Soil is having a tremendous source of unknown microorganisms, as bacteria, algae, protozoans, yeasts, molds, and microscopic worms are habitually found in this environment. Researchers or students primarily isolate bacteria, molds, or fungus on their primary culture plates, Based upon this variety early exposure can be allowed for staining, and microscopically observing some noticeable differences between prokaryotes and eukaryotes.
The microorganisms obtained from soil consists of bacteria, actinomycetes, fungi, algae, and these microorganisms only provide a source for biochemical agents viz., mineralization of complex organic compounds into simpler inorganic forms and their further adaptation to elements carrying required constituents.
Hence, microorganisms execute a vital role in preserving the fertility of the soil. As they play an important role in the nutrient cycling of various elements, which can be further reuptake by plants. Since carbon and nitrogen are the most important atom for plant nutrition, therefore, their recycling has been elaborated.1
Aim of this experiment is to perform the isolation, screening, and purification of microorganisms obtained from the soil.
|Chemicals||Soil sample (1g)|
Nutrient agar medium
Potato dextrose agar
|Glass wares||Petri plates,|
After the collection of soil samples near the area of any plant base, as microorganisms are found in abundant quantity, the first step is to make a broth by inoculating the soil sample by a serial dilution method, in which one gram of soil sample has to be transferred to a test tube having 10 ml of sterile distilled water which one can label as 10:1 dilution.
This is followed by a 30-minute agitation process on a rotary shaker, the suspension obtained after the first dilution is again diluted by taking one ml from the suspension in another flask containing nine ml water which is known as 10:2 dilution which is followed by successive dilution series to obtain dilution upto 10:5.2
The dilution series is followed by plating each of that dilution in sterilized Petri plates previously settled with nutrient agar media and potato dextrose agar medium and then incubated at 37°C overnight and for fungus at 25°C for 72 hrs. After examining the plates on the very next day, the results are noted which further gives detailed information for screening of different strains of bacteria.
For the purification of organisms, the primary step is the proper labeling of plates containing the appropriate dilution number. The streaking of every bigger zones from all dilution plates is carried out which is followed by staining of each zone using Gram’s staining, the color change and microscopic evaluation give the shape and structure of the microorganism.
Purification of an unknown organism from soil
This staining procedure is started by using sterilized plates, which are heat fixed after preparing smears. The stain kit contains crystal violet, Gram’s iodine(Mordant), alcohol-acetone mixture which is used as a decolorizer, and finally safranin as a counter stain.
The antimicrobial assay can be carried out by using two methods that is disc diffusion and well diffusion methods; nutrient agar media, petri plates, and several glass wares are sterilized by autoclaving.
These media are plated in Petri plates inside laminar airflow by swabbing the plates with microorganisms and discs are placed a minimum of 4 discs in a single plate which is disc diffusion, then using a cork borer well is bored in the middle of the plate after swabbing with microorganism, plates are then incubated overnight and the zone of inhibition was measured the next day.
The procedure for Gram’s staining: Smear has to be flooded with crystal violet for 2-3 mins and washed in the direct flow of tap water for 2 secs.
Then it was treated with mordant gram’s iodine for 1 min and washed in the direct flow of tap water for 2 secs which is flowed by the decolorizing agent was flooded for 30secs and added drop by drop until decolorizing agent in slides runs clear, the slide was counter-stained with safranin for 30secs and washed in the direct flow of tap water for 2 secs. This procedure ends with microscopic observation for the determination of morphological characteristics.
Fungus and bacteria which are obtained after primary screening are further optimized by observing various bioparametric characters such as temperature, pH, and antimicrobial assay as a result morphology of bacteria obtained. Often primary cultures contain the greater part of Bacillus colonies hiding other colonies. So one has to be skillful enough to select other bacterial colonies other than bacillus.
- Steubing PM. Isolation of an unknown bacteium from soil, Chapter 6, 1993: 83.
- Benson. Pure culture techniques in Microbiological Applications Lab Manual in general microbiology, The McGraw-Hill Companies., 2001;4:82-8.
- Vilain S, Luo Y, Hildreth MB, Brozel, VS. Analysis of the life cycle of the soil saprophyte Bacillus cereus in liquid soil extract and in soil. Appl Environ Microbiol. 2006;72(7):4970-7.
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Serial dilution method
Microscopically observing some noticeable differences between prokaryotes and eukaryotes after staining.
By streak plating method and/or spread plating method.