Isolation of Actinomycetes from Soil sample

Actinomycetes are a group of bacteria that have a distinctive filamentous and branching growth pattern, resembling fungi. They are gram-positive and mostly anaerobic, although some species can grow under aerobic conditions. Actinomycetes are widely distributed in nature, especially in soil, where they play an important role in decomposing organic materials such as plant residues, cellulose, lignin, and chitin.

Actinomycetes are also known for producing a variety of secondary metabolites, such as antibiotics, enzymes, pigments, and vitamins. Some of these compounds have beneficial applications in medicine, agriculture, and biotechnology. However, some actinomycetes can also cause diseases in humans and animals, such as actinomycosis, nocardiosis, and dermatophilosis. These infections usually occur when the bacteria gain access to the body through wounds or mucosal surfaces.

Actinomycetes are classified into several suborders based on their morphological and biochemical characteristics. Some of the most common genera include Actinomyces, Nocardia, Streptomyces, Dermatophilus, and Frankia. Each genus has its own distinctive features and ecological niches. For example, Actinomyces are commensal inhabitants of the oral cavity and the gastrointestinal tract of humans and animals; Nocardia are aerobic soil bacteria that can cause pulmonary infections; Streptomyces are prolific producers of antibiotics and other bioactive compounds; Dermatophilus are parasitic bacteria that cause skin lesions in livestock and humans; and Frankia are nitrogen-fixing bacteria that form symbiotic associations with certain plants.

In this article, we will focus on the isolation of actinomycetes from soil samples using different agar media. We will also describe the procedure for making soil slurry and preparing serial dilutions. We will then explain how to plate and incubate the dilutions on agar plates and observe the colony formation and diversity of actinomycetes. Finally, we will discuss how to isolate different colony types for further testing and screening for antibiotic production.

Actinomycetes are ubiquitous in soil and play an important role in decomposing organic materials, especially those that are resistant to degradation by other microorganisms. They also produce a variety of secondary metabolites, such as antibiotics, enzymes, pigments and hormones, that have potential applications in medicine, agriculture and industry.

However, the richness and diversity of actinomycetes in soil are not uniform and depend on various factors that affect their growth, survival and activity. Some of these factors are:

• Soil type: Different soil types have different physical and chemical properties, such as texture, pH, moisture, organic matter content, nutrient availability and mineral composition. These properties influence the distribution and abundance of actinomycetes in soil. For example, actinomycetes tend to be more prevalent in alkaline soils than acidic soils, and in soils with high organic matter content than low organic matter content.
• Geographical location: The geographical location of the soil sample affects the climate, vegetation and land use of the area. These factors influence the type and amount of organic materials that enter the soil and serve as substrates for actinomycetes. They also affect the temperature and rainfall patterns that regulate the moisture and aeration of the soil. For example, actinomycetes are more diverse and abundant in temperate regions than tropical regions, and in forest soils than agricultural soils.
• Cultivation: The cultivation of the soil involves practices such as plowing, tilling, fertilizing, irrigating and cropping. These practices alter the structure and composition of the soil and affect the microbial community. They may enhance or inhibit the growth and activity of actinomycetes depending on the type and intensity of cultivation. For example, moderate cultivation may increase the diversity of actinomycetes by creating more niches and introducing more organic materials into the soil. However, excessive cultivation may reduce the diversity of actinomycetes by disturbing their habitat and exposing them to adverse conditions such as drought, erosion and pollution.
• Organic matter: Organic matter is the main source of carbon and energy for actinomycetes in soil. It also provides other nutrients such as nitrogen, phosphorus and sulfur that are essential for their metabolism. The quality and quantity of organic matter in soil depend on the type and amount of plants and animals that contribute to it. Different types of organic matter have different degrees of resistance to decomposition by microorganisms. For example, lignin is more resistant than cellulose, which is more resistant than starch. Actinomycetes have the ability to degrade more resistant organic materials such as chitin, keratin and humus. Therefore, they are more diverse and abundant in soils with high content of these materials than low content.

These are some of the main factors that influence the richness and diversity of actinomycetes in soil. Understanding these factors can help us to select suitable soil samples for isolation of actinomycetes with desired characteristics or potential applications.

Actinomycetes are a group of filamentous bacteria that can decompose complex organic materials such as chitin, cellulose and lignin. They are also known for producing various antibiotics and other bioactive compounds. To isolate actinomycetes from soil sample, different agar media can be used depending on the purpose and preference of the researcher. Some of the commonly used agar media are:

• Actinomycetes Isolation Agar (AIA): This is a selective medium that contains sodium caseinate and asparagine as nitrogen sources, sodium propionate as a substrate for anaerobic fermentation, dipotassium phosphate as a buffer, sulphates as sources of sulphur and metallic ions, glycerol as an additional carbon source and cycloheximide and nystatin as antifungal agents. This medium inhibits the growth of most fungi and bacteria, while allowing the growth of actinomycetes. The colonies of actinomycetes appear as white to greyish powdery masses on AIA plates.

• Chitin Agar (CA): This is a differential medium that contains chitin as the sole carbon source. Chitin is a polysaccharide that forms the exoskeleton of insects and crustaceans. Only actinomycetes that can produce chitinase, an enzyme that hydrolyzes chitin, can grow on this medium. The colonies of chitinolytic actinomycetes appear as clear zones around them on CA plates.

• Starch Casein Agar (SCA): This is another differential medium that contains starch and casein as carbon and nitrogen sources respectively. Starch and casein are complex polymers that require specific enzymes to be degraded. Only actinomycetes that can produce amylase and protease, enzymes that break down starch and casein respectively, can grow on this medium. The colonies of starch-degrading and casein-degrading actinomycetes appear as clear zones around them on SCA plates.

These agar media can be used separately or in combination to isolate different types of actinomycetes from soil sample. By comparing the growth and morphology of the colonies on different media, the researcher can identify the diversity and characteristics of the actinomycetes present in the soil sample.

Actinomycetes Isolation Agar is a selective medium commonly used for the isolation of actinomycetes from soil samples. It contains the following components and their functions:

• Sodium caseinate: This is a protein derived from milk that serves as a nitrogen source for the growth of actinomycetes. It also provides some carbon and minerals.
• Asparagine: This is an amino acid that also acts as a nitrogen source for the actinomycetes. It can also be used as a carbon source by some species.
• Sodium propionate: This is an organic acid that is used as a substrate for anaerobic fermentation by some actinomycetes. It can also inhibit the growth of some bacteria and fungi that compete with actinomycetes in the soil.
• Dipotassium phosphate: This is a salt that provides the buffering system for the medium. It helps to maintain the pH at an optimal level for the growth of actinomycetes.
• Sulphates: These are salts that provide sulphur and metallic ions for the actinomycetes. Sulphur is an essential element for the synthesis of some amino acids and vitamins. Metallic ions are cofactors for some enzymes and proteins involved in metabolism and growth.
• Glycerol: This is a simple sugar that serves as an additional source of carbon for the actinomycetes. It can also enhance the solubility of some components in the medium.
• Cycloheximide and nystatin: These are antifungal agents that prevent the growth of fungi that may interfere with the isolation of actinomycetes. Cycloheximide inhibits protein synthesis by blocking mRNA translation, while nystatin disrupts the cell membrane by binding to ergosterol.

The composition of Actinomycetes Isolation Agar per liter of distilled water is as follows:

The medium is prepared by dissolving the components in distilled water, adjusting the pH to 7.0, autoclaving at 121°C for 15 minutes, and pouring into sterile Petri dishes.

Chitin agar is a selective medium that contains chitin as the sole carbon source. Chitin is a complex polysaccharide that forms the exoskeleton of insects, crustaceans and fungi. It is composed of repeating units of N-acetylglucosamine linked by β-1,4 glycosidic bonds. Chitin is resistant to degradation by most microorganisms, except for some specialized ones that produce chitinases, enzymes that can hydrolyze the glycosidic bonds and release monomers or oligomers of N-acetylglucosamine.

Actinomycetes are among the few microorganisms that can degrade chitin efficiently. They secrete extracellular chitinases that break down chitin into soluble products that can be transported and metabolized by the cells. Actinomycetes also have a high affinity for chitin and can attach to its surface by producing adhesive substances or appendages. By utilizing chitin as a carbon source, actinomycetes can gain an advantage over other soil microorganisms that cannot degrade it.

Chitin agar also contains peptone as a nitrogen source, potassium phosphate as a buffer, magnesium sulphate as a source of sulphur and trace elements, and agar as a solidifying agent. The pH of the medium is adjusted to 7.2-7.4. The medium is sterilized by autoclaving at 121°C for 15 minutes and then poured into sterile Petri dishes.

Chitin agar can support the growth of only actinomycetes because it inhibits the growth of other soil bacteria and fungi. The inhibition is due to several factors:

• The low availability of carbon in chitin agar limits the growth of heterotrophic bacteria that require more readily utilizable carbon sources.
• The high molecular weight and insolubility of chitin make it inaccessible to most bacteria and fungi that lack extracellular chitinases or surface attachment mechanisms.
• The presence of peptone in chitin agar may also suppress the growth of some bacteria and fungi by providing nitrogen in excess of their requirements, leading to ammonia accumulation and pH changes.
• The absence of other organic compounds in chitin agar may also prevent the growth of some bacteria and fungi that require specific growth factors or co-factors.

Therefore, chitin agar is an effective medium for isolating actinomycetes from soil samples, as it selects for their unique ability to degrade chitin and excludes other soil microorganisms that cannot. By using chitin agar, one can obtain pure cultures of actinomycetes with diverse morphological and physiological characteristics. These cultures can then be further tested for their antibiotic production or other biotechnological applications.

To isolate actinomycetes from soil sample, we need to first make a soil slurry and then prepare serial dilutions from it. A soil slurry is a suspension of soil particles in water that allows the microorganisms to be evenly distributed. A serial dilution is a method of reducing the concentration of microorganisms by transferring a known volume of the original sample to a new container and adding more water. This process is repeated several times until the desired dilution is achieved.

The steps for making soil slurry and preparing serial dilutions are as follows:

• Weigh 1 gram of dry soil and transfer it to a sterile 50 ml conical flask.
• Add 10 ml of sterile distilled water to the flask and close the cap tightly.
• Shake the flask vigorously for 2 minutes to mix the soil and water well. This is the soil slurry.
• Label four sterile test tubes as 10^-1^, 10^-2^, 10^-3^ and 10^-4^.
• Transfer 1 ml of the soil slurry to the test tube labeled 10^-1^ using a sterile pipette.
• Add 9 ml of sterile distilled water to the test tube and mix well by vortexing. This is the first dilution (10^-1^).
• Transfer 1 ml of the first dilution to the test tube labeled 10^-2^ using a new sterile pipette.
• Add 9 ml of sterile distilled water to the test tube and mix well by vortexing. This is the second dilution (10^-2^).
• Repeat the same procedure for the remaining two test tubes to obtain the third (10^-3^) and fourth (10^-4^) dilutions.
• Make duplicates of each dilution by transferring 1 ml of each dilution to another set of four test tubes labeled with the same numbers.

We have now prepared four serial dilutions from the soil slurry in duplicates. These dilutions will be used for plating and incubation on agar plates in the next step.

After preparing the soil slurry and the serial dilutions, the next step is to plate them on the agar media. This is done by spreading 1 ml of each dilution on a sterile agar plate using a glass spreader or a sterile cotton swab. The plates should be labelled with the dilution factor, the type of agar media, and the date of plating.

Two types of agar media are used for the isolation of actinomycetes from soil sample: Actinomycetes Isolation Agar (AIA) and Chitin Agar (CA). AIA is a selective medium that contains sodium caseinate, asparagine, sodium propionate, dipotassium phosphate, sulphates, glycerol, cycloheximide and nystatin. Cycloheximide and nystatin are antifungal agents that inhibit the growth of fungi and allow only actinomycetes to grow. CA is a differential medium that contains chitin, a complex sugar that is hydrolyzed by actinomycetes. CA supports the growth of only actinomycetes and prevents the growth of other soil bacteria and fungi.

The plates are incubated at 25°C for 7 to 14 days in an inverted position. During this period, the actinomycetes will grow and form colonies on the agar surface. The colonies may vary in size, shape, color, texture, and margin depending on the species and strain of actinomycetes. Some colonies may also produce pigments or diffusible substances that can change the color of the agar or form zones of inhibition around them.

The number of colonies formed in each dilution of both types of agar media are counted and recorded. The colony forming units (CFU) per gram of soil can be calculated by multiplying the number of colonies by the dilution factor and dividing by the volume plated. The CFU per gram of soil is an indicator of the richness or abundance of actinomycetes in the soil sample.

The plates are also observed under a microscope to detect diversity in colonies formed. The diversity can be measured by using indices such as richness, evenness, and Shannon index. Richness is the number of different types of colonies present on a plate. Evenness is the degree of similarity in the abundance of different types of colonies. Shannon index is a mathematical formula that combines both richness and evenness to give a single value of diversity.

The plates with different types of colonies are selected for further isolation and testing. The colonies are picked out with sterile forceps or toothpicks and streaked on glucose yeast extract agar (GYEA) plates to obtain pure single colonies. The GYEA plates are incubated at 25°C for another 7 to 14 days and then tested for antibiotic production.

After incubating the agar plates for 14 days, the colonies of actinomycetes can be observed and recorded. Actinomycetes colonies usually appear as white, gray, yellow, orange, red or brown masses of intertwined filaments with a powdery or velvety texture. Some colonies may also produce aerial hyphae that extend above the surface of the agar and form spores at their tips. The spores may have different shapes and colors depending on the species of actinomycetes.

To record the colony formation and diversity, the following steps can be followed:

• Count the number of colonies in each dilution plate and calculate the colony forming units (CFU) per gram of soil using the formula: CFU/g = (number of colonies x dilution factor) / volume plated. Record the CFU/g for each agar medium and compare the results.
• Observe the morphology and color of the colonies under a stereomicroscope or a magnifying glass. Note any differences in size, shape, texture, pigmentation and spore formation among the colonies. Record the characteristics of each colony type and assign them a code or a name for identification.
• Select a representative colony from each type and make a wet mount slide by scraping some of the colony material with a sterile toothpick and placing it on a drop of water on a glass slide. Cover with a cover slip and observe under a compound microscope. Note the shape, arrangement and branching pattern of the filaments and spores. Draw sketches or take photographs of the microscopic features and label them accordingly.
• Calculate the richness, evenness and diversity index of the actinomycetes community in each agar medium using appropriate formulas. Richness is the number of different colony types present in a sample, evenness is the measure of how equally distributed the colony types are in a sample, and diversity index is a single value that combines both richness and evenness. There are different methods to calculate these indices, such as Simpson`s index, Shannon`s index or Margalef`s index. Record and compare the values for each agar medium and discuss what they indicate about the actinomycetes community in soil.

By following these steps, you can observe and record the colony formation and diversity of actinomycetes isolated from soil sample on different agar media. This will help you to identify the potential species of actinomycetes present in soil and their ecological roles. It will also help you to select suitable candidates for further testing for antibiotic production.

After observing and recording the colony formation and diversity on the agar plates, the next step is to isolate different colony types for further testing. This is done to identify the actinomycetes species and their potential for antibiotic production.

To isolate different colony types, the following procedure is followed:

• Select a few representative colonies from each agar plate that show different morphological characteristics, such as color, shape, size, texture, and aerial mycelium formation.
• Using sterile forceps or a sterile toothpick, carefully pick up a small portion of each selected colony and transfer it to a new glucose yeast extract agar plate. Label the plate with the source and the colony type.
• Streak the transferred colony on the new plate using a sterile loop or needle. The streaking technique should be done in such a way that it produces isolated single colonies at the end of the streak. A common method is to divide the plate into four quadrants and streak the colony in a zigzag pattern across each quadrant, flaming the loop or needle between each quadrant.
• Repeat the above steps for each selected colony type from each agar plate.
• Incubate the new plates at 25°C for 7 to 14 days or until single colonies are visible.
• Observe and record the characteristics of the single colonies on the new plates. Compare them with the original colonies on the source plates to confirm their identity.

The isolated single colonies are now ready for further testing for antibiotic production. This can be done by using various methods such as disc diffusion assay, well diffusion assay, or broth dilution assay. The details of these methods are beyond the scope of this article, but they basically involve exposing different bacteria to the actinomycetes extracts or cultures and measuring their inhibition zones or growth rates. The results can help to determine which actinomycetes species have antibacterial activity and against which bacteria they are effective. This can lead to the discovery of novel antibiotics that can be used for medical purposes.

After obtaining pure single colonies of different actinomycetes, the next step is to test their ability to produce antibiotic substances that can inhibit the growth of other microorganisms. This can be done by using different methods, such as:

• Parallel streak method: This method involves streaking the actinomycetes and the test organisms (such as bacteria or fungi) on the same agar plate in parallel lines. After incubation, the presence or absence of zones of inhibition around the actinomycetes indicates their antibiotic activity .
• Cross-streak method: This method involves streaking the actinomycetes on one side of the agar plate and the test organisms on the other side in perpendicular lines. After incubation, the intersection of the streaks shows whether the actinomycetes can inhibit the growth of the test organisms.
• Well diffusion method: This method involves making wells in an agar plate that is uniformly inoculated with a test organism. A small amount of culture broth or extract of the actinomycetes is then added to each well. After incubation, the diameter of the zones of inhibition around the wells is measured to determine the antibiotic activity.
• Paper disc diffusion method: This method involves soaking paper discs with culture broth or extract of the actinomycetes and placing them on an agar plate that is uniformly inoculated with a test organism. After incubation, the diameter of the zones of inhibition around the discs is measured to determine the antibiotic activity.

The antibiotic activity can be quantified by calculating the antibiotic potency, which is defined as the reciprocal of the highest dilution of the culture broth or extract that still produces a zone of inhibition. The higher the potency, the more effective the antibiotic substance.

The antibiotic activity can also be characterized by determining its antibiotic spectrum, which is defined as the range of microorganisms that are susceptible to the antibiotic substance. The broader the spectrum, the more useful the antibiotic substance.

The antibiotic activity can also be influenced by various factors, such as:

• pH: The optimal pH for antibiotic production varies depending on the actinomycete and the antibiotic substance. Some antibiotics are more stable and active at acidic pH, while others are more stable and active at alkaline pH.
• Temperature: The optimal temperature for antibiotic production also varies depending on the actinomycete and the antibiotic substance. Some antibiotics are more stable and active at higher temperatures, while others are more stable and active at lower temperatures.
• Nutrients: The availability and composition of nutrients in the culture medium can affect the growth and metabolism of the actinomycete and hence its antibiotic production. Some nutrients may stimulate or inhibit antibiotic production, while others may serve as precursors or co-factors for antibiotic synthesis.

The testing for antibiotic production by actinomycetes is an important step in discovering new and effective antibiotics that can combat various infections caused by resistant microorganisms. By screening a large number of actinomycetes from different sources and using different methods, it is possible to identify potential candidates for further characterization and development.

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