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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+archaea+and+bacteria+dominate+stable+microbial+communities+in+North+America's+Largest+Hot+Spring.">Novel archaea and bacteria dominate stable microbial communities in North America's Largest Hot Spring.</a> Microb Ecol. 56 (2), 292-305 (2008).</li><li>Hugenholtz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+prokaryotic+diversity+in+the+genomic+era.">Exploring prokaryotic diversity in the genomic era.</a> Genome Biol. 3 (2), (2002).</li><li>Onyenwoke, R. U., Brill, J. A., Farahi, K., 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=Sporulation+genes+in+members+of+the+low+G+C+Gram-type-positive+phylogenetic+branch+(Firmicutes).">Sporulation genes in members of the low G+C Gram-type-positive phylogenetic branch (Firmicutes).</a> Arch Microbiol. 182 (2-3), 182-192 (2004).</li><li>Delmont, T. O., 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=Accessing+the+soil+metagenome+for+studies+of+microbial+diversity.">Accessing the soil metagenome for studies of microbial diversity.</a> Appl Environ Microbiol. 77 (4), 1315-1324 (2011).</li><li>Ricca, E., Henriques, A. O., Cutting, S. 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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=Small-Scale+DNA+Sample+Preparation+Method+for+Field+PCR+Detection+of+Microbial+Cells+and+Spores+in+Soil.">Small-Scale DNA Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil.</a> Appl Environ Microbiol. 64 (7), 2463-2472 (1998).</li><li>von Mering, 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=Quantitative+phylogenetic+assessment+of+microbial+communities+in+diverse+environments.">Quantitative phylogenetic assessment of microbial communities in diverse environments.</a> Science. 315 (8515), 1126-1130 (2007).</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=Stage+0+sporulation+gene+A+as+a+molecular+marker+to+study+diversity+of+endospore-forming+Firmicutes.">Stage 0 sporulation gene A as a molecular marker to study diversity of endospore-forming Firmicutes.</a> Environ Microbiol Rep. 5 (6), 911-924 (2013).</li><li>Siala, A., Hill, I. 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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Environment

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.

Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either "Contamination Detected" or "No Contamination Detected" within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

A Bending Test for Determining the Atterberg Plastic Limit in Soils

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a considerable subjective judgment from the operator that carries out the test is involved during its performance, which may affect the final result significantly. Different alternative methods have been put forward, but they cannot compete with the standard rolling test in speed, simplicity and cost.
In an earlier study by the authors, a simple method with a simple device to determine the PL was presented (the &#34;thread bending test&#34; or simply &#34;bending test&#34;); this method allowed the PL to be obtained with minimal operator interference. In the present paper a version of the original bending test is shown. The experimental basis is the same as the original bending test: soil threads which are 3 mm in diameter and 52 mm long are bent until they start to crack, so that both the bending produced and its related moisture content are determined. However, this new version enables the calculation of PL from an equation, so it is not necessary to plot any curve or straight line to obtain this parameter and, in fact, the PL can be achieved with only one experimental point (but two experimental points are recommended).
The PL results obtained with this new version are very similar to those obtained through the original bending test and the standard rolling test by a highly experienced operator. Only in particular cases of high plasticity cohesive soils, there is a greater difference in the result. Despite this, the bending test works very well for all types of soil, both cohesive and very low plasticity soils, where the latter are the most difficult to test via the standard thread rolling method.

The traditional standardized test for determining the plastic limit in soils is performed by hand, and the result varies depending on the operator. An alternative method based on bending measurements is presented in this study. This allows the plastic limit to be obtained with a clear and objective criterion.

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Evaluating the Effect of Environmental Chemicals on Honey Bee Development from the Individual to Colony Level

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be brought back to the beehive after the bees have foraged on flowers that have been sprayed with pesticides. Pesticide contaminated food can be exchanged between workers which then feed larvae and therefore can potentially affect the development of honey bees. Thus, residual pesticides in the environment can become a chronic damaging factor to honey bee populations and gradually lead to colony collapse. In the presented protocol, honey bee feeding methods are described and applied to either an individual honey bee or to a colony. Here, the insect growth regulator (IGR) pyriproxyfen (PPN), which is widely used to control pest insects and is harmful to the development of honey bee larvae and pupae, is used as the pesticide. The presenting procedure can be applied to other potentially harmful chemicals or honeybee pathogens for further studies.

Herein we present a method to feed pesticide contaminated food to both an individual honey bee and a beehive colony. The procedure evaluates the pesticide effect on individual honey bees by in vivo feeding of basic larval diet and also on the natural condition of beehive colony.

The presence of pesticides in the beekeeping environment is one of the most serious problems that impacts the life of a honey bee. Pesticides can be ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...