of 70% ethanol separately. From the different stock solutions, 20 μL of solution was impregnated to discs, respectively, and dried in the oven at 37°C to evaporate the solvent. Discs for methanol extract of C. sativum L. were done by the same procedure. Antibiotic disc, ceftriaxone  (30 μg), was used as positive control reference standard. Disc as negative control was prepared using the same solvent employed to dissolve the plant extract.

Antibacterial susceptibility test

Antibacterial susceptibility test was deter-mined by a standard disc diffusion technique using Muller-Hinton agar according to the recommendations of Clinical and Laboratory Standards Institute (CLSI).

A sterile cotton swab was dipped into bacterial suspension. Freshly grown liquid cultures of the test pathogens were seeded over the Muller-Hinton agar (MHA) plates with a sterile cotton swap. The swab was streaked in at least three directions through the angle of 60° over the surface of the Muller-Hinton agar to obtain uniform growth. A final sweep was made around the edge of the agar surface. After the inoculum has dried for a few minutes, the sterile filter paper discs impregnated with plant extracts were placed on the seeded MHA plates at equal distance with sterile forceps and gently pressed down to ensure contact with the medium. The plates were incubated at
37
°C for 24 hours. Following overnight incubation, zones of inhibition occurred around the discs. The inhibition zones were recorded as millimeters (mm).

Determination of minimum inhibitory concentration (MIC) and minimum bact-ericidal concentration (MBC)

Minimum inhibitory concentration (MIC) is defined as the lowest concentration of antimicrobial activity that will inhibit the visible growth of a microorganism after overnight incubation, and minimum bact-ericidal concentration (MBC) as the lowest concentration of antimicrobial activity that will prevent the growth of an organism after subculture on to antibiotic-free media. The determination of the MIC involves a semi-quantitative test procedure which gives an approximation to the least concentration of an antimicrobial needed to prevent microbial growth. MIC/MBC values can be determined by a number of standard test procedures. The most commonly employed methods are the tube dilution method and agar dilution method. Serial dilutions of the extracts were prepared from the highest concentration of 1000 mg/ml to the lowest concentration of 200 mg/ml. The test organisms are
then added to the dilution of the products, incubated and scored for growth.

Determination of minimum inhibitory con-centration and minimum bactericidal con-centration of ethanolic and methanolic extracts of Coriandrum sativum L.

The ethanolic and methanolic extracts of Coriandrum sativum L. were proceeded for minimum inhibitory concentration (MIC) by broth dilution method.14

Different concentrations of ethanolic and methanolic extracts of C. sativum L. ranging from 200 mg/ml to 1000 mg/ml. were tested on different test organisms. A series of ten tubes for each test organism were prepared. Each tube contains 20 μL of test organisms in 1 ml of Muller-Hinton broth. The different dilutions of 1 ml of ethanolic extract of C. sativum L. were added to the tubes. The eighth tube was used as control tube which contained Muller-Hinton broth, 70% ethanol with test organisms. The ninth tube was used as test extract control and the tenth tube, test organisms only. Then, the different dilutions of methanolic extract were also done and incubated at 37°C for 24 hours.
After incubation, minimum inhibitory con-centration (MIC) was recorded as tube with lowest concentration at which no visible turbidity was observed. For determination of minimum bactericidal concentration (MBC), one loopful from each tube of  above dilutions was streaked on Muller-Hinton agar plate and incubated at 37
°C for
24 hours.

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