Name: physical isolation of endospores from environmental samples by targeted lysis of vegetative cells
Uploaded: 2016-01-21T13:00:00.000+00:00
Duration: 9 min 38 s
Description: Watch this Scientific Journal Video about Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells at JoVE.com

Yazarlar Bağış No. 31003A-132358/1, 31003A_152972 ve sayılı 151948 İsviçre Ulusal Bilim Vakfı kabul ve Fundation Pierre Mercier la Science dökün.

Kimyasalların ve Ekipman 1. Hazırlık <ol><li> % 1 sodyum heksametafosfat (SHMP) (napo 3) n çözeltisi 500 ml yapmak ve otoklavda sterilize edilir. </li><li> Kapalı cam Petri kapları içinde otoklavlanarak sterilize nitroselüloz (NC) filtreleri ile (gözenek büyüklüğü 0.22 um, çap 47 mm). </li><li> Kapalı cam Petri kapları içinde otoklav ile Kuzey Carolina filtre ile (gözenek büyüklüğü 0.22 um, çap 25 mm) sterilize edin. </li><li> Tartılır ve (kapağı ile) steril 50 ml tüpler boş ağırlığı (numune başına bir tüp) not edin. </li><li> Tris-EDTA-tamponu (TE-tamponu) 1x hazırlayın: 10 mM Tris (tris (hidroksimetil) aminometan) ve 1 mM EDTA tampon solüsyonu yapmak. 8 pH ayarlayın ve otoklav ile sterilize. </li><li> 1 ml TE-tamponu içinde lizozim 0.02 g çözülerek lizozim çözeltisi (20 mg / ml) hazırlayın. İdeal olarak, her zaman taze olun. En fazla 1 hafta boyunca 4 ° C&#39;de saklayın. </li><li> 8 g / L sodyum klorid çözündürülmesiyle fizyolojik çözeltiyi hazırlayınDamıtılmış su (NaCI). Otoklav ile sterilize. </li></ol> Sedimentten Biyokütle 2. ayrılması <ol><li> Tortu numunesi 3 g etanol Alevli metal kepçe kullanarak 50 ml tüpler steril tartılmış önceden ekle. Kirlenmesini önlemek için temiz, UV steril biyogüvenlik kabini bu gerçekleştirin. </li><li> Steril bir büret kullanılarak dereceli numuneye% 1 steril (otoklav) SHMP çözeltisi 15 ml ekleyin. SHMP çözüm de kirlenmeyi önlemek için filtre edilebilir. </li><li> Bir sıvı dağıtıcı / homojenizör (örneğin, Ultra-Turrax homojenleştirici) ile tortu ve SHMP çözelti homojenize edilir. % 70 etanol-sterilize veya kullanmadan önce dağılım rotor otoklav. 17.500 rpm&#39;de 1 dakika için homojenizasyon çalıştırın. 2 dakika boyunca numune kalan verin ve rotor hızında 1 dakika için homojenizasyon tekrarlayın. </li><li> Numune 10 dakika bekletin. Bu aşamada, ağır parçacıklar (mineraller) razı olacak. Hücreleri ve bununla birlikte W numunenin herhangi bir organik bileşenlerHasta çözelti içinde kalır. Sediment pelet rahatsız etmemek için dikkat çekerken Daha sonra, temiz bir 50 ml tüp içine süpernatant solüsyonu (içeren hücre biyokütle) aktarın. </li><li> Sediman pelet steril bir büret mezun kullanarak% 1 steril (otoklav) SHMP solüsyonu tekrar 15 ml ekleyin. Sonra tekrar 2.3 ve 2.4 adımları tekrarlayın. Bu tekrarlama tortu mineral bileşeni hücreleri ve organik partiküllerin maksimum miktarını ayrılmasını sağlar. Bu ikinci ayrılık süpernatant ilk ayrılık süpernatanıyla birleştirilebilir. Not: Aşağıdaki adımlar tüm (hücre biyokütle içeren) süpernatant yapılır. Mineral bileşen (tortu topak) iptal edilebilir. </li><li> 1 dakika için 20 x g&#39;de santrifüjleyin. Biyolojik hücre malzeme hala çözelti içinde kalır Bu adım, küçük mineral parçacıkları çözmek için yeterli g-gücünü daha da arttırır. Santrifüj işleminden sonra, bir yüzer madde halinde çözeltisi içeren (Hücre biyokütle) aktarmaktemiz 50 ml tüp. Mineral pelet atın. </li><li> Numuneyı tartmak biyokütle içeren çözeltinin son hacmi belirlenir. Ağırlığı belirlemesi, dereceli bir silindire örnek aktarmak zorunda ve kirlenme riskini azaltır önler. </li></ol> Filtre Zarında Biyokütle 3. Koleksiyonu <ol><li> Filtrasyon ünitesi (47 mm çaplı membranlara) ve vakum pompası hazırlayın. Otoklav yoluyla veya% 70 etanol ile püskürtme ve Bunsen beki ile yanan tarafından (Pyrex cam varsa) ya filtrasyon ünitesi sterilize edin. Bu protokol ile devam etmeden önce soğumasını bekleyin. </li><li> Etanol-yakılmış sterilize forseps kullanarak filtrasyon ünitesi steril NC membranı ekleyin. </li><li> Vakum pompası kullanılarak membran üzerinde hücrelerin membran filtrasyon ünitesi üzerine (adım 2.6 dan itibaren) süpernatant numunenin yarısını ekleyin ve toplamak. </li><li> Sıvı tamamen filtre üzerinden geçtikten sonra, vakum pompası durdurmak ve dikkatle Ethan kullanarak membran kaldırmakol-alev forseps sterilize. Steril bir Petri kabı içine zarın yerleştirin. <ol><li> Etanol-yakılmış sterilize makas kullanarak yarısında zarı kesin. Ayrı bir 2 ml&#39;lik bir tüpe zar her yarım ekleyin. Membranın bir yarısı DNA ekstraksiyonu ve tüm bakteriyel eden analizi için kullanılır. Filtrenin diğer yarısı -80 ° C&#39;de saklanır ve yedeği olarak görev yapar edilecektir. </li></ol></li><li> Filtrasyon ünitesi üzerine yeni NC membranı yerleştirin ve vakum pompası kullanılarak (adım 2.6 dan itibaren) örnek hacmi ikinci yarısından itibaren biyokütle toplamak. </li><li> Sıvı tamamen filtre üzerinden geçtikten sonra, vakum pompası durdurmak ve dikkatle etanol-Alevli sterilize forseps kullanarak membran kaldırmak. Ayrı 2 ml tüp içine tüm membran yerleştirin. Bu örnek, bitki hücrelerinde endosporlar ayırmak için tedavi için kullanılır. Örnek kullanılana kadar -20 ° C&#39;de muhafaza edilebilir. </li></ol> Bitkisel Hücre 4. Liziz <ol><li> GerçekleştirmekTedavi önceden NC zarın (adım 3.6) toplanan biyokütle üzerinde vejetatif hücrelerden endosporlar ayırmak için. <ol><li> Membran donmuş ise, çözülme 10 dakika boyunca oda sıcaklığında bekletin. Daha sonra steril bir Petri kabı membran yerleştirmek ve etanol-Alevli steril makaslar kullanılarak küçük parçalar halinde (yaklaşık 4 kat) kesti. Sonra steril bir 2 ml tüp içine tüm membran filtre parçaları yerleştirin. </li></ol></li><li> Örnek zarı içeren tüp 1x TE (Tris-EDTA) tamponu 900 ul (1.5 bakınız) ekleyin ve vorteks ile iyice karıştırın. Bu adımda, biyokütle TE-tamponu çözeltisi içine zardan çıkarılır. </li><li> 10 dakika karıştırıldı ve 80 rpm&#39;de 65 ° C&#39;de bir kuluçka tüp yerleştirin. Daha sonra inkübatör tüp kaldırmak ve 15 dakika soğumasını bekleyin. </li><li> 2 mg / ml&#39;lik bir nihai konsantrasyona ulaşmak için taze hazırlanmış lisozim (1.5 bakınız) 100 ul ekle. Numune 37 ° C soğuduktan önce bu deg olabilir gibi, lizozim ilave etmeyinizenzim rade. </li><li> Lizozim için en uygun koşulları vejetatif hücreleri lize etmek için, 60 dakika karıştırıldı ve 80 rpm&#39;de 37 ° C&#39;de örnek inkübe edin. </li><li> Lisis tamamlandıktan sonra, 3 N sodyum hidroksit (NaOH) ve örnek 6% sodyum dodesil sülfat (SDS) çözeltisi 250 ul 250 ul ilave edin. Bu ekleyerek, numune hacmi 1,5 ml ulaşır ve 0,5 N NaOH ile nihai konsantrasyon% 1 SDS nihai konsantrasyon vardır. </li><li> 60 dakika karıştırıldı ve 80 rpm&#39;de, oda sıcaklığında bu karışımı inkübe edin. Taban ve deterjan ekleme nihai hücre parçalama yardımcı olacaktır. Vejetatif hücreler lize ise, bu deterjan konsantrasyonu, endosporlar zarar şekilde optimize edilmiştir. </li><li> (Pyrex cam ise)% 70 etanol ile püskürtme ve Bunsen beki ile yanan otoklav ile 25 mm çaplı zarlarını tutan, ya da steril filtrasyon ünitesi hazırlayın. Soğumaya bırakın. </li><li> Etanol-alev sterilize forseps kullanarak filtreleme ünitesi üzerindeki 0.2 mikron NC membranı (25 mm çapında) yerleştirin. &lt;/ li&gt; <li> Membran üzerine adım 4.7 numuneyi ekleyin ve vakum pompası kullanılarak sıvı filtre. Sıvı içinden geçtikten sonra, vakum pompası devre dışı bırakın. Bu zar üzerinde muhafaza edilmez Bu adımda, parçalanmış bitki hücre malzemesi çıkarılır. Sadece endosporlar membran üzerinde kalacaktır. </li><li> Kalıntı deterjanlar yıkayın ve vakum pompası kullanılarak sıvıyı filtrelemek üzere steril fizyolojik solüsyon 2 ml ilave edilir. </li><li> Sıvı tamamen süzülür olduğunda, vakum pompası kapatın. Filtrasyon ünitesi ile ilgili membran bırakın. </li></ol> 5. DNase Tedavi Not: Filtre membran üzerinde doğrudan DNaz tedavi uygulayın. Bu filtreleme ünitesi akmaması önemlidir ve vakum pompası kapatılır. <ol><li> Filtre membran üzerine steril su, 450 ul doğrudan DNase reaksiyon tampon maddesi (1x) ve 0.5 ul DNaz enzimi 50 ul ilave edin ve 15 dakika boyunca bekletildi. Mümkünse, biraz savaş bir odada bu sindirim yapmakOrtalama RT daha mer, enzimin, yukarıdaki 25 ° C ve sıcaklıklarda daha iyi olduğunda işe yaramaktadır. Not: Bunsen beki tutulması kenara filtrasyon ünitesi de sıcaklığını arttırır ve steril bir ortamda tutmak yararı vardır, bu nedenle örneklerin bulaşma riskini azaltmıştır. </li><li> DNaz sindirim tamamlandığında, numuneden enzimi kaldırmak için vakum pompası açın. </li><li> Ekleme ve fizyolojik çözelti 1 ml filtre edilerek artık enzimin yıkayın. </li><li> Sıvı tamamen geçtiyse, vakum pompasını kapatın ve steril forseps kullanarak ve steril Petri kabı içine yerleştirin endosporlar içeren filtre membran kaldırmak. Not: ayrılmış endosporlar örnek şimdi aşağı analiz için hazırdır. Çimlenme ve kültivasyonu için kullanıldığında -20 ° C&#39;de saklayın DNA ekstraksiyonu için ya da alternatif olarak 4 ° C &#39;de kullanıldığında. </li></ol>

<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bacterial spore formers: probiotics and emerging applications. , (2004).</li><li>Kuske, C. 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>

Yazarlar ifşa hiçbir şey yok.

Dış agresif fizikokimyasal faktörlerden (örneğin, sıcaklık ya da deterjanlar) ile endosporlar direnci çevre örneklerinde bitkisel hücrelerden bakteri endosporlar ayırmak için bir yöntem bulmak için kullanıldı. Bu bir tahribatsız şekilde çevresel örneklerden izole endosporlar ilk kapsamlı yöntemdir. Ölçmek algılamak veya numunelerde endosporlar analiz etmek için önceki metotlar, dipikolinik asit veya spesifik bir işaretleyici genler gibi endosporlar özel vekiller ölçümüne dayanma...

Bu çalışmanın amacı, çevre numuneleri içinde vejetatif bakteriyel hücrelerden bakteri sporları ayrılması için bir protokol sağlamaktır. Bakteri sporları oluşumu filum Firmicutes 1 ait olan bakteri grupları bir dizi bulunan bir hayatta kalma stratejisi genellikle açlık tetiklediği vardır. Endospore oluşturan bakteriler de suşları bir dizi patojenleri ve dolayısıyla tıbbi önemi (örneğin, Bacillus anthracis veya Clostridium difficile) vardır başlıca nedeni, incelenmiştir. Endospore oluşturan bakterilerin Çevre suşları hemen hemen her ortamda (toprak, su, sediment, hava, buz, insan bağırsak, hayvanlar gut, ve daha fazla) 1-3 izole edilmiştir. Bu nedenle, Firmicutes kültür koleksiyonları 4 ikinci en bol filum vardır. Çünkü onların cesur dış korteks ve koruyucu çekirdek proteinlerinin, endosporlar fr değişen aşırı çevre koşulları yaşayabiliryüksek radyasyon, aşırı sıcaklıklara ve zararlı kimyasallar 5 om kuruma. Bu olağanüstü direnci endosporlar 6-8 DNA ayıklamak için bir meydan okuma yapar. Çevresel sıralama çalışmalarında 9,10 gözardı edilmiş bu yüzden muhtemelen açıklıyor. Floresan antikorlar 11 ile çevre numuneleri içinde endosporlar hedeflenmesi gibi diğer yöntemler, toprak 12 ve tortu 13 dipikolinik asit ölçümü (DPA), 14 akış sitometrisi veya pastörizasyon ve daha sonra kültür 15,16 endosporlar almak veya ölçmek için kullanılmıştır Çevresel örnekleri. Son yıllarda, optimize edilmiş DNA ekstraksiyon yöntemleri yanı sıra, spesifik moleküler primerler endospor-özgü gen sekansları, 10,17-20 geliştirilmiştir hedef. Bu bakteriler 21 bu grup arasında daha biyolojik çeşitlilik ortaya çıkarmak için yardımcı olan ve aynı zamanda endosporlar tespiti için endüstride ve tıpta uygulamalara yol açmıştırSüt tozu 19, örneğin. Burada sunulan protokol hücreleri vejetatif göre bakteri sporları arasında (örneğin, ısı ve deterjanlar gibi), zararlı fizikokimyasal koşullara direnç farka dayanır. Bir numunede vejetatif hücrelerini yok etmek için, biz arka arkaya ısı, lizozim ve deterjan düşük konsantrasyonlarda geçerlidir. Sporlar yok etmek ama tüm vejetatif hücreleri lize etmek için değil, böylece bu tedavilerin zaman ve güç optimize edilmiştir. Bir çevre hücre havuzu bazı hücreler diğerlerinden daha dayanıklıdır, böylece tüm vejetatif hücrelerin tahrip olasılığını artırmak amacıyla, üç farklı tedaviler uygulanır. Bu yöntemin avantajı, ve yenilik tedaviden sonra endosporlar hala sağlam ve daha fazla alt analizler için kullanılabilir olmasıdır. Bunlar d azaltılması ve böylece DNA ekstraksiyon, kantitatif PCR (qPCR) ve amplikon veya özel endosporlar grubu hedef alan metagenomic dizileme (ve dahilÇeflitlilik) kapsama artırırken. Endosporlar de mansap yetiştirme veya floresan mikroskobu ile miktar tayini için kullanılabilir, akış sitometrisi veya DPA tespiti. Bu yöntemin önemli bir özelliği, tedavi edilen bir örnek ile işlem görmemiş bir numune ile karşılaştırılması, tek bir hücre vejetatif karşılık gelen bileşene ek olarak bir çevre numunede endosporlar miktar ve çeşitliliği anlamak olabilir.

<table><tbody><tr><td>Whatman nitrocellulose membrane filters, 0.2 &amp;#956;m pore size, 47 mm diameter</td><td>Sigma-Aldrich</td><td>WHA7182004 Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Sigma-Aldrich</td><td>252859 Sigma-Aldrich</td><td>CAS 77-86-1</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884 Sigma-Aldrich</td><td>CAS&amp;nbsp; 60-00-4&amp;nbsp;</td></tr><tr><td>Lysozyme from chicken egg white</td><td>Sigma-Aldrich</td><td>62971 Fluka</td><td>powder, CAS&amp;nbsp; 12650-88-3&amp;nbsp;</td></tr><tr><td>Ultra-Turrax homogenizer T18 basic</td><td>IKA</td><td>3720000</td><td></td></tr><tr><td>Glass filter holders for 47&amp;nbsp;mm membranes, Pyrex glass</td><td>EMD Millipore</td><td>XX10 047 00</td><td></td></tr><tr><td>Chemical duty vacuum pump</td><td>Millipore</td><td>WP6122050</td><td>220 V/50 Hz</td></tr><tr><td>Manifold sampling filtration for 25&amp;nbsp;mm membranes</td><td>Millipore</td><td>1225 Sampling Manifold</td><td>polypropylene</td></tr><tr><td>Dnase</td><td>New England Biolabs</td><td>M0303S</td><td>Rnase free</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S5881 Sigma-Aldrich</td><td>CAS&amp;nbsp; 1310-73-2&amp;nbsp;</td></tr><tr><td>Sodium dodecyl sulfphate (SDS)</td><td>Sigma-Aldrich</td><td>L3771 Sigma&amp;nbsp;</td><td></td></tr><tr><td>Whatman nitrocellulose membrane filters, 0.2 &amp;#956;m pore size, 25 mm diameter</td><td>Sigma-Aldrich</td><td>WHA7182002 Aldrich</td><td></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.

Burada sunulan sonuçlar, daha önce 10,21 yayınlanmıştır. Verilerin çevre yorumlanması ve tartışılması için bu makaleleri bakın. Genel prosedür Şekil 1 &#39;de özetlenmiş ve üç ana adımlar karşılık gelir: ilk olarak, birikinti ya da herhangi bir diğer çevresel matris biyokütle ayrılması; İkinci, vejetatif hücrelerin tahribi; ve üçüncü olarak, ayrılmış endosporlar alt baş analizi. Alt analizi çeşitliliği belirlemek için, DN...

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.

Bitkisel Hücreler Hedefli lizis ile Çevre Örneklerden endosporlar Fiziksel İzolasyonu

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

physical-isolation-endospores-from-environmental-samples-targeted

Research

JoVE Journal

Environment

14.1K Görüntüleme.  Macquarie University. 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.

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

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Kültürleme ve bakımı<em&gt; Clostridium difficile</em&gt; Bir Anaerobik Çevre

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

İmmünoloji ve Enfeksiyon

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

Sucul Habitatlardan Hidrokarbon Metabolize Edici Özelliğe Sahip Bakteri Türlerinin İzolasyonu, Çoğaltılması ve Tanımlanması

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

Çevre

Detecting Cortex Fragments During Bacterial Spore Germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

Bakteriyel Spor Filizlenmesinin sırasında Cortex Fragments algılama

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

Görselleştirme Germinosomes ve Bacillus subtilis sporlar iç zar

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...

Biyomühendislik

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Zararlı etkileri, düşük basınç plazma sterilizasyon Bacillus subtilis sporlar kullanarak canlı cep mikroskobu sağkalıma soruşturma

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Biyoloji

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

Microrespiration analizi ve mikrodiseksiyon kullanarak soruşturma Teliospore çimlenme

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Toprak Örneklerinden İntomopatojenik Mantarların İzolasyonu ve Seçimi ve Böcek Zararlılarına Karşı Mantar Virülansının Değerlendirilmesi

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

Entomopatojenik mantarları toprak örneklerinden izole etme yöntemlerinin karşılaştırılması

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

Isolation of Culturable Yeasts and Molds from Soils to Investigate Fungal Population Structure

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species. 
We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis. 
The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

Mantar Popülasyon Yapısını Araştırmak için Kültürlenebilir Maya ve Küflerin Topraklardan İzolasyonu

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

Mikoheterotrofik Bitkilerde Endofitik Mantarları Tanımlamak için İzolasyon, Karakterizasyon ve Toplam DNA Ekstraksiyonu

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...