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Isolation of Aspergillus niger strain from soil and Water Samples: Practical

Aim:

A) To isolate and purify Aspergillus niger strain from soil and water

B) To characterize (isolate citric acid production A. niger strain macroscopically and microscopically).

Theory:

Microbial habitats including soils, rivers, lakes, oceans on the surface of living and dead thing inside the organism, on man-made structures and everything in between mainly provide nutrient and protect cells from harsh conditions. Every environment is stratified in terms of degree of temperature, oxygen, nutrients and sunlight present. These stratification make up different niches to which a specific microorganism or a group of the billion of the years that microorganism have been on the earth. They have evolved to fit perfectly almost every niche.

Aspergillus niger is a cosmopolitan representative of microscopic filamentous fungi. Although the main source of this strain is soil, it frequently occurs in various other sources. The ability of Aspergillus niger to produce substances of various type, such as organic acid (eg. citric, oxalic, malic, lactic etc.) enzymes (eg. amylase, β-glucosidase, cellulase and lipase) has great use in the food, medicine, pharmaceutical and chemical industries. There are no universal screening method. The success of screening program depends on the selection of appropriate microorganism to be tested, the capacity of an industrial screening group for isolation of the microorganism and though testing is around 1000-2000 strains per year.

The success of a screening program depends upon both the kinds of organism used and the method for detection of activity. Currently the choice of strain has a 30-40% influence on the outcome, the test procedure a 60-70% influence. A gram of soil contain between 10⁶-10⁸ bacteria, 10⁴-10⁶ actinomycetes spores and 10²-10⁴ fungal spores and 10²-10⁴ fungal spores. Less than 1% of the words microorganism have been intensively studied. Above all the approximately 100,000 known fungi have been poorly studied so that a vast no. of new natural products can be expected from this group in future.

Requirements:

i. Czapadex broth
ii. Czapadex agar
iii. Garden soil sample
iv. Plants
v. Sterile saline
vi. Lactophenol cotton blue

Procedure:

1. The garden soil (1 gm) was collected and suspended in a definite amount of sterile water and mixed well.
2. Then 0.1 ml of soil sample was inoculated into Czapadex broth with 10 µg/ml concentration of streptomycin to inhibit the growth of bacteria.
3. The flasks were incubated at room temperature on shaker (150 rpm) for 7 days.
4. After 7 days of incubation 0.1 ml was drawn out of media and dilution from 10⁻¹ to 10⁻⁶ was prepared.
5. Out of this 10⁻⁴ solution was selected and spread on Czapadex agar.
6. The plates were incubated at room temperature for 7 days.
7. Single colonies from the plates were selected and purified by restreaking on other sterile Czapadex agar.
8. The pure Aspergillus colonies were selected and maintained at pure culture from further studies.
9. The microscopic characterization of Aspergillus niger was done by wet mount method.

Observation:

Cottony appearance of Aspergillus niger was observed.

Result:

After incubation fungal colony of Aspergillus niger had colony appearance which was initially white to yellow and then turning black. The reverse of the plate appeared to change from white to yellow after an incubation period of 7 days.

Conclusion:

The pure culture of Aspergillus niger was successfully isolated and preserved as pure strains for further citric acid production.