Bacillus thuringiensis (Bt) Isolation from Soil

Table of Contents

• Introduction to Bacillus thuringiensis

• Bacillus thuringiensis Isolation Principle

• Bacillus thuringiensis Isolation Procedure

• Bacillus thuringiensis Isolation Expected Results

Introduction to Bacillus thuringiensis

• Bacillus thuringiensis (Bt) is a Gram-positive, sporulating bacterium that produces insecticidal proteins with broad insect specificity during sporulation (Cry and Cyt) (Vip and Sip).
• Bt has been marketed as an environmentally beneficial alternative to chemical pesticides by these proteins.

Bacillus thuringiensis Isolation Principle

• A thermal shock treatment followed by selective germination of spores is a widely utilised approach for isolating Bt from soil. 
• Thermal shock is used to destroy any bacteria from the sample soil that are incapable of forming endospores. 
• The samples are then diluted numerous times to remove the humic substances from the soil and lower the overall colony forming units in the sample. 
• The diluted samples are then cultivated on nutrient agar to allow the spores to germinate on media rich in nutrients and at an ideal temperature. 
• The medium promotes the development of a wide variety of microorganisms, including Bacillus thuringiensis. 
• A series of selection assays are then used to isolate Bacillus thuringiensis from the diverse bacteria present in the crude soil sample population. 
• Sodium acetate selection test, Gram's staining, Amino black and Ziehl's carbol fuchsin staining, Endospore staining, Catalase test, testing for growth above 45oC, and examining for the presence of parasporal bodies are all common essential tests.

Bacillus thuringiensis Isolation Procedure

1. After removing the top 2-3 cm of soil, collect around 20 g of cultivated or non-cultivated soil sample with a tubular soil sampler.
2. Place samples in 50 ml (sterile) centrifuge tubes or zip-lock bags at 4 oC until isolated.
3. Soil samples of 1 g are suspended in 10ml 0.85% NaCl.
4. For 10 minutes, heat with shaking at 70°C.
5. 100l aliquots of suspension were placed on nutrient agar (0.5% Peptone, 0.3% beef extract, 0.5% NaCl, and 1.5% agar).
6. Plates should be incubated at 302°C for 48 hours.
7. Incubate bacterial colonies displaying a Bt-like phenotype that have been subcultured on new plates.
8. For preliminary identification, stain the culture with amino black and Ziehl's carbol fuchsin and examine it under a conventional light microscope.

Bacillus thuringiensis Isolation Expected Results

• Colonies are often matte white in colour, flat, dry, and with irregular boundaries.
• Cultures with parasporal crystals coloured black on microscopic examination may be important and should be saved.