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Antibacterial Bioassys

A large number of human, animal and plant diseases are caused by pathogenic bacteria. Infections due to bacteria are major cause of death in higher organisms. Unfortunately human struggle against pathogenic microbes is far from or due to many reasons. Most important of them is time to time discovery of new pathogens, appearance of microbial resistance and occurrence of fatal opportunistic infections. Remarkable diversity of chemicals present in biological samples leads to the search for new effective antimicrobial agents as they have tremendous potential against pathogens.

In order to assess the antibacterial potentials of the plants collected during the ethanobotanical surveys, Agar Well Diffusion Method was employed.

Method: Agar Well Diffusion Method

In this method wells are cut in seeded agar and the test sample is then introduced directly into these wells. After incubation the diameter of zone around the well is measured and compared with standard drug.

Biotechnology section at ICCBS, following strains of bacteria are used, the account of their correlation with various disorders are described below:

• Staphylococcus aureus: Gram +ve cocci cause boils, sinusitis and intoxication in human.

• Echerichia coli: Gram –ve bacilliassociated with infantile diarrhea, traveler’s diarrhea, normally present in the gut.

• Pseudomonas aeruginosa: It is an opportunistic pathogen causesurinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections.

• Salmonella typhi: a gram negative short rod causes typhoid fever.

• Bacillus subtilis: not a common pathogen produces different enzymes.

• Shigella flexenari: causes bloody dysentery.

Nature of Fractions Tested:
Crude plant extracts and Fractions from ethanobotanic surveys.

Materials and methods:

Media Preparation
Three types of media are prepared in this assay

1. Nutrient agar
a. Nutrient agar -------------------------28 gm
b. Distilled water -----------------------1 L
Medium is dissolved and autoclaved at 121° C for 15 min and then cooled to 45° C. Addition of 40-50 ml mediais made in sterile 14 cm diameter Petri plate and kept at room temperature.

2. Nutrient Broth
a. Nutrient broth----------------------0.8 gm
b. Distilled water --------------------100 mL
Dissolved the broth and dispense approx. 3 ml nutrient broth in screw capped test tubes and autoclaved 121° C for 15 min.

3. Soft Agar
a. Agar Agar------------------------0.8 gm
b. Distilled water -----------------100 mL
Dissolved the agar and dispense approx.7 ml soft agar in screw capped test tubes and autoclaved it at 121° C for 15 min and refrigerate it.

Test Sample preparation:
Crude extract: 3 mg/mL of DMSO

Procedure:

1. First day: inoculate single colony of bacterial culture in nutrient broth and incubate it at 37° C for 24 hrs.
2. Second day take soft agar tube, melt it and cool it up to 45° C then add 10 µl of fresh bacterial culture shake it well and then pour it on to the nutrient agar containing plate. Rotate the plate to make even distribution of the culture, allow solidifying the lawn.
3. Make wells by using 6mm-diameter sterile borer and Mark the well with sample code.
5. Add 100uL of sample in respective agar well plate according to bacterial culture.
6. Other wells supplemented with DMSO and reference antibacterial drug serving as positive and negative control.
7. Incubate the plates at 37° C for 24 hrs.
8. Next day note down the result in terms of zone of inhibition in mm.

Criteria
      -         = No activity
9-11 mm   = Non significant
12-14 mm = Low activity
15-17 mm = Good activity
Above 18 mm = Significant

Result Interpretation

Presence of antibacterial agent is indicted by the growth inhibition of the bacterial strains and appearance of zone of inhibition. i.e. observe a clear zone where the growth of bacteria had not occurred. The cultures were kept at 4° C prior to testing. They were sub-cultured in liquid nutrient broth and incubate at 37° C for 18-24 hrs and then used for the screening.

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