Evaluation of antibacterial activity of fungicides

Materials required:
Pure cultures of the test bacteria
Nutrient agar medium
Fungicides: Blitox, Kocide, Zineb, Dodine, Captan, Thiram, Carbendazim
Sterilized Whatman filter paper (No. 1) discs
Sterilized forceps
Sterilized Petri dishes
Sterilized cotton swab/ L-rod
Inoculation loop, etc.
Procedure:
Method 1
1. Inoculate the phytopathogenic bacterial isolates in Nutrient Broth and incubate at 28+2oC for
24-48 h.
2. Spread the culture of phytopathogenic bacteria on to Petri dishes containing Nutrient Agar
medium to make a lawn of bacteria [Using sterile cotton swab/ by spread plate method (100 µl of
culture containing 108 cfu/ml)].
3. Place the plate inside the refrigerator for 10 min.
4. Mark the quarters on the Petri dish and mark the central point of each quarter.
5. Prepare fungicide solutions to produce concentrations of 1500, 2000, 3000 ppm in test tubes
using sterile distilled water.
6. Dip the sterilized Whatman filter paper disc in fungicide solution and place it in the central point
of each quarter on the nutrient agar medium previously spread with phytopathogenic bacteria (after
draining the excess suspension by pressing against the wall of the tube).
7. Incubate the plates at 28+2oC for 24-48 h.
8. Observe the formation of inhibition zone and measure the diameter of the inhibition zone.
9. Calculate the area of the inhibition zone.
Method 2
1. Inoculate the phytopathogenic bacterial isolates in Nutrient Broth and incubate at 28+2oC for
24-48 h.
2. Add the fungicide to melted cool (~ 50oC) NA medium to produce concentrations of 1500, 2000,
3000 ppm.
3. Spread 100 µl broth culture onto the fungicide amended NA medium in Petri dishes. Keep
unamended NA medium inoculated with broth culture by spread plate method as control.
4. Incubate the plates at 28+2oC for 24-48 h.
5. Count cfu and compare with cfu in control plates