Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

The overall goal of this procedure is to separate bacterial endospores from complex microbial community samples, such as sediment This method helps answer key questions in the field of microbiology on the presence and abundance of endospores in natural samples. It is based on a difference in resistance between endospores and vegetative cells towards chemicals and heat. The main advantage of this technique is that the method is non destructive for endospores so they can be used for downstream experiments like cultivation, quantification, and metabolic testing.

Although we have designed this method to work with the ecology of endospore forming bacteria, it can also be used in fields such as exobiology or the food industry, in which spores can be either beneficial or detrimental. After preparing solutions and equipment according to the text protocol, under a UV sterilized biosafety cabinet use ethanol flamed metal scoops to add three grams of sediment sample to pre-weighed, sterile 50 milliliter tubes. Using a sterile, graduated burette add 15 milliliters of a sterile, filtered 1%sodium hexametaphosphate, or SHMP solution to the sample.

With a liquid disperser or homogenizer and a sterile dispersion rotor, homogenize the sample at 17, 500 RPM for one minute. Let the sample rest for two minutes and repeat the homogenization. Allow the sample to rest for 10 minutes for the heaviest particles to settle.

Transfer the supernatent containing the cell biomass into a clean 50 milliliter tube, taking care not to disturb the pellet. Add another 15 milliliter aliquot of SHMP to the sedimented pellet and repeat the homogenization before collecting the supernatant and combining it with the first aliquot. Centrifuge the sample at 20 times G for one minute and transfer the supernatant into a fresh 50 milliliter tube before discarding the pellet.

Once the filtration unit and vacuum pump have been prepared according to the text protocol, use ethanol flame to sterilize 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 on to the membrane and use the vacuum pump to filter the liquid, capturing the endospores on the membrane, while the vegetative cell material passes through. To wash off residual detergents add two milliliters of sterile physiological solution to the membrane and apply the vacuum pump. When the liquid is fully filtered turn off the pump and leave the membrane on the filtration unit.

To carry out DNase treatment directly on the membrane add 450 microliters of sterile water 50 microliters of 1X DNase reaction buffer and 0.5 microliters of DNase enzyme onto the membrane and let it stand at approximately 25 degrees Celsius for 15 minutes. The extracellular DNA previously released from lysed cells is digested enzymatically and then washed off. This ensures that the final endospore preparation is free of external DNA which is key for downstream analysis such as molecular analysis.

It is important to remember that there should be no leaking on the filter apparatus, and the sample doesn't dry out. After the incubation add one milliliter of physiological solution and turn on the pump to wash off the residual enzyme from the sample. Use sterile forceps to transfer the membrane containing endospores into a sterile petri dish.

Analyze the sample according to the text protocol. The protocol demonstrated in this video of targeted lysis of vegetative cells results in a purified endospore sample and a significant increase in the fraction of endospore forming firmicutes. In amplicon sequencing of the 16S ribosomal RNA gene firmicutes in untreated samples correspond to only 8%to 19%of the sequences.

In contrast, after treatment firmicutes represented 90.6%and 83.9%of the endosporin rich sample. The two major orders of endospore forming firmicutes, bacillales and clostridiales were enriched while non endospore forming firmicutes like lactobacillales were absent. The efficiency of the treatment was also demonstrated using pure cultures.

An endospore preparation and a vegetative cell culture were treated, all cells were incubated and growth was measured an optical density of 600 nanometers. As shown here, growth was observed for the endospore cultures, while no growth was observed in the treated E.Coli culture, indicating that vegetative cells are irreversibly damaged. Once mastered, and if solutions and equipment have been prepared previously, this technique should be done in about five hours.

It is important to remember to avoid all contamination by working under sterile conditions and ensuring sterility of the solutions and all equipment. Following this procedure, other methods like cultivation, isolation, and metabolic testing can be done in order to answer additional questions on the strain identification and metabolic potential of endospores. Also, downstream DNA extraction and metagenomic sequencing can be done to analyze the species diversity and gene presence.

After watching this video you should have a good understanding of how to separate endospores from environmental samples. Don't forget that working with detergents such as SDS can be extremely hazardous. Don't forget to wear personal protective equipment.