HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 26 of 115 24 h)and reported as nmol of MUF or MCA released per g of sediment dry weight h.The replicates must be generally run per each sediment samples. Solutions of 7-amino-4-methylcoumarin and 4-methylumbelliferone(0.1 to 1.0 uM)are used as standards for Leu-MCA,and for MUF-Glu and MUF-P respectively.Standard solutions should be freshly prepared using pre-filtered and autoclaved seawater. Aminopeptidase and B-glucosidase activities can be transformed into equivalents of C mobilised assuming that 1 nmol of substrate hydrolysed enzymatically corresponds to 72 ng of mobilised C. References: Deming JW,Baross JA(1993)The early diagenesis of organic matter:bacterial activity.P. 119-144.In M.H.Engel and S.A.Macko (ed.),organic geochemisry:principles and applications.Plenum Press,New York,NY Deming,JW,Baross JA(2000)Survival,dormacy and non-culturable cells in extreme deep- sea environments.In:Colwell RR,Grimes DJ(Eds)Nonculturable Microorganisms in the Environment.American Society for Microbiology Press,Washington DC pp 147-197 Hendel B,Marxen J(1997)Measurement of low-level extracellular enzyme activity in natural waters using fluorigenic model substrates.Acta Hydrochim Hydrobiol 25:253-258 Hoppe HG(1993)Use of fluorogenic model substrates for extracellular enzyme activity (EEA)measurement of bacteria.In:Kemp PF,Sherr BF,Sherr EB,Cole J(Eds)Handbook of methods in aquatic microbial ecology.Lewis Publishers,Boca Raton,Ann Arbor,1993,pp 423-431 Hoppe HG(1991)Microbial extracellular enzyme activity:a new key parameter in aquatic ecology.In:Microbial enzyme in aquatic environments(Chrost J Ed).Springer-Verlag,New York pp 60-79 Meyer-Reil LA(1987)Seasonal and spatial distribution of extracellular enzymatic activities and microbial incorporation of dissolved organic substrates in marine sediments.Appl Environ Microbiol 53:1748-1755 Meyer-Reil LA(1991)Ecological aspects of enzymatic activity in marine sediments.In R.J. Chrost(ed.),Microbial enzymes in aquatic environments.Springer-Verlag,Berlin,Germany pp84-95 Meyer-Reil LA,Koster M(1992)Microbial life in pelagic sediments:the impact of environmental parameters on enzymatic degradation of organic matter in deep-sea sediments. Mar Ecol Prog Ser 81:65-72 Poremba K(1995)Hydrolytic enzymatic activity in deep-sea sediments.FEMS Microbiol Ecol16:213-222 Contact: Roberto Danovaro,Department of Marine Sciences,Polytechnic University of Marche, Ancona.(e-mail:danovaro@univpm.it )
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 26 of 115 24 h) and reported as nmol of MUF or MCA released per g of sediment dry weight h-1. The replicates must be generally run per each sediment samples. Solutions of 7-amino-4-methylcoumarin and 4-methylumbelliferone (0.1 to 1.0 µM) are used as standards for Leu-MCA, and for MUF-Glu and MUF-P respectively. Standard solutions should be freshly prepared using pre-filtered and autoclaved seawater. Aminopeptidase and ß-glucosidase activities can be transformed into equivalents of C mobilised assuming that 1 nmol of substrate hydrolysed enzymatically corresponds to 72 ng of mobilised C. References: Deming JW, Baross JA (1993) The early diagenesis of organic matter: bacterial activity. P. 119-144. In M.H.Engel and S.A. Macko (ed.), organic geochemisry: principles and applications. Plenum Press, New York, NY Deming, JW, Baross JA (2000) Survival, dormacy and non-culturable cells in extreme deepsea environments. In: Colwell RR, Grimes DJ (Eds) Nonculturable Microorganisms in the Environment. American Society for Microbiology Press, Washington DC pp 147-197 Hendel B, Marxen J (1997) Measurement of low-level extracellular enzyme activity in natural waters using fluorigenic model substrates. Acta Hydrochim Hydrobiol 25:253-258 Hoppe HG (1993) Use of fluorogenic model substrates for extracellular enzyme activity (EEA) measurement of bacteria. In: Kemp PF, Sherr BF, Sherr EB, Cole J (Eds) Handbook of methods in aquatic microbial ecology. Lewis Publishers, Boca Raton, Ann Arbor, 1993, pp 423-431 Hoppe HG (1991) Microbial extracellular enzyme activity: a new key parameter in aquatic ecology. In: Microbial enzyme in aquatic environments (Chrøst J Ed). Springer-Verlag, New York pp 60-79 Meyer-Reil LA (1987) Seasonal and spatial distribution of extracellular enzymatic activities and microbial incorporation of dissolved organic substrates in marine sediments. Appl Environ Microbiol 53:1748-1755 Meyer-Reil LA (1991) Ecological aspects of enzymatic activity in marine sediments. In R.J. Chrost (ed.), Microbial enzymes in aquatic environments. Springer-Verlag, Berlin, Germany pp 84-95 Meyer-Reil LA, Köster M (1992) Microbial life in pelagic sediments: the impact of environmental parameters on enzymatic degradation of organic matter in deep-sea sediments. Mar Ecol Prog Ser 81:65-72 Poremba K (1995) Hydrolytic enzymatic activity in deep-sea sediments. FEMS Microbiol Ecol 16: 213-222 Contact: Roberto Danovaro, Department of Marine Sciences, Polytechnic University of Marche, Ancona. (e-mail: danovaro@univpm.it )
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 27 of 115 4.Counting Methods 4.1.Acridine orange direct counting(AODC)of prokaryotic cells in sediment This method aims to count the total number of prokaryotes present in sediment and the proportion that are growing,as indicated by cells that are dividing or have just divided. Acridine orange is used to differentially stain prokaryotes green when viewed under blue light by epifluorescence microscopy.Samples(mini-cores)are taken from the core using a sterile(autoclaved)5-ml plastic syringe from which the luer end has been removed.A 1 cm plug is ejected directly into a serum vial(previously furnaced at 450C)containing 9 ml of filter sterilized (0.2-um)2%formaldehyde in artificial seawater,crimp sealed,and shaken vigorously to disperse the sediment plug. In the laboratory the samples are processed using acridine orange staining and epifluoresence microscopy based on the general recommendations of Fry (1988).Generally,between 5 ul and 25 ul of formaldehyde-preserved sub sample are stained with acridine orange (50 ul of 1 g/l solution)in 10 ml of filter sterilized(0.1 um pore size)2%formaldehyde for three minutes and then vacuum filtered through a polycarbonate(0.2 um pore size)membrane.The membrane is then rinsed with a further 10 ml of 2%filter sterilized formaldehyde and mounted in a minimum of paraffin oil under a cover slip. The mounted membrane filters are viewed under incident illumination with a Zeiss Axioskop microscope fitted with a 50-W mercury vapour lamp,a wide-band interference filter set for blue excitation,a 100 X(numerical aperture =1.3)Plan Neofluar objective lens,and 10X eyepieces.The volume of sample stained and filtered should be adjusted to optimize filter coverage by particles at around 50-70%.This adjustment is very important as too much sediment on the slide will obscure too many cells,as particles will overlap,and so underestimate numbers grossly,whilst too little sediment makes counting very tedious. Sediment particles will appear orange/red and prokaryotic cells will glow with a bright green/blue light.Sometimes prokaryotes that are not on particles will appear as orange/red prokaryote shaped cells,these should be counted. Three replicate filters are prepared from each sample to minimize count variance(Kirchman et al.,1982).A minimum of 200 fields of view,or 200 bacterial cells are counted.The total number of bacteria and the numbers of dividing and divided cells are separately counted.The number of cells counted on opaque particles is doubled to account for cells hidden from view (Goulder,1977).Blank membranes are regularly counted and bacterial population size is calculated after subtraction of the appropriate blank. Total bacterial numbers are calculated from: 2CON+CoFF CDG+2CDD+ CoN x(CDG+2CDD) xA -BT x1000 2CON CoFF Vcr VIEW D
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 27 of 115 4. Counting Methods 4.1. Acridine orange direct counting (AODC) of prokaryotic cells in sediment This method aims to count the total number of prokaryotes present in sediment and the proportion that are growing, as indicated by cells that are dividing or have just divided. Acridine orange is used to differentially stain prokaryotes green when viewed under blue light by epifluorescence microscopy. Samples (mini-cores) are taken from the core using a sterile (autoclaved) 5-ml plastic syringe from which the luer end has been removed. A 1 cm3 plug is ejected directly into a serum vial (previously furnaced at 450°C) containing 9 ml of filter sterilized (0.2-µm) 2% formaldehyde in artificial seawater, crimp sealed, and shaken vigorously to disperse the sediment plug. In the laboratory the samples are processed using acridine orange staining and epifluoresence microscopy based on the general recommendations of Fry (1988). Generally, between 5 µl and 25 µl of formaldehyde-preserved sub sample are stained with acridine orange (50 µl of 1 g/l solution) in 10 ml of filter sterilized (0.1 µm pore size) 2% formaldehyde for three minutes and then vacuum filtered through a polycarbonate (0.2 µm pore size) membrane. The membrane is then rinsed with a further 10 ml of 2% filter sterilized formaldehyde and mounted in a minimum of paraffin oil under a cover slip. The mounted membrane filters are viewed under incident illumination with a Zeiss Axioskop microscope fitted with a 50-W mercury vapour lamp, a wide-band interference filter set for blue excitation, a 100 X (numerical aperture = 1.3) Plan Neofluar objective lens, and 10 X eyepieces. The volume of sample stained and filtered should be adjusted to optimize filter coverage by particles at around 50-70%. This adjustment is very important as too much sediment on the slide will obscure too many cells, as particles will overlap, and so underestimate numbers grossly, whilst too little sediment makes counting very tedious. Sediment particles will appear orange/red and prokaryotic cells will glow with a bright green/blue light. Sometimes prokaryotes that are not on particles will appear as orange/red prokaryote shaped cells, these should be counted. Three replicate filters are prepared from each sample to minimize count variance (Kirchman et al., 1982). A minimum of 200 fields of view, or 200 bacterial cells are counted. The total number of bacteria and the numbers of dividing and divided cells are separately counted. The number of cells counted on opaque particles is doubled to account for cells hidden from view (Goulder, 1977). Blank membranes are regularly counted and bacterial population size is calculated after subtraction of the appropriate blank. Total bacterial numbers are calculated from: 2C x A ON + COFF + CDG + 2CDD VIEW - BT x 1000 VCT CON x (CDG + 2CDD) 2CON + COFF D +
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 28 of 115 CoN and CoFF:Number of cells counted ON and OFF particles.For the purposes of this calculation transparent particles e.g.,diatom frustules,are not particles. CDG and CDD: Numbers of cells observed DIVIDING (a cell with an invagination)and DIVIDED(two adjacent cells of identical morphology with a distinct space between them).Cells counted in these two categories are not also tallied under ON and OFF particle categories. VIEW: The total number of fields of view observed during a cell count on a filter. A: Filter area ratio.Total countable area of the filter divided by the area of filter observed for one field of view. Br and Bp: Blank correction terms for the total cell number(Br)and the dividing and divided cell numbers(Bp).Calculated from counts of blank membranes and using the same equation with the omission of the correction term.In this instance Ver will equal 10050(10 mL of formaldehyde +50 uL of acridine orange)and D will equal 1. VCT: Volume of formaldehyde-preserved sample that is stained(uL) D Dilution factor of original sample expressed as a proportion,e.g.,1 cmof sediment in 9 ml of formaldehyde will give D=0.1 Numbers of dividing and divided cells are calculated from the same equation with the omission of the terms"2CoN CoFr"at the start of the equation,and the substitution of Bp for BT. The percentage of dividing and divided cells is calculated from the numbers of dividing and divided cells expressed as a percentage of the total bacterial numbers. Where total bacterial numbers approach the calculated detection limit,or numbers of cells counted approach the number of cells observed in the blanks,then this calculation becomes unreliable. References: Fry,J.C.(1988).Determination of biomass.In Austin,B.,(Ed.),Methods in Aquatic Bacteriology:Chichester(Wiley),27-72. Goulder,R.(1977).Attached and free bacteria in an estuary with abundant suspended solids. J.Appl.Bacteriol.,43:399-405 Kirchman,D.,Sigda,J.,Kapuscinski,R.and Mitchell,R.(1982).Statistical analysis of the direct count method for enumerating bacteria.Appl.Environ.Microbiol.44:376-382 Contact: Barry Cragg,Cardiff University,UK(e-mail:b.cragg@earth.cf.ac.uk
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 28 of 115 CON and COFF: Number of cells counted ON and OFF particles. For the purposes of this calculation transparent particles e.g., diatom frustules, are not particles. CDG and CDD: Numbers of cells observed DIVIDING (a cell with an invagination) and DIVIDED (two adjacent cells of identical morphology with a distinct space between them). Cells counted in these two categories are not also tallied under ON and OFF particle categories. VIEW: The total number of fields of view observed during a cell count on a filter. A: Filter area ratio. Total countable area of the filter divided by the area of filter observed for one field of view. BT and BD: Blank correction terms for the total cell number (BT) and the dividing and divided cell numbers (BD). Calculated from counts of blank membranes and using the same equation with the omission of the correction term. In this instance VCT will equal 10050 (10 mL of formaldehyde +50 µL of acridine orange) and D will equal 1. VCT: Volume of formaldehyde-preserved sample that is stained (µL) D: Dilution factor of original sample expressed as a proportion, e.g., 1 cm3 of sediment in 9 ml of formaldehyde will give D = 0.1 • Numbers of dividing and divided cells are calculated from the same equation with the omission of the terms “2CON + COFF” at the start of the equation, and the substitution of BD for BT. • The percentage of dividing and divided cells is calculated from the numbers of dividing and divided cells expressed as a percentage of the total bacterial numbers. Where total bacterial numbers approach the calculated detection limit, or numbers of cells counted approach the number of cells observed in the blanks, then this calculation becomes unreliable. References: Fry, J.C. (1988). Determination of biomass. In Austin, B., (Ed.), Methods in Aquatic Bacteriology: Chichester (Wiley), 27-72. Goulder, R. (1977). Attached and free bacteria in an estuary with abundant suspended solids. J. Appl. Bacteriol., 43:399-405. Kirchman, D., Sigda, J., Kapuscinski, R. and Mitchell, R. (1982). Statistical analysis of the direct count method for enumerating bacteria. Appl. Environ. Microbiol. 44:376-382. Contact: Barry Cragg, Cardiff University, UK (e-mail: b.cragg@earth.cf.ac.uk )
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 29 of 115 4.2. Fluorescent In Situ hybridization(FISH) The aim of FISH is to stain prokaryotic cells with a fluorescently tagged molecular probe so that different groups of organisms can be counted directly with epifluorescence microscopy with suitable filter sets to visualise the bacteria that have hybridized with the probe.Multiple probes can sometimes be used with fluorescent tags that can be seen with different filter sets. Field: .15 ml vials are prepared with 3 ml formaldehyde(4%formaldehyde in 0.2 um sterile filtered seawater) The sediment is sampled with a 5 ml capped syringe of which 1 ml is transferred to the formaldehyde and vortexed. Vials are left for 3-4 h for fixation at 4C and mixed well before 2 ml of the suspension is transferred to a 2 ml Eppendorf vial with a cut-off pipette tip. The Eppendorf vials are centrifuged(max rpm for 2 min)and the supernatant is discharged The pellet is resuspended in 1.5 ml 1xPBS(10 mM sodium phosphate pH 7.2; 130 mM NaCl)and centrifuged again.This washing step is repeated and the pellet resuspended in 1.5 ml 1xPBS/EtOH (1:1)and stored at-20C Laboratory: Sonication (optional but highly recommended for marine sediments) Dilute sample 1:10(75 ul fixed sample +675 ul 1xPBS:EtOH(1:1)for sediments Apply sonication at a setting of 20 s,amplitude 42 um,and <10 W while keeping the sample on ice. (MS73 probe,Sonopuls HD70,Bandelin,Berlin,Germany). Filtration and Hybridisation Mix 10-20 ul (sediments)of the sonicated sample with 5-10 ml 1xPBS. Put a cellulose nitrate filter(0,45 um,Sartorius on a filter-tower and place a GTTP polycarbonate filter(0,2 um,Millipore,Germany)on top of it,shining side up Add sample and apply vacuum;let the filter dry on paper tissue. Cut the filter in quarters and label them with a pencil(do not use edding).For each probe use only one of the quarters. For each probe prepare 2 ml hybridisation buffer: 360 ul NaCl(5 M) 40 ul Tris-HCI(1 M)pH=7,5 xul formamide (amount depends on the probe;the higher the stringency;optimum has to be tested in advance) ad 2 ml Milli-Q H2O 2 ul SDS(10%) Put filter on a glass slide Mix 13,5 ul of the hybridisation buffer +1,5 ul probe(50 ng/ul)per quarter-filter and and carefully pipette it on the filter.With the rest of the 2 ml hybridisation buffer moisten a 19
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 29 of 115 4.2. Fluorescent In Situ hybridization (FISH) The aim of FISH is to stain prokaryotic cells with a fluorescently tagged molecular probe so that different groups of organisms can be counted directly with epifluorescence microscopy with suitable filter sets to visualise the bacteria that have hybridized with the probe. Multiple probes can sometimes be used with fluorescent tags that can be seen with different filter sets. Field: • 15 ml vials are prepared with 3 ml formaldehyde (4% formaldehyde in 0.2 µm sterile filtered seawater) • The sediment is sampled with a 5 ml capped syringe of which 1 ml is transferred to the formaldehyde and vortexed. • Vials are left for 3-4 h for fixation at 4°C and mixed well before 2 ml of the suspension is transferred to a 2 ml Eppendorf vial with a cut-off pipette tip. • The Eppendorf vials are centrifuged (max rpm for 2 min) and the supernatant is discharged • The pellet is resuspended in 1.5 ml 1xPBS (10 mM sodium phosphate pH 7.2; 130 mM NaCl) and centrifuged again. This washing step is repeated and the pellet resuspended in 1.5 ml 1xPBS/EtOH (1:1) and stored at –20°C Laboratory: Sonication (optional but highly recommended for marine sediments) • Dilute sample 1:10 (75 µl fixed sample + 675 µl 1xPBS:EtOH (1:1) for sediments • Apply sonication at a setting of 20 s, amplitude 42 µm, and <10 W while keeping the sample on ice. (MS73 probe, Sonopuls HD70, Bandelin, Berlin, Germany). Filtration and Hybridisation • Mix 10-20 µl (sediments) of the sonicated sample with 5-10 ml 1xPBS. • Put a cellulose nitrate filter (0,45 µm, Sartorius ) on a filter-tower and place a GTTP polycarbonate filter (0,2 µm, Millipore, Germany) on top of it, shining side up. • Add sample and apply vacuum; let the filter dry on paper tissue. • Cut the filter in quarters and label them with a pencil (do not use edding). For each probe use only one of the quarters. • For each probe prepare 2 ml hybridisation buffer: 360 µl NaCl (5 M) 40 µl Tris-HCl (1 M) pH = 7,5 x µl formamide (amount depends on the probe; the higher the stringency; optimum has to be tested in advance) ad 2 ml Milli-Q H2O 2 µl SDS (10 %) • Put filter on a glass slide • Mix 13,5 µl of the hybridisation buffer + 1,5 µl probe (50 ng/µl) per quarter-filter and and carefully pipette it on the filter. With the rest of the 2 ml hybridisation buffer moisten a
HERMES Micro Ecol Methods Handbook-Sept 2005 Edition Page 30 of 115 piece of paper tissue and put it into a 50 ml falcon tube.The filter tissue protects the filters from drying during hybridization(humid chamber").Place the glass slide on the filter tissue.Incubate for 2 h at 46C. It is highly recommended to use separate slides for different probes to avoid mixing of the probes during hybridization! Prepare washing buffer in a 50 ml falcon tube and preheat in water bath to 48 C. 50 ml washing buffer per sample contains: x ul NaCl(5 M)depending on amount of formamide used during hybridization(see table) 1ml Tris-HCI(1 M)pH=7.5 500ul EDTA(0.5 M)pH=8,0(>20%FA used add 50 ml Milli-Q H2O 50 ul SDS(10%) Formamide concentrations in washing buffer: Formamide[%] NaCl [ul] NaCl in mol (used for hybridisation) in 50 ml washing buffer 0 9000 0,900 5 6300 0.636 10 4500 0,450 15 3180 0,318 20 2150 0,225 25 1490 0,159 30 1020 0,112 35 700 0,080 40 460 0,056 45 300 0,040 50 180 0,028 55 100 0,020 60 40 0,014 65 - 0,010 70 ---+350 ul EDTA only 0,007 75 250 ul EDTA only 0,005 80 ---+175 ul EDTA only 0.0035 After incubation transfer glass slide with the filters into the washing buffer and incubate for 15-20 min in the water bath at 48 C.Try to keep samples warm,i.e.work quickly! Wash filters sections in 50 ml of deionized water,and then dehydrate with absolute ethanol.Let sections air dry. The filter sections can now be counterstained(e.g.with the DNA stain 4',6'-diamidino-2- phenylindol,DAPI):Prepare glass slide with 10 ul DAPI(1 ug/ml)per filter.Dip the filter into it upside down and incubate for 3-10 min.Shield the filters from light during and after dying with DAPI. .Rinse filter first in H2O and afterwards in EtOH(80%)and dry on paper tissue.Make sure that it is completely dry before proceeding. Microscopy is performed after embedding the filters in Citifluor.Sections could also be stored at-20C until further processing.For sediments it is recommended to incubate the embedded filters at 4C overnight before microscopy
HERMES Micro Ecol Methods Handbook - Sept 2005 Edition Page 30 of 115 piece of paper tissue and put it into a 50 ml falcon tube. The filter tissue protects the filters from drying during hybridization (“humid chamber”). Place the glass slide on the filter tissue. Incubate for 2 h at 46 °C. It is highly recommended to use separate slides for different probes to avoid mixing of the probes during hybridization! • Prepare washing buffer in a 50 ml falcon tube and preheat in water bath to 48 °C. 50 ml washing buffer per sample contains: x µl NaCl (5 M) depending on amount of formamide used during hybridization (see table) 1 ml Tris-HCl (1 M) pH = 7.5 500 µl EDTA (0.5 M) pH = 8,0 (≥20% FA used add 50 ml Milli-Q H2O 50 µl SDS (10%) Formamide concentrations in washing buffer: Formamide [%] (used for hybridisation) NaCl [µl] in 50 ml washing buffer NaCl in mol 0 9000 0,900 5 6300 0,636 10 4500 0,450 15 3180 0,318 20 2150 0,225 25 1490 0,159 30 1020 0,112 35 700 0,080 40 460 0,056 45 300 0,040 50 180 0,028 55 100 0,020 60 40 0,014 65 --- 0,010 70 --- + 350 µl EDTA only 0,007 75 --- + 250 µl EDTA only 0,005 80 --- + 175 µl EDTA only 0.0035 • After incubation transfer glass slide with the filters into the washing buffer and incubate for 15-20 min in the water bath at 48 °C. Try to keep samples warm, i.e. work quickly! • Wash filters sections in 50 ml of deionized water, and then dehydrate with absolute ethanol. Let sections air dry. • The filter sections can now be counterstained (e.g. with the DNA stain 4´,6´-diamidino-2- phenylindol, DAPI): Prepare glass slide with 10 µl DAPI (1 µg/ml) per filter. Dip the filter into it upside down and incubate for 3-10 min. Shield the filters from light during and after dying with DAPI. • Rinse filter first in H2O and afterwards in EtOH (80 %) and dry on paper tissue. Make sure that it is completely dry before proceeding. • Microscopy is performed after embedding the filters in Citifluor. Sections could also be stored at –20°C until further processing. For sediments it is recommended to incubate the embedded filters at 4°C overnight before microscopy