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The following points highlight the top four methods used for isolation of microorganisms present on the seed. The methods are: 1. Seed Washings 2. Dry Seed Agar Plates 3. Blotter Technique 4. Collection of Seed Leachates.
Method # 1. Seed Washings:
Principle:
Maximum number of organisms can be brought into culture by inoculating the washings on agar plates.
Requirements:
1. Seeds—Phaseolus.
2. Distilled water.
3. Media for bacteria, actinomycetes and fungi.
4. Petri plates.
5. Water blanks.
Procedure:
1. Place one seed of Phaseolus in one ml of water blank.
2. Shake well in order to bring all organisms present on the surface of the seed into suspension.
3. For isolation of bacteria spread 0.1 ml of the suspension on nutrient agar and incubate at 37°C for 24-48 hours.
For actinomycetes and fungi, place the entire one ml suspension on the respective media (with 1 mg/l streptopenicillin added after autoclaving to prevent bacterial growth) along with the seed. Press the seed slightly on the agar, spread the washings on the agar plate and incubate at room temperature for 4-5 days.
4. Count the number of colonies which gives the number of spores per seed. Identify them.
Method # 2. Dry Seed Agar Plates:
Principle:
This method makes all microorganisms present on the seed to come in contact with the medium and grow.
Requirements:
1. Seeds of Phaseolus.
2. Agar plates with Nutrient agar and Ac- tinomycete and fungal media.
3. Incubator.
Procedure:
1. Incubate each plate aseptically with two seeds (dry).
2. Roll the seeds a little on the agar plate and gently press them to the agar medium.
3. Incubate at room temperature and observe and count the colonies.
4. Identify them.
Method # 3. Blotter Technique:
Principle:
Mostly cellulolytic microorganisms can be isolated by this method.
Requirements:
1. Absorbent cotton.
2. Sterile distilled water.
3. Filter paper.
4. Petri dishes.
5. Seeds – Phaseolus.
Procedure:
1. Spread a thin layer of absorbent cotton at the bottom of the Petri dish and then place two to three layers of filter paper.
2. Moisten with distilled water and autoclave.
3. Place two seeds of Phaseolus in each Petri dish after cooling and incubate at room temperature.
It takes about 10-15 days for cellulolytic microorganisms—mostly fungi to grow. Isolate them on cellulose agar and identify them.
Cellulose Agar:
Method # 4. Collection of Seed Leachates:
Principle:
Leachates that are exuded on the surface of seeds can be brought into solution by seed washings and analysed.
Requirements:
1. Seeds—Phaseolus.
2. Distilled water.
3. Shaker.
4. Water bath.
5. Chromatographic equipments.
Procedure:
1. Take 10 healthy seeds in 99 ml distilled water in a 250 ml flask and place it on a shaker (1200 rpm) for 30 minutes.
2. Decant solution, evaporate to one to 2 ml concentrate and analyse it chromatographically.