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 7 of 115 Data required as metadata for HERMES: The basic metadata information will allow proper identification of samples and the data required includes the following. Campaign/cruise Project name,institute(s),campaign/cruise name,basis(ship) Site Site label(site number),latitude/longitude,elevation(-below sea level,above sea level),date,time,area Event(core/sample/measurement) Core/sample/measurement label("event label"),latitude/longitude,gear,depth in water/depth in sediment,recovery instrument,date,time Data Full name of investigated parameters(method such as cell numbers,thymidine incorporation etc)and parameter units (following SI standard or internationally used/widely accepted format) Complete list of abbreviations used in the data table Short description of the analytical or calculating methods(laboratory device(s), analytical process,age model,...)reference(s)for the used method,principle investigator(name,address,email) Contact: Hannes Grobe,AWI,Bremerhaven,Germany,(e-mail:hgrobe@awi-bremerhaven.de
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 7 of 115 Data required as metadata for HERMES: The basic metadata information will allow proper identification of samples and the data required includes the following. Campaign/cruise • Project name, institute(s), campaign/cruise name, basis (ship) Site • Site label (site number), latitude/longitude, elevation (- below sea level, + above sea level), date, time, area Event (core/sample/measurement) • Core/sample/measurement label (“event label”), latitude/longitude, gear, depth in water/depth in sediment, recovery instrument, date, time Data • Full name of investigated parameters (method such as cell numbers, thymidine incorporation etc) and parameter units (following SI standard or internationally used/widely accepted format) • Complete list of abbreviations used in the data table • Short description of the analytical or calculating methods (laboratory device(s), analytical process, age model, …); reference(s) for the used method, principle investigator (name, address, email) Contact: Hannes Grobe, AWI, Bremerhaven, Germany, (e-mail: hgrobe@awi-bremerhaven.de )
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
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 10 of 115 Reagents: 10%(w/v)Trichloroacetic acid(TCA)in Milli-Q water. 5%(w/v)TCA solution. 95%(v/v)ethanol solution 1 M NaOH in Milli-Q water 2 M NaOH in Milli-Q water 'Acidifying solution'20%(w/v)TCA in 3.6 M HCI DNA solution 1 mg/ml in Milli-Q water(e.g.,Sigma D-6898 or D-1501) RNA solution 1 mg/ml in Milli-Q water(e.g.,Sigma R-7125) Kieselguhr(Sigma D-5384) Centrifugation: Centrifugations are carried out at 2000 x g and 3000 x g and the RPM required is calculated from: RPM= g×1,000,000 11.18XR Where:RPM=revolutions per minute;g=g-force;R=average sample radius in rotor(cm) References: Karl,D.M.,(1982)Selected nucleic acid precursors in studies of aquatic microbial ecology. Appl.Environ.Microbiol.,44:891-902 Craven D.B.and Karl,D.M.,(1984).Microbial RNA and DNA synthesis in marine sediments.Mar.Biol.83:129-139. Wellsbury,P.,Herbert,R.A.,and Parkes,R.J.,(1996).Bacterial activity and production in near-surface estuarine and freshwater sediments.FEMS Microbiol.Ecol.,19:203-214. Contact: Barry Cragg,School of Earth,Ocean and Planetary Sciences,Cardiff University,UK(e-mail: b.cragg@earth.cf.ac.uk 8
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 10 of 115 Reagents: 10% (w/v) Trichloroacetic acid (TCA) in Milli-Q water. 5% (w/v) TCA solution. 95% (v/v) ethanol solution 1 M NaOH in Milli-Q water 2 M NaOH in Milli-Q water 'Acidifying solution' 20% (w/v) TCA in 3.6 M HCl DNA solution 1 mg/ml in Milli-Q water(e.g., Sigma D-6898 or D-1501) RNA solution 1 mg/ml in Milli-Q water(e.g., Sigma R-7125) Kieselguhr (Sigma D-5384) Centrifugation: Centrifugations are carried out at 2000 x g and 3000 x g and the RPM required is calculated from: Where: RPM = revolutions per minute; g = g-force; R = average sample radius in rotor (cm) References: Karl, D.M., (1982) Selected nucleic acid precursors in studies of aquatic microbial ecology. Appl. Environ. Microbiol., 44:891-902 Craven D.B. and Karl, D.M., (1984). Microbial RNA and DNA synthesis in marine sediments. Mar. Biol. 83:129-139. Wellsbury, P., Herbert, R.A., and Parkes, R.J., (1996). Bacterial activity and production in near-surface estuarine and freshwater sediments. FEMS Microbiol. Ecol., 19:203-214. Contact: Barry Cragg, School of Earth, Ocean and Planetary Sciences, Cardiff University, UK (e-mail: b.cragg@earth.cf.ac.uk ) g x 1,000,000 11.18 x R RPM =