Isolation of Bacillus thuringiensis (Bt) from Soil sample

Bacillus thuringiensis (Bt) is a gram-positive, soil-dwelling bacterium that naturally produces a toxin that is fatal to certain herbivorous insects. Bt has been used as a biological pesticide since the 1920s and is commonly used in organic farming. Bt also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well as on leaf surfaces, aquatic environments, animal feces, insect-rich environments, and flour mills and grain-storage facilities.

Bt has many strains or subspecies that affect different insect groups, such as beetles, flies, mosquitoes, black flies, and moths. Each strain produces a specific type of protein crystal (delta endotoxin) during sporulation that binds to specific receptors on the membranes of mid-gut cells of the targeted insects, resulting in their rupture and death. Other organisms that lack the appropriate receptors in their gut are not affected by the Bt toxin and therefore are not harmed by Bt.

Isolation of Bt from soil samples involves a series of steps to select and identify Bt strains from the diverse bacterial population present in the soil. The main principle of isolation is to use a thermal shock treatment followed by selective germination of spores on nutrient agar plates. Thermal shock eliminates all bacteria that cannot produce endospores from the soil sample, while selective germination allows only spore-forming bacteria to grow on the plates. Further tests are then performed to confirm the presence of Bt based on its morphology, staining properties, catalase activity, growth temperature range, and parasporal crystal formation.

In this article, we will explain the procedure for isolation of Bt from soil samples in detail and discuss the expected results of this method. We will also highlight the importance and applications of Bt as a biopesticide for insect pest management.

Bacillus thuringiensis (Bt) is a bacterium that produces endospores, which are dormant and resistant forms of the cells that can survive harsh environmental conditions. Endospores can germinate and resume vegetative growth when they encounter favorable conditions, such as adequate nutrients and temperature. Therefore, one way to isolate Bt from soil samples is to use a thermal shock treatment followed by selective germination of spores.

The thermal shock treatment involves heating the soil samples at 70°C for 10 minutes. This step kills most of the bacteria that do not form endospores, such as E. coli, Pseudomonas, and Staphylococcus. The remaining bacteria in the soil samples are mostly endospore-forming bacteria, such as Bacillus and Clostridium. However, not all of them are Bt.

To select for Bt among the endospore-forming bacteria, the soil samples are diluted and plated on nutrient agar plates. The dilution reduces the amount of humic material and other contaminants in the soil samples, and also ensures that each colony on the plate originates from a single cell. The nutrient agar provides a rich medium for the spores to germinate and grow into vegetative cells. The plates are incubated at 30°C for 48 hours, which is an optimal temperature for Bt growth.

After incubation, the plates are examined for colonies that exhibit Bt-like phenotype. Bt colonies are typically white, flat, dry, and irregular in shape. They also produce parasporal crystals, which are proteinaceous inclusions that have insecticidal activity. The parasporal crystals can be detected by staining the colonies with amino black and Ziehl’s carbol fuchsin dyes. The crystals appear as black dots under a light microscope.

The colonies that show positive staining for parasporal crystals are sub-cultured on fresh plates to obtain pure cultures of Bt. These cultures can then be further characterized by other tests, such as Gram staining, endospore staining, catalase test, and growth above 45°C.

By using thermal shock treatment and selective germination of spores, Bt can be isolated from soil samples with high efficiency and specificity. This method is widely used by researchers and biotechnologists who study Bt or use it as a biopesticide.

The following steps describe the procedure for isolating Bt from soil samples using the thermal shock and selective germination method:

1. Step 1: Collect about 20 g of soil from a cultivated or non-cultivated area using a tubular soil sampler. Remove the top 2-3 cm of the soil layer to avoid contamination by other microorganisms. Store the samples in sterile 50 ml centrifuge tubes or zip-lock bags at 4°C until isolation.
2. Step 2: Suspend 1 g of soil in 10 ml of 0.85% NaCl solution and mix well. This will help to disperse the soil particles and release the spores into the solution.
3. Step 3: Heat the suspension at 70°C for 10 minutes with shaking. This will kill most of the vegetative bacteria that are not capable of forming endospores, leaving only the spores of Bt and other spore-forming bacteria.
4. Step 4: Plate 100 μl of the suspension onto nutrient agar plates (0.5% peptone, 0.3% beef extract, 0.5% NaCl and 1.5% agar). Spread the suspension evenly over the surface of the agar using a sterile glass rod or a cotton swab. This will dilute the suspension and reduce the number of colonies on each plate.
5. Step 5: Incubate the plates at 30±2°C for 48 hours. This will allow the spores to germinate and grow into colonies on the nutrient-rich medium.
6. Step 6: Examine the plates for colonies that have a Bt-like phenotype. These are usually matte white, flat, dry and with uneven borders. Sub-culture these colonies onto fresh nutrient agar plates to obtain pure cultures of Bt. Incubate the plates again at 30±2°C for 48 hours.
7. Step 7: Stain the pure cultures with amino black and Ziehl`s carbol fuchsin to detect the presence of parasporal crystals. Amino black is a dye that binds to proteins and stains them black, while Ziehl`s carbol fuchsin is a dye that binds to lipids and stains them red. The parasporal crystals are composed of protein and lipid, so they will appear as black dots surrounded by red halos under a light microscope. These crystals are responsible for the insecticidal activity of Bt.

This is how you can isolate Bt from soil samples using a simple and effective method. You can store the pure cultures of Bt in glycerol at -20°C for future use or further characterization.

The expected results of isolation of Bacillus thuringiensis (Bt) from soil sample depend on the morphological, biochemical and molecular characteristics of the isolated strains. The following are some of the possible outcomes and their implications:

• Morphological characteristics: The colonies of Bt on nutrient agar are usually matte white, flat, dry and with uneven borders. Under a phase contrast microscope, most of the Bt strains produce parasporal crystals during the sporulation phase, which are proteinaceous inclusions that contain the insecticidal toxins. However, some acrystalliferous strains may also exist, which do not produce visible crystals but may still have insecticidal activity. The shape and size of the crystals may vary depending on the type of toxin produced by the strain. For example, some strains produce bipyramidal crystals that are toxic to lepidopteran larvae, while others produce spherical or cuboidal crystals that are toxic to dipteran or coleopteran larvae. A novel stain called amino black can be used to dye the crystals black and distinguish them from the spores under a standard light microscope. Another stain called Ziehl’s carbol fuchsin can be used to confirm the presence of endospores, which are resistant structures that allow Bt to survive harsh environmental conditions.
• Biochemical characteristics: The Bt strains are gram-positive, endospore-forming and catalase-positive bacteria. They can grow at a wide range of temperatures, but some strains may have optimal growth above 45°C, which can be used as a selection criterion. The Bt strains can also utilize different carbon sources, such as sodium acetate, which can be used as another selection test. Some Bt strains may produce other metabolites that have antimicrobial or nematocidal activity, such as antibiotics or small RNAs.
• Molecular characteristics: The Bt strains can be identified and characterized at the molecular level by using different techniques, such as polymerase chain reaction (PCR), probe hybridization, restriction fragment length polymorphism (RFLP), DNA sequencing and bioinformatics analysis. The most common target for molecular identification is the 16S rRNA gene, which is a conserved region in the bacterial genome that can be amplified and sequenced to determine the phylogenetic relationship among Bt strains and other Bacillus species. Another target for molecular characterization is the cry gene, which encodes the insecticidal toxin. There are many different types and subtypes of cry genes, such as cry1, cry2, cry3, cry4, cry7 and cry8, each with a specific insect host range and mode of action. The presence and distribution of cry genes among Bt strains can be detected by using specific primers or probes that hybridize to the gene sequences. The diversity and novelty of cry genes among Bt strains can be assessed by comparing their sequences with those available in public databases, such as GenBank.

The expected results of isolation of Bt from soil sample can provide valuable information about the diversity and potential of Bt strains as biological control agents against various agricultural pests. By using a combination of morphological, biochemical and molecular methods, it is possible to isolate and characterize novel Bt strains with improved or unique insecticidal properties.

In this article, we have discussed the principle and procedure of isolating Bacillus thuringiensis (Bt) from soil samples. We have explained how thermal shock treatment and selective germination of spores are used to eliminate non-spore-forming bacteria and enrich for Bt in the samples. We have also described the steps involved in culturing, sub-culturing and staining the samples to identify Bt colonies and crystals. Finally, we have discussed the expected results of the isolation process and how to store the potential Bt strains for further analysis.

Bt is a widely used biopesticide that has many advantages over chemical pesticides, such as specificity, safety and biodegradability. Isolating Bt from soil samples is an important method to discover new strains with novel insecticidal properties and to improve the existing ones. By following the protocol outlined in this article, one can isolate Bt from soil samples with minimal equipment and materials. However, further tests and characterization are required to confirm the identity and activity of the isolated strains.

We hope this article has been informative and helpful for anyone interested in isolating Bt from soil samples. If you have any questions or feedback, please feel free to leave a comment below. Thank you for reading!

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