HERMES Micro Ecol Methods Handbook：微生物生态学方法手册 Handbook of Methods for Microbial Ecology（used to Study the Biodiversity and Function of Microbial Habitats）.pdf_大学文库

HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 2 of 115 Contents INTRODUCTION............... 1.1. ACKNOWLEDGEMENTS. 1.2. LIST OF CONTRIBUTORS 2.SAMPLE IDENTIFICATION DATA MANAGEMENT FOR MICROBIOLOGY SAMPLES........6 3.RATE AND ACTIVITY MEASUREMENTS.....8 3.1. THYMIDINE INCORPORATION 8 3.2 LEUCINE INCORPORATION........... ..11 3.3. SULPHATE REDUCTION...... 13 3.4. METHANOGENESIS FROM CO2+H2 AND FROM ACETATE .16 3.5. ANAEROBIC OXIDATION OF METHANE........ 18 3.6. IN VITRO SULPHATE REDUCTION WITH METHANE..... 21 3.6.1. In vitro rate determination from substrate concentration changes 2 3.6.2. In vitro rate determination from radio-labelled tracer turnover.............2.4 3.7. ASSESSMENT OF EXTRACELLULAR ENZYMATIC ACTIVITIES OF BENTHIC ASSEMBLAGES 25 4.COUNTING METHODS 27 4.1. ACRIDINE ORANGE DIRECT COUNTING(AODC)OF PROKARYOTIC CELLS IN SEDIMENT...............27 4.2 FLUoRESCENT IN SITU HYBRIDIZATION (FISH)29 4.3 CARD-FISH...... .32 5.BIOMASS AND BIOMARKER METHODS............................................................36 5.1. PHOPSPHOLIPIDS FOR BIOMASS ESTIMATION 36 5.2. PROTEIN FOR BIOMASS ESTIMATION. 38 5.3. PHOSPHOLIPID/BIOMARKER ANALYSIS..... …41 6. MOLECULAR METHODS FOR INVESTIGATING DIVERSITY:CLONE LIBRARIES .45 6.1. DNA EXTRACTION FROM SEDIMENTS..... .45 6.1.1. Modified Zhou et al (1996)method............. ……45 6.1.2. Modified FastDNA Spin kit(Q-BIO gene)DNA extraction method and tips for subsequent molecular diversity studies in low DNA sediments.............. .48 6.2. PCR… 6.3 SELECTING AND CONFIRMING EFFICACY OF 16S RRNA GENE PRIMERS.. 51 53 6.4. SELECTING AND USING PRIMERS FOR FUNCTIONAL GENES...5.6 6.4.1. Methyl coenzyme M reductase(mcrA)genes for methanogens...... 56 6.4.2 Quantitative Real-time PCR of the dsrAB genes from sulphate-reducers..........59 6.4.3. Other functional genes and small subunit rRNA groups.61 6.5. CLONING,CLONE SELECTION AND SEQUENCING...... .64 6.5.1. Contribution 64 6.5.2 Contribution 2........... 67 6.6. SCREENING CLONE LIBRARIES FOR ARTEFACTS,CHIMERAS AND COVERAGE 70 6.7 PHYLOGENETIC ANALYSES....... .79 7.MOLECULAR METHODS FOR INVESTIGATING DIVERSITY:PROFILING METHODS..........85 7.1. DENATURING GRADIENT GEL ELECTROPHORESIS(DGGE)FOR 16S RRNA GENES ..............85 7.1.1. Sequencing bands from DGGEgels8 7.1.2 Analysis of DGGE profile data................ .88 7.2. BENTHIC DIVERSITY PROFILING OF BACTERIA USING ARISA 92

HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 2 of 115 Contents 1. INTRODUCTION........................................................................................................................................4 1.1. ACKNOWLEDGEMENTS........................................................................................................................5 1.2. LIST OF CONTRIBUTORS......................................................................................................................5 2. SAMPLE IDENTIFICATION & DATA MANAGEMENT FOR MICROBIOLOGY SAMPLES.........6 3. RATE AND ACTIVITY MEASUREMENTS............................................................................................8 3.1. THYMIDINE INCORPORATION ...............................................................................................................8 3.2. LEUCINE INCORPORATION.................................................................................................................11 3.3. SULPHATE REDUCTION......................................................................................................................13 3.4. METHANOGENESIS FROM CO2 + H2 AND FROM ACETATE ...............................................................16 3.5. ANAEROBIC OXIDATION OF METHANE...............................................................................................18 3.6. IN VITRO SULPHATE REDUCTION WITH METHANE..............................................................................21 3.6.1. In vitro rate determination from substrate concentration changes.......................21 3.6.2. In vitro rate determination from radio-labelled tracer turnover..............................24 3.7. ASSESSMENT OF EXTRACELLULAR ENZYMATIC ACTIVITIES OF BENTHIC ASSEMBLAGES ...............25 4. COUNTING METHODS ..........................................................................................................................27 4.1. ACRIDINE ORANGE DIRECT COUNTING (AODC) OF PROKARYOTIC CELLS IN SEDIMENT ................27 4.2. FLUORESCENT IN SITU HYBRIDIZATION (FISH) ...............................................................................29 4.3. CARD-FISH.....................................................................................................................................32 5. BIOMASS AND BIOMARKER METHODS..........................................................................................36 5.1. PHOPSPHOLIPIDS FOR BIOMASS ESTIMATION ..................................................................................36 5.2. PROTEIN FOR BIOMASS ESTIMATION.................................................................................................38 5.3. PHOSPHOLIPID/BIOMARKER ANALYSIS.............................................................................................41 6. MOLECULAR METHODS FOR INVESTIGATING DIVERSITY: CLONE LIBRARIES................45 6.1. DNA EXTRACTION FROM SEDIMENTS ...............................................................................................45 6.1.1. Modified Zhou et al (1996) method .................................................................................45 6.1.2. Modified FastDNA® Spin kit (Q-BIO gene) DNA extraction method and tips for subsequent molecular diversity studies in low DNA sediments...............................................48 6.2. PCR...................................................................................................................................................51 6.3. SELECTING AND CONFIRMING EFFICACY OF 16S RRNA GENE PRIMERS ........................................53 6.4. SELECTING AND USING PRIMERS FOR FUNCTIONAL GENES .............................................................56 6.4.1. Methyl coenzyme M reductase (mcrA) genes for methanogens.............................56 6.4.2. Quantitative Real-time PCR of the dsrAB genes from sulphate-reducers...........59 6.4.3. Other functional genes and small subunit rRNA groups..........................................61 6.5. CLONING, CLONE SELECTION AND SEQUENCING..............................................................................64 6.5.1. Contribution 1 ......................................................................................................................64 6.5.2. Contribution 2 ......................................................................................................................67 6.6. SCREENING CLONE LIBRARIES FOR ARTEFACTS, CHIMERAS AND COVERAGE ................................70 6.7. PHYLOGENETIC ANALYSES ...............................................................................................................79 7. MOLECULAR METHODS FOR INVESTIGATING DIVERSITY: PROFILING METHODS..........85 7.1. DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) FOR 16S RRNA GENES .....................85 7.1.1. Sequencing bands from DGGE gels ..............................................................................87 7.1.2. Analysis of DGGE profile data.........................................................................................88 7.2. BENTHIC DIVERSITY PROFILING OF BACTERIA USING ARISA.........................................................92

HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 4 of 115 1.Introduction This handbook has been prepared by the Cardiff University HERMES scientists as fulfilment of a 6 month deliverable in the HERMES research project(funded by the European Commission's Framework Six Programme,under the priority Sustainable Development. Global Change and Ecosystems.Contract No.GOCE-CT-2005-511234-1).It is a handbook of methods for microbial ecology that will be used by the HERMES microbiology partners during their studies of biodiversity and function in a variety of coastal marine habitats that are part of the HERMES multidisciplinary research programme. All HERMES microbiologists have been asked to contribute methods that they will use or that they think will be useful to others.The resulting collection is comprehensive and representative of the variety of microbiological approaches that will be used by HERMES microbiologists.It is not expected to be an exhaustive collection of all the methods that will be used as microbiological approaches to ecology and biogeochemistry are evolving very rapidly and it would be unwise to restrict investigators to a standard set of approaches.Also it was beyond the scope of this handbook to include the chemical,sampling or statistical methodologies that all of us use to some extent.Providing these methods to all HERMES microbiologists will stimulate interaction,technical developments and the rapid education of PhD students,postdoctoral scientists and others who are new to this branch of science and who are contributing to the HERMES programme. The methods are written as protocols for use at the bench,during the planning of experiments and during data analysis.All the individual sections start with an introductory paragraph which states the aim of the method and/or a summary of the approach used.All sections also have relevant references which are collated in alphabetical order at the end of the handbook. At the end of each section is a contact name,brief statement of this persons location and their e-mail address to facilitate communication.In most cases the contact person has written the protocol,uses the approaches described routinely,has published studies using the methodology and is available for consultation in cases of difficulty.The protocols do not follow a standard format apart,from the common elements described here,instead in requesting,collating and editing the contributions I have tried to encourage the sections to be written in a way that suits the subject matter of the approach.I apologise if the editorial changes that I have made are not completely endorsed by the authors. Although this handbook was completed on 30 September 2005 it need not be static,as it will be possible to add material to the body of the work or as appendices as is thought appropriate in the future.No such book is ever complete or fully comprehensive,but rather aims to act as a signboard for us all to use on our journey through scientific discovery. Comments and suggestions for future development and improvement will be welcomed. John Fry Cardiff School of Biosciences,Cardiff University Cardiff,UK E-mail:fry@cardiff.ac.uk 30 September 2005

HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 4 of 115 1. Introduction This handbook has been prepared by the Cardiff University HERMES scientists as fulfilment of a 6 month deliverable in the HERMES research project (funded by the European Commission's Framework Six Programme, under the priority Sustainable Development, Global Change and Ecosystems. Contract No. GOCE-CT-2005-511234-1). It is a handbook of methods for microbial ecology that will be used by the HERMES microbiology partners during their studies of biodiversity and function in a variety of coastal marine habitats that are part of the HERMES multidisciplinary research programme. All HERMES microbiologists have been asked to contribute methods that they will use or that they think will be useful to others. The resulting collection is comprehensive and representative of the variety of microbiological approaches that will be used by HERMES microbiologists. It is not expected to be an exhaustive collection of all the methods that will be used as microbiological approaches to ecology and biogeochemistry are evolving very rapidly and it would be unwise to restrict investigators to a standard set of approaches. Also it was beyond the scope of this handbook to include the chemical, sampling or statistical methodologies that all of us use to some extent. Providing these methods to all HERMES microbiologists will stimulate interaction, technical developments and the rapid education of PhD students, postdoctoral scientists and others who are new to this branch of science and who are contributing to the HERMES programme. The methods are written as protocols for use at the bench, during the planning of experiments and during data analysis. All the individual sections start with an introductory paragraph which states the aim of the method and/or a summary of the approach used. All sections also have relevant references which are collated in alphabetical order at the end of the handbook. At the end of each section is a contact name, brief statement of this persons location and their e-mail address to facilitate communication. In most cases the contact person has written the protocol, uses the approaches described routinely, has published studies using the methodology and is available for consultation in cases of difficulty. The protocols do not follow a standard format apart, from the common elements described here, instead in requesting, collating and editing the contributions I have tried to encourage the sections to be written in a way that suits the subject matter of the approach. I apologise if the editorial changes that I have made are not completely endorsed by the authors. Although this handbook was completed on 30 September 2005 it need not be static, as it will be possible to add material to the body of the work or as appendices as is thought appropriate in the future. No such book is ever complete or fully comprehensive, but rather aims to act as a signboard for us all to use on our journey through scientific discovery. Comments and suggestions for future development and improvement will be welcomed. John Fry Cardiff School of Biosciences, Cardiff University Cardiff, UK E-mail: fry@cardiff.ac.uk 30 September 2005

HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 5 of 115 1.1. Acknowledgements I am very grateful to all those HERMES microbiologists and others who have willingly contributed to this handbook.I hope that we will all find it useful.We of course all thank the European Commission who have funded HERMES.I am especially grateful to Antje Boetius (MPL,Bremen)for her guidance and support during the book's planning and to all my colleagues,HERMES microbiologists and others,who have helped me in various ways. Thanks also go to the scientists working on the Metrol EU programme(EVK2-CT-2002- 00080)whose collection of methods formed the basis for some of the protocols presented here 1.2. List of contributors Kevin Ashelford,Cardiff School of Biosciences,Cardiff(e-mail:ashelford@cardiff.ac.uk ) Barry Cragg,School of Earth,Ocean and Planetary Sciences,Cardiff University,UK (e-mail: b.cragg@earth.cf.ac.uk Roberto Danovaro,Department of Marine Sciences,Polytechnic University of Marche, Ancona.(e-mail:danovaro@univpm.it ) Tim Ferdelman,Max Planck Institute for Marine Microbiology,Bremen,Germany (e-mail: tferdelm @mpi-bremen.de ) John Fry,Cardiff School of Biosciences,Cardiff University (e-mail:fry@cardiff.ac.uk ) Hannes Grobe,AWI,Bremerhaven,Germany,(e-mail:hgrobe@awi-bremerhaven.de Gwang Tae Kim,Cardiff School of Biosciences,Cardiff University (e-mail: kimgt@Cardiff.ac.uk ) Katrin Knittel,Max Planck Institute for Marine Microbiology,Bremen,Germany (e-mail: kknittel@mpi-bremen.de) Konstantinos Ar.Kormas,Dept.of Animal Production and Aquatic Environment,Univeristy of Thessaly,Volos,Greece (e-mail:kkormas@uth.gr ) Julie Leloup,Max Planck Institute for Marine Microbiology,Bremen,Germany,(e-mail: jleloup@mpi-bremen.de Helge Niemann,Max Planck Institute for Marine Microbiology,Bremen,Germany (e-mail: hniemann@mpi-bremen.de Richard Pancost,Organic Geochemistry Unit,University of Bristol,UK(e-mail: R.D.Pancost@bristol.ac.uk Paraskevi Polymenakou,Hellenic Center for Marine Research,Iraklion,Crete,Greece (e-mail:polymen@imbc.gr Henrik Sass,School of Earth,Ocean and Planetary Sciences,Cardiff University (e-mail: sassh@earth.cf.ac.uk Ingo Schewe,AWI,Bremerhaven,Germany (e-mail:ischewe@awi-bremerhaven.de ) Gordon Webster,Cardiff School of Biosciences,Cardiff University (e-mail: websterg@Cardiff.ac.uk Andrew Weightman,Cardiff School of Biosciences,Cardiff University (e-mail: Weightman@Cardiff.ac.uk Friedrich Widdel,Max Planck Institute for Marine Microbiology,Bremen,Germany(e-mail: fwiddel@mpi-bremen.de

HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 5 of 115 1.1. Acknowledgements I am very grateful to all those HERMES microbiologists and others who have willingly contributed to this handbook. I hope that we will all find it useful. We of course all thank the European Commission who have funded HERMES. I am especially grateful to Antje Boetius (MPI, Bremen) for her guidance and support during the book’s planning and to all my colleagues, HERMES microbiologists and others, who have helped me in various ways. Thanks also go to the scientists working on the Metrol EU programme (EVK2-CT-2002- 00080) whose collection of methods formed the basis for some of the protocols presented here. 1.2. List of contributors Kevin Ashelford, Cardiff School of Biosciences, Cardiff (e-mail: ashelford@cardiff.ac.uk ). Barry Cragg, School of Earth, Ocean and Planetary Sciences, Cardiff University, UK (e-mail: b.cragg@earth.cf.ac.uk ) Roberto Danovaro, Department of Marine Sciences, Polytechnic University of Marche, Ancona. (e-mail: danovaro@univpm.it ). Tim Ferdelman, Max Planck Institute for Marine Microbiology, Bremen, Germany (e-mail: tferdelm@mpi-bremen.de ). John Fry, Cardiff School of Biosciences, Cardiff University (e-mail: fry@cardiff.ac.uk ). Hannes Grobe, AWI, Bremerhaven, Germany, (e-mail: hgrobe@awi-bremerhaven.de ) Gwang Tae Kim, Cardiff School of Biosciences, Cardiff University (e-mail: kimgt@Cardiff.ac.uk ). Katrin Knittel, Max Planck Institute for Marine Microbiology, Bremen, Germany (e-mail: kknittel@mpi-bremen.de) Konstantinos Ar. Kormas, Dept. of Animal Production and Aquatic Environment, Univeristy of Thessaly, Volos, Greece (e-mail: kkormas@uth.gr ). Julie Leloup, Max Planck Institute for Marine Microbiology, Bremen, Germany, (e-mail: jleloup@mpi-bremen.de ) Helge Niemann, Max Planck Institute for Marine Microbiology, Bremen, Germany (e-mail: hniemann@mpi-bremen.de ) Richard Pancost, Organic Geochemistry Unit, University of Bristol, UK (e-mail: R.D.Pancost@bristol.ac.uk ) Paraskevi Polymenakou, Hellenic Center for Marine Research, Iraklion, Crete, Greece (e-mail: polymen@imbc.gr ) Henrik Sass, School of Earth, Ocean and Planetary Sciences, Cardiff University ( e-mail: sassh@earth.cf.ac.uk ) Ingo Schewe, AWI, Bremerhaven, Germany (e-mail: ischewe@awi-bremerhaven.de ). Gordon Webster, Cardiff School of Biosciences, Cardiff University (e-mail: websterg@Cardiff.ac.uk ) Andrew Weightman, Cardiff School of Biosciences, Cardiff University (e-mail: Weightman@Cardiff.ac.uk ) Friedrich Widdel, Max Planck Institute for Marine Microbiology, Bremen, Germany (e-mail: fwiddel@mpi-bremen.de )

HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 6 of 115 2.Sample identification data management for microbiology samples During HERMES we will collect a diversity of microbiological samples from a number of sites.Some groups will work on samples from pre-HERMES cruises and collaborative projects.One of the main tasks for every investigator in HERMES will be to keep and provide clear sample identification(the so-called metadata)for each data point.The HERMES database and metadata archive is PANGAEA (www.pangaea.de).To facilitate sample identification and data management,a few basic rules need to be considered. Sample storage: The procedures for sample storage are given in each method description.Most importantly: Keep all samples identifiable!They need a clear identification number,which gives a reference to the geographical position of the site where the samples have been obtained.You need to keep sample IDs and a station list for each cruise you have participated in,and for each sample you will receive. DNA samples need to be stored frozen (at-20C or below) RNA samples are very sensitive and need to be handled rapidly.They are stored at-80C. ● Samples for cell counts are stored in 2%formalin,in the cold and dark,and preferably in plastic vials,never frozen FISH samples are briefly (1-4 h)fixed in 2%formalin and then washed thoroughly and stored frozen in 50%ethanol/PBS. Samples for activity measurements are kept at in situ temperature until analysis. Samples for cultivation are best kept as bulk sediments in glass vials at in situ temperature in the dark.Aerobic samples need aeration,anaerobic samples anaerobic storage. Data storage Each data point needs a reference to the site and date where and when it was sampled. Usually,this information is provided by the station list of a scientific expedition,which you need to store and use to keep records of the station number and device with which the sample was obtained.It is very important also to keep track of the sediment horizon,which was sampled,and all subsequent handling(storage temperature,fixatives,dilutions etc). PANGAEA is the data base selected for HERMES.It has already defined parameters for most of the data generated through HERMES microbiologists,including a variety of biomass and activity measures.HERMES is also concerned with biodiversity of microbes.The global solution at the moment for storing information about gene and protein sequences are international databases such as GenBank http://www.ncbi.nih.gov/Genbank/.However, unfortunately most available databases provide poor geographical and environmental information.One of the goals in HERMES WP4 is to tackle this problem-and here you need to help by keeping metadata information available!

HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 6 of 115 2. Sample identification & data management for microbiology samples During HERMES we will collect a diversity of microbiological samples from a number of sites. Some groups will work on samples from pre-HERMES cruises and collaborative projects. One of the main tasks for every investigator in HERMES will be to keep and provide clear sample identification (the so-called metadata) for each data point. The HERMES database and metadata archive is PANGAEA (www.pangaea.de). To facilitate sample identification and data management, a few basic rules need to be considered. Sample storage: The procedures for sample storage are given in each method description. Most importantly: • Keep all samples identifiable! They need a clear identification number, which gives a reference to the geographical position of the site where the samples have been obtained. You need to keep sample IDs and a station list for each cruise you have participated in, and for each sample you will receive. • DNA samples need to be stored frozen (at -20°C or below) • RNA samples are very sensitive and need to be handled rapidly. They are stored at -80°C. • Samples for cell counts are stored in 2% formalin, in the cold and dark, and preferably in plastic vials, never frozen • FISH samples are briefly (1-4 h) fixed in 2% formalin and then washed thoroughly and stored frozen in 50% ethanol/PBS. • Samples for activity measurements are kept at in situ temperature until analysis. • Samples for cultivation are best kept as bulk sediments in glass vials at in situ temperature in the dark. Aerobic samples need aeration, anaerobic samples anaerobic storage. Data storage: Each data point needs a reference to the site and date where and when it was sampled. Usually, this information is provided by the station list of a scientific expedition, which you need to store and use to keep records of the station number and device with which the sample was obtained. It is very important also to keep track of the sediment horizon, which was sampled, and all subsequent handling (storage temperature, fixatives, dilutions etc). PANGAEA is the data base selected for HERMES. It has already defined parameters for most of the data generated through HERMES microbiologists, including a variety of biomass and activity measures. HERMES is also concerned with biodiversity of microbes. The global solution at the moment for storing information about gene and protein sequences are international databases such as GenBank http://www.ncbi.nih.gov/Genbank/ . However, unfortunately most available databases provide poor geographical and environmental information. One of the goals in HERMES WP4 is to tackle this problem – and here you need to help by keeping metadata information available!

HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 8 of 115 3.Rate and Activity Measurements 3.1. Thymidine incorporation Thymidine is one of the four bases of DNA.By measuring the rates of incorporation of tritiated thymidine into prokaryotes we can obtain a measure of population growth.There are a number of provisos of this method.Methanogenic Archaea and many sulphate-reducing bacteria do not incorporate thymidine,relying instead on de novo synthesis.Thus thymidine incorporation is more usefully a measure of growth in the heterotrophic population. Thymidine may be utilized by starving prokaryotes as a carbon source and be metabolized rather than incorporated into DNA.Incubation periods must consequently be short-typically a few hours. Field: Sediment subcores are sampled in 26 mm ID acrylic tubes with injection ports filled with silicone rubber. About 10 ul(~200 kBq)radioactively labeled'H-thymidine solution is injected into the sediment in 1-cm depth intervals and incubated for 3-12 h at in situ temperature. Activity is terminated by extruding the sediment sample into cold Trichloroacetic acid(TCA) in a 50 ml centrifuge tube and storing at 0-4C Alternatively,where individual 5 ml syringe mini-cores are used then 37 ul of tritiated thymidine (~750 Kbq)is injected along the centre line of the syringe and incubations are terminated as described above or by directly freezing the syringe for long term storage followed by defrosting in TCA when processing begins. Blank samples are prepared by adding 37 ul of tritiated thymidine to a well mixed slurry of sediment sample (5 ml)and TCA(5 ml)in a centrifuge tube at 0-4C Laboratory: Processing methodology is adapted from Wellsbury et al.(1996)as originally derived from Karl (1982)and Craven Karl (1984). DAY 1 1.If not already done(see above),transfer sample(5 ml)to 50 ml centrifuge tube containing 5 ml of 10%TCA at 0-4C.Mix thoroughly,and store in a fridge or cold room at 0-4C until extraction. 2.Centrifuge at 2000 g for 15 min at 2C 3.Decant and collect the supernatant in a Sterilin bottle.Add another 10 ml of 5%TCA at 0- 4C to the centrifuge tube,mix and centrifuge at 2000 g for 15 min at 2C.Decant and add the supernatant to the Sterilin bottle,repeat rinse for a third time with a further 10 ml of 5%TCA.Thoroughly mix the Sterilin bottle on a vortex mixer and count a 5 ml sub- sample of the combined supernatant.This is the UNINCORPORATED fraction.Discard remaining supernatant to sink and soak"Sterilin"bottle in Decon prior to disposal into bin

HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 8 of 115 3. Rate and Activity Measurements 3.1. Thymidine incorporation Thymidine is one of the four bases of DNA. By measuring the rates of incorporation of tritiated thymidine into prokaryotes we can obtain a measure of population growth. There are a number of provisos of this method. Methanogenic Archaea and many sulphate-reducing bacteria do not incorporate thymidine, relying instead on de novo synthesis. Thus thymidine incorporation is more usefully a measure of growth in the heterotrophic population. Thymidine may be utilized by starving prokaryotes as a carbon source and be metabolized rather than incorporated into DNA. Incubation periods must consequently be short – typically a few hours. Field: Sediment subcores are sampled in 26 mm ID acrylic tubes with injection ports filled with silicone rubber. About 10 µl (~200 kBq) radioactively labeled 3 H-thymidine solution is injected into the sediment in 1-cm depth intervals and incubated for 3-12 h at in situ temperature. Activity is terminated by extruding the sediment sample into cold Trichloroacetic acid (TCA) in a 50 ml centrifuge tube and storing at 0-4°C Alternatively, where individual 5 ml syringe mini-cores are used then 37 µl of tritiated thymidine (~ 750 Kbq) is injected along the centre line of the syringe and incubations are terminated as described above or by directly freezing the syringe for long term storage followed by defrosting in TCA when processing begins. Blank samples are prepared by adding 37 µl of tritiated thymidine to a well mixed slurry of sediment sample (5 ml) and TCA (5 ml) in a centrifuge tube at 0-4°C Laboratory: Processing methodology is adapted from Wellsbury et al. (1996) as originally derived from Karl (1982) and Craven & Karl (1984). DAY 1 1. If not already done (see above), transfer sample (5 ml) to 50 ml centrifuge tube containing 5 ml of 10% TCA at 0-4°C. Mix thoroughly, and store in a fridge or cold room at 0-4°C until extraction. 2. Centrifuge at 2000 g for 15 min at 2°C 3. Decant and collect the supernatant in a Sterilin bottle. Add another 10 ml of 5% TCA at 0- 4°C to the centrifuge tube, mix and centrifuge at 2000 g for 15 min at 2°C. Decant and add the supernatant to the Sterilin bottle, repeat rinse for a third time with a further 10 ml of 5% TCA. Thoroughly mix the Sterilin bottle on a vortex mixer and count a 5 ml subsample of the combined supernatant. This is the UNINCORPORATED fraction. Discard remaining supernatant to sink and soak “Sterilin” bottle in Decon prior to disposal into bin

HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 9 of 115 4.Rinse (re-suspend,vortex mix and centrifuge at 2000 g and 2C)sediment twice in 10 ml of 95%ethanol at 0-4C,collecting both supernatants in a new Sterilin bottle.Re-suspend sediment pellet in 7 ml of 95%ethanol and transfer to a 15 ml centrifuge tube.(This is best done by re-suspending the sediment initially in 5 ml of ethanol,tipping it into the 15 ml centrifuge tube,then re-suspending any residual sediment with a further 2 ml of ethanol before adding it to the 15 ml centrifuge tube).Centrifuge at 2000 g and 2C and add supernatant to that already acquired.Vortex mix and count a 5 ml sub-sample of the combined supernatant.This is the LIPID fraction.Discard remaining supernatant to sink and soak Sterilin bottle and large centrifuge tube in Decon prior to disposal into bin. 5.Leave the samples to dry off overnight under an extractor with the lids off the tubes.Max temp 37C. DAY 2 6.Add 7 ml of IM NaOH,mix and incubate in a water bath for 1 hr at 37C.Centrifuge at 2000 g for 15 min at 2C 7.Transfer 5 ml of supernatant to a new 15 ml centrifuge tube.Discard remaining supernatant to sink.Keep sediment pellet,this is the PROTEIN fraction(replace lid,ensure tube is suitably identified and store in freezer).Do not process for protein at this stage.GO TO STEP 13 8.To the 5 ml of supernatant add 1.5 ml of 'acidifying solution',50 ul of cold carrier DNA (0.05 mg)and 50 ul of cold carrier RNA(0.05 mg),and a small amount of Kieselguhr. Mix and cool on ice to 0-4C. 9.Centrifuge at 3000 g for 15 min at 2C.Count a 2 ml subsample of the supernatant.This is the RNA fraction.Discard remaining supernatant very carefully to sink ensuring that the tiny pellet at the bottom of the tube is not disturbed. 10.Rinse(re-suspend,vortex mix and centrifuge at 3000 g for 15 min at 2C)remaining pellet twice with ice cold 5%TCA carefully discarding the supernatant to sink and retaining the pellet. 11.Add 5 ml of 5%TCA,vortex mix and incubate at 100C in a water bath for 30 min.(You may need to loosen the caps to prevent the tubes bursting). 12.Cool on ice rapidly,centrifuge at 3000 g for 15 min at 2C.Count a 2 ml sample of the supernatant.This is the DNA fraction.Discard remaining supernatant to sink.Dispose of extracted pellet and soak centrifuge tube in Decon prior to disposal into bin. Protein extraction: 13.Sediment Pellet Rinse(re-suspend,vortex mix and centrifuge at 3000 g for 15 min at 2C)sediment pellet once with 5%TCA and once with 95%ethanol.Discard rinses to sink. 14.Add 5 ml of 2M NaOH,mix and incubate at 37C for 18 hrs.Centrifuge at 2000 g for 15 min at 2C 15.Count a 2 ml sub-sample of the supernatant.This is the PROTEIN fraction.Dispose of sediment to sink and soak tubes in Decon prior to disposal into bin

HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 9 of 115 4. Rinse (re-suspend, vortex mix and centrifuge at 2000 g and 2°C) sediment twice in 10 ml of 95% ethanol at 0-4°C, collecting both supernatants in a new Sterilin bottle. Re-suspend sediment pellet in 7 ml of 95% ethanol and transfer to a 15 ml centrifuge tube. (This is best done by re-suspending the sediment initially in 5 ml of ethanol, tipping it into the 15 ml centrifuge tube, then re-suspending any residual sediment with a further 2 ml of ethanol before adding it to the 15 ml centrifuge tube). Centrifuge at 2000 g and 2°C and add supernatant to that already acquired. Vortex mix and count a 5 ml sub-sample of the combined supernatant. This is the LIPID fraction. Discard remaining supernatant to sink and soak Sterilin bottle and large centrifuge tube in Decon prior to disposal into bin. 5. Leave the samples to dry off overnight under an extractor with the lids off the tubes. Max temp 37°C. DAY 2 6. Add 7 ml of 1M NaOH, mix and incubate in a water bath for 1 hr at 37°C. Centrifuge at 2000 g for 15 min at 2°C 7. Transfer 5 ml of supernatant to a new 15 ml centrifuge tube. Discard remaining supernatant to sink. Keep sediment pellet, this is the PROTEIN fraction (replace lid, ensure tube is suitably identified and store in freezer). Do not process for protein at this stage. GO TO STEP 13 8. To the 5 ml of supernatant add 1.5 ml of 'acidifying solution', 50 µl of cold carrier DNA (0.05 mg) and 50 µl of cold carrier RNA (0.05 mg), and a small amount of Kieselguhr. Mix and cool on ice to 0-4°C. 9. Centrifuge at 3000 g for 15 min at 2°C. Count a 2 ml subsample of the supernatant. This is the RNA fraction. Discard remaining supernatant very carefully to sink ensuring that the tiny pellet at the bottom of the tube is not disturbed. 10. Rinse (re-suspend, vortex mix and centrifuge at 3000 g for 15 min at 2°C) remaining pellet twice with ice cold 5% TCA carefully discarding the supernatant to sink and retaining the pellet. 11. Add 5 ml of 5% TCA, vortex mix and incubate at 100°C in a water bath for 30 min. (You may need to loosen the caps to prevent the tubes bursting). 12. Cool on ice rapidly, centrifuge at 3000 g for 15 min at 2°C. Count a 2 ml sample of the supernatant. This is the DNA fraction. Discard remaining supernatant to sink. Dispose of extracted pellet and soak centrifuge tube in Decon prior to disposal into bin. Protein extraction: 13. Sediment Pellet Rinse (re-suspend, vortex mix and centrifuge at 3000 g for 15 min at 2°C) sediment pellet once with 5% TCA and once with 95% ethanol. Discard rinses to sink. 14. Add 5 ml of 2M NaOH, mix and incubate at 37°C for 18 hrs. Centrifuge at 2000 g for 15 min at 2°C. 15. Count a 2 ml sub-sample of the supernatant. This is the PROTEIN fraction. Dispose of sediment to sink and soak tubes in Decon prior to disposal into bin