Isolation of Bacillus thuringiensis (Bt) from Soil sample

• • 11 min readby

Bacillus thuringiensis (Bt) is a Gram positive bacteria. It is a spore forming bacteria. It is found naturally in different environment like soil water dead insects and plant surface.

It is known for producing special protein crystal during sporulation phase. These protein crystal is known as delta endotoxin (Cry and Cyt proteins).

When these crystal are ingested by susceptible insect pests like caterpillars beetles and mosquitoes it is dissolved in insect midgut. It is converted into active toxin. It destroys the gut lining and finally insect is killed.

These toxins are highly specific to certain pests. It is safe for humans animals plants and beneficial insects. Due to this Bt is used as biological pesticide. It is considered as environmentally friendly pesticide.

The genes responsible for producing these insecticidal protein is known as cry genes. These genes are introduced into genetically modified crops like Bt corn and Bt cotton. It provides built in protection against agricultural pests.

Principle

The principle for isolation of Bacillus thuringiensis (Bt) from soil sample is based on selective spore germination and thermal shock. Soil contains many types of microorganisms. Due to this a selection method is used to target Bt.

In this method sodium acetate selection is used. High concentration of sodium acetate (0.25 M) is taken. In presence of sodium acetate Bt spores are inhibited from germination. Most of other unwanted Bacillus spores are germinated during short liquid incubation. It is converted into vegetative cells.

After incubation heat treatment is given. It is generally done at 80°C for 3 to 10 minutes. This heat shock kills the vegetative cells of competing bacteria. It also destroys non spore forming microbes. Bt spores remains dormant and it survives the heat.

Finally the surviving Bt spores are plated on nutrient rich acetate free agar medium. It provides proper condition for germination and multiplication. Bt forms identifiable colonies on the medium.

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

  • Soil sample (collected from 2 to 5 cm depth after removing top surface layer).
  • Sterile spatula or tubular sampler for collection.
  • Sterile plastic bag or sterile centrifuge tube for storage.
  • Refrigerator condition (4°C) for preserving sample till processing.
  • Soil suspension materials (1 g soil in sterile 0.85% NaCl normal saline or distilled water).
  • Test tubes and pipette for vigorous mixing and serial dilution.
  • Water bath for thermal shock (70°C to 80°C for 10 to 30 minutes).
  • Sodium acetate buffered broth (0.25 M) for selection incubation.
  • Alternative pretreatment facility (dry heat at 80°C up to 5 hours) when Bt population is low.
  • Enrichment medium like Glucose-Yeast-Salt (GYS) medium (for propagation before isolation).
  • Agar media for plating (Nutrient agar, LB agar, T3 sporulation medium, MYP agar).
  • Incubator for aerobic incubation (around 30°C or up to 37°C for 24 to 72 hours).
  • Staining reagents for parasporal crystal identification (Coomassie brilliant blue, Amino black, Ziehl’s carbol fuchsin, Smirnoff stain).
  • Microscope (light microscope or phase contrast microscope) with oil immersion (1000x) for observing endospore and protein crystal.

Procedure

Procedure for Isolation of Bacillus thuringiensis (Bt) from Soil sample
Procedure for Isolation of Bacillus thuringiensis (Bt) from Soil sample
  1. Soil sample is collected by removing the top 2–3 cm soil layer. The sample is taken by sterile tubular sampler or spatula. It is stored in sterile bag or tube at 4°C till processing.
  2. A portion of soil sample (1 to 5 g) is taken. It is suspended in sterile solution like 0.85% NaCl sterile distilled water or LB broth. The suspension is mixed properly.
  3. Selective enrichment is done (optional but recommended). Sodium acetate (0.25 M) is added into soil suspension. It is incubated on rotary shaker for about 4 hours at 30°C. In this step Bt spores are inhibited from germination. Other unwanted spore forming bacteria are germinated and it becomes vegetative cells.
  4. Heat shock treatment is given. The suspension is heated in water bath at 70°C to 80°C for 3 to 30 minutes. Non spore forming microbes are killed. Newly germinated vegetative cells are also destroyed. Bt spores survives the heat treatment.
  5. Serial dilution of heat treated sample is done in sterile saline or water. It reduces total colony number. It also helps in removing humic soil materials.
  6. Plating is done by taking aliquot (100 µl) from diluted sample. It is spread on solid media like nutrient agar LB agar or T3 medium. The plates are incubated aerobically at 30°C to 37°C for 24 to 48 hours.
  7. Colonies are observed. Colonies showing typical Bt characters are selected (matte white or creamish flat dry irregular margin or fried egg like). These colonies are sub cultured on fresh agar plate. Pure culture is obtained.
  8. Microscopic confirmation is done. Smear is prepared from pure culture. It is observed under phase contrast microscope or after staining (Coomassie brilliant blue amino black Ziehl’s carbol fuchsin). Gram positive rod shaped cells endospore and parasporal crystal protein are confirmed.

Limitations

  • Bt occurs in very low concentration in soil. Due to this recovery becomes inefficient. Sometimes isolation is failed by standard method like sodium acetate selection or dry heat pretreatment.
  • Soil contains large number of competing microorganism. It becomes difficult to target Bt spores. Background population is very high (up to 10^9 bacteria per gram).
  • Thermal shock and sodium acetate treatment do not isolate only Bt. Other related endospore forming bacteria are also selected like Bacillus cereus Bacillus anthracis and Bacillus sphaericus.
  • Differentiation of Bt from Bacillus cereus is difficult. Colony morphology Gram staining and basic biochemical test is not enough for proper identification.
  • Soil matrix creates interference. Physical debris biological impurities and chemical constituents like humic materials obstruct isolation. It also interferes in further analysis.
  • The whole screening process is time consuming. Yield is low in many cases. Number of novel or highly active insecticidal isolate obtained are less.

Precautions

  • The top 2–3 cm soil layer should be removed before sampling. It reduces surface contaminants and debris.
  • Sterile tools are used for collection (spatula or tubular sampler). The soil is transferred into sterile airtight container or zip lock bag.
  • The sample should be maintained at 4°C during transport and storage. It helps in preserving microbial population.
  • All isolation work should be done in BSL-2 facility. Soil sample of Bacillus cereus group may contain opportunistic pathogen or Bacillus anthracis.
  • Personal protective equipment is worn (eye protection gloves single or double and snap front lab coat with cinched cuffs).
  • Aerosol producing steps like vortexing sonication and centrifugation should be performed in Class II biosafety cabinet. Sealed safety cups and rotors are used when required.
  • Food and drink is not allowed inside laboratory. Accidental ingestion of bacteria or toxin should be avoided.
  • Spore effective disinfectant is used. Work surface and spill is decontaminated by 1:10 bleach dilution (1% sodium hypochlorite). Contact time is maintained for 10 to 30 minutes.
  • All biological waste is autoclaved at 121°C for minimum 30 to 60 minutes before disposal.
  • In case of accidental splash exposed mucous membrane is flushed for 15 minutes using emergency eyewash station. Medical consultation is taken.
  • Aseptic technique is followed in all steps. Outside contamination in culture media is prevented.
  • Representative colonies of different morphologies should be picked in initial plating. It should not be restricted only to fried egg colony. Sodium acetate and heat shock may change colony morphology temporarily.
  • Atypical isolate should be handled carefully. Non motile non hemolytic isolate may be B. anthracis. It should be destroyed by autoclaving or it is submitted to specialized pathology laboratory for identification.

References

  1. Alemu, F., Tamiru, T., Berhane, N., & Gemeda, T. (2024). Isolation and characterization of Bacillus thuringinesis from soil and water and laboratory testing of their insecticidal activity against Spodoptera furgiperda (Lepdoptera: Noctuidae) in Gondar, North Western Ethiopia. EAS Journal of Biotechnology and Genetics, 6(6), 108-125. https://doi.org/10.36349/easjbg.2024.v06i06.001
  2. Ammons, D., Rampersad, J., & Khan, A. (2002). Usefulness of staining parasporal bodies when screening for Bacillus thuringiensis. Journal of Invertebrate Pathology, 79(2), 203-204.
  3. Aryal, S. (n.d.). Isolation of Bacillus thuringiensis (Bt) from soil sample. Microbe Notes.
  4. Carolina Knowledge Center. (n.d.). Carbol fuchsin, Ziehl-Neelsen.
  5. Chaudhary, A. V., Patel, K. D., Bhanshali, F., & Ingle, S. (2013). A new enrichment method for isolation of Bacillus thuringiensis from diverse sample types. Applied Biochemistry and Biotechnology.
  6. Chhetri, G. G., Ghimire, P., Gautam, I., & Banjara, M. R. (2024). To isolate and test the novel bacteria Bacillus thuringiensis and Bacillus sphaericus from the soil sample of Nepal as a biological mosquito control tool.
  7. Environment, Health and Safety. (2023). Bacillus cereus biological agent reference sheet (BARS). Cornell University.
  8. Food Standards Australia New Zealand. (2013). Bacillus cereus.
  9. Handayani, K., Ekowati, C. N., & Sumardi. (2023). Study of morphology and protein crystal character. Atlantis Press, 281-293.
  10. Kashyap, R. R., & Brahma, D. (2025). Isolation and identification of two bacterial species (Genus: Bacillus) from flacherie-infected larvae of Samia ricini. Research Journal of Biotechnology.
  11. Kassogue, A., Maiga, K., Traore, D., Dicko, A. H., Fane, R., Guissou, T., Faradji, F. A., Valicente, F. H., & Hamadoun, A. (2015). Isolation and characterization of Bacillus thuringiensis (Ernst Berliner) strains indigenous to agricultural soils of Mali. African Journal of Agricultural Research, 10(28), 2748-2755. https://doi.org/10.5897/AJAR2015.9848
  12. Konecka, E., Baranek, J., Hrycak, A., & Kaznowski, A. (2012). Insecticidal activity of Bacillus thuringiensis strains isolated from soil and water. The Scientific World Journal, 2012, 710501. https://doi.org/10.1100/2012/710501
  13. Malik, K., & Riasat, R. (2014). Study of combined effect of locally isolated Bacillus thuringiensis and turmeric powder on red flour beetle (Tribolium castaneum). International Journal of Current Microbiology and Applied Sciences, 3(4), 760-773.
  14. Manoj, M., & Tamilvendan, K. (2025). Characterization of Bacillus thuringiensis isolated from central and southern dry zones of Karnataka. Mysore Journal of Agricultural Sciences, 59(1), 172-186.
  15. Mukhija, B., & Khanna, V. (2018). Isolation, characterization and crystal morphology study of Bacillus thuringiensis isolates from soils of Punjab. Journal of Pure and Applied Microbiology, 12(1), 189-193. https://doi.org/10.22207/JPAM.12.1.24
  16. Orbit Biotech. (n.d.). Isolation of Bacillus thuringiensis (Bt) from soil sample.
  17. Porcar, M., & Juárez-Pérez, V. (2003). PCR-based identification of Bacillus thuringiensis pesticidal crystal genes. FEMS Microbiology Reviews, 26(5), 419–432. https://doi.org/10.1111/j.1574-6976.2003.tb00624.x
  18. Rabha, M., Sharma, S., Acharjee, S., & Sarmah, B. K. (2017). Isolation and characterization of Bacillus thuringiensis strains native to Assam soil of North East India. 3 Biotech, 7(5), 303. https://doi.org/10.1007/s13205-017-0935-y
  19. Rezaei, R., Moazamian, E., & Montazeri-Najafabady, N. (2023). Parasporin-4, a novel apoptosis inducer of breast cancer cells produced by Bacillus thuringiensis. Molecular Biology Reports.
  20. Santana, M., Moccia, V., & Gillis, A. (2008). Bacillus thuringiensis improved isolation methodology from soil samples. Journal of Microbiological Methods, 75(2), 357-358. https://doi.org/10.1016/j.mimet.2008.06.008
  21. Schneider, K. R., Goodrich Schneider, R., Silverberg, R., Kurdmongkoltham, P., & Bertoldi, B. (2017). Preventing foodborne illness: Bacillus cereus. UF/IFAS Extension.
  22. Sharif, F., & Alaeddinoĝlu, N. G. (1988). A rapid and simple method for staining of the crystal protein of Bacillus thuringiensis. Journal of Industrial Microbiology and Biotechnology, 3(4), 227-229. https://doi.org/10.1007/BF01569580
  23. Silvestri, E. E., Perkins, S. D., Feldhake, D., & Schaefer III, F. W. (2014). Recent literature review of soil processing methods for recovery of Bacillus anthracis spores.
  24. Sun, Y., Fu, Z., Ding, X., & Xia, L. (2008). Evaluating the insecticidal genes and their expressed products in Bacillus thuringiensis strains by combining PCR with mass spectrometry. Applied and Environmental Microbiology, 7(21), 6811–6813. https://doi.org/10.1128/AEM.01085-08
  25. Tallent, S. M., Knolhoff, A., Rhodehamel, E. J., Harmon, S. M., & Bennett, R. W. (2020). Bacteriological analytical manual chapter 14: Bacillus cereus. U.S. Food and Drug Administration.
  26. Tefera, M. M., Berhane, N., & Abuhay, T. E. (2025). Isolation and characterization of Bacillus thuringiensis (BT) strains and evaluate their insecticidal activity against malaria vectors.
  27. Thermo Fisher Scientific. (n.d.). LB broth and LB agar.
  28. Tohidi, F., Toulami, S., Tolami, H. F., Tameshkel, F. S., Nourani, A., Seyedinasab, S. R., Nazemi, A., & Jafarpour, M. (2013). Isolation and molecular identification of cry gene in Bacillus thuringiensis isolated from soils by semi-conserve PCR. European Journal of Experimental Biology, 3(2), 452-456.
  29. Travers, R. S., Martin, P. A. W., & Reichelderfer, C. F. (1987). Selective process for efficient isolation of soil Bacillus spp. Applied and Environmental Microbiology, 53(6), 1263–1266.
  30. Turku Bioscience Centre. (2009). Coomassie blue staining.
  31. Xian, H., Li, C., Li, Y., Zheng, G., & Wang, Y. (2021). Fermentation culture method of Bacillus thuringiensis (Patent No. CN107090420B). Google Patents.
  32. Zhao, X., Begyn, K., Delongie, Y., Rajkovic, A., & Uyttendaele, M. (2023). UV-C and wet heat resistance of Bacillus thuringiensis biopesticide endospores compared to foodborne Bacillus cereus endospores. Food Microbiology, 115, 104325. https://doi.org/10.1016/j.fm.2023.104325

Start Asking Questions