2.3. Antimicrobial Assays

An anti-microbial substance either kills or inhibits the growth of microorganisms, such as fungi, bacteria. Plants has been investigated scientifically for antimicrobial activity, and a large number of plant products have been shown to inhibit the growth of pathogenic microorganisms. Antimicrobial activity of the crude extracts can be determined by these two methods:

1. Agar well method
2. Disc diffusion assay method

A. Agar well method

Nutrient agar can be prepared for bacteria according to the manufacturers' instructions. Immediately after autoclaving, allow the media to cool at 45°C to 50°C in water bath. Pour the freshly prepared cooled media (approximately 4 mm depth) into flat-bottomed Petri dishes (90mm in diameter). Spread about 0.2ml of the test inoculum of bacteria uniformly on the surface of the solidified agar media using a sterile inoculation L-shaped glass rod. Make four equidistant wells of 5mm diameter and 4mm in depth on the agar using a sterile cork borer. Make two more wells for positive and negative controls at the middle of the agar. Fill about 25 µl of the plant extracts (concentrations ranged from 25–200mg/ml) and controls into the wells. The positive controls will be antibiotic (e.g. Ampicillin). The negative controls will be DMSO for organic solvent extracts and distilled water for aqueous extracts. Label the wells to correspond with the code numbers of the test crude extracts and controls. Store the treated plates in a refrigerator at 4°C for at least six hours to allow diffusion of the extracts into the agar while arresting the growth of the test microbes. The plates were then incubated for 24 hours at favorable condition for a particular organism. The test was carried out in triplicates. Antimicrobial activity was determined by measuring the diameters of zones of inhibition in mm.

 

 

 

 

 

 

 

 

 

B. Disk diffusion assay

The preparation of media and inoculation of the test microbes are same as described in the agar well method. However, instead of punching out wells on the agar, sterile 5 mm Whatman No. 1 filter paper discs will be used in the disc diffusion method. Soak the discs into the dissolved crude extracts for minimum of two hours. Blank discs impregnated generally with Ampicillin (10µg/disc) for gram positive bacteria, Gentamicin (15µg/disc) for gram negative bacteria and Fluconazole (0.4mg/disc) for Candida are used as positive controls. For negative control, discs will be soaked in DMSO and distilled water, for organic solvent and water extracts, respectively. By use of a sterile forceps, place four seeded discs of the plant extracts equidistantly onto each of the inoculated plates. Place two extra discs for positive and negative controls at the middle of plate. Store the treated plates in a refrigerator at 4°C for minimum of six hours and then transfer to incubator for 24 hours at favorable conditions for a particular organism (37°C for bacteria and for 48 hours at 30°C for Candida). Perform the test in triplicates. Antimicrobial activities were determined by measuring the diameters of zones of inhibition in mm.

 

 

 

 

 

 

 

 

 

2.4. Anthelmintic Assay

Plant-derived compounds have played significant role in the field of anthelmintic drugs, such as Santonin , the main active substance isolated from wormwood (Artemisia maritima L). The protocol for anthelmintic screening using Caenorhabditis elegans (Maupas) developed by Simpkin and Coles (1981). The test is carried out in a 24-well tissue culture plate with a well volume of 2.5 ml. Each well is filled with 2 ml of M₉ liquid medium. M₉ solution consists of 6g Na₂HPO₄, 3 g KH₂PO₄, 5g NaCl, and 0.25g MgSO₄. 7H₂O dissolved in 1000 ml water and autoclaved at 120°C for 20 min. Extract solutions, dissolved in DMSO, are added to make 500 ppm solutions, and then 10 μl C. elegans suspensions containing 30 to 40 larvae are subsequently introduced into each cell. The plates are incubated at 20°C for 5 day, and the number of dead nematodes is recorded using a phase contrast microscope, and anthelmintic activity is graded as shown below:

- Nematode counts and the motility of the nematodes correspond to the control

+ 0 to 20% fewer nematodes than the control; nematodes move slowly

++ Slightly higher nematode counts than the initial counts; nematode counts 20% less than the

control; nematodes move very slowly

+++ Same nematode counts as the initial counts, all dead

Active fractions/compounds are repeated with lower concentrations and compared to known anthelmintics, such as santonin.