Isolation of Microorganisms from Soil: Principle and Procedure

Isolation of Microorganisms from Soil is a culture-based technique used to separate and grow different microorganisms present in the soil sample on suitable culture media.

Soil contains a large number of microorganisms, mainly bacteria, fungi and actinomycetes. Actinomycetes are filamentous bacteria. The number and types of microorganisms are not the same in all soil samples. They vary with the soil type, moisture, pH, organic matter and the location from where the soil is collected.

In this method, the soil sample is first made into a soil suspension which is then diluted, if required, and inoculated onto a suitable culture medium. The microorganisms capable of growing under the given conditions develop visible colonies on the medium. Soil may also contain potentially harmful microorganisms. The isolation is carried out under suitable laboratory biosafety conditions.

For the isolation of bacteria from soil, the serial dilution method is commonly used along with the plating technique. The soil suspension is diluted successively to reduce the number of microorganisms in a particular volume. When the diluted sample is plated on the agar medium, the microorganisms may grow as separate colonies instead of continuous growth.

A colony may arise from a single viable cell or a group of cells. Such growth is counted as a colony-forming unit (CFU).

The colonies may have different sizes, shapes, colours and textures. These are some of the visible characters used for the preliminary examination of the isolated microorganisms. However, the formation of a separate colony does not necessarily confirm the identity of the microorganism or that it is a pure culture.

Not all soil microorganisms can be grown on ordinary laboratory media. Different groups require different nutrient media and cultivation conditions. The culture-based method can isolate only those microorganisms which are able to grow under the selected conditions.

Objectives of Isolation of Microorganisms from Soil

The main objectives of isolation of microorganisms from soil are as follows-

• To isolate and grow the culturable microorganisms present in the soil sample using suitable nutrient media.
• To obtain separate and distinguishable microbial colonies from a mixed population of soil microorganisms.
• To study the microbial diversity of soil by observing the differences in colony size, shape, colour and other visible characteristics.
• To obtain individual microbial isolates which can be further studied or developed into pure cultures under appropriate laboratory biosafety conditions.
• To estimate the number of culturable microorganisms present in the soil as colony-forming units (CFU), if enumeration is required.
• To identify the isolated microorganisms by further laboratory tests, when identification is included in the practical.

Principle of Isolation of Microorganisms from Soil

The isolation of microorganisms from soil is based on the separation of microorganisms from a mixed soil population by dilution and their cultivation on a suitable solid culture medium. The soil contains different microorganisms, many of which are present along with the soil particles. On preparing the soil suspension, some of these microorganisms become distributed in the liquid.

The original soil suspension may contain a large number of microorganisms which can produce crowded or overlapping growth when inoculated onto an agar medium. By serial dilution, the number of microorganisms present in a given volume is reduced. When the diluted suspension is grown on the solid medium, the viable cells or groups of cells which are sufficiently separated multiply and form visible colonies.

Each colony may develop from a single viable microbial cell or a group of cells. It is counted as one colony-forming unit (CFU).

The growth of these microorganisms depends on the culture medium and the conditions provided for their growth. Temperature and oxygen availability also affect the microbial growth. Different microorganisms have different growth requirements. Some may grow and produce colonies, while others may not grow under the same conditions.

A separate colony does not always represent a pure culture. It may contain more than one type of microorganism. The purity of the isolated colony has to be examined further.

Requirements

The following materials and equipment are required for the isolation of microorganisms from soil-

• Soil Sample- The soil sample containing different types of microorganisms which are to be isolated.
• Sterile Sample Containers- These are used to collect and transport the soil samples without contamination from external sources.
• Sterile Distilled Water or Normal Saline- It is used for the preparation of soil suspension and serial dilution of the sample. The diluent is selected according to the isolation method.
• Sterile Test Tubes or Dilution Bottles- To prepare and hold the different dilutions of soil suspension.
• Micropipettes, Sterile Pipettes and Pipette Tips- The required volume of soil suspension is measured and transferred using sterile pipettes during the dilution and inoculation process.
• Sterile Petri Dishes- Used to hold the solid culture media on which the microorganisms are grown.
• Culture Media- Nutrient agar or other suitable agar media are used for the cultivation of soil microorganisms. The media provide nutrients required for microbial growth. Different microorganisms may require different types of culture media.
• Sterile Glass Spreader or Inoculating Loop- These are used to inoculate or spread the soil suspension over the culture medium. The instrument used depends on the isolation method.
• Incubator- It maintains the required temperature for microbial growth. It is used when the isolation method requires controlled incubation.
• Vortex Mixer or Other Mixing Equipment- For mixing the soil suspension and its dilutions, depending on the method used.
• Marker Pen and Labels- Used for marking the soil samples, dilution tubes and culture plates.
• Laboratory Coat, Gloves, Eye Protection and Closed-Toe Shoes- These are used for personal protection while handling the soil samples and microbial cultures. The protective equipment required is selected according to the laboratory risk assessment.
• Biological Safety Cabinet– It is used to provide additional containment when the procedure involves the production of potentially infectious aerosols. Its requirement depends on the risk assessment.
• Laboratory Disinfectant and Biohazard Waste Containers- Disinfectants are used for cleaning and disinfecting the working surfaces, while biohazard waste containers are used to collect the contaminated laboratory materials for safe disposal.
• Sterilization or Decontamination Facilities- These are required for the approved treatment of contaminated culture media, instruments and other laboratory waste.
• Colony Counter– Used for counting the microbial colonies when enumeration is required.
• Approved Laboratory Procedure and Risk Assessment- A laboratory risk assessment and an approved procedure are required before handling unknown soil microorganisms. Appropriate biosafety facilities and trained laboratory supervision are necessary. Further handling or subculturing of unknown environmental isolates generally requires biosafety level 2 (BSL-2) practices, depending on the risk assessment.

Culture Media for Soil Microorganism Isolation

Different types of culture media are used for the isolation of microorganisms from soil. The medium is selected depending on the type of microorganism to be isolated and its nutritional requirements. Bacteria, fungi and actinomycetes may require different media for their growth. No single culture medium can support the growth of all soil microorganisms.

Some of the commonly used culture media for soil microorganisms are given below-

Factors Affecting the Selection of Culture Media

• Nutrient Composition- Different microorganisms have different nutritional requirements. Some soil bacteria grow well on general-purpose nutrient media, while others may grow better on low-nutrient or soil-extract-based media.
• pH of the Medium- The pH affects the growth of microorganisms. An acidic medium may favour the growth of certain fungi, whereas many soil bacteria grow better at near-neutral pH. The required pH depends on the microorganism to be isolated.
• Selective Agents- Certain culture media may contain selective agents such as antibiotics to inhibit the growth of unwanted microorganisms. These agents may also inhibit some of the microorganisms intended for isolation. The selective agents are used only when required by the approved laboratory method.
• Type of Microorganism- The culture medium is selected according to whether bacteria, fungi or actinomycetes are to be isolated. Some media favour particular groups of microorganisms while restricting the growth of others.

Methods of Isolation of Soil Microorganisms

The following are the common methods used for the isolation of soil microorganisms–

1. Serial dilution agar plate method- This is the most common method for isolation and counting of soil bacteria, fungi and actinomycetes. A known amount of soil is mixed with sterile water or saline and shaken properly to detach microorganisms from soil particles. Then the soil suspension is diluted step by step, usually as 10⁻¹, 10⁻², 10⁻³ and so on.A measured amount from suitable dilution is transferred into sterile Petri plates or on agar surface. After incubation, separated colonies are formed. Each colony is assumed to develop from one viable cell or one colony forming unit (CFU). Nutrient agar is commonly used for bacteria, Czapek-Dox agar or Sabouraud agar for fungi and glycerol yeast agar or actinomycete isolation agar for actinomycetes.
2. Pour plate method– In this method, diluted soil suspension is added into sterile Petri plate and then cooled molten agar is poured over it. The plate is rotated gently so that the inoculum spreads throughout the medium.Colonies develop both inside the agar and on the surface of agar. It is useful for isolation and enumeration of microorganisms from soil. But heat-sensitive organisms may be affected because the inoculum comes in contact with warm molten agar.
3. Spread plate method– In this method, a small measured volume of diluted soil suspension is placed on the surface of solid agar medium. It is spread uniformly with a sterile L-shaped glass rod or spreader.The colonies grow only on the surface of the agar plate. This makes colony picking easier. Spread plate method is preferred when surface colonies are needed for observation, purification and further subculture.
4. Streak plate method– Streak plate is mainly used for obtaining pure culture from a mixed colony growth. A loopful of soil suspension or a selected colony is streaked over the agar surface in different sectors.The number of cells decreases with each streaking area. At the last streaked region, individual cells become separated and form isolated colonies. These isolated colonies are picked and transferred to fresh medium to get pure culture.
5. Enrichment culture method- Enrichment culture is used when the required microorganism is present in low number or is hidden among many fast-growing soil microbes. The soil sample is added to a liquid or solid medium that favours the desired organism.The medium, temperature, oxygen level, pH, light, salt concentration or nutrient source is selected according to the organism. For example, nitrogen-free medium is used for nitrogen-fixing bacteria and cellulose medium is used for cellulolytic microorganisms. After enrichment, the culture is streaked or plated on suitable agar medium to isolate colonies.
6. Selective medium method- Selective media contain nutrients or inhibitory substances that allow one group of soil microorganisms to grow better than others. Antibiotics, dyes, salts, low pH, high pH or special carbon source may be used.This method is used for isolation of particular groups such as Azotobacter, Rhizobium, actinomycetes, fungi or phosphate-solubilizing bacteria. The unwanted organisms are suppressed. The desired colonies are then picked and purified.
7. Differential medium method- Differential media are used when different microorganisms grow on the same plate but show different colony characters. The difference may appear as colour change, clear zone, pigment formation, acid production or substrate breakdown.This method helps in selecting useful colonies from mixed soil population. For example, phosphate-solubilizing bacteria may form a clear halo around colonies on insoluble phosphate medium. Cellulose degraders may show a clearing zone after suitable staining.
8. Soil plate method- Soil plate method is mostly used for isolation of soil fungi. In this method, a very small amount of soil is placed directly in a sterile Petri dish and mixed with cooled agar medium.Soil particles remain distributed in the agar. Fungal spores and hyphal fragments grow out from soil particles and form colonies. It is useful because some fungi remain attached with soil particles and may be missed during ordinary dilution plating.
9. Pretreatment method- Soil is treated before plating to suppress unwanted organisms and favour a selected group. Heat treatment, drying, chemical treatment, pH treatment or antibiotics may be used.Heat-shock treatment is used for isolation of spore-forming bacteria such as Bacillus. Many vegetative cells are killed, but endospores remain viable and germinate after plating. Dry heat or air-drying treatment is also used in actinomycete isolation because many actinomycetes tolerate dry condition better than many non-sporing bacteria.
10. Baiting method- Baiting method is used when the target organism grows toward or infects a specific bait material. The bait is placed in moist soil sample and incubated for a fixed period. Insect larvae may be used for isolation of entomopathogenic fungi such as Beauveria and Metarhizium. Plant pieces, pollen, hair, cellulose strips or other substrates may be used for other special groups. After growth appears on the bait, the organism is transferred to fresh agar medium.
11. Dilution-to-extinction method- This method is used for isolation of slow-growing, low-abundance or easily overgrown soil microorganisms. The soil cell suspension is highly diluted so that each tube or well receives very few cells. The diluted suspension is inoculated into liquid medium, often in microtiter plates. Wells showing growth are transferred to fresh medium and checked for purity. It reduces competition from fast-growing organisms and may recover microorganisms that do not form colonies easily on ordinary agar plates.

Procedure for Isolation of Microorganisms from Soil

The isolation of soil microorganisms is usually done by serial dilution and plating method in a microbiology laboratory. The work must be done by using aseptic technique.

The exact amount of soil, volume of diluent, dilution range, medium, incubation temperature, incubation time and subculture step are not same for all organisms. These are selected according to the target microorganism, approved protocol and laboratory risk assessment.

Collection of Soil Samples

• The soil sample is collected from a selected site according to the aim of the work. It may be garden soil, field soil, rhizosphere soil, compost soil, polluted soil or any other required soil source.
• A representative sample is collected from one or more points of the same area, when the whole site has to be studied. Surface leaves, stones, sticks and loose unwanted materials are removed first.
• Sterile spatula, sterile scoop or other approved sampling tool is used for collection. The sample should not touch hand, bench, clothes, open air dust or non-sterile surface.
• The collected soil is transferred into a sterile bottle, sterile sampling bag or sterile container. The container is closed immediately.
• The container is labelled properly with sample code, place, date, time and collector name. Soil condition is also written, such as dry, moist, rhizosphere attached, manure-rich, treated or polluted. These records are needed because microbial growth depends on the original condition of the soil.

Preparation of Soil Suspension

• A suitable sterile diluent is taken in a sterile flask or tube. Sterile distilled water, sterile saline, phosphate-buffered saline (PBS) or other approved diluent may be used.
• A measured amount of soil is added into the diluent under aseptic condition. The container is closed before mixing.
• The soil and diluent are shaken, vortexed or mixed as given in the protocol. This makes the soil particles break up and releases the microorganisms attached to soil particles into the liquid.
• Soil is not dissolved chemically in this step. The soil particles are only dispersed. The liquid part becomes a microbial suspension.
• Large particles may be allowed to settle for a short time when it is required. The upper suspension is used carefully for serial dilution, without disturbing the coarse settled soil too much.

Serial Dilution of Soil Sample

• Sterile dilution tubes are arranged in order. Each tube is labelled before transfer, such as 10⁻¹, 10⁻², 10⁻³, 10⁻⁴ and further as required.
• A measured portion of soil suspension is transferred into the first dilution tube and mixed properly. From this tube, a measured portion is again transferred into the next sterile dilution tube. The same step is continued to prepare graded dilution.
• Each dilution tube is mixed before taking the next transfer. Soil microorganisms may remain unevenly distributed if the tube is not mixed properly.
• Sterile pipette or sterile tip is used for every transfer. Used tip or pipette should not be put back into the stock suspension.
• The dilution selected for plating must give clear and countable or interpretable colonies after incubation. The highest dilution is not automatically selected. A very high dilution may give no colony or very few colonies, while a low dilution may give crowded growth.

Inoculation and Incubation

• The culture medium is selected according to the microorganism that has to be isolated. General nutrient media may support many heterotrophic bacteria. Other media are used for fungi, actinomycetes, nitrogen-fixing bacteria, phosphate-solubilizing bacteria or other selected soil groups.
• A measured amount from the selected dilution is inoculated on the selected medium by the approved plating method. The plate is handled near the aseptic working area and the lid is opened only for the required time.
• In spread plating, the inoculum is spread on the agar surface with a sterile spreader. In pour plating, the inoculum is mixed with suitable molten agar as written in the protocol. The plate is then kept undisturbed until the inoculum is absorbed or the agar becomes set.
• The plates are incubated in controlled condition. Temperature, time, oxygen level, light, humidity and plate position depend on the target organism and culture medium. One fixed incubation condition is not suitable for all soil microorganisms.
• Plates containing unknown soil microorganisms are kept closed during incubation and observation. Any heavy sporulation, unusual colony, leakage, contamination of outer plate surface or suspected hazardous growth is handled only by following laboratory biosafety rule.

Isolation and Assessment of Microbial Colonies

• After incubation, the plates are observed for discrete colonies. Colony size, shape, margin, elevation, colour, opacity, surface texture, pigment and any clear zone around the colony may be noted.
• A well-separated colony is selected only when further handling is permitted in that laboratory. A separated colony on a plate only shows visual separation. It does not confirm that the culture is pure.
• The selected colony may be checked by approved purity methods in a properly equipped laboratory. Re-streaking on fresh medium, microscopic observation, staining, colony uniformity and other identification tests may be used according to the protocol.
• The colony code, source soil sample, dilution used, medium, incubation condition and colony character are recorded. These records are needed for comparing the isolate with the original plate.
• Subculturing of unknown environmental microorganisms is not done casually. It needs suitable biosafety control, trained handling, sterile technique, proper waste decontamination and laboratory authorization. The next step depends on the target organism, approved protocol and risk assessment.

Observations and Results

In soil microorganism isolation practical, observation is written from the actual plates only. Colony growth, no growth, colony number, colony type, culture medium and dilution used are recorded.

No result is assumed.

Colony Morphology

• Visible growth may appear as separate colonies, crowded colonies, spreading growth, mixed colonies or no visible growth. Plates are observed without unnecessary opening, especially when unknown soil organisms or fungal growth is present.
• Colony morphology is written from the visible characters present on the agar medium. The common characters are size, shape, margin, elevation, colour, opacity and surface appearance.
• Colony size may be written as pinpoint, small, medium or large. It may also be measured in millimetre (mm), if measurement is required in the practical.
• Colony shape is seen from the upper side of the plate. It may be circular, irregular, filamentous, rhizoid or punctiform.
• Margin means the outer edge of the colony. It may be entire, undulate, lobate, curled, serrated or filamentous. This is a descriptive character only.
• Elevation is observed from the side view of the colony. Colonies may be flat, raised, convex, pulvinate, umbonate or slightly depressed in the centre.
• Colour and surface characters are also recorded. Colonies may be white, cream, yellow, orange, pink, green, grey, black or differently pigmented. The surface may be smooth, rough, shiny, dull, dry, wrinkled, mucoid, powdery or granular.
• Fungal colonies may show different appearance from ordinary bacterial colonies. They may be cottony, woolly, powdery, spreading or mycelial. The upper surface and reverse side of fungal colony may also show different colour.
• Colony morphology is not reliable taxonomic identification. A colony may look different because of medium, incubation condition, age of culture and mixed growth. Genus or species name is not confirmed only from colony colour or shape.

Specimen Observation Table Format

This is only a recording format. The entries are filled from the actual observed plates. Do not write imaginary colony results in this table.

Interpretation of Isolation Results

• Visible colony growth means that some culturable microorganisms have grown under the medium and incubation condition used. It does not show all microorganisms present in the soil.
• Different colony appearances may indicate different colony types in the soil sample. This is a preliminary observation. It is not a direct proof of different species.
• Different culture media may give different growth. One medium may show more bacterial colonies, another medium may favour fungal colonies or actinomycete-like colonies. Some media may suppress many organisms and allow only selected groups to grow.
• No visible growth is also recorded as a result. It may occur due to unsuitable medium, unsuitable incubation condition, very high dilution, absence of culturable cells in the plated portion, inhibitory material or technical error. It should be written as “no visible growth”, not as “soil has no microorganisms”.
• Discrete colonies are useful for selecting colonies for later work. A discrete colony means the colony is separated visibly from other colonies on the plate.
• A discrete colony does not prove culture purity. Pure culture confirmation needs further approved checks, such as repeated streaking, uniform colony appearance, microscopy, staining and other identification tests in a properly equipped laboratory.
• Colony-forming unit (CFU) is used for viable plate count. One CFU may develop from one cell, a pair of cells, a chain, a cluster or a small clump. So CFU is not always equal to exact single cell number.
• CFU per gram of soil can be calculated only when the required data are present. Colony count, dilution factor, plated volume, original soil mass and dilution preparation details are needed.
• The selected dilution must give interpretable colonies. A plate with too many colonies is difficult to count. A plate with very few colonies may give weak count value. The highest dilution is not automatically the best plate for result interpretation.
• Unknown environmental colonies are not opened, picked, subcultured or stored casually. Further handling needs proper biosafety control, authorization, aseptic work area and approved waste decontamination.

Precautions for Isolation of Microorganisms from Soil

The following precautions should be taken during the isolation of microorganisms from soil-

• Collect the soil samples using suitable sampling instruments and sterile containers. Care should be taken to avoid contamination from other sources.
• The collection site, date and other sample details should be properly recorded on the sample container.
• Use sterile culture media, dilution tubes, pipettes and inoculating instruments throughout the experiment.
• Proper aseptic techniques must be maintained during sample preparation, dilution and inoculation to prevent contamination of the soil sample and culture media.
• Use a separate sterile pipette or pipette tip for each dilution to prevent cross-contamination between the dilution tubes.
• The volume of sample and diluent must be measured accurately during serial dilution. Each dilution tube should be labelled correctly.
• Select the culture medium according to the type of microorganism to be isolated.
• The culture plates should be handled carefully to avoid contamination and accidental exposure.
• Incubation temperature and period should be maintained according to the growth requirements of the microorganisms and the approved laboratory procedure.
• Avoid producing aerosols and splashes while mixing the soil suspension or handling the microbial cultures.
• Unknown environmental cultures should remain sealed during observation in biosafety level 1 (BSL-1) teaching laboratories. They must not be opened or subcultured without authorization and appropriate biosafety level 2 (BSL-2) facilities and practices.
• Wear laboratory coats, gloves, eye protection and closed-toe shoes as required by the laboratory risk assessment.
• Disinfect the working surfaces before and after the experiment. All contaminated materials must be properly decontaminated before disposal.
• Wash hands after handling the samples and before leaving the laboratory.
• The isolation of unknown soil microorganisms should be carried out under trained laboratory supervision, following an approved procedure and the required biosafety precautions.

Applications of Soil Microorganism Isolation

The major applications of soil microorganism isolation are as follows-

• Used to isolate and identify soil bacteria, fungi and actinomycetes for studying microbial diversity.
• To study microorganisms involved in nitrogen fixation, phosphate solubilization and organic matter decomposition.
• Used to isolate and screen plant growth-promoting microorganisms for biofertilizer production.
• To isolate soil microorganisms having biocontrol activity against plant pathogens.
• Soil actinomycetes such as Streptomyces are isolated for screening antibiotics and other bioactive compounds.
• Used to isolate microorganisms producing industrial enzymes such as cellulase and amylase.
• To study microorganisms capable of degrading petroleum hydrocarbons and other organic pollutants for bioremediation.
• Used to isolate cellulose-degrading microorganisms for improving the composting process.
• To study the growth, nutritional requirements and metabolic activities of isolated soil microorganisms.
• Used to compare culturable microbial populations under different soil and environmental conditions. It does not represent all microorganisms present in soil.

Limitations of Soil Microorganism Isolation

The major limitations of soil microorganism isolation are as follows-

• Most soil microorganisms cannot be grown on ordinary laboratory media. Only a small fraction of the soil microbial diversity can be isolated.
• The culture media may favour the growth of certain microorganisms while inhibiting others.
• Some microorganisms require specific temperature, pH, oxygen availability and incubation period for their growth.
• Rapidly growing microorganisms may overgrow the slow-growing microorganisms, restricting their growth.
• The number and types of microorganisms vary with soil location, depth and sampling time. A single soil sample may not represent the entire microbial population.
• Improper serial dilution may produce overcrowded colonies or very few colonies on the agar medium.
• The colony-forming unit (CFU) count does not always give the actual number of microbial cells. A group of cells may form a single colony.
• A well-separated colony may still contain more than one type of microorganism. Further examination is required to confirm the purity and identity of the isolate.
• Contamination during sample collection, dilution or inoculation may affect the isolation results.
• Isolation of slow-growing microorganisms takes a longer time. Further purification and identification also require additional laboratory work.
• Soil may contain potentially pathogenic microorganisms. Their isolation and handling require trained personnel, suitable biosafety facilities and a laboratory risk assessment.

References

1. Emmert, E. A. B. (2013). Biosafety guidelines for handling microorganisms in the teaching laboratory: Development and rationale. Journal of Microbiology & Biology Education, 14(1), 78–83. https://doi.org/10.1128/jmbe.v14i1.531
2. Parker, N., Schneegurt, M., Tu, A.-H. T., Lister, P., & Forster, B. M. (2016). Microbiology. OpenStax. https://openstax.org/books/microbiology/pages/1-introduction
3. Timofeeva, A. M., Galyamova, M. R., & Sedykh, S. E. (2023). Plant growth-promoting soil bacteria: Nitrogen fixation, phosphate solubilization, siderophore production, and other biological activities. Plants, 12(24), 4074. https://doi.org/10.3390/plants12244074