Isolation of Endospores from an Environmental Sample by Targeted Lysis of Vegetative Cells

Take an environmental sample containing vegetative cells and endospores of an endospore-forming bacterium, along with other bacteria.

Endospores have a protective coat that helps them survive in adverse conditions.

Using a vacuum filtration unit, filter the sample to trap the cells and endospores on a membrane.

Place the membrane in a dish and cut it into small pieces.

Transfer the pieces into a tube, add a buffer, and agitate the tube to release the cells and endospores.

Incubate with agitation at a high temperature to weaken the vegetative cell structures without harming the endospores.

Cool the sample, then add lysozyme and incubate with agitation. Lysozyme disrupts the cell wall of the vegetative cells, causing them to rupture.

Add sodium hydroxide and a detergent, and incubate to degrade the cellular macromolecules and debris while preserving the resilient endospores.

Filter the sample to isolate the endospores. Wash the endospores to remove excess reagents, preparing them for further analysis.

Once the filtration unit and vacuum pump have been prepared according to the text protocol, use ethanol-flamed sterilized forceps to add sterile nitrocellulose membrane to the filtration unit. A