This article details a protocol for isolating endospores from environmental samples containing vegetative cells of endospore-forming bacteria. The method involves filtration, incubation, and chemical treatment to ensure the preservation of endospores while degrading 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. Add half of the supernatant sample just prepared onto the membrane filtration unit and use the vacuum pump to collect the cells on the membrane. When the liquid has fully passed through the filter, stop the vacuum pump.
Use sterilized forceps to carefully remove the membrane and place it into a sterile petri dish. With a fresh piece of sterile membrane, repeat the filtration with the remaining half of the solution. To separate endospores from vegetative cells in the biomass just collected, thaw the membrane if it was frozen, and use ethanol-flamed sterilized scissors to cut it into smaller pieces.
Transfer the membrane pieces into a sterile two milliliter tube and add 900 microliters of 1X TE buffer. Mix thoroughly by vortexing to release the biomass from the membrane. Incubate the tube at 65 degrees Celsius and 80 RPM for 10 minutes.
Then, remove the tube from the incubator and let it cool down for 15 minutes. Add 100 microliters of freshly prepared lysozyme to a final concentration of two milligrams per milliliter and incubate the sample at 37 degrees Celsius and 80 RPM for 60 minutes to lyse the vegetative cells. After lysis is complete, add 250 microliters of 3 normal sodium hydroxide and 250 microliters of 6% SDS solution to the sample. Incubate at room temperature and 80 RPM for 60 minutes.
Next, prepare a sterile filtration unit that holds 25 millimeter diameter membranes by autoclaving. After the filtration unit cools, place a 25 millimeter diameter 0.2 micrometer nitrocellulose membrane onto it.
Add the sample onto the membrane and use the vacuum pump to filter the liquid, capturing the endospores on the membrane, while the vegetative cell material passes through.
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