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The following points highlight the top two techniques used for screening of antibiotic producing organisms. The techniques are: 1. Crowded Plate Technique 2. Determination of Antimicrobial Spectrum Giant Colony Technique.
1. Crowded Plate Technique:
Principle:
When soil or suspension with lower dilutions is inoculated, large number of microorganisms grow on the agar plate. Those which are capable of producing antibiotics will inhibit the growth of the adjacent forms by producing a clear zone of inhibition.
Requirements:
1. Two soil samples, one which is seeded with Streptomyces griseus to serve as positive control.
2. Agar plates of glucose asparagine agar/ Potato Dextrose agar (two sets) and slants.
3. Water blank 99 ml in 250 ml flask (2).
4. Sterile pipettes.
5. Incubator.
6. Bunsen flame.
7. Inoculation loops.
8. Glass marking pencil.
Procedure:
1. Weigh both soil samples (lg each) and add to labelled 99 ml water blanks and shake well.
2. Using sterile pipettes transfer 1 ml of this dilution each to the labelled agar plates and rotate the plates gently to spread the dilution evenly on the agar plate.
3. Incubate at room temperature (28°- 30°C). Observe for inhibition zones on both plates.
4. Isolate the colonies that show inhibition, transfer them to the agar slants. Incubate and preserve them as antibiotic producing organisms and use them for finding the antimicrobial spectrum.
2. Determination of Antimicrobial Spectrum Giant Colony Technique:
Principle:
Several microorganisms (pathogens especially) are used as test organisms in order to find out the zone of inhibition.
Requirements:
a. Cultures isolated with antibiotic activity.
b. Petri plates with same agar (PDA/glucose Asparagine agar).
c. Cultures of test organisms.
d. Inoculation needle.
e. Glass marking pencil.
Procedure:
1. Label the plates with soil sample source.
2. Aseptically inoculate the centre of the agar plate with the antibiotic producing organisms and incubate them at 28°30°C (room temperature) for 72-96 hours (3-4 days).
3. Mark the test organism (1 to 6) by drawing a line.
4. Inoculate test organisms marked 1 to 6 perpendicular to the giant colony of the antibiotic producing organism by sheaking till the colony but without touching it.
5. Incubate at 37°C for 24 hours.
Observe the colonies that (streaks) grow farther away from the organism as well as the ones which are not inhibited by the antibiotic produced by the giant colony.