Name: removal of exogenous materials from the outer portion of frozen cores to investigate the ancient biological communities harbored inside
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This work was funded through the U.S. Army Engineer Research and Development Center, Basic Research Program Office. Permission for publishing this information has been granted by the Chief of Engineers.

1. Equipment Preparation and Permafrost Core Collection
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	<li>Equipment preparation and field sample collection and preservation gear
	<ol>
		<li>Assemble auger for sample collection by inserting the drive adapter into the top of the barrel and rotating the lever to lock it in place. Pin the adapter tube onto the drive adapter and pin the motor onto the adapter tube. Insert the cutters on the barrel.</li>
		<li>Wear light duty suits, nitrile gloves, and masks to reduce any contamination to the samples. Wear ear pro...

<ol>
	<li>Solomon, S., 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=.">.</a> Climate Change 2007: The Physical Science Basis. , (2007).</li><li>Marchenko, S., Romanovsky, V., Tipenko, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+Modeling+of+Spatial+Permafrost+Dynamics+in+Alaska.">Numerical Modeling of Spatial Permafrost Dynamics in Alaska.</a> Proc. Ninth Int. Conferen. Permafr. 29, 1125-1130 (2008).</li><li>Pachauri, R. K., Meyer, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Climate Change 2014: Synthesis Report. Contributions of Working Groups I, II, and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. , (2007).</li><li>Osterkamp, T. E., Romanovsky, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+warming+and+thawing+of+discontinuous+permafrost+in+Alaska.">Evidence for warming and thawing of discontinuous permafrost in Alaska.</a> Permafr. Periglac. Process. 10 (1), 17-37 (1999).</li><li>Wolken, J. 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=Evidence+and+implications+of+recent+and+projected+climate+change+in+Alaska's+forest+ecosystems.">Evidence and implications of recent and projected climate change in Alaska's forest ecosystems.</a> Ecosphere. 2 (11), 1-35 (2011).</li><li>Hinzman, L. D., Kane, D. L., Gieck, R. E., Everett, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrologic+and+thermal+properties+of+the+active+layer+in+the+Alaskan+Arctic.">Hydrologic and thermal properties of the active layer in the Alaskan Arctic.</a> Cold Reg. Sci. Technol. 19 (2), 95-110 (1991).</li><li>Hinzman, L. D., Goering, D. J., Kane, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+distributed+thermal+model+for+calculating+temperature+profiles+and+depth+of+thaw+in+permafrost+regions.">A distributed thermal model for calculating temperature profiles and depth of thaw in permafrost regions.</a> J. Geophys. Res.: Atmos. 103 (D22), 28975-28991 (1998).</li><li>Osterkamp, T. E., Jorgenson, J. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+pathways+of+organic+carbon+from+permafrost+to+arctic+rivers+in+a+changing+climate.">Mobilization pathways of organic carbon from permafrost to arctic rivers in a changing climate.</a> Geophys. Res. Lett. 34 (13), L13603 (2007).</li><li>Katayama, T., 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=Phylogenetic+analysis+of+bacteria+preserved+in+a+permafrost+ice+wedge+for+25,000+years.">Phylogenetic analysis of bacteria preserved in a permafrost ice wedge for 25,000 years.</a> Appl. Environ. Microbiol. 73 (7), 2360-2363 (2007).</li><li>Katayama, T., 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=Glaciibacter+superstes+gen.+nov.,+sp.+nov.,+a+novel+member+of+the+family+Microbacteriaceae+isolated+from+a+permafrost+ice+wedge.">Glaciibacter superstes gen. nov., sp. nov., a novel member of the family Microbacteriaceae isolated from a permafrost ice wedge.</a> Int. J. Syst. Evol. Microbiol. 59, 482-486 (2009).</li><li>Waldrop, M. P., White, R., Douglas, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+identification+of+cold-adapted+fungi+in+the+Fox+Permafrost+Tunnel,+Alaska.">Isolation and identification of cold-adapted fungi in the Fox Permafrost Tunnel, Alaska.</a> Proc. Ninth Int. Conferen. Permafr. , 1887-1891 (2008).</li><li>Douglas, T. A., 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=Biogeochemical+and+geocryological+characteristics+of+wedge+and+thermokarst-cave+ice+in+the+CRREL+Permafrost+Tunnel.">Biogeochemical and geocryological characteristics of wedge and thermokarst-cave ice in the CRREL Permafrost Tunnel.</a> Alaska Permafr. Periglac. Process. 21 (2), 120-128 (2011).</li><li>Willerslev, E., 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=Diverse+plant+and+animal+genetic+records+from+Holocene+and+Pleistocene+sediments.">Diverse plant and animal genetic records from Holocene and Pleistocene sediments.</a> Science. 300 (5620), 791-795 (2003).</li><li>Bellemain, E., 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=Fungal+palaeodiversity+revealed+using+high-throughput+metabarcoding+of+ancient+DNA+from+Arctic+permafrost.">Fungal palaeodiversity revealed using high-throughput metabarcoding of ancient DNA from Arctic permafrost.</a> Environ. Microbiol. 15 (4), 1176-1189 (2013).</li><li>Steven, B., Pollard, W. H., Greer, C. W., Whyte, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+diversity+and+activity+through+a+permafrost/ground+ice+core+profile+from+the+Canadian+high+Arctic.">Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic.</a> Environ. Microbiol. 10 (12), 3388-3403 (2008).</li><li>Lorenzen, E. D., 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=Species-specific+responses+of+Late+Quaternary+megafauna+to+climate+and+humans.">Species-specific responses of Late Quaternary megafauna to climate and humans.</a> Nature. 479 (7373), 359-364 (2011).</li><li>Wilhelm, R. C., Radtke, K., Mykytczuk, N. C. S., Greer, C. W., Whyte, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+at+the+wedge:+The+activity+and+diversity+of+Arctic+ice+wedge+microbial+communities.">Life at the wedge: The activity and diversity of Arctic ice wedge microbial communities.</a> Astrobiol. 12 (4), 347-360 (2012).</li><li>Sheriden, P. P., Miteva, V. I., Brenchley, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+analysis+of+anaerobic+psychrophilic+enrichment+cultures+obtained+from+a+Greenland+glacier+ice+core.">Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a Greenland glacier ice core.</a> Appl. Environ. Microbiol. 69 (4), 2153-2160 (2003).</li><li>Rivkina, E., 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=Biogeochemistry+of+methane+and+methanogenic+archaea+in+permafrost.">Biogeochemistry of methane and methanogenic archaea in permafrost.</a> FEMS Microbiol. Ecol. 61 (1), 1-15 (2007).</li><li>Juck, D. F., 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=Utilization+of+fluorescent+microspheres+and+a+green+fluorescent+protein-marked+strain+for+assessment+of+microbiological+contamination+of+permafrost+and+ground+ice+core+samples+from+the+Canadian+High+Arctic.">Utilization of fluorescent microspheres and a green fluorescent protein-marked strain for assessment of microbiological contamination of permafrost and ground ice core samples from the Canadian High Arctic.</a> Appl. Environ. Microbiol. 71 (2), 1035-1041 (2005).</li><li>Griffiths, R. I., Whiteley, A. S., O'Donnell, A. G., Bailey, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+method+for+coextraction+of+DNA+and+RNA+from+natural+environments+for+analysis+of+ribosomal+DNA-+and+rRNA-based+microbial+community+composition.">Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA- and rRNA-based microbial community composition.</a> Appl. Environ. Microbiol. 66 (12), 5488-5491 (2000).</li><li>T&#246;we, S., 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=Improved+protocol+for+the+simultaneous+extraction+and+column-based+separation+of+DNA+and+RNA+from+different+soils.">Improved protocol for the simultaneous extraction and column-based separation of DNA and RNA from different soils.</a> J. Microbiol. Methods. 84 (3), 406-412 (2011).</li><li>Nadkarni, M. A., Martin, F. E., Jacques, N. A., Hunter, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+bacterial+load+by+real-time+PCR+using+a+broad+range+(universal)+probe+and+primers+set.">Determination of bacterial load by real-time PCR using a broad range (universal) probe and primers set.</a> Microbiol. 148, 257-266 (2002).</li><li>Takai, K., Horikoshi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+and+quantification+of+members+of+the+archaeal+community+by+quantitative+PCR+using+fluorogenic+probes.">Rapid detection and quantification of members of the archaeal community by quantitative PCR using fluorogenic probes.</a> Appl. Environ. Microbiol. 66 (11), 5066-5072 (2000).</li><li>Fogel, G. B., Collins, C. R., Brunk, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prokaryotic+genome+size+and+SSU+rDNA+copy+number:+Estimation+of+microbial+relative+abundance+from+a+mixed+population.">Prokaryotic genome size and SSU rDNA copy number: Estimation of microbial relative abundance from a mixed population.</a> Microb. Ecol. 38, 93-113 (1999).</li><li>Bodilis, J., Nsigue-Meilo, S., Besaury, L., Quillet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+copy+number,+intra-genomic+heterogeneities+and+later+transfers+of+the+16S+rRNA+gene+in+Pseudomonas.">Variable copy number, intra-genomic heterogeneities and later transfers of the 16S rRNA gene in Pseudomonas.</a> PLOS One. 7, e35647 (2012).</li><li>Caporaso, J. G., 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=QIIME+allows+analysis+of+high-throughput+community+sequencing+data.">QIIME allows analysis of high-throughput community sequencing data.</a> Nature Methods. 7, 335-336 (2010).</li><li>Sellmann, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Geology of the USA CRREL permafrost tunnel, Fairbanks, Alaska. US Army Cold Reg. Res. Eng. Lab. Technical Rep. 199. , (1967).</li><li>Sellmann, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Additional information on the geology and properties of materials exposed in the USA CRREL permafrost tunnel. US Army CRREL Special Rep. , (1972).</li><li>Christner, B. C., Mikucki, J. A., Foreman, C. M., Denson, J., Priscu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glacial+ice+cores:+A+model+system+for+developing+extraterrestrial+decontamination+protocols.">Glacial ice cores: A model system for developing extraterrestrial decontamination protocols.</a> Icarus. 174 (2), 572-584 (2005).</li><li>Mackelprang, 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=Metagenomic+analysis+of+a+permafrost+microbial+community+reveals+a+rapid+response+to+thaw.">Metagenomic analysis of a permafrost microbial community reveals a rapid response to thaw.</a> Nature. 480 (7377), 368-371 (2011).</li><li>Champlot, S., 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=An+efficient+multistrategy+DNA+decontamination+of+PCR+reagents+for+hyper+sensitive+PCR+applications.">An efficient multistrategy DNA decontamination of PCR reagents for hyper sensitive PCR applications.</a> PLoS One. 5 (9), e13042 (2010).</li><li>Yergeau, E., Hogues, H., Whyte, L. 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The cryosphere offers access to preserved organisms that persisted under past environmental conditions. Though the recovered taxa may not represent the complete historic community, those recovered from analysis of glacial ice and permafrost samples can yield valuable historic information about select time periods15-16. For instance, meaningful biological information has been drawn from ice studies investigating anaerobic activity in the Greenland ice sheet20 and permafrost studies investigating carb...

The cryosphere (e.g., permafrost soils, ice features, glacial snow, firn, and ice) offers a glimpse into what types of organisms persisted under past environmental conditions. Since these substrates can be tens to hundreds of thousands of years old, their microbial communities, when preserved frozen since deposition, reflect ancient environmental conditions. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is of utmost importance as climate projections for the 21st century indicate the potential for a pronounced warming in Arctic and sub-Arctic regions1. Specifically, Interior Alaska and Greenland are expected to warm approximately 5 &#176;C and 7 &#176;C, respectively by 21002,3. This is expected to significantly impact soil and aquatic microbial communities, and therefore, related biogeochemical processes. The warmer temperatures and altered precipitation regime are expected to initiate permafrost degradation in many areas2-5 potentially leading to a thicker, seasonally thawed (active) layer6,7, the thawing of frozen soils, and the melting of massive ice bodies such as ground ice, ice wedges, and segregation ice8. This would dramatically change the biogeochemical attributes in addition to the biodiversity of plants and animals in these ecosystems.
Glacial ice and syngenetic permafrost sediment and ice features have trapped chemical and biological evidence of an environment representing what lived there at the time the features formed. For example, in Interior Alaska, both Illinoisan and Wisconsin aged permafrost are present and this permafrost in particular provides unique locations dating from modern to 150,000 years before the present (YBP) that contain biological and geochemical evidence of the impact of past climatic changes on biodiversity. As a result, these sediments provide a record of the biogeochemistry and biodiversity over many thousands of years. Since the area has low sedimentation rates and has never been glaciated, undisturbed samples are accessible for collection and analysis, either drilling vertically into the soil profile or drilling horizontally in tunnels. More importantly, extensive records exist that especially highlight the unique biogeochemical features of permafrost in this region9-14. Specifically, the application of DNA analysis to estimate presence and extent of biodiversity in both extant and ancient ice and permafrost samples enables exploration of the linkage of ancient environmental conditions and habitat to occupation by specific organisms.
Previous studies have identified climatic impacts on mammals, plants and microorganisms from samples dating to 50k YBP11, 15-19, though each study used a different methodology to collect and decontaminate the permafrost or ice cores. In some instances, the drilling cores were sterilized16, 20-21, though the specific methodology did not clarify whether foreign nucleic acids were also eliminated from the samples. In other studies, bacterial isolates15 (e.g., Serratia marcescens) as well as fluorescent microspheres22 have been used to measure the efficacy of decontamination procedures.
This experiment was part of a larger study investigating microbial communities from permafrost samples dating back to approximately 40k YBP. The specific objective of this portion of the study was to successfully decontaminate ice and permafrost cores. To our knowledge, no methodology has integrated the use of solutions designed to eliminate foreign nucleic acids and associated nucleases from the outer portion of the frozen cores. This is despite the fact that these solutions are commonly used to decontaminate laboratory equipment for molecular experiments.
Once the cores were decontaminated, genomic DNA was extracted using the protocols developed by Griffiths et al.23 and T&#246;we et al.24, quantified using a spectrophotometer, and diluted with sterile, DNA-free water to achieve 20 ng per reaction. Bacterial 16S rRNA genes were amplified with primers 331F and 797R and probe BacTaq25 and archaeal 16S rRNA genes were amplified with primers Arch 349F and Arch 806R and probe TM Arch 516F26 under the following conditions: 95 &#176;C for 600 sec followed by 45 cycles of 95 &#176;C for 30 sec, 57 &#176;C for 60 sec, and 72 &#176;C for 25 sec with final extension at 40 &#176;C for 30 sec. All qPCR reactions were conducted in duplicate. The 20 &#181;l reaction volumes included 20 ng DNA, 10 &#181;M of primers, 5 &#181;M of the probe, and 10 &#181;l of the qPCR reaction mix. Standards for bacterial and archaeal qPCR were prepared using genomic DNA from Pseudomonas fluorescens and Halobacterium salinarum, respectively. Both were grown to log phase. Plate counts were conducted and DNA was isolated from the cultures. Genomic DNA was quantified with a spectrophotometer with the assumption of one and six copies of the 16S rRNA gene per genome for H. salinarum and P. fluorescens, respectively27-28. Copy numbers of the bacterial and archaeal genes were calculated based on the standard curve, log-transformed to account for unequal variances between treatments, and assessed by ANOVA.
Community composition was determined by sequencing the 16S rRNA gene using flow cells and bridge amplification technologies and analyzing the communities with &#39;quantitative insights into microbial ecology&#39; (QIIME)29. Forward and reverse reads were joined together and then sequences were filtered, indexed, and high quality representatives were selected for de novo operational taxonomic units (OTU) assignment through sequence alignment with a reference database. Aligned sequences were compared to a separate reference database for taxonomic assignment. A phylum level OTU table was created to determine general community composition.

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			<td height="42" style="height:42px;width:439px;">Auger</td>
			<td style="width:300px;">Snow, Ice, and Permafrost Research Establishment (SIPRE), Fairbanks, AK</td>
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			<td></td>
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			<td style="width:161px;">B01445</td>
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			<td height="21" style="height:21px;">ATCC Agar 181- Tryptone</td>
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			<td height="21" style="height:21px;">ATCC Agar 181- Agar</td>
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			<td height="42" style="height:42px;width:439px;">Halobacterium salinarum</td>
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			<td style="width:161px;"></td>
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			<td height="42" style="height:42px;width:439px;">Pseudomonas fluorescens&#160;</td>
			<td style="width:300px;">American Type Culture Collection (ATCC), Manassas, VA</td>
			<td style="width:161px;"></td>
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			<td height="21" style="height:21px;width:439px;">Microbial DNA Isolation Kit</td>
			<td style="width:300px;">MoBio Laboratories, Carlsbad, CA</td>
			<td style="width:161px;">12224-50</td>
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			<td height="21" style="height:21px;width:439px;">Ear Protection</td>
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			<td height="21" style="height:21px;width:439px;">Hard Hat</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Kimwipes</td>
			<td style="width:300px;">Kimberly-Clark Professional, Roswell, GA</td>
			<td style="width:161px;">34705</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">Glass Wool</td>
			<td style="width:300px;">Pyrex</td>
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			<td></td>
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			<td height="21" style="height:21px;width:439px;">Ruler</td>
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			<td style="width:161px;">N/A</td>
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			<td height="21" style="height:21px;width:439px;">Weighing Tin&#160;</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">08-732-100</td>
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			<td height="21" style="height:21px;width:439px;">Sodium chloride</td>
			<td style="width:300px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:161px;">S-9625</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">Potassium chloride</td>
			<td style="width:300px;">JT Baker, Phillipsburg, NJ</td>
			<td style="width:161px;">3040-04</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:439px;">Potassium phosphate, monobasic</td>
			<td style="width:300px;">JT Baker, Phillipsburg, NJ</td>
			<td style="width:161px;">&#160;3246-01</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">Potassium phosphate, dibasic</td>
			<td style="width:300px;">JT Baker, Phillipsburg, NJ</td>
			<td>3252-01</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">Sodium phosphate dibasic, anhydrous</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">BP332-500</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">50 ml Centrifuge Tubes</td>
			<td style="width:300px;">Corning, Corning, NY</td>
			<td style="width:161px;">4558</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">2 ml Microcentrifuge Tubes</td>
			<td style="width:300px;">MoBio Laboratories, Carlsbad, CA</td>
			<td style="width:161px;">1200-250-T</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">2 ml Ceramic Bead Tubes (1.4 mm)</td>
			<td style="width:300px;">MoBio Laboratories, Carlsbad, CA</td>
			<td style="width:161px;">13113-50</td>
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			<td height="21" style="height:21px;width:439px;">Scoopula</td>
			<td style="width:300px;">Thermo Fisher Scientific, Wilmington, DE</td>
			<td style="width:161px;">1437520</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">Balance</td>
			<td style="width:300px;">Ohaus, Parsippany, NJ</td>
			<td style="width:161px;">E12130</td>
			<td></td>
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			<td height="21" style="height:21px;">Diethylpyrocarbonate (DEPC)</td>
			<td style="width:300px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:161px;">D5758</td>
			<td></td>
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			<td height="21" style="height:21px;">Hexadecyltrimethylammoniabromide (CTAB)&#160;</td>
			<td style="width:300px;">Acros Organics, NJ</td>
			<td style="width:161px;">22716-5000</td>
			<td></td>
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			<td height="21" style="height:21px;">Polyethylene glycol 8000&#160;</td>
			<td style="width:300px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:161px;">P5413-1KG</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">Phenol-chloroform-isoamyl alcohol (25:24:1) (pH 8)&#160;</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">BP1752-400</td>
			<td></td>
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			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:300px;">Eppendorf, Hauppauge, NY</td>
			<td style="width:161px;">5417R</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Chloroform-isoamyl alcohol (24:1)</td>
			<td style="width:300px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:161px;">C0549-1PT</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">TE Buffer</td>
			<td style="width:300px;">Ambion (Thermo Fisher), Wilmington, DE</td>
			<td style="width:161px;">AM9860</td>
			<td></td>
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			<td height="42" style="height:42px;width:439px;">Pipets</td>
			<td style="width:300px;">Rainin, Woburn, MA</td>
			<td style="width:161px;">Pipet Lite XLS, 2, 10, 200, 1nd 1000ul pipets</td>
			<td></td>
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			<td height="84" style="height:84px;width:439px;">Pipet tips</td>
			<td style="width:300px;">Rainin, Woburn, MA</td>
			<td style="width:161px;">Rainin LTS presterilized, low retention, filtered tips, 10, 20, 200, 1000ul</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">Vortexor</td>
			<td style="width:300px;">Scientific Industries, Bohemia, NY</td>
			<td style="width:161px;">G-560</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">Vortex Adaptor</td>
			<td style="width:300px;">MoBio Laboratories, Carlsbad, CA</td>
			<td style="width:161px;">13000-V1</td>
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			<td height="21" style="height:21px;width:439px;">Clear Bottle</td>
			<td style="width:300px;">Corning, Corning, NY</td>
			<td style="width:161px;">C1395500</td>
			<td></td>
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			<td height="21" style="height:21px;width:439px;">Amber Bottle</td>
			<td style="width:300px;">Corning, Corning, NY</td>
			<td style="width:161px;">C5135250</td>
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			<td height="21" style="height:21px;width:439px;">Bottle Top Filters, 0.22um</td>
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			<td style="width:161px;">430513</td>
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			<td style="width:300px;">Becton, Dickenson and Company, Franklin Lakes, NJ</td>
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			<td height="21" style="height:21px;width:439px;">Millex Syringe filters, 0.22 &#956;m</td>
			<td style="width:300px;">EMD Millipore, Billerica, MA</td>
			<td style="width:161px;">SLGV033RB</td>
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			<td height="21" style="height:21px;width:439px;">70% Ethanol</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">BP2818-500</td>
			<td>diluted &#38; filter sterilized</td>
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			<td height="21" style="height:21px;width:439px;">Isotemp 100 L Oven</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">151030511</td>
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			<td height="21" style="height:21px;width:439px;">Cell Spreader</td>
			<td style="width:300px;">Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:161px;">08-100-10</td>
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			<td height="21" style="height:21px;width:439px;">Disposable Inoculating Loops</td>
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</table>

removal of exogenous materials from the outer portion of frozen cores to investigate the ancient biological communities harbored inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The presented method could be used to decontaminate frozen samples collected from various cryosphere environments, from glacial ice to permafrost. Here, we present data specifically collected from ice and permafrost samples collected from the Engineering Research and Development Center - Cold Regions Research and Engineering Laboratory (ERDC-CRREL) Permafrost Tunnel located in Fox, AK (Figure 1A and 1B). The Permafrost Tunnel extends approximately 110 m i...

Watch this Scientific Journal Video about Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside at JoVE.com

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. The overall goal of this procedure, is to uncover hidden diversity by successfully collecting and removing exogenous material from frozen cores. This method can help answer valuable historic questions about select time periods by identifying organisms recovered from glacial ice and permafrost.Collecting an aspetic core in the field is challenging given the high probability of contaminate introduction. Therefore the main advantage of this technique is that contaminate removal occurs in a controlled laboratory setting. Visual demonstration of this protocol is critical as the scraping and decontamination steps can be kind of hard to learn.It's really a two person job, so it requires a lot of patience and team work especially. Demonstrating this procedure will be two support scientists from my laboratory, Karen Foley, and Robert Jones. This protocol requires meticulous cleanliness.Use an oven set to 450 degrees Celsius to bake the following for four hours. Sterile nucleic acid free metal racks, glassware, metal forceps, and glass wool. Use a wrap of heavy foil to prevent re-contamination.Next, filter sterilize 85%ethanol and ultra-pure water using 0.22 micron filters. These liquids must stay at or below four degrees Celsius or they will crack the sample, making it easier to contaminate. Store the ethanol at minus 20 degrees Celsius.And store the water at four degrees Celsius. Then, sterilize a plastic ruler with 70%ethanol, followed by DNA decontamination solution and RNase decontamination solution. Wipe each solution off immediately after it has been applied.Then store the ruler in a sterile, nucleic acid free Whirl-Pak bag. Next, prepare the cold room space by first scrubbing the walls, floors and a metal work bench therein with 1%bleach. Then set the room temperature to approximately minus 11 degrees Celsius.Once prepared, wear light-duty suits, nitrile gloves and masks when in the cold room to reduce any chance of contamination of the room or the samples. This procedure requires that two people work in tandem. This can be challenging considering the many opportunities for re-contamination.To ensure success, we monitor each other, communicate, and change gloves frequently. With everything prepared, bring the sterilized materials and samples into the cold room, and set them up on the bleach scrubbed table. This includes prepared cultures of S.marcescens.First the permafrost core is placed on a prepared sheet of aluminum foil. Next, the outside of the core is lightly inoculated with a dilute culture of S.marcescens using a sterile foam plug. Then, set the core on a sterile metal rack above a tray.Now, the steel microtome blade is wiped sterile with 70%ethanol, DNA decontamination solution, and RNase decontamination solution. Next, researcher A cleans their nitrile gloves in the same manner. Then A holds the core at a 45 degree angle above the tray.B gently scrapes about five millimeters off the outside of the entire core, including the ends, using the sterile blade, while A turns the core after each scrape. As needed, the blade must be re-cleaned with the three solutions. Next, B quickly and carefully pours filter-sterilized 95%ethanol over the core while A turns the core.Then, the core is quickly rinsed with water. The metal rack over the tray is now replaced, and the core is set on the new rack. A now cleans their nitrile gloves and then places the core on a clean rack above the tray.Meanwhile, B sprays the entire core with DNA decontamination solution, followed by a rinse with filter-sterilized water. Next, B sprays the entire core with RNase decontamination solution followed by another rinse with the water. Lastly, the core is placed in a sterile Whirl-Pak bag.Begin with placing a metal rack over a glass dish in a bio hood with laminar flow. Then place the core on the metal rack. Allow two to five millimeters of the outer surface of core to thaw over about 10 minutes.During this time, turn the core 90 degrees every two minutes. After the thawing, grip some glass wool with forceps and swab the entire surface of the core to collect bacteria. Then, inoculate the two plates using the glass wool.Next, measure outer dimensions of the core using the ruler but do not touch the core. Also inoculate two additional plates with the original culture that was previously used to dope the outside of the core to verify the inoculae was viable. Then, transfer the core into a large sterile bag and store it at minus 80 degrees Celsius.Incubate the inoculated plates at 23 degrees Celsius for one week, and then examine them for S.marcescens colonies which turn red at this temperature. Though this protocol has a series of critical steps, one of the most important ones is to check for the growth of Serratia Marcescens. If there are no visible S.marcescens colonies then proceed with the next thawing phase.IF there are S.marcescens colonies, then scrape another five millimeters off the core and repeat the procedure. Check whether S.marcescens grew from the original culture exposed to the cold temperatures of the cold room. Samples were collected from ice and permafrost from a 110 meter tunnel located in Fox, Alaska.The tunnel provided access to ice-rich silt and alluvium. Samples were collected in triplicate. At facilities in New Hampshire, the described protocols were carried out.The 16S rRNA gene was sequenced from bacteria grown from the cores. Pseudomonas species dominated but members related to Planctomycetia were also present. And, of particular interest was the absence of Serratia species, which shows that the decontamination protocol sufficiently removed exogenous DNA.After watching this video, you should have a good understanding of how to successfully decontaminate ice and permafrost cores. Once mastered, the cold room processing steps can be done in 30 to 45 minutes if it is performed properly. When trying this procedure, it's critical to be mindful of everything and everywhere that you touch.When in doubt, just change your gloves and re-sterilize. Following this procedure, other methods such as amplicon sequencing and shotgun metagenomics can be performed to answer additional questions, like what was the species composition for that time period, or what functional attributes dominated under those environmental conditions.

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Method for the Isolation of Francisella tularensis Outer Membranes

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other bacterial pathogens, the extremely low infectious dose (&lt;10 CFU), rapid disease progression, and high morbidity and mortality rates suggest that the virulent strains of Francisella encode for novel virulence factors. Surface-exposed molecules, namely outer membrane proteins (OMPs), have been shown to promote bacterial host cell binding, entry, intracellular survival, virulence and immune evasion. The relevance for studying OMPs is further underscored by the fact that they can serve as protective vaccines against a number of bacterial diseases. Whereas OMPs can be extracted from gram-negative bacteria through bulk membrane extraction techniques, including sonication of cells followed by centrifugation and/or detergent extraction, these preparations are often contaminated with periplasmic and/or cytoplasmic (inner) membrane (IM) contaminants. For years, the "gold standard" method for the biochemical and biophysical separation of gram-negative IM and outer membranes (OM) has been to subject bacteria to spheroplasting and osmotic lysis, followed by sucrose density gradient centrifugation. Once layered on a sucrose gradient, OMs can be separated from IMs based on the differences in buoyant densities, believed to be predicated largely on the presence of lipopolysaccharide (LPS) in the OM. Here, we describe a rigorous and optimized method to extract, enrich, and isolate F. tularensis outer membranes and their associated OMPs.

Method for the Isolation of Francisella tularensis Outer Membranes

A protocol for separating inner and outer membranes from Francisella tularensis by spheroplasting, osmotic lysis, and sucrose density gradient ultracentrifugation.

Francisella tularensis is a Gram-negative intracellular coccobacillus and the causative agent of the zoonotic disease tularemia. When compared with other ...

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Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Volume Segmentation and Analysis of Biological Materials Using SuRVoS (Super-region Volume Segmentation) Workbench

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of interest. When considering the analysis of complex biological systems, the segmentation of three-dimensional image data is a time consuming and labor intensive step. With the increased availability of many imaging modalities and with automated data collection schemes, this poses an increased challenge for the modern experimental biologist to move from data to knowledge. This publication describes the use of SuRVoS Workbench, a program designed to address these issues by providing methods to semi-automatically segment complex biological volumetric data. Three datasets of differing magnification and imaging modalities are presented here, each highlighting different strategies of segmenting with SuRVoS. Phase contrast X-ray tomography (microCT) of the fruiting body of a plant is used to demonstrate segmentation using model training, cryo electron tomography (cryoET) of human platelets is used to demonstrate segmentation using super- and megavoxels, and cryo soft X-ray tomography (cryoSXT) of a mammalian cell line is used to demonstrate the label splitting tools. Strategies and parameters for each datatype are also presented. By blending a selection of semi-automatic processes into a single interactive tool, SuRVoS provides several benefits. Overall time to segment volumetric data is reduced by a factor of five when compared to manual segmentation, a mainstay in many image processing fields. This is a significant savings when full manual segmentation can take weeks of effort. Additionally, subjectivity is addressed through the use of computationally identified boundaries, and splitting complex collections of objects by their calculated properties rather than on a case-by-case basis.

Volume Segmentation and Analysis of Biological Materials Using SuRVoS Super-region Volume Segmentation Workbench

Segmentation of three-dimensional data from many imaging techniques is a major bottleneck in analysis of complex biological systems. Here, we describe the use of SuRVoS Workbench to semi-automatically segment volumetric data at various length-scales using example datasets from cryo-electron tomography, cryo soft X-ray tomography, and phase contrast X-ray tomography techniques.

Segmentation is the process of isolating specific regions or objects within an imaged volume, so that further study can be undertaken on these areas of ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an ...

Efficient In Vitro Cultivation of Mouse Cochlear Hair Cells

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. 
After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 &#176;C, and gently suspended in culture medium using a 200 &#181;L pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached &gt;90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

Author Spotlight: Advancements in Cultivating Mouse Hair Cells for Auditory Research 

Herein, we present a detailed protocol for isolating and culturing primary cochlear hair cells from mice. Initially, the organ of Corti was dissected from neonatal (aged 3-5 days) murine cochleae under a microscope. Subsequently, cells were enzymatically digested into a single-cell suspension and identified using immunofluorescence after several days in culture.

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for ...