Principle
Bacillus thuringiensis produces spores, as do all members of the genus Bacillus. But they also produce proteinaceous bodies near the spores called parasporal crystals. Parasporal crystals kill the larvae of various insect groups (especially Lepidopterans) and are often specific to hosts of different B. thuringiensis strains.
After ingestion of the crystal, it is activated in the larval gut by a protease enzyme. The result is cytolysis of larval cells and, presumably, a ready nutrient source for the endospore. Some insecticides contain the Bt toxin (as it is commercially known). The genes encoding the toxin (most of which are on plasmids) have also been genetically engineered into crop plants as a natural alternative to insecticide use.
Purpose
Production of parasporal crystals is a unique ability (among Bacillus species) of Bacillus thuringiensis. This stain is a means of rapid identification of the species.
Requirement
- one-week old sporulation medium or TSA cultures of Bacillus thuringiensis, Bacillus subtilis.
- clean microscope slide
- spore stain kit (optional)
- parasporal crystal stains
- methanol
- 0.5% basic fuchsin
- fume hood
Procedure
- If desired, perform a spore stain on the two specimens to verify that they are producing spores. If they aren’t, keep incubating the cultures for a couple of days until they are.
- Prepare a heat fixed emulsion of the two species on one slide.
- Add methanol for 30 seconds, then pour it off.
- Add the 0.5% basic fuchsin and gently heat until steam rises.
- Remove the slide from the steam, wait 2 minutes, and repeat the steaming.
- Allow the slide to cool, and then rinse with water.
- Observe under oil immersion. If positive, darkly stained, angular crystals will be apparent.
Result
The dark objects (arrows) at the end of some cells are parasporal crystals. (X1000).
