Isolation of Bacillus thuringiensis (Bt) from Soil sample
A common, Gram-positive, and sporulating bacterium called Bacillus thuringiensis (Bt) produces insecticidal proteins with selectivity for a variety of insects during sporulation (Cry and Cyt) and vegetative growth (Vip and Sip). These proteins have promoted Bt as a more sustainable pesticide than chemical ones.
Principle of Bacillus thuringiensis Isolation
Thermal shock treatment followed by selective spore germination are the methods that are frequently used to separate Bt from soil. All bacteria unable to produce endospores are eradicated from the sample soil using thermal shock. After that, the samples are multiplied many times to lower the amount of colony-forming units and to remove the humic substances from the soil. In order to give the spores an opportunity to germinate on medium with enough nutrients and at the right temperature, the samples are then grown on nutritional agar. A wide variety of microorganisms, including Bacillus thuringiensis, can grow well in the media. Thus, further selection procedures are used to isolate Bacillus thuringiensis from the diverse population of microorganisms present in the basic soil sample. Common and significant tests include Gram’s staining, Amino black and Ziehl’s carbol fuchsin staining, Endospore staining, Catalase test, determining whether growth occurs above 45 °C, and determining whether parasporal bodies are present, among others.
The steps involved in isolating Bacillus thuringiensis
- After removing the top 2-3 cm, collect around 20 g of cultivated or uncultivated soil with a tubular soil sampler.
- Until isolation, place samples at 4oC in 50 ml (sterile) centrifuge tubes or zip-top bags.
- 1 g of soil samples should be suspended in 10 ml of 0.85% NaCl.
- 10 minutes of shaking heating at 70°C.
- Aliquots of 100l of suspension should be spooned onto nutrient agar (1.5% agar, 0.5% peptone, 0.3% beef extract, and 0.5% sodium chloride).
- For 48 hours, incubate plates at 30°C.
- Subculture bacterial colonies displaying a Bt-like phenotype once more on brand-new plates, then incubate.
- Use Ziehl’s carbol fuchsin and amino black to stain the culture, then examine it under a regular light microscope to make a preliminary identification.
Expected Outcomes of Bacillus Thuringiensis Isolation
- Most colonies have an overall matte white color that is flat, dry, and has uneven borders.
- Cultures that exhibit parasporal crystals that are stained black under a microscope may be important and need to be stored.
