Isolation of Bacillus thuringiensis (Bt) from Soil sample

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  • Bacillus thuringiensis (Bt) is an ubiquitous, Gram-positive and sporulating bacterium that synthesizes insecticidal proteins with specificity against a wide range of insects during sporulation (Cry and Cyt) and vegetative growth (Vip and Sip).
  • These proteins have portrayed Bt as an environment-friendly alternative to chemical insecticides.

Isolation of Bacillus thuringiensis (Bt) from Soil sample

Principle of Isolation of Bacillus thuringiensis

Widely used methodologies to isolate Bt from soil consist of a thermal shock treatment followed by selective germination of spores. Thermal shock is employed to eliminate all bacteria incapable of producing endospores from the sample soil. The samples are then diluted many folds to eliminate the amount of humic material within the soil and to reduce the overall colony forming units within the sample. The diluted samples are then cultured on nutrient agar in order to give the spores chance to germinate on media with adequate nutrients and at optimal temperature. The media offers favourable growth conditions for a wide range of bacteria including Bacillus thuringiensis. A series of selection tests are thus further employed to identify Bacillus thuringiensis alone from the range of bacteria present in the crude soil sample population. Common important tests include Sodium acetate selection test, Gram’s staining, Amino black and Ziehl’s carbol fuchsin staining, Endospore staining, Catalase test, checking for growth above 45oC and looking for presence of parasporal bodies among others.

Procedure of Isolation of Bacillus thuringiensis

  1. Obtain about 20 g of cultivated or non-cultivated soil sample with a tubular soil sampler after removing the 2-3 cm of the top layer.
  2. Place samples at 4 ºC in 50 ml (sterile) centrifuge tubes or zip-lock bags until isolation.
  3. Suspend the soil samples of 1 g in 10ml 0.85% NaCl.
  4. Heat with shaking at 70°C for 10 min.
  5. Plate aliquots of 100μl of suspension onto nutrient agar (0.5% Peptone, 0.3% beef extract, 0.5% NaCl and 1.5% agar).
  6. Incubate plates at 30±2°C for 48h.
  7. Sub-culture bacterial colonies exhibiting Bt-like phenotype for single-colony isolation again on fresh plates and incubate.
  8. Stain the culture with amino black and Ziehl’s carbol fuchsin and examine under a standard light microscope for preliminary identification.

Expected Results of Isolation of Bacillus thuringiensis

  • Colonies mostly appear matte white colour, flat, dry and with uneven borders.
  • Cultures that show parasporal crystals dyed black on microscopic observation may be of importance and require storing.

Reference

  1. (PDF) Isolation and Characterization of Bacillus thuringiensis Isolated from Soil and their Possible Impact on Culex pipiens Larvae. Available from: https://www.researchgate.net/publication/299845038_Isolation_and_Characterization_of_Bacillus_thuringiensis_Isolated_from_Soil_and_their_Possible_Impact_on_Culex_pipiens_Larvae [accessed Nov 04 2018].
  2. Travers, R.S., Martin, P.A. and Reicheferfer, C.F. 1987. Selective process for efficient isolation of soil Bacillus spp. Applied and Environmental Microbiology. 53, 1263-1266.
  3. http://biopublisher.ca/index.php/bt/article/html/2019/
  4. https://www.sciencedirect.com/science/article/pii/S0167701208002352
  5. https://aem.asm.org/content/aem/53/6/1263.full.pdf
  6. https://core.ac.uk/download/pdf/39664802.pdf

About Author

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Sagar Aryal, PhD

Dr. Sagar Aryal is a microbiologist, researcher, and science communicator from Nepal. He is also co-founder and Research Head of the Department of Natural Products at the Kathmandu Research Institute for Biological Sciences (KRIBS), where he leads research on natural product discovery and microbial biodiversity. Dr. Aryal earned his Ph.D. in Microbiology from Tribhuvan University, Kathmandu, Nepal, in 2025, supported by a DAAD Research Grant and a University Grants Commission (UGC) Nepal Ph.D. Fellowship. His research interests include Actinomycetes, Myxobacteria, microbial taxonomy, and natural products, particularly the discovery of bioactive compounds with potential pharmaceutical applications. He isolated and identified Nocardia nepalensis, a novel bacterial species from Nepal, contributing to understanding microbial diversity in the region. Dr. Aryal has published more than 16 research articles in international journals and contributed three book chapters to established academic publications. He has presented scientific research findings and developed international research collaborations through visits to more than 18 countries across Asia, Europe, North America, and Oceania. From 2023 to 2025, he served as the American Society for Microbiology (ASM) Young Ambassador to Nepal. Since December 2025, he has served as an Editorial Board Member of Scientific Reports, a Nature Portfolio journal. In recognition of his scientific contributions, Dr. Aryal received the NAST Science and Technology Youth Award 2025 from the Nepal Academy of Science and Technology. He previously received the Frs. Locke and Stiller Research Award (LSRA) for the 2017-2018 cycle from St. Xavier's College, Kathmandu, and a Nepal Academy of Science and Technology (NAST) Research Grant for 2017-2018. Alongside his scientific career, Dr. Aryal has more than 15 years of experience in blogging, content writing, and search engine optimization (SEO).