In order to obtain microorganisms which can be used for obtaining a product of industrial importance, the following steps are to be followed: 1. Collection of Soil Samples 2. Collection of Root Exudates.
Collection of Soil Samples:
Microorganisms are more abundant in the upper zone known as ‘A’ horizon which extends between 5-45 cm. Their distributions vary in different areas, e.g. samples taken a few inches apart will have varying numbers and forms. They are attached to clay, humus and organic matter and are seldom free in soil solutions. Hence random samples should be collected even from a particular area after removing the uppermost layer of 5-8 cm and a composite mixture be made.
Composite soil samples from A’ horizon will yield maximum number of microorganisms.
1. Sterile sickle or any other iron loop.
2. Sterile vials or soil augers.
3. Sterile containers/plastic bags.
1. Remove 4-8 cm soil from surface.
2. Push in soil augers or sterile vials into ‘A’ horizon (5-45 cm).
3. Collect at least five random samples in sterile containers/plastic bags and keep in cool places while transporting.
4. Mix all five samples in sterile containers, weigh and use for dilution technique.
Collection of Rhizosphere Soil:
Rhizosphere soil is that portion of the soil which is adjacent to the root system of plants and influenced by the root system. This region has the maximum population of organisms because of root exudates and dead and decaying parts of roots and root hairs.
From rhizosphere region more number of organisms can be isolated since this region is rich in root exudates that contain sugars, amino acids, vitamins, organic acids, etc.
1. Sterile tools.
2. Sterile vials.
3. Sterile containers/plastic bags.
4. An actively growing plant (Phaseolus) from field.
1. Make the soil around the root system (10-12 cm away from the base of the plant) loose by means of a sterile iron tool.
2. Herbaceous plants (Phaseolus) can be uprooted and placed in sterile containers. For trees, the soil just adjacent to the feeder roots should be collected. In both cases collect at least five random samples.
3. Remove soil particles sticking to the root system of plants and make a composite mixture of the random samples.
4. Weigh this soil and use for isolation of microorganisms.
Rhizoplane is the immediate external surface of plant roots together with any closely adhering particle of soil or debris. This region has the maximum concentration of exudates and hence harbours nutritionally fastidious organisms.
1. For isolation, use either the roots of same plant or others.
2. Weigh the roots and use them for serial root washings.
Collection of Root Exudates:
In a natural environment the exudates produced by roots are quickly utilised by microorganisms. Not only that, there will be components of microbial metabolisms too. Hence it is necessary that the plants be grown aseptically in an inorganic nutrient solution.
Large Test Tube Method:
The roots exude substances into quartz and/or filter paper in the tube which is collected, concentrated and analysed chromatographically.
1. Seeds – Phaseolus.
2. HgCl2, 0.2%.
3. Sterile Petri plates and also ones with filter papers.
4. Acid washed and autoclaved quartz sand.
5. Plant nutrient solution:
k2hpo4 – 1.0 g
KC1 – 7.0 g
CaS04 – 2.5 g
MgS04 7H20 – 2.5 g
Ca3 (P04)2 – 0.5 g
FeP04 – 0.5 g
KN03 – 2.0 g
Grind all salts together, mix and take 1.5 g of this mixture and dissolve in 1000 ml distilled water.
1. Long tubes (20 X 3 cm) with sterile quartz.
2. Long tubes with sterile filter paper platform.
3. Black paper.
4. Filter paper.
6. Water bath.
7. Chromatographic assembly with filter paper.
8. Sterile distilled water.
9. Bunsen flame or Laminar clean air- flowhood.
11. Growth chamber.
1. Keep Phaseolus seeds in 0.2% HgCl2 for 5-6 minutes, wash several times with distilled water (sterile) and place them in sterile Petri plates with filter paper moistened with plant nutrient solution and incubate.
2. After 24-48 hr. the germinated seeds are transferred to long tubes (20×3 cm) with acid washed autoclaved quartz sand, wet with plant nutrient solution and cap them. Cover the sand region of the tube (into which roots will grow) with black paper and incubate at room temperature (28°- 30°C) in a growth chamber.
3. Transfer the germinated seeds with a sterile forceps to the tubes (20 X 3 cm) on the filter paper platforms moistened with nutrient solution, cap and cover the lower region of the tube with black paper.
4. Keep them in a growth chamber at room temperature (28o-30oC).
5. Allow plants of both tubes to grow. The period depends on the plant. Phaseolus requires two weeks.
6. Remove the plants from both sand and filter paper tubes, cut off the crown, wash root system thoroughly along with sand and filter paper in sterile beakers with sterile distilled water.
7. Centrifuge the washings to remove root debris and concentrate the supernatant on a water bath or under vacuum to get a one ml concentrate for every ten plants.
8. Analyse them chromatographically for sugars, amino acids, etc.