Name: physical isolation of endospores from environmental samples by targeted lysis of vegetative cells
Uploaded: 2016-01-21T13:00:00
Description: Watch this Scientific Journal Video about Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells at JoVE.com

The authors acknowledge the Swiss National Science Foundation for Grant No. 31003A-132358/1, 31003A_152972 and No. 151948, and Fundation Pierre Mercier pour la Science.

1. Preparation of Chemicals and Equipment&#160;
<ol>
	<li>Make 500 ml of a 1% sodium hexametaphosphate (SHMP) (NaPO3)n solution and sterilize by autoclaving.</li>
	<li>Sterilize nitrocellulose (NC) filters (pore size 0.22 &#181;m, diameter 47 mm) by autoclaving in closed glass Petri dishes.</li>
	<li>Sterilize NC filters (pore size 0.22 &#181;m, diameter 25 mm) by autoclaving in closed glass Petri dishes.</li>
	<li>Weigh and note the empty weight of sterile 50 ml tubes (with cap on) (one tube per ...

<ol>
	<li>Nicholson, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Bacillus+endospores+in+the+environment.">Roles of Bacillus endospores in the environment.</a> Cell Mol Life Sci. 59 (3), 410-416 (2002).</li><li>Lester, E. D., Satomi, M., Ponce, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microflora+of+extreme+arid+Atacama+Desert+soils.">Microflora of extreme arid Atacama Desert soils.</a> Soil Biol Biochem. 39 (2), 704-708 (2007).</li><li>Wilson, M. S., Siering, P. L., White, C. L., Hauser, M. E., Bartles, A. 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R., Gray, T. R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Populations+of+spore-forming+bacteria+in+an+acid+forest+soil,+with+special+reference+to+Bacillus+subtilis.">Populations of spore-forming bacteria in an acid forest soil, with special reference to Bacillus subtilis.</a> J General Microbiol. 81 (1), 183-190 (1974).</li><li>Brandes Ammann, A., Kolle, L., Brandl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+bacterial+endospores+in+soil+by+terbium+fluorescence.">Detection of bacterial endospores in soil by terbium fluorescence.</a> Int J Microbiol. , 435281 (2011).</li><li>Fichtel, J., Koster, J., Rullkotter, J., Saas, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+variations+in+endospore+numbers+within+tidal+flat+sediments+revealed+by+quantification+of+dipicolinic+acid.">High variations in endospore numbers within tidal flat sediments revealed by quantification of dipicolinic acid.</a> Geomicrobiol J. 25 (7-8), (2008).</li><li>Cronin, U. P., Wilkinson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+flow+cytometry+in+the+study+of+Bacillus+cereus.">The potential of flow cytometry in the study of Bacillus cereus.</a> J Appl Microbiol. 108 (1), 1-16 (2010).</li><li>de Rezende, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersal+of+thermophilic+Desulfotomaculum+endospores+into+Baltic+Sea+sediments+over+thousands+of+years.">Dispersal of thermophilic Desulfotomaculum endospores into Baltic Sea sediments over thousands of years.</a> ISME J. 7 (1), 72-84 (2013).</li><li>Hubert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermophilic+anaerobes+in+Arctic+marine+sediments+induced+to+mineralize+complex+organic+matter+at+high+temperature.">Thermophilic anaerobes in Arctic marine sediments induced to mineralize complex organic matter at high temperature.</a> Environ Microbiol. 12 (4), 1089-1104 (2010).</li><li>Garbeva, P., van Veen, J. A., van Elsas, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predominant+Bacillus+spp.+in+agricultural+soil+under+different+management+regimes+detected+via+PCR-DGGE.">Predominant Bacillus spp. in agricultural soil under different management regimes detected via PCR-DGGE.</a> Microb Ecol. 45 (3), 302-316 (2003).</li><li>Brill, J. A., Wiegel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+between+spore-forming+and+asporogenic+bacteria+using+a+PCR+and+southern+hybridization+based+method.">Differentiation between spore-forming and asporogenic bacteria using a PCR and southern hybridization based method.</a> J Microbiol Methods. 31 (1-2), 29-36 (1997).</li><li>Rueckert, A., Ronimus, R. S., Morgan, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+real-time+PCR+assay+targeting+the+sporulation+gene,+spo0A.,+for+the+enumeration+of+thermophilic+bacilli+in+milk+powder.">Development of a real-time PCR assay targeting the sporulation gene, spo0A., for the enumeration of thermophilic bacilli in milk powder.</a> Food Microbiol. 23 (3), 220-230 (2006).</li><li>Bueche, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+endospore-forming+firmicutes+by+quantitative+PCR+with+the+functional+gene+spo0A.">Quantification of endospore-forming firmicutes by quantitative PCR with the functional gene spo0A.</a> Appl Environ Microbiol. 79 (17), 5302-5312 (2013).</li><li>Wunderlin, T., Junier, T., Roussel-Delif, L., Jeanneret, N., Junier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endospore-enriched+sequencing+approach+reveals+unprecedented+diversity+of+Firmicutes+in+sediments.">Endospore-enriched sequencing approach reveals unprecedented diversity of Firmicutes in sediments.</a> Environ Microbiol Rep. 6 (6), 631-639 (2014).</li><li>Nicholson, W. L., Law, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+purification+of+bacterial+endospores+from+soils:+UV+resistance+of+natural+Sonoran+desert+soil+populations+of+Bacillus+spp.+with+reference+to+B.+subtilis+strain+168.">Method for purification of bacterial endospores from soils: UV resistance of natural Sonoran desert soil populations of Bacillus spp. with reference to B. subtilis strain 168.</a> J Microbiol Methods. 35 (1), 13-21 (1999).</li></ol>

The resistance of endospores to external aggressive physicochemical factors (e.g., temperature or detergents) was used to devise a method to separate bacterial endospores from vegetative cells in environmental samples. This is the first comprehensive method to isolate endospores from environmental samples in a non-destructive manner. Previous methods to quantify, detect or analyze endospores in samples were based on the measurement of specific proxies for endospores such as dipicolinic acid or specific marker ge...

The goal of this work is to provide a protocol for the separation of bacterial endospores from vegetative bacterial cells in environmental samples. The formation of bacterial endospores is a survival strategy, usually triggered by starvation, found in a number of bacterial groups belonging to the phylum Firmicutes1. Endospore-forming bacteria are well studied, mainly because a number of strains are pathogens and hence of medical importance (e.g., Bacillus anthracis or Clostridium difficile). Environmental strains of endospore-forming bacteria have been isolated from virtually every environment (soil, water, sediment, air, ice, human gut, animals gut, and more)1-3. Therefore, Firmicutes are the second most abundant phylum in culture collections4.
Because of their hardy outer cortex and protective core proteins, endospores can survive extreme environmental conditions ranging from desiccation to high radiation, extreme temperatures and harmful chemicals5. This remarkable resistance makes it a challenge to extract DNA from endospores6-8. This likely explains why they have been overlooked in environmental sequencing studies9,10. Other methods, such as targeting of endospores in environmental samples by fluorescent antibodies11, quantification of dipicolinic acid (DPA) in soil12 and sediment13, flow cytometry14 or pasteurization and subsequent cultivation15,16 have been used to retrieve or quantify endospores in environmental samples. In recent years, optimized DNA extraction methods as well as specific molecular primers to target endospore-specific gene sequences have been developed10,17-20. This has helped to reveal more biodiversity among this group of bacteria21 and has also led to applications in industry and medicine for the detection of endospores, for example in milk powder19.
The protocol presented here is based on the difference in resistance to harmful physicochemical conditions (such as heat and detergents) of bacterial endospores relative to vegetative cells. To destroy vegetative cells in a sample, we consecutively apply heat, lysozyme and low concentrations of detergents. The time and strength of these treatments have been optimized so as not to destroy spores, but to lyse all vegetative cells. Some cells in an environmental cell pool are more resistant than others, so in order to increase the probability of destroying all vegetative cells, we apply three different treatments. The advantage and novelty of this method is that the endospores after the treatment are still intact and can be used for further downstream analyses. These include DNA extraction, quantitative PCR (qPCR) and amplicon or metagenomic sequencing (targeting specifically the group of endospores and thus reducing diversity, while increasing coverage). The endospores could also be used for downstream cultivation or quantification by fluorescence microscopy, flow cytometry, or detection of DPA. An important feature of this method is that by comparing an untreated sample with a treated sample, one can deduce the quantity and diversity of endospores in an environmental sample in addition to the component corresponding to vegetative cells.

<table border="0" cellpadding="0" cellspacing="0" width="596">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Whatman nitrocellulose membrane filters, 0,2 um pore size, 47 mm diameter</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td colspan="2" style="width:285px;">WHA7182004 Aldrich&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tris(hydroxymethyl)aminomethane</td>
			<td style="width:95px;">Sigma-Aldrich</td>
			<td>252859 Sigma-Aldrich</td>
			<td>CAS 77-86-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884 Sigma-Aldrich</td>
			<td>CAS&#160; 60-00-4&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysozyme from chicken egg white</td>
			<td>Sigma-Aldrich</td>
			<td>62971 Fluka</td>
			<td>powder, CAS&#160; 12650-88-3&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-Turrax homogenizer T18 basic</td>
			<td style="width:95px;">IKA</td>
			<td>3720000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass filter holders for 47 mm membranes, pyrex glass</td>
			<td style="width:95px;">EMD Millipore</td>
			<td>XX10 047 00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chemical duty vacuum pump</td>
			<td style="width:95px;">Millipore</td>
			<td style="width:119px;">WP6122050</td>
			<td>220 V/50 Hz</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Manifold sampling filtration for 25 mm membranes</td>
			<td style="width:95px;">Millipore</td>
			<td>1225 Sampling Manifold</td>
			<td>polypropylene</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dnase</td>
			<td style="width:95px;">New England Biolabs</td>
			<td>M0303S</td>
			<td>Rnase free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S5881 Sigma-Aldrich</td>
			<td>CAS&#160; 1310-73-2&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium dodecyl sulfphate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771 Sigma&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Whatman nitrocellulose membrane filters, 0,2 um pore size, 25 mm diameter</td>
			<td>Sigma-Aldrich</td>
			<td colspan="2">WHA7182002 Aldrich</td>
		</tr>
	</tbody>
</table>

physical isolation of endospores from environmental samples by targeted lysis of vegetative cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

The results presented here have been published earlier10,21. Please refer to those articles for the environmental interpretation and discussion of the data.
The overall procedure is summarized in Figure 1 and corresponds to three main steps: first, the separation of biomass from sediment or any other environmental matrix; second, the destruction of vegetative cells; and third, the downstream analysis of the separated endospores. Downstream analysis could consist, fo...

Watch this Scientific Journal Video about Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells at JoVE.com

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

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

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.

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