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1. Preparation of Chemicals and Equipment
1. Make 500 ml of a 1% sodium hexametaphosphate (SHMP) (NaPO3)n solution and sterilize by autoclaving.
2. Sterilize nitrocellulose (NC) filters (pore size 0.22 µm, diameter 47 mm) by autoclaving in closed glass Petri dishes.
3. Sterilize NC filters (pore size 0.22 µm, diameter 25 mm) by autoclaving in closed glass Petri dishes.
4. Weigh and note the empty weight of sterile 50 ml tubes (with cap on) (one tube per sample).
5. Prepare Tris-EDTA-buffer (TE-buffer) 1x: make a solution of 10 mM Tris (tris(hydroxymethyl)aminomethane) and 1 mM EDTA buffer. Adjust pH to 8 and sterilize by autoclaving.
6. Prepare lysozyme solution (20 mg/ml) by dissolving 0.02 g of lysozyme in 1 ml TE-buffer. Ideally, make it fresh every time. Store at 4 °C for no more than 1 week.
7. Prepare a physiological solution by dissolving 8 g/L sodium chloride (NaCl) in distilled water. Sterilize by autoclaving.
2. Separation of Biomass from Sediment
1. Add 3 g of sediment sample to pre-weighed sterile 50 ml tubes using ethanol-flamed metal scoops. Perform this in a clean, UV-sterilized biosafety cabinet to avoid contamination.
2. Add 15 ml of a 1% sterile (autoclaved) SHMP solution to the sample using a sterile graduated burette. The SHMP solution can also be filtered to avoid contamination.
3. Homogenize the sediment and SHMP solution with a liquid disperser/homogenizer (e.g., Ultra-Turrax homogenizer). 70% ethanol-sterilize or autoclave the dispersion rotor prior to use. Run the homogenization for 1 min at 17,500 rpm. Let the sample rest for 2 min and repeat the homogenization for 1 min at the same rotor speed.
4. Let the sample stand for 10 min. At this step, the heaviest particles (minerals) will settle. The cells and any organic components of the sample, however, will remain in solution. Afterwards, transfer the supernatant solution (containing cell biomass) into a clean 50 ml tube, while taking care not to disturb the sediment pellet.
5. To the sediment pellet, add again 15 ml of a 1% sterile (autoclaved) SHMP solution using a sterile graduated burette. Then repeat steps 2.3 and 2.4. This repetition ensures the separation of the maximum amount of cells and organic particles from the mineral component of the sediment. The supernatant of this second separation can be merged with the supernatant from the first separation.
Note: The following steps are all done on the supernatant (containing cell biomass). The mineral component (sediment pellet) can be discarded.
6. Centrifuge the sample at 20 x g for 1 min. This step increases the g-force enough to settle small mineral particles while the biological cell material still remains in solution. After centrifugation, transfer the supernatant solution (containing cell biomass) into a clean 50 ml tube. Discard the mineral pellet.
7. Determine the final volume of the solution containing biomass by weighing the sample. The weight determination avoids having to transfer the sample to a graduated cylinder and reduces the risk of contamination.
3. Collection of Biomass on Filter Membrane
1. Prepare a filtration unit (for 47 mm diameter membranes) and a vacuum pump. Sterilize the filtration unit either by autoclaving or (if Pyrex glass) by spraying it with 70% ethanol and flaming it with a Bunsen burner. Let it cool down before continuing with the protocol.
2. Add a sterile NC membrane to the filtration unit using ethanol-flamed sterilized forceps.
3. Add half of the supernatant sample (from step 2.6) onto the membrane filtration unit and collect cells on the membrane using the vacuum pump.
4. When the liquid has fully passed through the filter, stop the vacuum pump and carefully remove the membrane using ethanol-flame-sterilized forceps. Place the membrane into a sterile Petri dish.
1. Cut the membrane in half using ethanol-flamed sterilized scissors. Add each half of the membrane to a separate 2 ml tube. One-half of the membrane will be used for DNA extraction and analysis of the entire bacterial community. The other half of the filter will be stored at -80 °C and serves as a backup.
5. Place a new NC membrane onto the filtration unit and collect biomass from the second half of the sample volume (from step 2.6) using the vacuum pump.
6. When the liquid has fully passed through the filter, stop the vacuum pump and carefully remove the membrane using ethanol-flamed, sterilized forceps. Place the entire membrane into a separate 2 ml tube. This sample will be used for the treatment to separate endospores from vegetative cells. The sample can be stored at -20 °C until use.
4. Lysis of Vegetative Cells
1. Perform the treatment to separate endospores from vegetative cells on the biomass previously collected on an NC membrane (step 3.6).
1. If the membrane was frozen, leave it at RT for 10 min to thaw. Then, place the membrane in a sterile Petri dish and cut it (approximately 4 times) into smaller pieces using ethanol-flamed sterilized scissors. Then, place all membrane filter pieces into a sterile 2 ml tube.
2. Add 900 µl of 1x TE (Tris-EDTA) buffer (see 1.5) to the tube containing the sample membrane and mix thoroughly by vortex. At this step, the biomass is removed from the membrane into the TE-buffer solution.
3. Place the tube in an incubator at 65 °C for 10 min and 80 rpm. Afterwards, remove the tube from the incubator and let it cool down for 15 min.
4. Add 100 µl of freshly prepared lysozyme (see 1.5) to reach a final concentration of 2 mg/ml. Do not add the lysozyme before the sample has cooled down to 37 °C, as this could degrade the enzyme.
5. Incubate the sample at 37 °C for 60 min and 80 rpm, the optimal conditions for the lysozyme to lyse vegetative cells.
6. After lysis is complete, add 250 µl of 3 N sodium hydroxide (NaOH) and 250 µl of 6% sodium dodecyl sulfate (SDS) solution to the sample. By adding this, the sample volume reaches 1.5 ml, and there is a final concentration of 0.5 N NaOH and a final concentration of 1% SDS.
7. Incubate this mix at RT for 60 min and 80 rpm. Adding the base and detergents will help in the final cell lysis. The concentration of these detergents has been optimized so as not to harm the endospores while lysing vegetative cells.
8. Prepare a sterile filtration unit that holds 25 mm diameter membranes by autoclaving, or (if Pyrex glass) spraying it with 70% ethanol and flaming it with a Bunsen burner. Let it cool down.
9. Place a 0.2 µm NC membrane (25 mm diameter) on the filtration unit using ethanol-flame sterilized forceps.
10. Add the sample from step 4.7 onto the membrane and filter the liquid using the vacuum pump. When liquid has passed through, turn off the vacuum pump. At this step, the lysed vegetative cell material is removed, as it is not retained on the membrane. Only endospores will remain on the membrane.
11. Add 2 ml of sterile physiological solution to wash off residual detergents and filter the liquid using the vacuum pump.
12. When the liquid has fully filtered, turn off the vacuum pump. Leave the membrane on the filtration unit.