Isolation of Bacillus thuringiensis (Bt) from Soil sample

Bacillus thuringiensis (Bt) is a Gram-positive, facultatively anaerobic bacterium that belongs to the B. cereus group. It is the organism that produces the proteinaceous parasporal crystals during sporulation, and these crystals are called delta-endotoxins. These are known as Cry and Cyt toxins, and it is the major insecticidal component. It is widely distributed in soil, water, dead insects, stored grains, and on plant surfaces. The vegetative cells are rod-shaped, and the colonies are usually rough and spreading with irregular margins. The spores are ellipsoidal and non-swollen, and these are found in subterminal position in the cell. It is the main microbial insecticide used against Lepidoptera, Diptera, and Coleoptera insects, and it has been used for many years in agriculture and public health.

To isolate Bacillus thuringiensis from soil, the sodium acetate selective inhibition method is mostly used. It is the process where the spores of Bt are kept dormant, while the other contaminants are forced to germinate. In this method, about 1 g of soil is mixed in nutrient broth containing sodium acetate at around 0.25 M to 0.35 M concentration. The mixture is incubated at 37°C for 4 hours, and it is shaken frequently. It is the step where non-target spores germinate into vegetative cells. Then the culture is heated at 80°C for about 7 minutes, and this treatment kills the vegetative cells and non-spore-forming microbes. The Bt spores are resistant to heat, so these survive. After this heat shock, the culture is plated on nutrient agar and incubated at 30°C. The colonies appear after 3–4 days, and then they start sporulation along with crystal formation.

The isolated colonies are examined for parasporal crystals, and it is confirmed by staining techniques. The Coomassie brilliant blue staining is used, and the dark blue crystals are seen near the spores under the microscope. The Schaeffer-Fulton endospore stain is also used to show the spores which stain green inside the pink vegetative cells. For final identification, PCR is used to detect the cry genes, and the pathogenic members of the B. cereus group like B. anthracis are ruled out by checking the virulence markers.

Principle

The principle for the isolation of Bacillus thuringiensis (Bt) from soil is based on selective inhibition and selective survival of its spores. It is the process where sodium acetate is used to stop the germination of Bt spores while allowing the germination of other contaminant spores present in the soil. These germinated contaminants then become vegetative cells which cannot tolerate heat. The soil sample is first suspended in nutrient broth containing sodium acetate (0.25–0.35 M), and this condition keeps the Bt spores in dormant state during the initial incubation period at 37°C. The non-Bt spore-formers grow into vegetative cells in this step, and these cells become sensitive to heat treatment.

In the next step, the culture is heated at 80°C for about 7 minutes. It is the heat-shock process where all vegetative cells and non-spore-forming bacteria are destroyed. The Bt spores survive because these are heat-resistant and remain unaffected during this treatment. After this dual selection, the surviving spores are highly enriched in Bt population. These spores are then plated on nutrient agar and allowed to grow at around 30°C so that germination and sporulation take place, and the parasporal crystal formation can be seen. This principle isolates all crystal-forming spore bacteria, and later staining methods are used to confirm the presence of the characteristic Bt crystal.

Requirements for Isolation of Bacillus thuringiensis (Bt) from Soil sample

I. Isolation Requirements

• 1 g soil sample in sterile condition
• Nutrient broth for soil suspension
• Sodium acetate solution (0.25 M–0.35 M)
• Incubation tubes and shaker for frequent mixing
• Incubator maintained at 37°C
• Water bath set at 80°C for heat-shock treatment
• Sterile nutrient agar plates
• Incubator at 30°C for colony growth and sporulation

II. Post-Isolation Confirmation Requirements

• Microscope for examining colony and spore structure
• Reagents for Gram staining
• Schaeffer–Fulton endospore staining set
• Coomassie brilliant blue stain for parasporal crystal detection
• Clean slides, loops, and staining racks
• Biochemical test media for motility and group-specific reactions
• PCR reagents and primers for cry gene detection
• Markers to exclude pathogenic members of B. cereus group
• Sterile nutrient broth with 50% glycerol for long-term storage

Procedure

1. Preparation of soil suspension– About 1 g of soil is taken and mixed in 10 ml sterile nutrient broth (or LB broth). Sodium acetate is added so that its final concentration become nearly 0.25 M to 0.35 M. It is the step where the chemical selection is started in the suspension.

2. Incubation– The mixture is incubated at 37°C for about 4 hours. It is shaken regularly in this step so that the spores of other organisms germinate properly into vegetative cells. These vegetative cells will be destroyed in the next step.

3. Heat shock treatment– After incubation, about 1 ml of suspension is heated at 80°C for 7 minutes. This temperature destroy all vegetative cells and non-spore-forming organisms. Bt spores remain in dormant stage and survive the heat. After heating, the tubes are cooled so that further heat damage is prevented.

4. Plating of spores– Serial dilutions are prepared from the heat-treated sample. Proper dilutions are plated on Nutrient Agar or LB Agar and incubated at 30°C for 4 days. It is during this period that dormant Bt spores germinate slowly and start sporulation later.

5. Colony selection– After incubation, colonies showing typical Bacillus-like characters are selected. These colonies usually appear white or cream coloured, rough and irregular.

Microscopic Examination

6. Gram staining and endospore staining– Selected colonies are Gram stained where the cells appear as Gram-positive rods. Endospore staining (Schaeffer–Fulton method) is done to observe the spores. The spores are mostly ellipsoidal and subterminal. These spores do not cause swelling of the cell.

7. Crystal protein staining– Smears are made from cultures grown for 48–60 hours. These are heat fixed and stained with Coomassie Brilliant Blue for about 3 minutes. On observation, dark blue parasporal crystals are seen. These crystals can appear bipyramidal or cuboidal in shape and help in identifying Bt.

Biochemical and Molecular Differentiation

8. Motility test– Motility is tested to differentiate Bt from non-motile Bacillus anthracis. Most Bt strains show active motility.

9. Detection of cry genes– PCR is performed to confirm the presence of cry genes. These cry genes indicate insecticidal property and are commonly present in Bt.

10. Screening for virulence markers– It is necessary to test for virulence plasmid markers like pagA, lef, and capB to ensure the isolate is not related to B. anthracis. PCR or qPCR is usually used for this step.

11. Maintenance of pure culture– The confirmed Bt isolates are preserved in sterile nutrient broth containing about 50% glycerol and stored at –20°C for long-term use.