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1. ueseesssensnnenenn 21 Detection OF Arten N re an Dedede couche voce Cabot E e A E a 21 Detection of nitrate Goleman jossan a a a ar a 22 Determination of ammonia ammonium Solorzano 1969 uuu002ssssessnsenesennenenenneenennnnenn 23 Determination 0f nitrite ves an een irn RI 23 3 Analysis of sulphur and phosphate COMPOUNAG sscccsssssssscccssssssccssssssscssssseseees 24 Colorimetric determination of sulphide Cline 1969 uussurssssesssnssnnnsnnnnnnennnnnnnnnn 25 Determination of dissolved sulphide ee eeeeesecseceseceseceseceseeeseecseecsaesseceseesseeeaecaeesaeenseeees 25 Turbidometric determination of inorganic sulphate Tabatabai 1974 eee esceeeceeeeeeeee ees 26 Sulphate T pid test 22 eetet ire aa ant vies eek aa a K A EaR S neS 26 Photometric analysis of thionates Kelly et al 1969 Fitz and Cypionka 1990 27 Colorimetric determination of sulphite Pachmayr 1960 es eesececeseceeeeeeeeeeeceeeeeeesaeeeeeees 28 Colorimetric determination of sulphur Chan and Suzuki 1993 seeesnnee 28 Photometric determination of orthophosphate 22 2200220ensesneesnnesnnensnennnnnnnnnnesnnennsennnnnnnn 29 Photometric determination of sulphide in culture of sulphate reducing bacteria Cord 29 R wish 1989 2222 2232 Ra an Na late bats Ree as Resa haat leee A e aa Analysis of sulphate by ion chromatography
2. Assay Put 2400 ul sample into a glass test tube Add 600 ul sulphanilamide solution and vortex Measure the extinction at 545 nm against water Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 3 Analysis of sulphur and phosphate compoun 3 Analysis of sulphur and phosphate compounds Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg ds 25 3 Analysis of sulphur and phosphate compounds Colorimetric determination of sulphide Cline 1969 Reagent lg N N dimethyl phenyl diammonium dichloride DMPD and 1 5 g FeCl 6 HO are dissolved in HCI 25 The solution is filled up to a final volume of 50 ml using HCI 25 Attention Reagent is strongly corrosive and carcinogenic Prevent skin contact always use a pipettor and pay attention to adhesive remains Rinse used vessels and pipettes as soon as possible Procedure 0 4 ml of reagent is added to 15 ml screw cap tubes Subsequently 5 ml of sample are added the tubes are closed and thoroughly mixed Wait at least 20 minutes before measuring the extinction at 670 nm If measured values are higher than one samples have to be diluted Calibration curve A NaS solution is used as standard In a volurimetric flask a NaOH pellet is dissolved in 500 ml H O and the solution is flushed with N for at least 20 minutes using a cannula Subsequently a precisely weighed amount of 0 6 g NaS 9 H O which
3. 55 6 Processing of nucleic acids Freeze amp Thaw DNA extraction from growing cultures Procedure 1 Harvesting cells a Transfer 2 ml of liquid culture into a sterile 2 ml reaction tube Eppendorf centrifuge for 30 min at 4 C and discard the supernatant b Pick up a colony from agar plates agar shakes and transfer it into a 1 5 ml reaction tube 2 Add 100 pl of TRIS buffer 50 mM pH 7 4 or water used for PCR mix until you have a cell suspension 3 Run five freeze amp thaw cycles 80 C ethanol bath 85 C heater 3 min each 4 It is recommended to use 2 ul of the fresh DNA extract for PCR 50ul reaction Since this is a quick amp dirty procedure no DNA stabilisation no DNA purification etc extracts should be used for molecular biological purpose as soon as possible kept on ice or in the fridge and immediately frozen away after use DNA RNA extraction from sediment samples The extraction of nucleic acids is a necessary prerequisite for using them as template for PCR Besides the cell disruption purifying and protecting extracted nucleic acids from degrading enzymes is of key importance In the context of sediment samples freeze thawing cannot be applied to decompose bacterial cells because of the sediment matrix which lowers the efficiency significantly The main problem associated with alternative disruption methods is protecting the nucleic acids Mechanical and shear forces easily destabil
4. 13 14 15 16 17 18 20 21 22 23 24 25 26 27 28 29 30 33 35 36 manufacturer Keep under inert gas to be specified by the manufacturer 42 During fumigation spraying wear suitable respiratory equipment appropriate wording to be specified Keep container tightly closed 43 In case of fire use indicate in the space the precise type of fire fighting equipment If water increases the risk add Never use water Keep container dry 45 In case of accident or if you feel unwell seek medical advice immediately show label where possible Keep container in a well ventilated place 46 If swallowed seek medical advice immediately and show this container or label Do not keep the container sealed 47 Keep at temperature not exceeding E C to be specified by the manufacturer Keep away from food drink and animal feeding stuffs 48 Keep wetted with appropriate material to be specified by the manufacturer Keep away from incompatible materials to be indicated by 49 Keep only in the original container the manufacturer Keep away from heat 50 Do not mix with to be specified by the manufacturer Keep away from sources of ignition No smoking 51 Use only in well ventilated areas Keep away from combustible material 52 Not recommended for interior use on large surface areas Handle and open container with care 53 Avoid exposure obtain special instruct
5. ICBM Carl von Ossietzky Universit t Oldenburg 92 9 Unrelated experimental procedures Molecular diffusion coefficients for various ions and gases in aqueous solutions CRC Handbook of Chemistry and Physics 73 Ed The values refer to water at 25 C except oxygen and sulphide solute D m s ammonia NH 1 96 10 methane CH 1 49 10 nitrite NO 1 91 10 nitrate NO 1 90 10 oxygen O 2 13 10 sulfide HS 1 61 10 HS 1 96 10 sulphate SO 1 07 10 D sea water values at 25 C Fick s law of diffusion for sediments C porosity J e D rn D a apparent diffusion coefficient for sediment OX 8C x vertical concentration J diffusion flux The apparent diffusion coefficient for sediments differs from the coefficient for aqueous solutions Dga D 0 a 9 1 In Extraction of pore water The extraction of pore water is done by centrifugation of the sediment with a filter Fig The vessels for the filters are provided by the AG Organische Geochemie The centrifuge tubes and the appropriate teflon vessels are washed with 2 HCl and thereafter with ethanol The Teflon vessels are provided with membrane filters 0 4 um pore diameter and filled with ca 10 g sediment The centrifuge tubes are tarred by weighing Centrifugation at 3500 rpm for four minutes Then the centrifugate is decanted into a syringe and filtrated 0 2 um pore diameter into an Eppi cap Methods
6. water bath preheated to 42 C water bath preheated to 65 C water bath icy water 2 Bunsen burner 150 ml bottle completed medium sterile Iml 10ml pipettes Procedure The agar is liquidated and kept at 65 C 3 ml of agar are added to the single test tubes which are kept at 42 C Next 6 ml completed medium are added to each test tube One series of test tubes is removed from the water bath and placed in a rack at room temperature From the culture of interest one drop 0 5 ml is added to the first test tube slew the tube slightly Transfer one drop 0 5 ml from the first tube to the second one Place the first tube in the cold water bath icy water and so on Before each transfer wipe the corresponding tube to prevent water drops from being transferred unwanted dilution Before incubating the tubes at designated temperatures N CO 80 20 v v is introduced Obtained single colonies can be collected for microscopic investigation and for getting pure cultures using a 1ml syringe and a proper cannula Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 78 8 Physiological characterisation 8 Physiological characterisation Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 79 8 Physiological characterisation Catalase test Reagent 5 H20 solution produced by diluting a 30 stock solution with distilled water Store in
7. Carl von Ossietzky Universit t Oldenburg 26 27 28 Very Toxic by inhalation in contact with skin and if swallowed 26 28 Very Toxic by inhalation and if swallowed 27 28 Very Toxic in contact with skin and if swallowed 36 37 Irritating to eyes and respiratory system 36 37 38 Irritating to eyes respiratory system and skin 36 38 Irritating to eyes and skin 37 38 Irritating to respiratory system and skin 39 23 Toxic danger of very serious irreversible effects through inhalation 39 23 24 Toxic danger of very serious irreversible effects through inhalation in contact with skin 39 23 24 25 Toxic danger of very serious irreversible effects through inhalation in contact with skin and if swallowed 39 23 25 Toxic danger of very serious irreversible effects through inhalation and if swallowed 39 24 Toxic danger of very serious irreversible effects in contact ith skin 39 24 25 Toxic danger of very serious irreversible effects in contact ith skin and if swallowed 39 25 Toxic danger of very serious irreversible effects if swallowed 39 26 Very Toxic danger of very serious irreversible effects hrough inhalation 39 26 27 Very Toxic danger of very serious irreversible effects hrough inhalation and in contact with skin 39 26 27 28 Very Toxic danger of very serious irreversible effects hrough inhalation in contact with skin and if swallowed 39 26 28 Very Toxic da
8. IV Loading the gel and electrophoresis Produce 17 loading buffer directly in the electrophoresis chamber using the stock solution 50xTAE You just have to exchange 5 1 loading buffer for further runs In order you have to remove 5 1 used buffer and produce 5 1 new buffer using the stock solution 50 x TAE and fill up the DGGE chamber Attach the chamber cover Put the casting equipment with the gel into the electrophoresis chamber at least 1 5 hours before loading the gel Connect the plugs of the electrophoresis and the pump to the casting equipment and turn on the Low Voltage LV supply Let the buffer heat up to 60 C While heating up the buffer you have to prepare the PCR samples to load the gel later on Possibly you have to purify your samples Purification of PCR products Final volume max 40 ul Load the gel using a Hamilton syringe with max 40 ul for every single gel slot Flush the syringe after the loading of each sample Pipette 5 ul loading buffer into the empty slots Pipette 10 ul DGGE standard mixture of 16S rRNA products of pure cultures Turn on High Voltage HV up to 100 V The running time is 20 hours V Analysing the DGGE gel Turn off the voltage Take the gel out of the DGGE brackets and release the screws Take off the upper glass plate carefully Take off the DGGE spacer and mark one edge of the gel by cutting them in order to remember the loading direction l
9. Obtained single colonies can be collected for microscopic investigation and for getting pure cultures using a 1ml syringe and a proper cannula Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 20 2 Analysis of nitrogen compounds 2 Analysis of nitrogen compounds Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 21 2 Analysis of nitrogen compounds Detection of ammonium Chaney and Marbach 1962 Solutions A Dissolve 3 g Phenol 3 mg Na nitroprusside Na pentacyanonitrosylferrate III in 100 ml distilled H2O solution can be stored in the fridge for two weeks B Dissolve 2 g NaOH in 80 ml distilled H2O allow the solution to cool down and add 0 5 ml NaClO 13 Fill up with distilled H O to a final volume of 100 ml Procedure 1 ml of solution A is added to 10 ml of sample and mixed Next 1 ml of solution B is added and the mixture is again blended Subsequently the preparation is incubated for one hour at room temperature in the dark The extinction is measured at 635 nm against an ammonium free blank Possible precipitates can be removed by centrifugation prior to measuring the extinction The assay is very sensitive used glass vessels need to be very clean Calibration curves are generated based on NH4 2SO4 using standards with concentrations ranging from 0 to 100 uM The principle behind the assay is depicted in Figure 2 De
10. Procedure 160 ul Tris buffer are mixed with 40 ul test reagent in a polystyrene cuvette by rotating the cuvette slowly The emission of this blank is measured using a luminometer The cuvette is removed 10 ul cell extract are added new volume 210 ul and the cuvette is mixed and the light emission measured again Next the measuring is calibrated by adding 5 ul of ATP standard two times 2 5 pmol to the same cuvette new volumes 215 and 220 ul By this internal standardisation mistakes which can be caused by the different components of the single samples are excluded Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 9 Unrelated experimental procedures 9 Unrelated experimental procedures Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 87 9 Unrelated experimental procedures Determination of methane concentrations via Gas Chromatography Material overheated glown out 50 ml bottles or Bellco tubes with defined volume sterile rubber stoppers 1M NaOH gastight syringes methane standard 100 ppmv gaschromatograph Varian 3400 Preparation To stabilize the system the standby modus of the GC should run 24h 48h before the first measurement The nitrogen valve on the floor must be opened first The nitrogen pressure within the GC should be adjusted to 0 8 bar Now the GC can be turned on and method 4 must be started By pushing reset
11. Solution I phenol solution 5 10 ml tannic acid 2g AIK SO 12 H O saturated 10 ml solution Solution I saturated solution of crystal violet 12 g 100 ml MeOH Staining solution I 10 1 Total cell count Nowadays a combination of epifluorescence microscopy and membrane filtration has established as standard method for the direct counting of microorganisms Before or after filtration microorganisms are stained using fluorescence dyes like acridine orange or DAPI 4 6 Diamidino 2 phenyle indole Acridine orange binds to the phosphate groups of nucleic acids Stained bacteria fluoresce green partially orange Foreign particles appear red orange or yellow DAPI binds double stranded DNA Stained bacteria fluoresce pale blue Foreign particles appear yellow For filtration membrane filters consisting of polycarbonate or aluminium oxide Anodisc are used The pore size should not exceed 0 2 um Materials filtration top part Millipore 20 mm glass filter retainer 0 2 um Anodisc filters Whatman Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 41 5 Microscopy and cell counts Reagents 0 5 TWEEN 80 sterile filtrated 0 22 um sterile filtrated ddH 0 0 22 um sterile filtrated PBS buffer 130 mM NaCl 5 mM NaH PO pH 7 2 fixative 4 paraformaldehyde 0 1 Triton X100 in 1 x PBS pH 7 25 DAPI solution 10 pg ml steril mounting solution DABCO 25 m
12. the cells are lysed mechanically by a beadbeater at 5000 rpm for min wait 30 sec before beadbeating a second time centrifugation at 15 000 rpm for 15 min at 4 C transfer supernatant in a sterile 1 5 ml Eppi and add 250 ul PPS invert 10 times and centrifuge for 5 min split the supernatant in two sterile 1 5 ml Eppis and add 500 ul Binding Matrix Suspension respectively vortex the Matrix Suspension before addition shake the samples 2 min by hand and wait ten minutes so that the particles can sediment remove 350 ul of the supernatant and transfer 750 ul of the suspended solution to a spin filter centrifuge 2 min remove filtrate add the remaining supernatant to the supernatant of the second aliquot and transfer it to the filter centrifugation for 2 min remove supernatant add 500 ul SEWS M and resuspend cautiously centrifugation for 2 min Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 58 6 Processing of nucleic acids remove filtrate centrifugation for 2 min place the spin filter into a new catch tube keep the old tube so that the filter can be placed there later dry the open tube for 5 min under the clean bench add 50 ul DES to the filter and centrifuge for 1 min transfer the extract to a sterile Eppicap and repeat the DES step Usually less than 50ml are needed for amplification 30ul can be transferred to a sterile
13. the error message disappears The content of the nitrogen bottle must be controlled regularly About 2h before measurement the synth air and hydrogen bottles must be opened The nitrogen pressure within the GC must be corrected upwards to 5 7 bar Now open the outlets for synth air and hydrogen The pre adjustments for synth air and hydrogen are synthetic air hydrogen 4 2bar 2 8bar 300 ml min 30 ml min These two gases are the fuel gases for the Flame Ionisation Detector FID Pay attention that the hydrogen valve is opened after the valve for synth air and that afterwards method 2 is started immediately After additional two hours the base line should be near zero and the measurements can be started Calibration For calibration use method 2 and a methane standard of 100 ppmv The standard injection volume is 20u1 that means 20ul of the standard is equal to 100ppmv The volumes listed in the table below are injected into Injector A Three parallels are measured for calculating the calibration line and the correlated standard deviation Before measuring a standard or a sample the syringe must be purged at least thrice with the substance that should be measured Volume ppmv 1 5 2 10 3 25 7 5 37 5 10 50 15 75 20 100 25 125 30 150 40 200 50 250 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 88 9 Unrelated experimental procedures Methane measurement After calibration the syr
14. 45 5 Microscopy and cell counts Figure 7 Reduction of triphenyl tetrazolium chloride Reagents CTC 5 Cyano 2 3 ditolyl tetrazolium chloride 50 mM dissolve 011 g in 4 6 ml particle free distilled water Store the solution in the dark at cool conditions Attention metabolic poison Procedure 1ml of bacterial cell suspension are mixed with 40 ul of CTC colution final concentration 2 mM As control a reference sample is killed using formaldehyde final concentration 2 CTC is added to the reference sample after 5 minutes Samples are incubated for 4 hours at room temperature In the following samples are stained using DAPI filtered and counted using the DAPI and Rhodamin filter set Figure 8 Determining actively respiring cells in a sediment sample taken from Bornhorster lake Left hand side DAPI Middle CTC Right hand side overlay Fluorescence in situ Hybridisation FISH using rRNA probes to detect distinct phylogenetic groups Introduction Fluorescence in situ hybridization FISH with rRNA targeted oligonucleotide probes has developed into an invaluable molecular tool in the late 80 s Giovannoni er al 1988 DeLong et al 1998 Amann et al 1990 and is now a well established technique It is a method for the rapid and specific in situ identification of prokaryotes bacteria and archaea on different phylogenetic levels independent of whether or not they can be cultured FISH is a staining technique using fi
15. 72 C because the DNA binding dyes can only bind into double stranded DNA Because of the application of binding dsDNA dyes unspecific products for instance primer dimers can be detected To eliminate primer dimers a temperature of 80 C will be set up at the end of the run and melt on the primer dimers Furthermore the presentation of the raw data can be chosen normal High Sensitive Low Sensitive you can also choose all of them Melt At the end of the PCR program a melting curve will be recorded Therefore the initial temperature often 50 C will be raised to 99 C using 1 C steps At the same time the fluorescence of all samples will be detected Before running the melting program the initial temperature of the melting curve has to be hold constantly for at least 1 min Samples Choose the names of the samples You can choose among SAMPLE NTC non template control STANDARD and NONE Set up the concentration or the copy numbers of your standards Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 72 6 Processing of nucleic acids CYCLING Figure 18 Adjusting the settings Start You can see the PCR profile again before starting the run Furthermore the experiment will be saved automatically You can have a view on the raw data of the PCR run fluorescence detection temperature profile during the entire run After the run completed Choose the window analysis Th
16. In order to stop the reaction samples are cooked for 3 minutes and cooled down to 25 C Next 1 ml of ammonium glycine buffer is added particles are allowed to settle down and the supernatant is transferred and centrifuged for 15 minutes at 13000 rpm Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 84 8 Physiological characterisation Determination of B glucosaminidase activity in microplates Necessary equipment 96 well plates automatic multichannel pipettes microplate reader Prepare a substrate analogue solution 10 fold amount Prepare 11 of a 0 14 M sodium chloride solution Procedure 200 ul from each sample are added in triplicate to a microplate and mixed with 20 ul of a substrate analogue solution As sample blank 20 ul of sodium chloride solution are added to 200 ul of sample In order to set up a photometric blank 200 ul of sodium chloride solution are mixed with 20 ul of substrate analogue solution The microplates are covered and incubated at 30 C for 24 hours After the incubation the pH of the single wells is adjusted to 10 using 50 ul glycine buffer The plates are analysed using a microplate reader at an excitation wavelength of 355 nm and emission wavelength of 460 nm Calibration curve For preparing standards between 10 and 70 ul MUF stock solution at least three standards are filled up with sterile double distilled water in a glown out vessel to 200 uland 20 ul of su
17. KH PO 136 09 Se 0 20 0 20 0 20 NH Cl 53 49 0 5 0 0 25 0 25 0 25 KCl 74 55 0 4 0 0 30 0 30 0 30 CaCl 2 HO 147 02 0 1 0 0 15 0 15 0 15 MgCl 6 H O 203 30 15 0 50 2 00 3 00 NaCl 58 44 342 13 20 resazurine 0 5 mg ml ml 0 50 0 50 0 50 After autoclaving and cooling down add from sterile stock solutions under N exposure Trace element solution SL10 1 0 ml Vitamin solution V 7 1 0 ml Se W solution 0 1 mM 2 10 M 0 2 ml NaHCO 1 M gt 30 mM 30 ml Dithionite crystalline a little until decolouration lt 17 mg pH adjust with sterile 1 M HCl or NaCO P not required for all strains Attention Protective vessel do not touch hot containers 9 6 8 7 0 for medium F 7 0 7 3 for medium B and medium M Annotation The only difference between media F B and M is the concentration of NaCl and MgCl By adding concentrated salts NaCl 5 M MgCl 0 2 M 30 salt one is able to create medium B and M from a medium with a lower salt content 45 ml 1 F medium for F gt B 68 ml 1 F medium for F gt M 23 ml 1 B medium for B gt M Often used electron donors and electron acceptors mM H 80 CO 20 acetate 2 mM lactate 20 mM Na SO 10 mM Na gt S 0 10 mM Na gt S 0 SmM NaNO 10 mM Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 15 Mineral base medium for marine aerobic bacteria before autoclaving Tabl
18. METHODS SCRIPT AG CYPIONKA v0 1 last update 16 04 2010 Table of contents Version HISIOTY uralten 5 Disposal GUnelimes vases csiacesascsaieesseisesicasicdecissscsssdsaraieust iedisnedecustecassvassessalesestscusiaacesusnssttoseise 6 R S Samen 7 Hazard code nee dusdasidesicetuaidcciscipussuss sos bos vestens tesos sonsos eios sias serasi 10 1 Media preparation eesseseescesoeseesoeeoeecoroeesercoesercorseecorsoesorsorsoeeoesoreoeeceroreeeroeeeesoesoesoreese 12 Preparation of growth media tiressi oeeie niee eee e ea eaer EE EE EEEE SE aine 13 Mineral base medium for many anaerobic bacteria Cypionka and Pfennig 1986 14 Mineral base medium for marine aerobic bacteria ussseessssensssnnsnnennnnnnnenenn nn 15 Preparation of trace element solutions Tschech and Pfennig 1984 uusesssenennen 15 Preparation of Selenite Wolframate Solution Widdel 1980 uuessesssenseenenne 16 Preparation of vitamin solutions neei ei e a ii a iiia 16 Setting up HPG agar platesn ceiien gl e E iS 17 Preparation of oxic medium and agar plates urssessnesnnessnessnnsensensnesnnesnnennonnnnnnnnennnen nn 18 Agar dilution series noi ieoi e eia 2 eer a asr ER gol edad e a reiasa 19 2 Analysis of nitrogen compoundSsS sssescesssccccesssececsssooeessoococesssocoeessssecesssoosessssesssssossee 20 Detection of ammonia ammonium Chaney and Marbach 1962
19. 1969 and modified for water samples in the range of 0 01 to 2 mM of ammonia Notes Glassware should be very clean rinsed with 0 1 M HCl followed by rinsing with distilled water Make sure that water used for preparing for reagents and standards is ammonia free Stock reagents A Phenol alcohol reagent 10 g phenol in 95 Ethyl alcohol to a final volume of 100 ml toxic B Dissolve 1 g Na nitroprusside in 200 ml distilled H20 Store in the dark for not more than 1 month toxic C Alkaline complexing agent Dissolve 100 g trisodium citrate and 5 g NaOH in distilled H gt O To a final volume of 500 ml D Oxidizing solution prepare fresh daily Mix 25 ml hypochlorite solution 15 with 100 ml of solution C Assay Mix 2 ml sample Porewater with 0 08 ml of solution A 0 08 ml of soultion B and 0 2 ml of solution D in a small clean test tube Carefully vortex the mixture Incubate the mix for 3 hours in the dark at room temperature Vortex every hour during incubation Read the absorbance using a spectrophotometer set to 630 nm Standards for calibration 0 100 uM with NH4 2SO Attention 1 NH4 2SO4 2 x NH4 Determination of nitrite Reagents toxic carcinogenic Sulphanilamide solution store in a dark bottle at 4 C Add 150 of ortho phosphoric acid carefully to 700 ml of water Add 10 g sulphanilamide stir and warm up a little bit Add 0 5 g naphthyl ethylenediamine dihydrochloride and fill up to 1000 ml
20. 2005 Therefore the quantitative PCR is one potential method to quantify specific targets and thus the number of organisms in the sample There are several Real time PCR detection chemistries e Application of specific primers in combination with DNA binding dyes SYBRGreen I which emit fluorescence when bound to dsDNA As the double stranded PCR product accumulates during cycling more dye can bind and emit fluorescence e Application of fluorescence labelled specific probes For instance FRET and TaqMan probes Molecular Beacons etc We are using SYBRGreen for detection Procedure Q PCR amplification is performed in a volume of 25 uL containing 12 5 uL of the DyNAmo HS SYBR Green qPCR Kit Finnzymes Oy Espoo Finland and 10 uL of the 1 10 diluted DNA templates Thermal cycling is performed using a Rotor Gene RG 3000 four channel multiplexing system Corbett Research Sydney Australia with the following parameters 95 C initial hold for 15 min to activate the Tag polymerase followed by 50 cycles of amplification with each cycle consisiting of denaturation at 94 C for 10s followed by 20 s of annealing at primer specific temperatures and an extension step of 30 s at 72 C Fluorescence was measured at the end of each amplification cycle for 20 s at primer specific temperatures Wilms et al 2006 To verify the results every quantification was repeated three times at the same concentrations of all chemicals and templates Me
21. ICBM Carl von Ossietzky Universit t Oldenburg 37 5 Microscopy and cell counts Cell number determination of MPN dilution series For the quantification of viable cell a MPN dilution series with appropriate medium is prepared in three parallels For this purpose the culture is diluted stepwise 1 10 and incubated at least for one week The number of wells or tubes that show microbial growth can be correlated to the MPN index which refers to guideline values Table 8 MPN index number of MPN Index confidence interval 95 positive tubes with cells ml upper lower 100 pl 10pl Iyl 0 0 0 1 ard NRF OO Si e S amp S NNNNNNY NNRFrR OO aie iS 3 3 3 3 3 3 3 3 3 3 3 3 WWWNNNFrRRK OOO NR ONFrF CONF ONKF CO Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 38 5 Microscopy and cell counts Quantification of MPN dilution series with SybrGreen I Preparation of staining solution The SybrGreenl stock solution 10 000 x is diluted in TAE buffer pH 7 4 containing 1 of a ascorbic acid solution 1 M The working solution is prepared freshly each day and must offer a five times higher concentration compared to the desired final assay concentration Quantification 200 ul sample from each well of the 2 ml 96 well plates and 50 ul SybrGreen I solution are transferred to black microtiter plates and mixed cautiously Fluorescence is measured after 2 hours of i
22. 1000 ul EDTA 0 5M 500 ul SDS 10 50 ul MQ water to 50 ml Add SDS finally in order to avoid precipitation Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 53 6 Processing of nucleic acids 6 Processing of nucleic acids Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 54 6 Processing of nucleic acids Isolation of DNA Introduction Nucleic acids can be isolated using multiple techniques The method of choice depends on the nature of the nucleic acids to be isolated and the intended purpose Some simple techniques rely on enzymatic digestions e g lysozyme or mechanical decompositions to break up cells e g freeze thaw beadbeater sonication Since nucleic acids obtained by these methods are usually strongly contaminated extraction methods based on organic solvents can be applied to process isolated nucleic acids further The origin of taken samples is crucial regarding the cell disruption Bacterial cells from environmental samples for example are partially protected by a sediment matrix from mechanical forces and enzyme activity The usage of a beadbeater for disrupting bacterial cells cultivated under laboratory conditions would expose them to too much shear forces Besides the generation of heat would destabilise destroy present nucleic acids In this case the cell disruption via freeze thawing which breaks cells physically by sequent cyc
23. BCL 2 gen DNA 11 1 11 BCL 2 gen DNA 11 1 12 BCL 2 gen DNA 1 1 13 BCL 2 gen DNA 1 1 14 BCL2 gen DNA 1 1 Figure 22 Result window Result window The result window shows the Ct values and there standard deviation CT STD DEV the standard defined concentrations GIVEN CONC and the calculated concentrations CALC CONC m Ct b In optimal case the GIVEN CONC And the CALC CONC should have the same value so the CV value Variation coefficient will be 0 The result file can be exported to Excel using the right mouse button Melt The melting curve shows the melting characterization of the PCR samples after finishing the PCR run So you can distinguish between specific and unspecific products primer dimer Amplification products OF dT Primer dimer oa os o4 o 2 resto Figure 23 Melting curve Agarose gel electrophoresis After finishing the pCR run 5 ul of PCR products 3 ul loading dye will be plotted on a 1 5 agarose gel see agarose gel electrophoresis Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 7 Isolation of anaerobes and sediment bacteria 7 Isolation of anaerobic and sediment bacteria Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 76 7 Isolation of anaerobes and sediment bacteria Enrichment and isolation of abundant heterotrophic sediment bacteria Z hlung und Dir
24. E e ee alee eA 40 Determination of the total cell count with SybrGreen I Lunau et al 2004 41 Total cell counts of water samples Hobbie et al 1977 uuenseesssenssssessneennnennneensnnennnnnnnnnn 42 Counting of growing cells using nalidixic acid Kogure et al 1979 uu22uersneensersneenneenn 43 Life Dead Staining Boulos et al 1999 2ucsssessesssssnesssessnnennsnennnnnnnnennnnensnnnnnsnsnnnnnenn 43 Counting of actively respiring CellS eee secseceseceeeeseeeseecsaeceaeeseeeseeeeeecaaecnaecsaeeaeeeseeeaaeenaees 44 Fluorescence in situ Hybridisation FISH 0sssnsesssseesssnnnnnensnnennnennnnnennn nennen 45 CARD Catalysed Reporter Deposition FISH Sekar et al 2003 modified by Ishii et al 48 DOO PD IE N EEE Mate Ae neh aaah Ga wa HER 6 Processing of nucleic acdsa usa 52 isolation on DNAS ee ne en 53 Freeze and Thaw DNA extraction from growing cultures u zucsnessnesnnenseenseennnennnennnennnennnn 54 DNA RNA extraction from sediment samples ccsssssssssseseeeseeseseseeececeeeenensesneceuenesenenenenenes 54 Rapid test for quantifying DNA uursuersseessesssessnensnnsnonsnssnnenennnnnnnnnnnennnosnnnennensnnnsnenn ons 56 DNA extraction from sediments with Fast DNA Spin Ate cr aonsta tener eaten 56 DNA extraction from liquid sariiples ict scicss cicaxelabicanlin Sordi acersihentetats wekekssioctbalaneres sales tel aveceaseh
25. Push the traffic light button gt Push Test name Reinhard Versuch 6 Start the measurement by pushing the button Start test run Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 60 6 Processing of nucleic acids 7 Reporting Push the Excel button gt choose your test name Reinhard Versuch gt Recording of the raw data DNA extraction from aquifer sediments modified from Lueders et al 2004 and Gabor et al 2003 Protocol modified from Lueders T Manefield M and Friedrich M W 2004 Environ Microbiol and Gabor et al 2003 FEMS Microbiol Ecol e Add 0 2 ml 1 PCR cup full of 1 1 mixed 0 1 mm and 0 7 mm Zirconia Silica beads www biospec com to 2 ml bead beating vial with screw cap autoclave e Add 0 8 1 g sediment sample usually 0 5 0 7 ml make a note of precise weight alternatively add 2 mm slices of a 0 2 um filter membrane cut with a sterile scalpel e add 750 ul PTN buffer pH 8 vortex 10 sec also turning over e add 40 ul Lysozyme 50mg ml in TE stable only for a few weeks at 20 C and 10 ul Proteinase K 10 mg ml in TE e Mix well manually incubate 15 min at 37 C in Thermomixer turn over amp shake occasionally e Add 100 ul SDS 20 mix manually and incubate for 15 min at 65 C amp 500 rpm in Thermomixer e Add 100 ul Phenol Chloroform Isoamylalcohol 25 24 1 pH8 e Check that vial is fill
26. Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 93 9 Unrelated experimental procedures Sediment Filter 0 4 pm Siebplatte gefiltertes Porenwasser Figure 32 Filter used for the extraction of pore water Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 94 REFERENCES Amann R amp Ludwig W 2000 Ribosomal RNA targeted nucleic acid probes for studies in microbial ecology FEMS Microbiol Rev 24 5 555 565 Amann R I Krumholz L amp Stahl D A 1990 Fluorescent Oligonucleotide probing of whole cells for determinative phylogenetic and environmental studies in microbiology J Bacteriol 172 2 762 770 Balch W E Fox G E Magrum L J Woese C R amp Wolfe R S 1979 Methanogens Re evaluation of a unique biological group Microbiol Rev 43 2 260 296 Bergmeyer H U ed 1983 Methods of enzymatic analysis Vol 3 Wiley VCH Blaut M amp Gottschalk G 1984 Coupling of ATP synthesis and methane formation from methanol and molecular hydrogen in Methanosarcina barkeri Eur J Biochem 141 1 217 222 Boulos L Prevost M Barbeau B Coallier J amp Desjardins R 1999 LIVE DEAD R BacLight TM application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water J Microbiol Meth 37 1 77 86 Bradford M M 1976 Rapid and sensitive metho
27. a cool and dark place Do not touch with naked hands Contaminations e g dust provoke corrosion Procedure Place some bacterial cell material from the middle of a fresh colony on a clean slide Add one drop of the diluted H O solution using a clean Pasteur pipette Formation of gas O2 shows catalase activity Oxidase test Reagents H 0 100 ml Ascorbic acid 0 18 Tetramethyl p phenylendiamin HC1 1 0g Attention carcinogenic Reagents will be provided freshly prepared every day Do not touch with naked hands and prevent spilling After using vessels and pipettes are immediately cleaned Procedure Soak a strip of filter paper cellulose with some drops of the reagent not too wet Add some bacterial material taken from a colony with an inoculation loop on the strip and rub it using a rounded glass rod Blue colouration indicates oxidase activity As control results are compared with reference strains of known activity e g E coli Determining the substrate spectrum of aerobic isolates The substrate test is the centre of the physiological characterisation of bacterial isolates Bacterial growth is tested in respect of 59 carbon compounds final concentration Complex substrates Peptone 0 05 Casamino Acids 0 05 Yeast extract 0 005 Polymer substrates Cellulose 0 05 Starch 0 1 Chitin 0 05 Xylane 0 05 Laminarin 0 05 Disaccharides Saccharose 5 Cellobiose 5 Maltose 5 Tre
28. and 0 25 ml of solution C The mixture is incubated for 30 minutes at 26 28 C Next 0 25 ml acetone and after 5 minutes 0 25 ml HCI are added Nitrite which is yielded through reduction is determined as already described see 2 Analysis of nitrogen compounds Detection of nitrite Calibration curves are generated based on KNO using standards with concentrations ranging from 0 to 200 uM Principle of the reaction Nitrate is reduced to nitrite However nitrite is not the only product yielded Out of this reason it is necessary to generate calibration curves based on nitrate Calibration curves generated regarding nitrite are not sufficient Abb Abb 2 Nitrit Bestimmung NHo Abb 3 Sulfid Bestimmung Figure 2 Reaction mechanisms with respect to some photometric assays Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 23 2 Analysis of nitrogen compounds Determination of ammonia ammonium Solorzano 1969 There are several colorimetric methods available for determining NH4 concentrations in water samples and soil extract Here we use the indole blue method detecting both NH and NH The method is based on the reaction of NH in alkaline solution with phenate to produce a blue colour indole blue in the presence of a strong oxidizing agent such as hypochlorite The procedure described is in accordance to Solorzano Limnol Oceanogr 14 799 801
29. by manufacturer 20 21 When using do not eat drink or smoke 24 25 Avoid contact with skin and eyes Hazard codes Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 10 E explosive F highly flammable F flammable oxidising T very toxic T toxic Xn harmful O Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 11 C corrosive Xi irritant N dangerous for the environment Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 12 1 Media preparation 1 Media preparation Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 13 1 Media preparation Preparation of growth media The growth media are prepared in special glass vessels according to Widdel 1980 Figure 1 The chemicals are weighed in and dissolved in the below described succession see schemata The bottling tube is covered with tinfoil and the connecting piece for gas inflow offering a cotton filter is closed by a rubber stopper The lateral screw caps are also closed but for autoclaving one of them is screwed on a little in order to prevent the vessel from bursting during heating The medium is autoclaved 30 minutes at 121 C After autoclaving supplementary solutions are added through one of the lateral connecting pieces a sterile addition gt DAN D NN ji squee
30. extracts the same The plate is developed in a shaded chamber If the eluent has risen roughly 16 cm mark with a pencil shortly dry the plate with nitrogen and develop the plate a second time Dry the plate Mark bands on the dried plate carefully using a spatula cover the plate with a glass plate and copy the pattern rapidly to a transparent foil and paper Note down the colours of the bands and identify reference organisms based on the obtained pattern Determination of chlorophylls Oelze 1985 Steenbergen and Korthals 1982 2 5 ml of a bacterial culture are filtered out using glass fibre f 25 mm or membrane filters pore size 0 2 um 25 mm if bacterial cells are tiny After transferring the cells to 3 ml of acetone chlorophylls are extracted over night in the dark at 4 C The extinction is measured against pure extraction reagent at 771 nm Bchl a 663 nm Bchl c 652 nm Bchl d and 647 nm Bchl e The pigment concentration is calculated according to the Lambert Beer law E c d Fe E extinction at absorbance maximum c pigment concentration d width of cuvette 1 cm g extinction coefficient at absorbance maximum for Bchl a 92 3 ml mg cm Bchl c 92 6 ml mg cm Bchl d 98 0 ml mg cm same for Bchl e Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 36 5 Microscopy and cell counts 5 Microscopy and cell counts Methods Script AG Cypionka
31. must be sealed with tape A standard gel contains 1 5 w v agarose which is dissolved in 40 ml 1xTAE buffer The agarose is dissolved in a microwave oven until the solution is clear The solution is then poured into the tray and a comb is inserted so that gaps are developed for the PCR products After 20 min the gel becomes solid and the comb and tape can be removed cautiously 1x TAE buffer 40 mM tris hydroxymethyl aminomethane 1 mM EDTA pH 7 4 adjusted with acetate Figure 10 Placing the comb Electrophoresis The tray is transferred to the electrophoresis chamber which contains 1x TAE buffer Make sure that the gel is covered entirely with buffer 1 ul 6x loading buffer is pipetted on a piece of Parafilm and mixed with 5ul PCR product This mixture is then transferred to the gel 1 5ul standard solution concentration 125ng ul are applied to the gel The Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 62 6 Processing of nucleic acids electrophoreses is carried out at 100V for 40 min or 90V for 45min Pay attention that the correct polarity is chosen Loading buffer 0 25 w v brome phenol blue 40 v v glycerine in 1x TAE After the electrophoresis the gel is stained for 20 min in an ethidium bromide solution 0 8ug ml The documentation is done with a transilluminator Figure 11 Loading the gel Purification of PCR products The PCR products will be purified us
32. of sediment is suspended in 10 ml Tris HCl buffer in combusted gold head tubes and 100 ul Tween 80 solution are added The sample is sonicated five times for 30 seconds 1 ml Tris HCl buffer is placed in a sterile Eppendorf tube and mixed with 5 ul of sample maximal 5 ul Next 10 ul SybrGreen I solution are added mix before 15 ul propidium iodine solution are added mix The sample is incubated for 10 minutes in the dark Subsequently the sample is filtered out on a polycarbonate filter Anodisc Clean the filtration tower and place Tris HCl buffer before adding the sample After filtration the filtration tower is rinsed again with Tris HCl buffer The filter is dried in the dark and is embedded on a microscopic slide Preparations can be stored in the dark at room temperature or in the refrigerator Filters are counted using the BP 450 490 filter set Counting of actively respiring cells Principle An artificial electron acceptor is lipophilic and membrane permeable due to hydrophobic side groups Due to the positive charge of cells offering a membrane potential the artificial electron acceptor is accumulated Within the cells the acceptor is transformed into its crystallised state due to electron transfers Crystallised states can be coloured e g TTC or fluorescing e g CTC Vey 2H42e Yen HCl amp zz n nfc N N TTC TPF Abb 15 Reduktion von Triphenyltetrazoliumchlorid TTC zur Triphenylformazan TPF
33. once without nalidixic acid Samples are incubated for 8 24 hours Subsequently samples are fixed using paraformaldehyde and the total cell counts are determined as well as the number of non natural elongated cells If obtained cell numbers are low cells should be concentrated through filtering them out using filters with a pore size of 0 2 um Life Dead Staining Boulos et al 1999 Principle SybrGreen I and propidium iodine are intercalating agents that fluoresce if being exposed to UV light Figure 7 SybrGreen I enters all cells while propidium iodine only enters dead cells due to its charge since dead cells offer no intact cell membrane Corresponding cells appear orange if being excited at 450 490 nm Figure 7 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 44 5 Microscopy and cell counts Figure 6 Intercalating substances Left hand site incorporation Right hand side Life Dead Staining Reagents all solutions are prepared in combusted Pfennig bottles 12 hours 180 C 11 Tris HCl buffer 10 mM pH 8 sterile filtrated Tween 80 0 5 sterile filtrated only needed for examining sediment samples SybrGreen I 1 200 dissolved in Tris HCl buffer propidium iodine 1 mg ml Tris HCl buffer mounting solution 1 1 mix of glycerine and Tris HCl buffer pH adjusted to 8 900 ul of this solution is mixed with 100 ul Phenol diamine keep refrigerated Procedure 1 g
34. seal test tubes with glass marbles immediately cool down icy water add 2ml of reagent B mix incubate for 30 minutes at 37 C water bath if solutions become turbid centrifuge down particles Measure extinction at 546 nm generate a calibration curve using BSA standards stock solution 50 mg ml ranging from 0 bis 10 mg preparation Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 34 4 Determination of proteins and bacterial pigments Bradford protein assay 1976 The assay is based on the binding of Coomassie Brilliant Blue G 250 to protein When binding to protein occurs the absorbance maximum of the dye shifts from 465 nm to 595 nm Therfore absorbance can be measured photometrically at 595 nm The assay is quick and reliable since a visible colour change occurs after 2 min and the extinction coefficient of a dye albumin complex solution is constant over a 10 fold concentration range Furthermore both hydrophobic and ionic interactions stabilize the anionic form of the dye i e there is no or neglible disturbance by natrium and kalium ions or carbohydrates like sugars Disturbances are only known from concentrated detergent like Sodiumdodecylsulfate SDS Trition X 100 or commercially available solutions Controls are recommended Bradford reagent Dissolve 100 mg Coomassie Brilliant Blue G 250 in 50 ml 95 ethanol add 100 ml 85 w v phosphoric acid Dilute to 1 liter when th
35. slides The ethanol baths can be used several times Hybridisation of the samples You need 7 ul hybridisation buffer for every single sample prepare fresh and 1 ul probe 850 ng ul Prepare the master mix for the appropriate amount of sample and preheat them 46 C Afterwards pipette the hybridisation solution 8 ul for every sample into the slots Put the microscope slides carefully into the hybridisation chambers and incubate them 46 C 2 h Stop the reaction Put two microscope slides in one Falcon tube back on back with cold water sway it and discard the water Remove non specific probes Put two microscope slides in one Falcon tube back on back with preheated washing buffer and sway them 48 C 20 min Discard the washing buffer flush the microscope slides with ddH20 and dry them on air Incorporation and counter staining Pipette 1 ul of Vecta Shield solution into every single slote Put a cover glas onto the microscope slide and microscope after ca 5 min or store the slides in the dark Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 48 5 Microscopy and cell counts CARD Catalyzed Reporter Deposition FISH Sekar et al 2003 modified by Ishii et al 2004 In environmental samples single oligonucleotides carrying only one fluorochrome may not provide enough fluorescence signals to detect cells with low ribosome content Pernthaler et al 2002 The Catalyzed Reporter Dep
36. solution to a final volume of 11 B formaldehyde 32 Procedure Using distilled water the sample is filled up to a volume of 8 9 ml Next 1 ml of reagent A and 0 1 ml of reagent B are added mix After 10 minutes the extinction is measured at 570 nm against a sulphite free preparation Colorimetric determination of sulphur Chan and Suzuki 1993 Solutions 1 10 ml distilled water 190 ml acetone 2 0 2 g NaCN 125 ml of solution 1 3 0 4 g FeCl 6 H O 5 ml distilled water 4 acetone 5 petroleum ether 6 6 4 mg S in 10 ml DMSO final concentration 20 mM 7 3 2 mg S in 10 ml petroleum ether Procedure prepare a S standard series using solution 6 white precipitate and buffer ranging from 5 1000 uM 0 uM blank extraction 0 5 ml bacterial suspension standard 1 0 ml of solution 5 in Eppendorf tubes mix for 30 seconds centrifugation 14 000 rpm 10 minutes Eppendorf centrifuge supernatant becomes clear preparation 0 5 ml supernatant 1 0 ml of solution 2 in Eppendorf tubes mix and allow reaction to occure 2 minutes Measurement 0 95 ml solution 4 0 05 ml solution 3 0 50 ml sample in Eppendorf tubes mix a brownish precipitate develops centrifugation 14 000 rpm 10 minutes Eppendorf centrifuge measure extinction at 464 nm Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 29 3 Analysis of sulphur and phosp
37. the anaerobe growing cells will be kept locked until the end of the experiments even if the OD gt 0 3 They will be measured against a tube containing water Measurements are performed using a photometer which can handle tubes Tubes are adjusted in a way that the measured OD is as low as possible The precise position is marked For further measurements tubes are always set in the same position Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 71 7 Isolation of anaerobes and sediment bacteria Isolation of anaerobes from agar deeps Preparation of so called shaking tubes 300 ml double distilled water and 12 g grinded agar are added to a 500 ml Schott bottle determine the empty weight including lid and magnetic stir bar The suspension is stirred for 10 minutes Afterwards the agar is allowed to settle down 20 minutes The water is carefully decanted The performed washing step is necessary to remove growth inhibiting ingredients and hydrolysis products The procedure is repeated five times Subsequently the agar is filled up on a balance 300 g empty weight of the bottle and autoclaved 20 minutes 120 C Place used pipettes in a Erlenmeyer flask filled hot water and boil them out Agar dilution series Needed material equipment per series 7 properly labelled sterile test tubes and suitable rubber stoppers keep some backup stoppers ready 5 times washed sterile agar 4
38. using the LiCor DNA Sequencing System 4200 of MWG Biotech Preliminaries The sequencing reaction via Sanger is a PCR like method using just one primer Since the use of several cycles of temperatures is also necessary it is spoken of Cycle Sequencing In order to encode one single sequence four different approaches are necessary each of them containing another didesoxynucleotide ddNTP chain abruption nucleotide The reaction approach contains up to 3 of the general dNTP s All ddNTP s are incorporated just like the general dNTP s by using the DNA polymerase However the ddNTP s induce a chain abruption because of the lack of the OH group on the 3 end Depending on a statical distribution of the incorporation of ddNTP s you will obtain a mixture of DNA fragments with a Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 63 6 Processing of nucleic acids distinct length ending with a particular nucleotide e g the nucleotide of the ddATP approach is an A see figure beneath The four different approaches will be plotted next to each other on a gel and in the following the mixture of DNA fragments will be separated due to length by electrophoretic separation short fragments running faster than the long ones The detection of the DNA bands is carried out via computer controlled laser and is possible because of the flourescence excitation of the bands The comparison of the band
39. was washed using distilled water and dried is added to the volurimetric flask The flask is closed with a rubber stopper The prepared stock solution contains approximately 5 mM sulphide 5 nmol l using 0 6 g NaS 9 H O and is stable for 1 day if kept under nitrogen For generating calibration curves 5 ml H gt O are placed in 12 test tubes that are closed using rubber stoppers and flushed with nitrogen for 20 minutes Using a Hamilton syringe 0 2 5 10 20 and 50 ul 0 250 nM each in duplicate of the stock solution are added Mix and directly add 0 4 ml of the reagent using a 1 ml syringe After 20 minutes the extinction is measured at 670 nm against a sulphide free blank In order to calculate the concentration of the NaS solution the molecular weight of NaS 9 H20 is used For very precise measurements it is necessary to add an aliquot of the NaS solution to an exactly adjusted acidic J KJ solution Next the excessive iodine is titrated using a Na gt S gt 0 solution The principle behind the assay is depicted in Figure 2 Determination of dissolved sulphide Ruwisch 1985 Sulphide is the final product of dissimilatory sulphate reduction The presence of dissolved sulphide in cultures can be rapidly proven by its colloidal precipitation as CuS in a copper sulphate reagent and quantified photometrically Copper reagent HCI 50 mM CuSO 5 mM Chemical reaction CuSO H2S gt CuS HS0 Procedure Remove 0 2 ml of
40. 6 0 Application use 1 ml of the respective solution per litre of medium SL9 like SL11 instead of EDTA Di Na Nitrilotacetic acid NTA 12 8 g Preparation of Selenite Wolframate Solution Widdel 1980 Table 4 Selenite Wolframate Solution distilled H O 1000 ml NaOH 0 48 Na SeO 5 H O 6 mg Na WO 2 H2O 8 mg Preparation of vitamin solutions Table 5 Vitamin solutions 7 Vitamins solution 10 Vitamins solution distilled H O 180 ml 1000 ml Biotin solution 20 ml Biotin 10 mg Nicotinic acid 20 mg 25 mg Thiamine Dichloride 10 mg 25 mg p Aminobenzoic acid 10 mg 25 mg Ca D Pantothenic acid 5 mg 25 mg Pyridoxamine Dihydrochloride 50 mg 50 mg Cyanocobalamine Vit B12 10 mg 5 mg Folic acid 10 mg Riboflavin 25 mg Lipoic acid Thioctic acid 25 mg according to Pfennig 1978 gt 5_times concentrated according to Balch et al 1979 10 mg Biotin in 100 ml H2O slight heat supply facilitates dissolving After sterile filtration prepared solutions are filled in sterile screw cap vials Vials are stored in the dark under cool conditions Application 1 ml per litre medium 7 Vitamins solution 2 ml per litre medium 10 Vitamins solution Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 17 1 Media preparation Setting up HPG agar plates Preparing the medium The recipe of the HPG medium is based on the mineral base medium for marine aerob
41. Eppendorfcap After precipitation with 90ul ethanol sodium acetate the DNA can be stored at 70 C DNA extraction from liquid samples Water samples have been filtered using 0 2 um polycarbonate filter In an Eppendorf tube 0 5 g zirkonia pearls 20 ul SDS 25 600 ul phosphate buffer and 600 ul phenol chloroform isoamylalcohol have been added to the filter In order to disrupt the cells and to remove cell remnants and foreign particles from the bacterial DNA the Eppendorf tube was thoroughly vortexed Next the tube was incubated for 10 minutes at 60 C using a water bath and again vortexed for 3 minutes The organic phenol phase was separated from the aqueous phase by centrifugation for 10 minutes 10000 rpm at room temperature The aqueous phase containing the DNA was transferred into a new Eppendorf tube 300 ul phosphate buffer have been added to the original sample which was vortexed for 1 minute and again centrifuged for 10 minutes The aqueous supernatant was unified with the already transferred one 1 ml phenol chloroform isoamylalcohol was added to remove the remaining impurities and the solution was vortexed and centrifuged as already described As long as a white precipitate was visible at the interphase the procedure was repeated by adding 1 ml phenol chloroform isoamylalcohol again Subsequently the aqueous solution was allowed to precipitate over night after adding 30 ul sodium acetate and 2 25 ml isopropanol By centrifug
42. Folin C reagent Merck is mixed with 2 moieties of distilled water e NaOH 0 3 M Procedure 10 ml cell suspension are centrifuged in the refrigerated centrifuge 6000 g 10 minutes and washed with saline solution 0 6 The pellet is thoroughly resuspended in saline solution and is filled up to a volume of 10 ml The protein content is determined in triplicate using 1 ml of the suspension respectively In order to digest cells 0 5 ml of 0 3 M NaOH are added and the samples are incubated at 60 C for 90 minutes water bath After cooling down 5 ml of copper reagent are added while shaking The samples are kept in the dark for 10 minutes 0 5 ml of Folin s reagent are added mix and the samples are kept for further 30 minutes in the dark Subsequently the samples are centrifuged 6000g 10 minutes and the extinction is measured at 623 nm against a blank Serum albumine is used for preparing a standard series ranging from 10 200 g per preparation Determination of proteins according to Schmitt 1969 modified Biuret protein assay according to La Rivi re 1958 Reagents A NaOH 4M 160 g l B K Na tartrate 5g NaOH 4g CuSO 5 H O lg KJ 2 5g in H O 400 ml Procedure 10 ml of cell suspension are washed with saline solution 0 9 and centrifuged the supernatant is discarded resuspend pellet in 5 ml saline solution 0 9 add 0 5 ml of reagent A mix cook samples for 10 minutes water bath
43. G Dewaal E C amp Uitterlinded A G 1993 Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S ribosomal RNA Appl Environ Microbiol 59 3 695 700 Oelze J 1985 Methods in Microbiology Academic Press pp 257 284 Pachmayr 1960 Vorkommen und Bestimmung von Schwefelverbindungen in Mineralwasser Technical report Universit t M nchen Pernthaler A Pernthaler J amp Amann R 2002 Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria Appl Environ Microbiol 68 6 3094 3101 Pfennig N 1978 Rhodocyclus purpureus gen nov and sp nov a ring shaped vitamin B12 requiring member of family Rhodospirillaceae Int J Syst Bacteriol 28 2 283 288 Pfennig N amp Wagener S 1986 An improved method of preparing wet mounts for photomicrographs of microorganisms J Microbiol Meth 4 5 6 303 306 Schonhuber W Fuchs B Juretschko S amp Amann R 1997 Improved sensitivity of whole cell hybridization by the combination of horseradish peroxidase labeled oligonucleotides and tyramide signal amplification Appl Environ Microbiol 63 8 3268 3273 Sekar R Pernthaler A Pernthaler J Warnecke F Posch T amp Amann R 2003 An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridiza
44. OLD button Standard curve This standard curve shows the Ct values plotted against chosen units concentration copy numbers You can chance the standard curve by adding or deleting standard values The blue points indicate the standard and the red points the unknown samples R value Correlation coefficient Number between 1 and 1 defines the relation of 2 variables y mx b in the curve value between 0 99 and 0 99 Standard Curve Export to JPEG conc 10 7 0 230 CT 11 461 Singlet Concentration Type Floating Import Curve Reset 10 2 10 3 10 4 10 5 10 6 10 Concentration Figure 21 Standard curve CONC Defines the correlation between Ct value and target concentration of the template TYPE FLOATING The standard curve will be calculated again by changing the threshold TYPE FIXED The standard curve stays unchanged by changing the threshold For instance by using the standard curve also for other PCR runs IMPORT CURVE You can choose and import a standard curve from another PCR run and use it for your current calculations Methods Script AG Cypionka ICBM Carl von Ossietzky Universitat Oldenburg 74 6 Processing of nucleic acids HE Quant Results Cycling A FAM A BCL 2 gen DNA 1 1 2 BCL 2 gen DNA 1 1 3 BCL2 gen DNA 1 1 4 BCL 2 gen DNA 1 1 5 BCL 2 gen DNA 1 1 6 BCL2 gen DNA 1 1 7 BCL 2 gen DNA 1 1 8 BCL2 gen DNA 11 1 9 BCL 2 gen DNA 1 1 10
45. Put fixed sediment sample on ice and sonicate it for 20 sec with lowest intensity 2 Vortex sample and dilute 20 50 ul of the supernatant in 10 ml 1x PBS buffer 3 Filtrate the diluted sample onto a polycarbonatefilter 0 2um 4 Wash with sterile 1x PBS 5 Let filter air dry 3 Embedding 6 Boil Agarose 0 1 0 2 and let it cool down to 35 40 C 7 Dip filters in Agarose and place filters face up onto filter paper and air dry 10 min 4 Permeabilization 8 Incubate filter in fresh lysozyme solution 37 C gt 60 min 9 Wash in ddH O 5 Inactivation of endogenous peroxidases 10 Incubate in H20 0 15 in methanol for 30 min at RT 11 Wash with ddH O 5 10 ml 1min 12 Wash with 96 Ethanol 5 10 ml 1 min and let filters air dry 6 Hybridization 13 Cut filter sections 14 Mix hybridization buffer with probe working solution 40041 buffer 1 3ul probe in a 0 5 ml vial Place filter sections in reaction vial 3 4 sections per vial 15 Incubate at 46 C for at least 1 5h up to 24h 7 Washing 16 Transfer filter sections to prewarmed washing buffer 48 C and wash for 15 min 17 Remove excess liquid with blotting paper but don t let filters run dry 8 CARD 18 Incubate in 1x PBS 10 ml 1 5 min RT 19 Dab filters on blotting paper but do not let filter run dry 20 Incubate in substrate mix Mix 200ul PBS with lul H20 30 Mix tyramide with 20ul DMF Dimethylformamide Mix 1 5 ml Amplification buffer 15
46. R 2004 An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments FEMS Microbiol Ecol 50 3 203 212 Kelly D P Chambers L A amp Trudinge P A 1969 Cyanolysis and spectrophotometric estimation of trithionate in mixture with thiosulfate and tetrathionate Anal Chem 41 7 898 901 Kogure K Simidu U amp Taga N 1979 Tentative direct microscopic method for counting living marine bacteria Can J Microbiol 25 3 415 420 La Riviere J W M 1958 On the microbial metabolism of the tartaric acid isomers Technical report University of Delft Netherlands Liaaen Jensen S amp Schmidt K 1963 Die Carotinoide der Thiorhodaceae 3 Die Carotinoide von Chromatium warmingii migula Arch Mikrobiol 46 2 138 149 Lowry O H Rosebrough N J Farr A L amp Randall R J 1951 Protein measurement with the folin phenol reagent J Biol Chem 193 1 265 275 Lueders T Manefield M amp Friedrich M W 2004 Enhanced sensitivity of DNA and rRNA based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients Environ Microbiol 6 1 73 78 Lunau M Lemke A Walther K Martens Habbena W amp Simon M 2005 An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy Environ Microbiol 7 7 961 968 Muyzer
47. Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 82 8 Physiological characterisation Fluorophore stock solution 10 mg 4 methylumbelliferone are dissolved in 5 ml ethylene glycol monomethyl ether and 5 ml double distilled water are added glown out vessel 100 ul of this solution are filled up to a final volume of 100 ml final concentration 1 ug ml using sterile double distilled water in a glown out vessel The solution is stored in the dark at 4 C Glycine buffer 0 75 g glycine and 0 58 g sodium chloride are dissolved in 100 ml of sterile double distilled water glown out vessel The pH is adjusted to 10 using 1 M sodium hydroxide Procedure Per sample three parallels are set up 100 ul of substrate analogue solution are added to 1 ml of sample Blank 1 ml of sterile double distilled water in a glown out vessel 100 ul of substrate analogue solution Samples are incubated at 25 C for two hours using a water bath In order to stop the reaction 75 ul of 0 1 M glycine buffer pH 10 are added The samples are well mixed and measured at an excitation wavelength of 360 nm and an emission wavelength of 440 nm against a blank using a spectrofluorophotometer If a high background fluorescence is detected the blank is prepared by mixing 1 ml of sample with 100 ul of sterile double distilled water and treated the same way as the samples The blank is measured against a fluorometric blank 3 3 ml of sterile d
48. Verunreinigungen anorganisch 31434 Used filters and soaked up material charcoal etc Verbrauchte Filter und Aufsaugmassen Aktivkohle 31435 Used filters and soaked up material with harmful impurities Verbrauchte Filter und Aufsaugmassen mit sch dlichen Verunreinigungen 52102 Acids acidic solutions etchants acidic S uren S uregemische Beizen sauer 52402 Bases alkaline solutions etchants alkaline Laugen Laugengemische Beizen basisch 52403 Ammonia solution Ammoniak L sung 52713 Concentrates and semi concetrates containing cyanide Konzentrate und Halbkonzentrate cyanidhaltig 55220 Solvent mixtures including halogenated organic solvents L semittelgemische inklusive halogenierte organische L sungsmittel 55370 Solvent mixtures without halogenated organic solvents L semittelgemische ohne halogenierte organische L sungsmittel 57124 Ion exchange resins Ionenaustauscherharze 57127 Plastic containers with harmful leftovers Kunststoffbeh ltnisse mit sch dlichem Restinhalt 59301 Fine chemicals Feinchemikalien 59302 Chemicals leftovers organic Laborchemikalienreste organisch 59303 Chemicals leftovers inorganic Laborchemikalienreste anorgansich R S Statements Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg Indication of particular risks R 1 Explosive when dry 2 Risk of explosion by shock friction fire or othe
49. al thionates is possible by performing the cyanolysis at different temperatures and using CuSO4 as catalyst Using iron ID obtained thiocyanates can be quantified photometrically 0 C I S406 3 CN H 0 gt 5 03 SO 2 HCN SCN 0 C CuSO4 I 505 CN SO SCN 100 C CuSO4 III S306 3 CN HO gt SO SO 2 HCN SCN Only the concentration of tetrathionate is determined by measuring preparation I Measuring preparation II allows the determination of tetrathionate thiosulphate yielded from cyanolysis and thiosulphate already present in the preparation Preparation II identifies all present thionates Solutions 1 NaH PO NaOH buffer 1M pH 7 4 2 KCN 1 25M 3 CuSO 5 HO 0 375 M 4 Fe NO3 3 H O 1 5M dissolved in 4 M HCIO increased volume due to dissolving gt 100 ml HCIO 92 g Fe NO gt 150 ml total volume As standards 1 mM solutions of thiosulphate tetrathionate and trithionate are prepared daily Procedure 0 06 ml of solution 1 are placed in each test tube Up to 2 25 ml of sample can be added Distilled water is used to fill up the preparations to a final volume of 2 31 ml A standard is prepared as mixture of all three thionates For each preparation a blank is set up 3 Preparations I I ID Preparation I Preparation I is cooled down to 0 C for 10 minutes before 0 06 ml solution 2 and 0 06 distilled water are added mix The preparation is
50. and mark Generate numeric data report and press OK Mark only P in the next window Choose device A and the files will be saved as text files Analysing the data Using Data Plot is the easiest way to plot and integrate the data Preparing the samples Material 20 ml autoclaved glass bottles 23mm diameter N20 opening rubber stoppers and hooks medium page or sterilized sea water Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 91 9 Unrelated experimental procedures inoculum e g sediment substrate optionally inhibitor For anaerobic growth experiments use the anaerobic chamber to fill the bottles with medium and sediment Put the filled and closed sample and reference ampoules attached to a hook respectively slowly into the equilibration position of the microcalorimeter Make sure that the ampoules are clean and dry After at least half an hour the ampoules can be lowered to the measuring position again slowly The microcalorimeter is so sensitive that it can even measure frictional heat caused by vibrations When the heat flow is constant approximately after two days the substrate can be added Determination of physicochemical gradients Various physical and chemical gradients of the water column and the upper 50 cm of the sediment are determined The resolution should be 1 cm The oxygen profile is determined by an oxygen electr
51. ater on Putthe gel bracket into the staining tank and fill in the staining solution Transfer the gel into the staining tank by inverting the glas plate with the gel and holding it into the staining solution Use 1x Sybr Gold staining solution in order to stain the DNA light sensitive Cover the staining tank with the another black tank Shake slightly 1 2 hours Transfer the staining solution back into the bottle the solution can be used several times Cover the gel with ddH O and incubate it 15 minutes Document and save the gel parameters on the UV blue light table using a documentation program Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 69 6 Processing of nucleic acids Wear cloves and a lab coat during staining procedures Figure 15 Staining tank Quantitative PCR The quantitative PCR also called Real Time PCR is the technique of collecting data throughout the PCR process as it occurs thus combining amplification and detection into a single step This is achieved using a variety of different fluorescent chemistries that correlate PCR product concentration to fluorescence intensity Higuchi et al 1993 The time point where the target amplification is first detected depends on the amount of targets in the template This value is referred to as cycle threshold C the time at which fluorescence intensity is greater than the background signal Wong et al
52. ation 13000 rpm 30 minutes 4 C present DNA was pelleted The supernatant was discarded and the pellet washed with 1 5 ml ethanol 70 The solution was centrifuged again 13000 rpm 10 minutes 4 C the supernatant removed and the pellet dried for 3 minutes using the Speed Vac The precipitated and dried DNA was taken up in 50 ul PCR water ready for use Quantification of DNA using PicoGreen lul of sample is mixed with 899 ul TE buffer For quantification 100 ul PicoGreen reagent PicoGreen 1 40 in TE buffer are added per sample Using a blank and a standard blank 900 ul TE buffer 100 ul PicoGreen reagent standard 899ul TE buffer 1 ul herring sperm 100 ng ul 100 PicoGreen reagent the DNA content is determined By means of a spectrofluorophotometer Shimadzu the emission was measured at 540 nm and the extinction at 460 nm Measured values refer to ng ul In this context the prepared standard should not differ Before the measurements samples have to vortexed and incubated in the dark for 5 minutes During the whole procedure gloves and lab coats should be worn Waste is discarded in the F rbeabfall Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 59 6 Processing of nucleic acids DNA Quantification via a Microtiter plate reader Since it is necessary to know the accurate amount of DNA for sequencing Real Time PCR and DGGE you have to quantify them The quantification i
53. bstrate analogue solution are added A standard series is prepared along with each test series and treated the same way as the samples After incubating them at 30 C for 24 hours the pH of each well is adjusted to 10 The plates are analysed at an excitation wavelength of 355 nm and an emission wavelength of 460 nm Determination of ATP Bergmeyer 1983 The determination of ATP is based on the reaction of ATP with luciferin and oxygen in presence of the enzyme luciferase Luciferase ATP Luciferin O gt Oxyluciferin AMP PP CO Light If the reagents besides from ATP are present in excess the light emission is proportional to the amount of ATP present Using a calibrated measuring system the measured light intensity can be related to the ATP content of the sample The light emission is determined using a luminometer LKB Wallac The intended intensity is adjusted by an internal C emitter A recorder at the outlet of the luminometer collects measured values in mV In order to enable the determination of the ATP content of cells it is necessary to disrupt them first Extraction of ATP Blaut and Gottschalk 1984 100 ul of 3 M perchloric acid stored on ice are pipetted into an Eppendorf tube 100 ul of sample are added and the solution is shaken shortly The shaking is repeated every half an hour samples stored on ice After 1 1 2 50 ul of 1 M Tres buffer pH 7 4 and 112 ul of 3 M KOH are added to adjust the
54. buffer using a syringe Finally mount the cover and the electric cable carefully You can find further information in DNA Sequencing and Genetic Analysis Manual section Gel preparation and Electrophoresis Electrophoresis settings Voltage 1500 V Amperage 35 mA Power 45 Watt Temperature 50 C Motor speed 2 Signal Channel 2 Frames 25 Loading the gel Stop the electrophoresis after the prerun is done Take off the cover of the upper buffer chamber and spill the gel slots a second time Attach the comb 64 sharkstooth comb to the gel Denature the samples 70 C 3 5 minutes and store them on ice before plotting them onto the gel using a 8 channel Hammilton syringe max 0 8 ul sample for every single slot Cover the buffer chamber and start the electrophoresis The run lasts 5 10 hours The generated data will be stored automatically Denotation of the bases and ambiguities according to the IUPAC glossary IUPAC Meaning Complement A A T C C G G G C T U T A M A C K R A G Y W A T W S C G S Y C T R K GT M V A C G B H A C T D D A G T H B C G T V N G A T C N Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 66 6 Processing of nucleic acids DGGE Denaturing Gradient Gel Electrophoresis The DGGE is a molecular technique used to separate different DNA sequences of the same length from each other The separation is carried out via a polyacrylamidegel composed of a grad
55. cesceseceseceseceseeeseeeseecsaeeaecesecseeeaecaecaeeaeeees 30 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg LBs 4 Determination of proteins and bacterial pigments ssccssssssscssssssssccsssssssceees 32 Lowry protein ss y 2 28 228 Rah Asal ee NS eR a Ge a a 33 Determination of proteins according to Schmitt 22ue220essuenseesnnesnnesseensnennnennnennennnnnnnnnnn 33 Bradford protem assa ys cet nenn nike 34 Determination of carotinoides Eichler and Pfennig 1986 uuseensennneennennsnnen 35 Determination of chlorophylls Oelze 1985 Steenbergen and Korthals 1982 35 5 Microscopy and cell counts nasnas ehesten 36 Cell number determination of MPN dilution series 2200002220sssensneessnnennennnnenennn nennen 37 Quantification of MPN dilution series with SybrGreen L eeeesseeeseeeeeseeeeseeerssreserrrsresresressesres 38 Determination of bacterial counts and cfu from water and sediment samples Cavalli Sforza 38 VOTZ e E IR ER ET NS eea OE oE ET Preparation of slides coated with agarose for microscopy Pfennig and Wagender 1986 modified after Cy pionka meca iina a E E R E EE AEE E NEE a E E a K Determination of gram type gram differentiation Bartholomew 1962 Gregersen 1978 39 Hlagell st ning Ryu 1937 oiee E R a ania 40 Total cell count ze as 2 ee Ra Rete ad Ae ohne a a e
56. culture from the culture vessel using a syringe Inject 0 1 ml culture free of gas bubbles into 4 ml of copper reagent dispensed into glass tubes Vortex and transfer the solution into a cuvette The absorbance is immediately measured at 480 nm using a photometer the colloidal CuS solution remains stable for 20 40s Copper reagent free of sulphide serves as blank Sulphide standard preparation Washed crystals of Na S 9H 0 13 g are dissolved in 50 ml anoxic water to serve as a stock solution 1M Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 26 3 Analysis of sulphur and phosphate compounds the final concentration should be determined via titration An anoxic dilution series of dissolved sulphide is prepared in the desired range 0 30mM An aliquot of the stock solution is anaerobically transferred into a Hungate tube containing anoxic water After shaking an aliquot of this mixture transferred into a second tube and so on Calibration curves should be linear up to an absorbance of 0 5 Turbidometric determination of inorganic sulphate Tabatabai 1974 Reagents A Dissolve 10 g citric acid H gt O in 80 ml distilled water Mix the solution with 120 ml of glycerine 95 B Dissolve 0 5 g BaCl 2 H O and 5 g citric acid H gt O in 50 ml distilled water Procedure 2 ml of sample centrifuged if necessary are mixed with 2 ml of reagent A keep an eye on cords 0 5 ml of solutio
57. d for quantitation of microgram quantities of protein utilizing principle of protein dye binding Anal Biochem 72 1 2 248 254 Cavalli Sforza L 1972 Biometrie Urban amp Fischer Chan C W amp Suzuki I 1993 Quantitative extravtion and determination of elemental sulfur and stoichiometric oxidation of sulfide to elemental sulfur by Thiobacillus thiooxidans Can J Microbiol 39 12 1166 1168 Chaney A amp Marbach E 1962 Chemical Analyses Vol 8 John Wiley and Sons Colorimetric determination of nonmetals Cline J D 1969 Spectrophotometric determination of hydrogen sulfide in natural waters Limnol Oceanogr 14 3 454 458 Cordruwish R 1985 A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfate reducing bacteria J Microbiol Meth 4 1 33 36 Cypionka H amp Pfennig N 1986 Growth yields of Desulfotomaculum orientis with hydrogen in chemostat culture Arch Microbiol 143 4 396 399 Danovaro R Dell anno A Trucco A Serresi M amp Vanucci S 2001 Determination of virus abundance in marine sediments Appl Environ Microb 67 3 1384 1387 DeLong E F Wickham G S amp Pace N R 1989 Phylogenetic stains ribosomal RNA based probes for the identification of single cells Science 243 4896 1360 1363 Eichler B amp PFfennig N 1986 Characterization of a new platelet forming purple sulfur bacterium Am
58. d with water pH 4 0 and 7 5 phenol chloroform saturated with water pH 4 0 and 7 5 SDS extraction solution 9 6 ml 20 sodium dodecyl sulphate SDS 2 4 ml 0 5 M sodium acetate pH 7 5 and 66 4 ml ddH O pipetted into a 100 ml Pfennig bottle and autoclaved TE buffer pH 8 0 10 mM Tris Base 1 mM EDTA prepared in ddH O pH adjusted using HCl and autoclaved all solutions are set up with DEPC treated ddH O to eliminate DNases and RNases autoclaving is done for 20 minutes at 121 C Cell disrupting using the beadbeater 1 g sediment 1 g zirkonia pearls and 1 ml SDS extraction solution are transferred into one cryo vial Using a beadbeater samples are shaken for 1 minute 5000 rpm The sediment and the zirkonia pearls are centrifuged down and the supernatant is transferred to a 1 5 ml Eppendorf tube and stored on ice try to prevent the accidental transfer of sediment 500 ul phenol are added to the to the cryo vial After shaking the vial again using the beadbeater sediment and pearls are again centrifuged down and the supernatant is unified with the previous transferred one The procedure is repeated again this time adding 250 ul SDS extraction solution and 250 ul phenol pH 7 5 For a combined DNA RNA extraction the supernatant is splitted before being subjected to a phenol chloroform extraction Phenol chloroform extraction Phenol pH 4 for RNA pH 7 5 for DNA is added in a 1 1 ratio to nucleic acid solution After revers
59. e 2 Mineral base medium for marine aerobic bacteria distilled H O NaCl MgCl 6 H O CaCl 2 H O KCl NaSO Trace element solution SL 10 Selenite Wolframate Solution 1000m HEPES KBr 0 84 M 24 32 g H3BO 0 4 M 108 SrCh 0 15 M 158 NH CI 0 4 M 0 66 g KH gt PO 0 04 M 4g NaF 0 07 M 1 ml 0 2 ml 1 Media preparation 2 38 g 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml KBr H3BO3 SrCl NHsCl KH PO NaF are added from sterile stock solutions Before autoclaving the pH of the medium is adjusted to 7 2 7 4 using 4 M NaOH after autoclaving the medium is allowed to cool down before the following solutions are added NaHCO solution 10 Vitamine solution 5 times conc 0 2 gin 10 ml H O 2 ml Preparation of trace element solutions Tschech and Pfennig 1984 Table 3 Trace element solutions distilled H O HCI 25 EDTA Di Na salt FeSO 7 H O FeCl 4 H O CoCl 6 HO MnCl 2 H O ZnCl NiCl 6 HO Na MoO 2 H O H BO CuCl 2 H O D first dissolve FeCl in HCl SL10 1000 ml 10 ml 1 5g 190 mg 100 mg 70 mg 24 mg 36 mg 6 mg 2 mg SL11 1000 ml 5 2g 1 5g 190 mg 100 mg 70 mg 24 mg 36 mg 6 mg 2 mg SL12 1000 ml 3 08 lig 190 mg 50 mg 42 mg 24 mg 18 mg 300 mg 2 mg Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 16 1 Media preparation before filling up with water adjust pH to
60. e dye has completely dissolved Final concentrations are 0 01 w v Coomassie Brilliant Blue G 250 4 7 w v ethanol and 8 5 8w v phosphoric acid The Bradford reagent should be light brown reddish in color In case of blue components filtrate the solution using a round filter Standard procedure 1 Transfer up to 5 ml of homogenised growing culture into 15 ml centrifuge tubes Centrifuge for 15 min 4000rpm 4 C Decant supernatant and freeze the pellet until the Bradford assay is carried out 2 Preheat a water bath 100 C 3 Add 500 ul bidestilled water and 500 ul NaOH 0 5 M to the cell pellet and mix thoroughly 4 Cook the suspension for 10 min at 100 C 5 Transfer 200 ul in three parallels into 1 5 ml Eppendorf reaction tubes Mix with 800 ul of Bradford reagent 6 Incubate for 30 min at room temperature 7 Transfer the whole volume into a half micro disposable cuvette 8 Measure the absorbance at 595 nm using the photometer Don t forget blind controls 9 Note all values and use the mean value for further calculations Calibration curve A calibration curve is made from bovine serum albumin BSA A stock solution 1mg ml is diluted with bidistilled water in triplicates to prepare standard solutions with concentrations ranging from 0 to 8 ug protein 200ul Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 35 4 Determination of proteins and bacterial pigments Determinatio
61. e gel solution doesn t reach the comb during the casting Preliminarily you have to remove the cannula and let the residual solution drop out into a beaker Add APS and TEMED to the 0 solution Absorb the solution using a 10 ml syringe and put a cannula onto it Pipette the solution carefully on one side onto the gel Change sides Take care that the gradient doesn t reach the comb Cast the gel up to the border of the plates so that the gel will almost overflow Wait at leat 2 4 hours until the gel is polymerized Spill all beakers tubes and equipment you have used with ddH O Stock solutions 0 and 80 Substance Stock solution 0 denaturing Stock solution 80 denaturing Urea 50 4 g Formamide 48 ml 50 x TAE pH 7 4 3 0 ml 3 0 ml Acrylamide Bisacrylamide 22 5 ml 22 5 ml Fill up with ddH O 150 ml 150 ml Gel solutions e g gradient of 50 70 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 68 6 Processing of nucleic acids Substanes Gel Low P High e Stock solution 0 9 ml 9 ml 3 ml Stock solution 80 15 ml 21 ml End volume 9 ml 24 ml 24 ml Add on the verge of use TEMED 8 ul 17 ul 17 ul APS 10 100 ul 86 ul 86 ul If you use another gradient you have to mix up the stock solutions in a different proportions to each other The end volume of the gel solutions need to be a volume of 24 ml The amounts of TEMED and APS stay the same no matter which gradient you use
62. e window will be opened by pressing the button Analysis in the toolbar You can perform new analysis or accomplish older ones You can choose among COMP QUANTITATION ALLELIC DISC MELT and QUANTITATION If Cycling A Cy5 you choose one of them the program will bring up a list of possible channels used mE a a for your analysis Double click on the channel name and the analyses will be shown up Cycling AJDE Cycling AJOE Set 2 Cycling A ROX af Cycling A ROX Set 3 Figure 19 Analysis software Quantitation Double click on the function Quantitation and 3 windows will be opened the main window showing the curves ofthe raw data the standard curve window and the results window Main window The raw data will be recorded in a linear and logarithmical scale Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 73 6 Processing of nucleic acids __ gt Ailend Threshold CT Calculation Threshold foo Sf e E pE PT aema S E E pree gamae se panarama tij EEF ER ma Figure 20 Graphical presentation of recorded in a linear and logarithmic manner Ct Calculation The Ct value cycle time value is the point where the threshold passes the amplification curve and indicates the starting point of the exponential amplification The CT value can be correlated to the initial copy numbers but can also be set up automatically using the AUTO FIND THRESH
63. eatd 57 Quantification of DNA using PicoGreen asien ee else hie 57 DNA quantification using a Microtiterplate reader 58 DNA extraction from aquifer sediments modiefied from Lueders et al 2004 and Gabor et 59 3 2003 has aie Oe ae ed ed Agarose lectr pheresis an a een Reg es itive tte ee ea See eae 60 Purification of PER products 2 et ereen nae ae Tei aaeei EEE aE ae EERTE 61 DNA sequencing according to Sanger Chain termination method rsuersessseesseeneeenne 61 Electrophoresis using the LiCor DNA sequencing system 4200 uunsuessnersnersnesnnennennnennnennnen 63 Denaturating gradient gelelectrophoresis DGGE 22222u2200rsneesneennennneennnennennn essen 65 Q antitative PER A GPCR as nein a aa a 68 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 4 7 Isolation of anaerobes and sediment bacteria ssesseseesoeseesoesecsoesoesoesoecoesorsoeeceesoreessee 74 Enrichment and isolation of abundant heterotrophic sediment bacteria unen 75 Isolation of anaerobes from agar CeeDPS eee esesseceeeeeeeeeeeeeseeeseesseceseceseeeaeeeneesaeeseeeseeeneeeseeeees 76 8 Physiological characterisation esessssssssssssnsssssnnnssnssssnnsnsnssnnnnsnssnssnnnssnssnsnnnsnsnssnnnnsse 77 Catalase t st 25 2 2 Gail lle EHRE ao IHR oe es 78 Oxidase testerit ii HR gl eeu ed covet faa sad E eater 78 Determining the spectrum of sub
64. ed maximally to 1 5 ml with minimally 500 ul headspace remaining e Bead beating 45 sec at 6 m s e Spin down 5 min at 7500 rpm 6000 g amp 4 C take 600 ul supernatant place in 2 ml vial at 4 C e Add up to 300 ul PTN buffer to sediment so that 400 ul supernatant remain e Bead beating 20 sec at 6 5 m s e spin down 5 min at 7500 rpm 6000 g amp 4 C take 300 ul and pool supernatants 900 ul keep at 4 C e Extract by vigorous shaking manually with 1 vol 900 ul PCI spin 4 min at 14000 u min 4 C e take 800 ul supernatant place in 2 ml Phase Lock Gel Heavy tube Eppendorf e Extract with 1 vol 800u1 Chloroform Isoamylalcohol 24 1 spin 4 min at 14000 u min 4 C e take 650 ul supernatant mix thoroughly with 2 volumes 1300 ul PEG e Incubate for up to 2 h min 30 min at 4 C pellet DNA by spinning over 30 min at 14000 rpm amp 20 C e Remove PEG with pipette or decant if pellet is clearly visible e Add 150 ul cold 20 C 70 EtOH gently wash pellet should be visible and sticking to the wall e spin 5 min at 14000 rpm 4 C e Remove EtOH carefully by pipetting dry DNA briefly at room temperature lt 5 min e elute each DNA precipitate in 30 ul EB buffer 25 100 ul possible depending on yield Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 61 6 Processing of nucleic acids e mix by flipping briefly spin down transfer to 0 5 ml Eppendorf Cup
65. ektisolierung 1 ml Verd nnungsreihe 1g Erde 9 ml 0 1 ml Drigalski Spatel saxon YOY UY aus 10 3 104 105 10 10 7 g Erde SS S amp S e amp F 1 Ausstrich 13 Striche 3 2 Ausstrich 63 853 2 Reinkulturen Figure 24 Counting and direct isolation Procedure Liquid dilutions and agar plates are checked for growth OD measurements microscopy of colony cells suspend the colony in 50 ul Tris buffer transfer 5 ul of them onto a microscope slide and put a cover slip on it use the 40x lens The liquid cultures and agar plates having the highest growth are used to set up subscultures via the 13 streak method If subcultures are grown the form and colour of the colony as well as the cell types will be compared to the initial culture microscopically New subcultures are set up to yield single and pure cultures The growth is checked daily the presence of colonies is recorded and colony types are specified Pure cultures will be harvested and used for physiological characterization OD measurement The OD measurements are done using the same photometer for all measurements Aerobically grown cultures are examined using the one cuvette method The photometer is adjusted to zero by using water After drying the cuvette using a filter paper samples are measured in the same cuvette The OD should not exceed 0 3 otherwise 70 mM phosphate buffer will be used for diluting samples properly The screw covered tubes containing
66. f the channel Sometimes it is impossible to enter a new name The easiest way is to change the name of an already existing file Method name Use the right mouse button and choose Browse file and enter a name for your method When you have filled in the parameters of the first channel do not press enter just change in the field Channel 1 to 2 and the parameters for channel 1 will disappear When you have defined the parameters of all four channels press Close To close a window choose the upper left corner not the right one as usual Go to menu Control and the submenu Experiment control to start the monitoring Mark Start Experiment for all four channels and then press OK If you want to monitor an experiment for a longer period than a week end the running experiment and start a new one with the same parameters except results name and combine the two data sets in Data plot This is necessary because the virtual memory is too little Go to menu Plot and submenu Define Screen Plot and insert the names of the Results name see above of the four running channels Update the plot by pressing Scale to show all End the experiment in menu Control and submenu Experimental control Saving the data on a disc Menu File Open Results file click on the file to be saved Ignore the now opening window Go to menu File Export
67. ffect on the hybridization Further information can be gained from review articles e g Amman amp Ludwig 2000 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 46 5 Microscopy and cell counts Equipment Microscopic slides Epoxy coated 8 chambers 6mm Hybridisation chambers Greiner tubes or falcon tubes Hybridisation oven Water bath Block thermostat 3 Staining chambers Fluorescence microscope with DAPI and Cy3 filter Washing chambers Greiner tubes or falcon tubes Chemicals and reagents Oligonucleotide probes Cy3 labelled Fixed cells of pure cultures to test the specificity of the probes Stock solution NaCl Stock solution 5 M autoclaved 1000 ml 292 2 g NaCl 100 ml 29 22 g NaCl Tris HCI Stock solution 1 M pH 7 2 autoclaved 1000 ml 157 6 g Tris HCl 500 ml 78 8 g Tris HCl 250 ml 39 4 g Tris HCl 100 ml 15 76 g Tris HCl SDS Stock solution 1 sterile filtrated 100 ml 1g SDS Hybridisation buffer with 35 formamide Formamide 17 5 ml 3 5 ml 350 ul 175 ul NaCl 5 M 9 ml 1 8 ml 180 ul 90 ul Tris HCL 1 M 1 ml 0 2 ml 20 ul 10 ul SDS 1 0 5 ml 0 1 ml 1 Oul Sul H20 videst 22 ml 4 4 ml 440 ul 220 ul Washing buffer with 35 formamide 80 mM NaCl NaCl 5 M 16 ml 8 ml Tris HCL 1 M 20 ml 10 ml SDS 1 10 ml 5 ml H3O bpidest ad 1000 ml ad 500 ml Concentration of NaCl in the washing buffer using different concentrations of formamide in the
68. g diazabicyclo octane 1 ml PBS 9 ml glycerine staining solution lml fixative 930 ul ddH3O sterile filtrated 70ul DAPI solution Procedure 100 ul TWEEN 80 are added to sediment samples fixed with glutardialdehyde 6 ml 3 and the samples are sonicated 5 x 5 seconds 10 ml PBS buffer are placed in combusted test tubes Samples are vortexed and 10 ul are transferred to the buffer Solutions were vortexed immediately before performing the filtration using Anodisc membrane filters and underlaid glass fibre filters Test tubes and the filtration tower were rinsed using sterile filtrated water The membrane was dry filtered and carefully removed Between every filtration the filtration tower was thoroughly cleaned using ddH O DAPI solution was made up in a small weighing dish The filter was applied to the staining solution with the sample featuring side facing upwards and stained for five minutes in the dark The back of the filter was stained in the dark using kitchen roll paper The filter was embedded on a microscopic slide using DABCO A cover slip was applied and the filter was enumerated Determination of the total cell count with SybrGreen I Lunau et al 2004 Dissolution of the cells with methanol and staining with SybrGreen I To break the particles in the initial sample apart and to remove adherent bacteria the samples are treated with 100 methanol in 1 5 ml Eppendorf reaction tubes final concentration 10 v
69. halose 5 Monosaccharides Arabinose 5 Rhamnose 5 Xylose 5 Fructose 5 Glucose 5 Mannose 5 Sugar derivatives Mannitol 5 Gluconate 5 Glucosamine 5 Carboxylic acids Formiate 5 Acetate 5 Propionate 1 Butyrate 2 5 Valerate 0 5 Capronate 0 5 Caprylate 0 5 Crotonate 0 2 Dicarboxylic acids Malonate 5 Succinate 10 Fumarate 5 Malate 5 Tartrate 2 Other organic acids Glycolate 5 Pyruvate 5 Lactate 10 2 Ketoglutarate 5 Citrate 2 Alcohols Methanol 2 Ethanol 5 Propanol 5 Butanol 5 Glycol 5 Glycerine 5 Tween 80 0 001 Amino acids Alanine 2 Arginine 2 Asparagine 2 Cysteine 2 Glutamine 2 Isoleucine 2 Phenylalanine 2 Tryptophane 2 Proline 2 Amines Betaine 2 Aromatic compounds _Benzoate 2 Salicyclic acid 2 Heterocyclic compounds Niacin 2 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 80 8 Physiological characterisation The assay is set up in 200 ul microplates see below aseptically 140 ul medium 40 ul substrate from a master plate and 20 ul of the bacterial cell suspension are pipetted into one well The plates are incubated at 20 C As control one well is set up without cells and one without substrate Important cells are washed before the inoculation to prevent the entry of foreign substrates 1 5 ml liquid culture are centrifuged down the supernatant is carefully
70. hate compounds Photometric determination of orthophosphate Reagents A molybdenum sulphuric acid reagent 14 4 ml of concentrated HSO d 1 84 are dissolved in 30 ml of distilled water After cooling down the following ingredients are added 1 g sulfamic acid in 10 ml distilled HO 1 25 g NH4 s MO7O24 4 H20 in 20 ml distilled H20 34 4 mg antimony potassium tartrate in 10 ml distilled H O distilled water is used to fill up the solution to a final volume of 100 ml B 1 0 g ascorbic acid in 10 ml distilled H2O prepare daily anew Procedure 10 ml of a filtered water sample are mixed with 0 4 ml of reagent A and 0 25 ml of reagent B After at least 10 minutes the extinction is measured at 865 nm in 1cm cuvettes against water Calibration curve Using KH PO standards are prepared ranging from 0 2 40 pmol l For comparison also prepare standards with concentrations of 400 and 4000 umol l Principle Molybdenum within the formed molybdenum posphoric acid is reduced to Mo IV by ascorbinic acid as reducing agent which which forms together with the remaining Mo VI a blue compound consisting of different valency stages Photometric determination of sulphide in cultures of sulphate reducing bacteria Cord Ruwisch 1985 This quick method allows the quantitative analysis of dissolved sulphide in just a few minutes The presence of sulphide can be measured based on the rapid formation of colloidal precipitation as CuS in a c
71. he primer you use After the completion of the program you add 5 ul formamide loading buffer to each approach and spin up Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 64 6 Processing of nucleic acids the samples by using the centrifuge Before plotting the samples onto the gel you have to denature them using a temperature of 70 C 5 min Afterwards you put the tubes immediately on ice water in the dark until you start plotting the gel Principle of a sequencing reaction _ known Sanger 5 unknown sequence sequence 3 LL fluorescence 3 NWS tabetea sequencing primer sequencing reaction in 4 tubes Sequencing approach with ddATP buffer polymerase MgCl dNTPs primerannealing 3 each TTGAACAGCCTGACGC a nn Tece P Bi primer ddGTP ddTTP ddCTP ddATP gt integration of ddATP T 15 bp TTGAACAGCCTGACGC G AcltsccH A chain abruption fragmentlength 6 bp C m A v c S TTGAACAGCCTGACGC c AcTTGTcGGcadtccch H 6 bp chain abruption fragmentlength 15 bp A EE C mm G c TTGAACAGCCTGACGC AACTTGTCGGACTGCGH sequencing gel chain abruption fragmentlength 16 bp Figure 12 Scheme of the sequencing principle after Sanger Electrophoresis using the LiCor DNA Sequencing System 4200 Prearrange the glass plates In order to carry out the electrophoresis you have to put
72. hybridization buffer Formamide in the NaCl in the hybridization buffer washing buffer mM 0 900 5 636 10 450 15 318 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 47 5 Microscopy and cell counts 20 225 25 159 30 112 35 80 40 56 45 40 50 28 55 20 60 14 65 10 70 7 75 5 80 3 5 Procedure Prepare microscope slides Coat the slots of the microscopic slides 8 chambers 6mm with coating solution and air dry them Plot the samples Plot the cell or sediment suspension 3 4 ul into the slots of the coated microscope slides and air dry them 15 min If you investigate environmental samples you can use up to 10 ul of sample Lysozyme treatment use only for gram positive bacteria Pipette 10 ul of lysozyme solution 10 mg ml PBS buffer 1 lysozyme and incubate at room temperature for 15 min use a cup as dust protector Wash off the lysozyme solution using ddH O Prepare hybridisation chamber falcon tube Put parts of pulp into the tube and pipette hybridization buffer 1 5 ml in Afterwards put the tube into the hybridization oven for 30 min and incubate at 46 C in order to adjust a continuous vapour pressure Ethanol treatment in the staining chamber Put the microscope slides into the first ethanol bath 50 3 min Afterwards put the slides into the second ethanol bath 80 3 min and finally put the slides into the third ethanol bath 96 98 3 min Air dry the
73. ial growth t to period examined h Turbidity measurements of microbial growth The turbidity OD436 nm against water is measured to estimate the growth yield The photometer is set to zero using water 3 ml cuvette glas cuvettes are preferred and the OD of the bacterial suspension is measured Values should not exceed 0 3 otherwise aerobic cultures should be diluted prior to the measurement using 70 mM phosphate buffer Cultures grown under anaerobic conditions are not diluted They are grown in tubes and measured against a tube filled with water When the tubes are measured for the first time the minimum absorption for each tube is found by turning the tube around in the photometer The corresponding positions are marked on the tubes and the same position is used after adjusting to measure the OD throughout the experiment Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 81 8 Physiological characterisation Taking samples Immediately after inoculation and at least every 60 minutes thereafter 1 ml samples are taken from aerobic cultures and investigated regarding the OD and cell counts max once every hour From anaerobic samples only the OD is measured The OD and maybe other parameters of slowly growing strains should be checked once a day or even a larger time interval during lag phase Temperature is an important parameter for biological turnover rates and should therefore remain c
74. ic bacteria As the medium is mixed with agar immediately before pouring the plates containing salts are only dissolved in 700 ml water before autoclaving Table 6 HPG medium distilled H2O 700 ml HEPES 2 38 g KBr 0 84 M 1 ml NaCl 24 32 g HBO 0 4 M 1 ml MgCl 6 H O 10g SrCl 0 15 M 1 ml CaCl 2 HO 1 5g NH Cl 0 4 M 1 ml KCl 0 66 g KH PO 0 04M iml Na SO 4g NaF 0 07 M 1 ml Trace element solution SL 10 1 ml Na Lactate 1 M 5 ml Selenit Wolframat Solution 0 2 ml Yeast extract 0 03 g Peptone 0 06 g Before autoclaving the pH of the medium is adjusted to 7 2 7 4 using 4 M NaOH The medium is filled in Schott bottles blue caps and autoclaved Supplementary solutions are added under sterile conditions clean bench after autoclaving and allowing the medium to cool down NaHCO solution 0 2 g in 10 ml H O 10 Vitamin solution 5 times concentrated 2 ml Glucose solution 0 5 M 0 2 ml Na Thiosulphate 1 M 1 ml Keep an eye on working carefully and aseptically It is not necessary to check adjust the pH of the finished medium Pouring the plates Before the medium is mixed with liquid agar 4 at least 5 times washed it is preheated to approximately 50 C water bath After adding the agar the medium is thoroughly mixed no cords should be visible before pouring the plates under sterile conditions using a clean bench Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenb
75. ient of denaturing agent urea and formamide Diverse DNA sequences run in a different way from each other resulting in a separation due to the amount of GC and generating melting domains Muyzer et al 1993 Figure 13 DGGE apparatus I Materials DGGE Equipment Ingeny a Gel Urea Formamide 50x TAE buffer pH 7 4 242 g Trisbase 57 1 ml conc Acetic acid 100 ml 0 5 M EDTA pH 7 4 fill up to 850 ml using ddH 0 pH 7 4 fill up to 11 using ddH 0 Acrylamide Bisacrylamide 40 37 5 1 ddH 0 10 Ammoniumpersulfate APS Tetramethylethylendiamine TEMED Loading buffer Ethanol 70 3x50 ml Beaker Cannula yellow and tubes 2Glasplates 1x block out at the top 1x block out at the bottom Spacer U shaped casting equipment electrode chamber Comb 20 48 choppers Srew conservation Gradient mixer DGGE Equipment Magnet stirrer Figure 14 Gradient mixer c Staining 2 black tanks 1000 ml 1 x Sybr Gold dye solution 100 ul Sybr Gold 10 000 x concentrated will be diluted in 1000 ml 1 x TAE buffer pH 7 4 1000 ml ddH O Gel beam UV permeable Shaker Methods Script AG Cypionka ICBM Carl von Ossietzky Universitat Oldenburg 67 6 Processing of nucleic acids II Prearrangement Work neatly Clean the glas plates the spacer and the comb using ddH 0 and ethanol Combine the glas plates with the spacer and the screw protecti
76. ilter black Whatman and rinse 3 times with 3 ml TAE puffer Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 42 5 Microscopy and cell counts alternatively 20 ul of the supernatant is distributed equally on a slide that is placed on a 20 20 mm square template 8 ul SybrGreen staining solution is given on a cover slip and the dried filter alternative the square sample is covered with it Preparation of the mounting medium In a 25 ml polypropylene tube Falcontube 2 4 g of polyvinylalcohol 4 88 moviol 4 88 Fluka Switzerland are added to 6 g of glycerol and vigorously mixed at room temperature Moviol does not dissolve completely Thereafter 6 ml double distilled water is added and the solution is stirred for two more hours at room temperature Some of the moviol still remains undissolved By adding 14 ml of TAE buffer and mixing for another 2h at 50 C moviol is completely dissolved Finally the solution is filtered through a 0 2 um Nalgene filter and aliquots of 200 1 000 ul are stored frozen at 20 C Preparation of the staining solution The mounting medium is added to the SybrGreen I stock solution The dilution depends on the sample fixation procedure Samples which are fixed with formalin are diluted 1 15 and those that such are fixed with glutardialdehyde are diluted 1 200 In addition a freshly prepared 1 M ascorbic acid solution dissolved in TAE buffer pH 7 4 is added a
77. imes To reduce the conductivity of the eluent which enhances the sensitivity the column is followed by a suppressor unit In this unit cations are replaced by H which bind to the strongly conductive carbonate ions and form the less conductive carbonic acid The suppressor column must be regenerated with sulphuric acid regularly The data is recorded by a thermic plotter which shows peak height and area The concentrations are determined by the peak area of several calibration standards Conditions column LCA A09 eluent 5 mmol I Na CO 5 w v EtOH flow rate 2 ml min temperature 40 C Eluent preparation 1 Degassing of 950 ml dd H O 2 Addition of Sodiumcarbonate and ethanol swaying cautiously Operation of the ion chromatograph 1 Connection of the eluent to the pump Pressure compensation of the eluent must be assured 2 Degassing of the eluent inlet Open the air escape valve of the pump head purge 3 Before switching the eluent flow to the system the suppressor column must be regenerated first Oherwise the valve of the suppressor unit does not enable the eluent to flow When the baseline stops drifting the application of the samples can start Three standards 50 uM 250 uM 1000 uM are used for the calibration The calibration curve is not exactly linear During the analysis the standards should be measured once in a while For every charge of eluent the standards must be measured again Different calibrati
78. ing the solution ten times and a centrifugation 13000 rpm 2 minutes the aqueous phase usually the supernatant is transferred to a new 1 5 ml Eppendorf tube The organic and interphase contain the proteins and humic acids which should be removed by the extraction procedure Phenol chloroform pH 4 RNA pH 7 5 DNA is added in a 1 1 ratio to the aqueous phase Again the solution is reversed and centrifuged and the supernatant transferred to a new Eppendorf tube Finally chloroform is added in a 1 1 ratio and the solution is treated as before The obtained aqueous solution is transferred into a 2 ml Eppendorf tube Next an ethanol precipitation is done to remove salts that would impair the PCR and to take up the nucleic acids in a suitable buffer Ethanol precipitation Precipitation solution is added in 2 6 1 ratio to the sample prepare freshly sodium acetate precipitates over time and the nucleic acids are allowed to precipitate overnight at 20 C alternatively 4 hours at 4 C The nucleic acids are centrifuged down 15000 rpm 30 minutes Pay attention to the orientation of the Eppendorf tubes to ensure the same orientation for the next centrifugation step After the centrifugation the supernatant is discarded and 500 ul ethanol 70 are added for washing the samples After a second centrifugation step 15000 rpm 10 minutes the supernatant is again discarded and the samples are centrifuged for 5 10 minutes using the Speed Vac at
79. ing the PCR Kombi purification kit Seqlab and a centrifuge Before starting with the purification procedure place a Spin filter into a 2 ml Receiver vessel 1 Binding of the PCR products Mix 500 ml Binding buffer thoroughly with the PCR sample by pipetting or vortexing Transfer that solution completely onto a Filter cartridge and centrifuge for 2 minutes at 10 000 x g Discard the Receiver vessel with the flow through If the volume of the PCR reaction is higher than 50 ul split the PCR Mix and add 500 ul Binding buffer to each part Load both mixes one after the other onto the Filter cartridge Centrifuge the first part for 1 minute and discard the filtrate Centrifuge the second part of the mixture for 2 minutes Then carry on with the elution step 2 Elution of the PCR products Place the Filter cartridge into an Elution vessel Pipett at least 10 ul Elution buffer or ddH O directly onto the centre of the Filter membrane For concentrating the PCR fragments it is possible to perform the elution with a lower volume of Elution buffer than the volume of the initial PCR mixture The minimum volume is 10 ul Incubate for 1 minute best is 5 to increase the normally already high final DNA yield further an extended incubation time for up to 5 minutes is recommended at room temperature then centrifuge for another minute at 5 000 x g The Elution vessel now contains the purified PCR product DNA sequencing via Sanger Chain abruption method
80. inge must be purged thrice with air and then air can be used as blank value Homogenize the headspace of the sample and inject 20ul headspace immediately after sampling into the injector repeat the last step twice Determination of methane concentrations essential parameters for calculation sample vessel volume head space volume GC area corrected GC area ppmv methane sediment volume Phage dislodgment and extraction from sediments samples Danovaro et al 2001 0 5 ml sediment 4 ml ddH O 1 ml sodium pyrophosphate final concentration 10mM incubation for 15 min with a shaking speed of 100 rpm sonication for 3 min interrupted for 30 sec every minute samples were shaken manually for 1 min and centrifuged for 1 min at 1000 rpm 800xg supernatants are filtered 0 2 um 0 45 um 0 2 um ON pases 8 ya storage at 4 C Sodium pyrophosphate working solution 100 ml ddH O 1 463 g sodium pyrophosphate x10H O pH 8 0 Sterilizing at 121 C 20 min Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 89 9 Unrelated experimental procedures Microcalorimetry Introduction Calorimetry is the science of measuring the heat production of physical chemical or biological reactions digital voltmeter DVM ampoule lifter a ampoule lifter heat exchanger sampie flow measuring cylinder water thermostat heat exchange con sealed ampoule at equilibra
81. ion before use When using do not eat or drink 56 Dispose of this material and its container to hazardous or special waste collection point When using do not smoke 57 Use appropriate container to avoid environmental contamination Do not breathe dust 59 Refer to manufacturer supplier for information on recovery recycling Do not breathe gas fumes vapour spray appropriate wording to 60 This material and or its container must be disposed of as e specified by the manufacturer hazardous waste A void contact with skin 61 Avoid release to the environment Refer to special instructions safety data sheet Avoid contact with eyes 62 If swallowed do not induce vomiting seek medical advice immediately and show this container or label In case of contact with eyes rinse immediately with plenty of 63 In case of accident by inhalation remove casualty to fresh air water and seek medical advice and keep at rest Take off immediately all contaminated clothing 64 If swallowed rinse mouth with water only if the person is conscious After contact with skin wash immediately with plenty of to e specified by the manufacturer Do not empty into drains Never add water to this product Take precautionary measures against static discharges This material and its container must be disposed of in a safe way Wear suitable protective clothing Combination of safety precautions 1 2 Keep locked up and out of the reach of children 24 25 Av
82. ise and destroy nucleic acids so that further investigations are not possible Moreover DNases and RNases are set free due to cell disruption Purifying extracted nucleic acids is necessary since sediment samples offer many substances which impair the PCR A possible way to purify nucleic acids is the phenol chloroform extraction Commercially available kits are expensive alternatives which are not inevitably more efficient A precipitation is done to absorb the nucleic acids in a certain volume and a proper buffer After finishing the extraction and if needed quantification nothing stands in the way anymore to amplify nucleic acids using PCR RT PCR Equipment beadbeater rack sterile cryo vials lids cold trap pump for the Speed Vac Speed Vac sterile Eppendorf tubes 1 5 ml und 2 0 ml sterile zirkonia pearls 0 1 mm Biofuge 13 R water bath Biofuge 15 R Reagents chloroform DEPC treated ddH O 0 1 diethyl pyrocarbonate DEPC Dissolve in ddH O and mix well Incubate over night at 37 C and autoclave subsequently DEPC dissolves completely in CO and ethanol DNase 1 U ul DNase buffer pH 7 5 40 mM Tris Base 6 mM MgCl prepared in ddH O pH adjusted via HCI and autoclaved Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 56 6 Processing of nucleic acids ethanol 70 precipitation solution 125 ml ethanol absolute 5 ml sodium acetate 3M phenol saturate
83. lack solution 0 1 As supporting filter a cellulose membrane filter is used The Nuclepore filter is dried on a microscopic slide fluorescence free immersion oil is added and a cover slip applied The filter is examined under the microscope In general per filter 10 squares are counted Each square should offer 30 50 bacteria cell number ml y A a V y mean cell number per square A filtration area um a square area um V filtration volume ml Counting of growing cells using nalidixic acid Kogure et al 1979 Principle Determining the total cell count does not allow to draw conclusions regarding the physiological state of the cells Out of this reason the direct viable count was developed This method involves the addition of an antibiotic to a bacterial culture which suppresses the doubling of DNA Although bacteria can grow they cannot divide to double It is assumed that viable cells are growing The number of growing cells is taken as direct viable count Reagents Nalidixic acid Dissolve 1 mg in 10 ml distilled water and filter the solution Yeast extract Dissolve 10 g in 1 1 of water and autoclave the solution Procedure 10 ml of a water sample are spiked with sterile yeast extract to get a final concentration of 50 mg l Next nalidixic acid is added to end up in a concentration of 20 ug l As controls reference samples are set up once without yeast extract and nalidixic acid and
84. les of freezing and thawing would be the method of choice In the context of decomposing gram positive cells it has to be taken into account that the cell wall is significant thicker compared to gram negative bacteria due to a higher number of peptidoglycane layers In this case a pre treatment by means of lysozyme is necessary to reduce the mechanical stability Glycosidic bonds between N acetylglucosamine and N acetylmuramic acid which form the backbone of murein are cleaved Equipment Biocentrifuge Freezer 70 C Heating block Reagents Lysozyme solutions 0 8 mg ml 10 mM Tris HCl pH 8 10 SDS solution 9 6 ml 20 SDS 2 4ml 0 5M sodium acetate pH 7 5 66 4 ml ddH O autoclaved 3M sodium acetate solution Procedure Frozen samples 20 C stored in Eppendorf tubes are thawed and centrifuged for 30 minutes 19000 rpm 4 C supernatants are discarded Per sample 100 ul lysozyme solution and 100 ul Tris HCl are added Samples are reversed ten times and subsequently incubated for 10 minutes stored on ice Next 40 ul SDS solution and 60 ul sodium acetate solution are added The incubation is continued for one hour and samples are still stored on ice For freeze thawing samples are snap frozen 70 C for 3 minutes and subsequently boiled up five times Afterwards a phenol chloroform extraction is performed Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg
85. medium temperature Thereby remaining ethanol is removed At least 10 minutes before using the Speed Vac the cold trap should be turned on to protect the affiliated pump generates the needed negative pressure from liquids The nucleic acids are taken up in 50 ul TE buffer after being dried After incubating them for 30 minutes at room temperature samples can be stored ready to be used at 4 C For long term storage nucleic acids can be taken up in precipitation solution 2 6 1 and stored at 70 C RNA extraction and DNase treatment In order to get pure RNA it is necessary to prevent remove DNA contaminations For extracting RNA the phenol chloroform extraction is done at a pH of 4 At this value DNA partially precipitates in the interphase The RNA still dissolves completely in the aqueous phase Since DNA is still present after extraction purification and precipitation the sample is treated with DNases Therefore the RNA samples are taken up Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 57 6 Processing of nucleic acids in 500 ul DNase buffer instead of TE buffer 5 ul DNase are added and the samples are incubated for 60 minutes at 37 C using a water bath Subsequently the phenol chloroform extraction is repeated to remove DNases In order to check the presence of DNA afterwards a PCR is done Summary cell disruption using a beadbeater splitting of the samples in DNA and RNA subsample
86. n B is added and the mixture is thoroughly blended to prevent cords After 30 45 minutes the mixture is blended again and the extinction is measured at 436 nm against a sulphate free control To improve the validity of the measurements always measure in duplicate triplicate using different diluted samples Always measure standards covering the expected concentration range along with the test series Calibration curve Using standards ranging from 0 1 5 0 umol sulphate In order to generate depth profiles sulphide and particle free water samples 1 1 are used Principle Citric acid acidifies the preparation and complexes the Ba ions Crystals offering a high optical density arise based on the complexed ions and sulphate Glycerine slows down the sedimentation of yielded crystals Sulphate rapid test Procedure 1 ml of culture is acidified with 2 drops of HCI and shaken 2 drops of BaCl are added Immediately observed turbidity indicates sulphate a slowly establishing turbidity suggests thiosulphate gt sulphur Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 27 3 Analysis of sulphur and phosphate compounds Photometric analysis of thionates Kelly et al 1969 Fitz and Cypionka 1990 The photometric analysis of thiosulphate trithionate and tetrathionate relies on the alkaline cyanolysis of thionates ending up in thiocyanate equivalents The differentiation between individu
87. n child Possible risk of impaired fertility Possible risk of harm to the unborn child May cause harm to breast fed babies Harmful May cause lung damage if swallowed Repeated exposure may cause skin dryness or cracking Vapours may cause drowsiness and dizziness Possible risk of irreversible effects 39 27 28 Very Toxic danger of very serious irreversible effects in contact with skin and if swallowed 39 28 Very Toxic danger of very serious irreversible effects if swallowed 42 43 May cause sensitization by inhalation and skin contact 48 20 Harmful danger of serious damage to health by prolonged exposure through inhalation 48 20 21 Harmful danger of serious damage to health by prolonged lexposure through inhalation and in contact with skin 48 20 21 22 Harmful danger of serious damage to health by prolonged exposure through inhalation and in contact with skin and if swallowed 48 20 22 Harmful danger of serious damage to health by prolonged exposure through inhalation and if swallowed 48 21 Harmful danger of serious damage to health by prolonged exposure in contact with skin 48 21 22 Harmful danger of serious damage to health by prolonged lexposure in contact with skin and if swallowed 48 22 Harmful danger of serious damage to health by prolonged lexposure if swallowed 48 23 Toxic danger of serious damage to health by prolonged lexposure through inhalation Methods Script AG Cypionka ICBM
88. n of carotinoides Eichler and Pfennig 1986 a Harvesting bacteria Well grown cultures are centrifuged 9000 rpm 20 minutes the supernatant is carefully removed and discarded b Extraction of carotinoides Carotinoides are unstable if exposed to light and air Extraction reagent ethanol 100 acetone 1 1 The pellet is resuspended in the remaining supernatant if necessary add 0 2 ml distilled water and transferred to a 10 ml centrifuge beaker Next 8 ml of extraction reagent are added mix well nitrogen is introduced and the vessel is thoroughly sealed using a butyl rubber stopper Extraction is allowed to take place for approximately 1 hour at room temperature Mix again and centrifuge down 15 min cell remnants remove the stopper before Concentrate the coloured supernatant at 25 C using a rotary evaporator under a black rag Resuspend the bottom in 0 75 ml extraction reagent Transfer the solution into a tube introduce nitrogen and seal the tube with a butyl rubber stopper Store in the dark c Thin layer chromatography eluent petroleum benzine acetone 9 1 The chromatography chamber is lined with blotting paper nitrogen is introduced and the chamber is closed 100 ml eluent are poured into the chamber and allowed to saturate for 1 hour The extract is placed to dry on the starting line of a silica gel thin layer plate under nitrogen exposure place 100 ul along 3 cm besides 50 ul along 3 cm Treat reference
89. ncubation in the dark Fluorescence intensities are determined in a microplate reader Fluostar Optima at 485 nm excitation and 520 nm emission All measurements are carried out in three reading cycles with integration of 20 flashes 0 5 s delay time between reading and without shaking before each cycle Determination of bacterial counts and colony forming units cfu from water and sediment samples Cavalli Sforza 1972 Depth profiles sediment surface tidewaywater and other depth layers of living cell counts aerobes and anaerobes are generated For the determination of living cell counts dilution series of tidewaywater and sediment samples are generated using artificial seawater Tidewaywater 1 10 1 100 sediment 1 100 1 1000 1 100000 and 100 ul are transferred and spread on agar plates see figure Every dilution factor is made up in duplicate One is incubated under oxic one under anoxic conditions The progress of colony formation is recorded daily Therefore the colonies are counted distinguished macroscopically as well as microscopically as colony forming units and specified morphologically 1 ml Verd nnungsreihe 0 1 ml Drigalski Spatel sein YOY OY aus 103 10 10 1068 107g Figure 5 Preparation of dilution series The living cell counts will be determined using the following equation x LC X n Zi Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 39 5 Microsco
90. nd amino acids from animal or plant proteins Substrate analogue stock solution prepare 100 ml of a 2 mM L leucine 7 amino 4 methylcoumarine solution in sterile double distilled water in a glown out vessel 12 hours at 180 C storage at 20 C in the dark necessary concentrations are prepared after thawing Fluorophore stock solution prepare 1 1 of a0 1 mM 7 amino 4 methylcoumarine solution in 10 ml ethylene glycol monomethyl ether and 990 ml sterile double distilled water in a glown out vessel aliquotted and stored at 20 C in the dark necessary concentrations are prepared after thawing Ammonium glycine buffer prepare 1 1 of a 0 2 M ammonium hydroxide 0 05 M glycine solution using sterile double distilled water in a glown out vessel adjust the pH to 10 5 using 5 M sodium hydroxide H3C HN H O H CHCH C Z N O O 3 2 a A HN AE AO sa HC HN H Leucin Aminopeptidase i 2 A 2 H3CHCH C C COOH Sy ae TS ae Nu na CH3 CH3 L Leucin 7 Amido 4 Methylcoumarin 7 Amino 4 Methylcoumarin L Leucin Figure 28 Principle of the assay for determining leucine aminopeptidase activity Procedure 1 ml of sample 1 ml of substrate analogue solution for calibration 1 ml of sample 1 ml fluorophore solution Blank 1 ml of sterile double distilled water in a glown out vessel 1 ml of substrate analogue solution for calibration 1 ml of sample 1 ml sterile double distilled water
91. nd if swallowed 23 24 Toxic by inhalation and in contact with skin 23 24 25 Toxic by inhalation in contact with skin and if swallowed 23 25 Toxic by inhalation and if swallowed 24 25 Toxic in contact with skin and if swallowed 26 27 Very Toxic by inhalation and in contact with skin Reacts violently with water liberating extremely flammable Contact with water liberates toxic extremely flammable gas oq 35 36 Causes severe burns Irritating to the eyes 37 Irritating to the respiratory system 38 39 40 41 42 43 44 45 46 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 Irritating to the skin Danger of very serious irreversible effects Limited evidence of a carcinogenic effect Risk of serious damage to eyes May cause sensitization by inhalation May cause sensitization by skin contact Risk of explosion if heated under confinement May cause cancer May cause heritable genetic damage Danger of serious damage to health by prolonged exposure May cause cancer by inhalation Very Toxic to aquatic organisms Toxic to aquatic organisms Harmful to aquatic organisms May cause long term adverse effects in the aquatic environment Toxic to flora Toxic to fauna Toxic to soil organisms Toxic to bees May cause long term adverse effects in the environment Dangerous for the ozone layer May impair fertility May cause harm to the unbor
92. nger of very serious irreversible effects hrough inhalation and if swallowed 39 27 Very Toxic danger of very serious irreversible effects in ontact with skin 48 23 25 Toxic danger of serious damage to health by prolonged xposure through inhalation and if swallowed 48 24 Toxic danger of serious damage to health by prolonged xposure in contact with skin 48 24 25 Toxic danger of serious damage to health by prolonged xposure in contact with skin and if swallowed 48 25 Toxic danger of serious damage to health by prolonged xposure if swallowed 50 53 Very Toxic to aquatic organisms may cause long term dverse effects in the aquatic environment 68 20 22 Harmful possible risk of irreversible effects through inhalation and if swallowed 68 22 68 21 Harmful possible risk of irreversible effects if swallowed skin Indication of safety precautions S1 Keep locked up N Keep out of the reach of children 3 Keep in a cool place A Keep away from living quarters 5 Keep contents under appropriate liquid to be specified by the Methods Script AG Cypionka ICBM Harmful possible risk of irreversible effects in contact with 48 23 24 Toxic danger of serious damage to health by prolonged lexposure through inhalation and in contact with skin 48 23 24 25 Toxic danger of serious damage to health by prolonged exposure through inhalati
93. nother water bath Bubble free agar solution is mixed with medium Don t shake No cords After cooling down the medium is transferred into sterile Erlenmeyer flasks and the plates can be poured sterile conditions clean bench Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 19 1 Media preparation Agar dilution series Needed material equipment per series 7 properly labelled sterile test tubes and suitable rubber stoppers keep some backup stoppers ready 5 times washed sterile agar 4 water bath preheated to 42 C water bath preheated to 65 C water bath icy water 2 Bunsen burner 150 ml bottle completed medium sterile Iml 10ml pipettes Procedure The agar is liquidated and kept at 65 C 3 ml of agar are added to the single test tubes which are kept at 42 C Next 6 ml completed medium are added to each test tube One series of test tubes is removed from the water bath and placed in a rack at room temperature From the culture of interest one drop 0 5 ml is added to the first test tube slew the tube slightly One drop is transferred 0 5 ml from the first tube to the second one The first tube is placed cold water bath icy water and so on Before each transfer the corresponding tube is wiped to prevent water drops from being transferred unwanted dilution Before incubating the tubes at designated temperatures N CO 80 20 v v is introduced
94. ode therefore an undisturbed sediment core is needed The profiles of ammonia nitrate and nitrite are analysed photometrically Sulphate is determined via ion chromatography HPLC and methane via gas chromatography GC Determination of the oxygen profile by a needle electrode The measurement of the oxygen profile is done by using a steel needle electrode of the cathode type which needs an external reference electrode This kind of oxygen electrode consists of a platinum wire which is isolated by glas with a gold tip Fig The electrode is surrounded by a steel capillary The sensible tip is located in the capillary tip ca 500 um The oxygen electrode is polarised by a voltage source to a level of 750mV The power flow that results from the reduction of oxygen is measured by a picoamperemeter O 2e 2H 20H The relation between oxygen concentration and power is linear at a voltage of 750 mV Thus a calibration with only two standards is useful gt 100 O air saturated water tap water gt 0 O oxygen free water oxygen free sediment layer Ze sel Mikromanipulator E DPX Membran Picoampere meter U Platindraht I i Glasummantelung T A D Wandler mE l PC 486 i A BE Goldspitze Figure 31 left hand side schematic set up right hand side electrode tip Methods Script AG Cypionka
95. oebobacter pedioformis sp nov Arch Microbiol 146 3 295 300 Fitz R M amp Cypionka H 1990 Formation of thiosulfate and trithionate during sulfite reduction by washed cells of Desulfovibrio desulfuricans Arch Microbiol 154 4 400 406 Francis C A Beman J M amp Kuypers M M M 2007 New processes and players in the nitrogen cycle the microbial ecology of anaerobic and archaeal ammonia oxidation ISME Journal 1 1 19 27 Gabor E M de Vries E J amp Janssen D B 2003 Efficient recovery of environmental DNA for expression cloning by indirect extraction methods FEMS Microbiol Ecol 44 2 153 163 Giovannoni S J DeLong E F Olsen G J amp PACE N R 1988 Phylogenetic group specific oligodeoxynucleotide probes for identification of single microbial cells J Bacteriol 170 2 720 726 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 95 Goltermann H L amp Clymo R S ed 1971 Methods for chemical analysis of fresh waters Blackwell Scientific Publications Oxford Edinburgh Gregersen T 1978 Rapid method for distinction of gram negative from gram positive bacteria Eur J Appl Microbiol 5 2 123 127 Hobbie J E Daley R amp Jasper S 1977 Use of Nuclepore filters for counting bacteria by fluorescence microscopy Appl Environ Microb 33 5 1225 1228 Ishii K Mussmann M MacGregor B J amp Amann
96. oid contact with skin and eyes 3 7 Keep container tightly closed in a cool place 27 28 After contact with skin take off immediately all contaminated clothing and wash immediately with plenty lof to be specified by the manufacturer 3 9 14 Keep in a cool well ventilated place away from 29 35 Do not empty into drains dispose of this container and it s incompatible materials to be indicated by manufacturer material in a safe way 3 9 14 49 Keep only in the original container in a cool well ventilated 29 56 Do not empty into drains dispose of this material and its place away from incompatible materials to be indicated container to hazardous or special waste collection point by manufacturer 3 9 49 Keep only in the original container in a cool well ventilated 36 37 Wear suitable protective clothing and gloves place 3 14 Keep in a cool place away from incompatible materials 36 37 39 Wear suitable protective clothing gloves and eye face to be indicated by the manufacturer protection 7 8 Keep container tightly closed and dry 36 39 Wear suitable protective clothing and eye face protection 7 9 Keep container tightly closed and in a well ventilated place 37 39 Wear suitable gloves and eye face protection 7 47 Keep container tightly closed and at a temperature not e 47 49 Keep only in the original container at temperature not lexceeding C to be specified by manufacturer exceeding C to be specified
97. on Mount the glass plates into the inductor Put the comb between the glas plates Stick together the tubes of the gradient mixer Put the magnetic stirrer into the first chamber and the contrary volume tube into the retral chamber Start the magnetic stirrer Prepare 3 beakers APS TEMED as well as the stock solutions refrigerator III Casting the gel Set up the gel solution in the beakers under the fume hood and mix them slightly Add APS and TEMED on the verge of casting the gel Add APS and TEMED to the 0 solution later on Transfer the lower concentrated solution into the posterior chamber of the gradient mixer Open the plug quickly in order to remove air bubbles Transfer the higher concentrated solution into the anterior chamber of the gradient mixer Don t put the cannula on the tube yet because of the resistance will be too high Afterwards put the cannula onto the tube Put the gradient mixer onto the magnet stirrer Hold the tube as high as possible at least higher than the gradient mixer is situated Connect both chambers by turning over the plug Clamp he cannula between the both glas plates and let the gel run between them Thereby you should notice the mixture of both solutions in the anterior chamber of the gradient mixer Formation of striae Let the complete solution drop out of the tube Thereby you have to take care that the cannula doesn t dip into the gel and that th
98. on in contact with skin and if swallowed 48 23 25 Toxic danger of serious damage to health by prolonged exposure through inhalation and if swallowed 48 24 Toxic danger of serious damage to health by prolonged exposure in contact with skin 48 24 25 Toxic danger of serious damage to health by prolonged lexposure in contact with skin and if swallowed 48 25 Toxic danger of serious damage to health by prolonged lexposure if swallowed 50 53 Very Toxic to aquatic organisms may cause long term adverse effects in the aquatic environment 51 53 Toxic to aquatic organisms may cause long term adverse effects in the aquatic environment 52 53 Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment 48 20 Harmful danger of serious damage to health by prolonged exposure through inhalation 48 20 21 Harmful danger of serious damage to health by prolonged lexposure through inhalation and in contact with skin 48 20 21 22 Harmful danger of serious damage to health by prolonged Jexposure through inhalation and in contact with skin and if swallowed 48 20 22 Harmful danger of serious damage to health by prolonged exposure through inhalation and if swallowed 48 21 Harmful danger of serious damage to health by prolonged exposure in contact with skin 48 21 22 Harmful danger of serious damage to health by prolonged exposure in contact with skin and if swallowed 48 22 Ha
99. on curves are determined for the different concentration ranges Usually the suppressor unit must be exchanged after five or six samples By switching the suppressor unit on the eluent flows to another suppressor column When the baseline stops drifting the application of the samples can start again Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 3j 3 Analysis of sulphur and phosphate compounds Injekticns adim ventil m mE Zluent Versauie Trenns ule Integrator Suppressor s ule Leitfahigkeits Detektor i Figure 3 Schematic assembly of the ion chromatograph 1 ei IE 4 537 5 A A z a e Sm amp Figure 4 Chromatogram example for a bacterial culture Peaks chloride 1 5 min phosphate 4 5 min sulphate 5 4 min The upper value of the data set offers the area and the lower one hi the peak height Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 4 Determination of proteins and bacterial pigments Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 33 4 Determination of proteins and bacterial pigments Lowry protein assay 1951 Reagents e copper reagent Dissolve 0 1 g CuSO 5 H2O in 20 ml K Na tartrate solution 1 1 ml of the solution is mixed with 50 ml of NaCO solution 2 The solution is prepared daily anew e Folin s reagent 1 moiety of
100. on for Sedimente en een es 91 REFERENCES onient sereine sasinen sentesi eesihasiek senie 93 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg Version history v0 1 16 04 2010 translation of all methods completed overview of R S statements given overview of hazard codes given safety advices regarding individual chemicals added disposal guideline added retrieved 01 04 2010 http www sigmaaldrich com sigma aldrich help help welcome risk and safety statements html retrieved 01 04 2010 http www chemie de tools sicherheit Gefahrensymbole php3 according to the Merck ChemDat Disposal guidelines Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg Different classes of waste are usual products of even standard laboratory procedures Resulting wastes are separated based on individual characteristics and collected in properly labelled containers Classes of waste and respective coded can be found in the table below Pieces of information regarding individual chemicals can be found in respective material and safety data sheets MSDS If there are any questions get in touch with responsible members of staff Code Class 18710 Paper filters including harmful impurities organic Papierfilter mit sch dlichen Verunreinigungen organisch 18711 Paper filters including harmful impurities inorganic Papierfilter mit sch dlichen
101. onstant In no case leave cultures standing at room temperature During the exponential growth phase decrease the intervals at which samples are taken OD data sets are plotted semi logarithmically Determination of B glucosidase activity B glucosidase cellulase hydrolysis the B 1 4 glycosidic bonds between the glucose units of cellulose Principle The glucosidases within a sample hydrolyse the non fluorescent substrate analogue 4 methylumbelliferyl B D glucoside The cleaved 4 methylumbelliferone is detected fluorometrically i CH HOH2C P Pa A a ao ye se yp nied HOH2C ssl Glucosidase 5 rei a je OH m a Ou 53 PR FEW Z as er pes 3 SO HO s lt SG SG HO 70H HO H 4 Methylumbelliferyl D Glucosid 4 Methylumbelliferon D Glucose Figure 26 Principle of the assay for determining B glucosidase activity Equipment Reagents spectrofluorophotometer 4 methylumbelliferyl B D glucoside test tubes including a rack 4 methylumbelliferone MUF Vortexer glycine magnetic stirrer sodium chloride pH electrode sodium hydroxide automatic pipettes ethylene glycol monomethyl ether 25 C water bath centrifuge Substrate analogue stock solution 3 mg 4 methylumbelliferyl B D glucoside are completely dissolved in 0 5 ml ethylene glycol monomethyl ether and 0 5 ml sterile double distilled water are added in a glown out vessel The substrate analogue solution has to be prepared freshly each day Methods
102. opper sulphate reagent The precipitation can subsequently be determined by measuring the absorbance with a photometer at 480 nm Reagent Add 1 25g of CuSO and 6 51 ml HCl 25 4 4 ml of 37 to water and fill up to 1000 ml Assay Fill 4ml of the CuSO reagent into small glass test tubes For sampling the culture take 0 3 ml of clear culture liquid avoid sediment particles with a nitrogen flushed 1 ml syringe Release gas bubbles if necessary Put the tip of the needle into the CuSO reagent and inject 0 1 ml of the culture Vortex shortly and tranfer the soltuion into a plastic cuvette Measure the absorption at 480nm against the pure CuSO solution Standards 1 2 5 10 20 mM of sulphide solution must be prepared with anoxic water For storage the headspace of used vessels is flushed with nitrogen Methods Script AG Cypionka ICBM Carl von Ossietzky Universitat Oldenburg 30 3 Analysis of sulphur and phosphate compounds Analysis of sulphate by ion chromatography Basic principle The column for the anion chromatography consists of polystyrole divinylbenzol carrier material with attached positive molecules Anions in the sample bind to these positive molecules and are displaced by carbonate ions which are provided by the eluent This is repeated several times when the sample flows through the column The frequency and the period of the absorption are specific for each anion which results in different retention t
103. osition CARD FISH uses horseradish peroxidase HRP labelled oligonucleotides to increase the intensity of fluorescence signals CARD is based on the deposition of a large number of labelled tyramine molecules by peroxidase activity HRP reacts with hydrogen peroxide and the phenol part of labelled tyramide to produce a quinine like structure bearing a radical on the C2 group This activated tyramide then covalently binds to tyrosine residues in the target cell Each HRP conferred by a probe catalyzes the deposition of many labelled tyramides FISH signals are up to 20 fold brighter with HRP labelled probes than with conventional single labelled probes Sch nhuber er al 1997 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 49 5 Microscopy and cell counts Hybridization Nature Reviews Microbiology Figure 9 Catalysed reporter deposition Amman and Fuchs 2008 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 50 5 Microscopy and cell counts Procedure according to Ishii et al 2004 1 Fixation Dilute 0 5 ml fresh culture or 0 5 cm sediment in 1ml 3 formaldehyde Incubate over night at 4 C Centrifuge at 16 000g for 5 minutes Pour off supernatant and resuspend the sample with 1x PBS Repeat the washing step twice Store washed sample in a 1 1 mix PBS ethanol at 20 C until further processing 2 Sonication and filtration 1
104. ouble distilled water The fluorescence of the blank is subtracted from the fluorescence of examined samples Calibration curve For preparing a standard series between 10 and 70 ul MUF stock solution are added to 3 3 ml of sterile double distilled water in a glown out vessel 750 ul glycine buffer are added the solutions are well mixed and measured against a blank 3 3 ml sterile double distilled water 750 ul glycine buffer The standard series has to be measured along with each test series The determination of glucosaminidase leucine aminopeptidase and f glucosidase activity using microtiterplates is done with slight modifications to the procedure described Determination of B glucosaminidase activity B glucosaminidase chitinase hydrolyses the 1 4 glycosidic bond between the N acetyl glucosamine units of chitin CH3 CH3 HOH2C4 FIR HOH C J T Glucosaminidase T gt Home OH fi No CRI See BR OSS ER FETT A Holm 0 Se o 5o HO gt 0 O HO NH ce 2 O HO G O 4 Methylumbelliferyl N Acetyl D Glucosaminid 4 Methylumbelliferon N Acetyl D Glucosamin Figure 27 Substrate analogue 4 methylumbelliferyl N acetyl B D glucosaminide Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 83 8 Physiological characterisation Determination of leucine aminopeptidase activity Leucine aminopeptidase cleaves peptone which is made up by a mixture of peptides a
105. pH to a value between 7 and 8 The potassium concentration in the neutralised extract is sufficient to precipitate the perchlorate which would interfere with the test reaction The precipitate is centrifuged down shortly Luciferase assay For the assay the following reagents are needed Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 85 8 Physiological characterisation Tris buffer EDTA tris acetate pH 7 75 12 12 g of Tris hydroxy methyl aminomethane and 0 74 g Na2 EDTA are weighed in and filled up with distilled water to a volume of 900 ml The pH is adjusted to 7 75 using 2 M acetic acid and the solution is filled up to a final volume of 1 1 using distilled water Test reagent The reagent 1243 102 Monitoring Kit LKB contains I firefly luciferase II D luciferin II 50 mg bovine serum albumine IV 0 5 mM magnesium acetate V 0 1 uM inorganic pyrophosphate The freeze dried test reagent ready to use mixture is dissolved in 10 ml ultrapure water 1 ml aliquots are distributed to cryo vials and are stored in liquid nitrogen until usage P standard 0 5 uM 30 26 mg ATP ATP Na H 3 H2O Boehringer Mannheim are dissolved in 100 ml Tris buffer see above and 1 ml aliquots are prepared and stored in liquid nitrogen For each test series P standard is thawed and diluted in three steps 1 1000 using Tris buffer This ATP standard offers a concentration of 0 5 uM 0 5 pmol ul
106. placed in a water bath for 20 minutes icy water Preparation II Prepartion II is cooled down to C for 10 minutes before 0 06 ml of solution 2 are added mix After incubating for 10 minutes 0 06 ml of solution 3 are added mix the preparation is kept for additonal 10 minutes in the water bath icy water Preparation III After adding 0 06 ml of solution 2 the preparation is cooked for 45 minutes water bath The test tubes are closed using glass marbles Subsequently the test tubes are cooled down to 0 C 10 minutes before 0 06 ml of solution 3 are added mix The preparations are kept in the water bath for additional 10 15 minutes icy water Finally 1 ml of solution 4 is added mix As soon as the test tubes have reached room temperature the extinction is measured at 460 nm According to given reaction equations the concentrations of thionates are determined as follows Concentration tetrathionate preparation I Concentration of thiosulphate preparation II 2 x preparation I Concentration of trithionate preparation III preparation II Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 28 3 Analysis of sulphur and phosphate compounds Colorimetric determination of sulphite Pachmayr 1960 Solutions A decolourised fuchsine solution 400 mg fuchsine is dissolved in distilled water and 125 ml concentrated sulphuric acid Distilled water is used to fill up the
107. py and cell counts x average count of colony forming units in the volume of the inoculum C Cy Ci number of counted colonies on the plates Ny No n plates dilution series Zi Z2 Zi dilution factors Example Dilution 10 303 290 285 colonies 10 32 21 colonies Average cell number x 931 3 10 2 107 2 91 108 Preparation of slides coated with agarose for microscopy Pfennig and Wagener 1986 modified after Cypionka 100ml of an agarose solution 2 w v are heated prepared in a Schott flask with blue cap in the microwave The solution should be free from cords A preheated water bath 45 C is used to keep the solution liquid In order to get a smooth agarose film clean microscopic slides are preheated free from fluff on a clean flat underground by using infrared light 2 ml of agarose solution are dispensed in zigzag lines onto one slide by using a clean glass pipette Avoid drainage of the agarose solution Before usage the agarose slides are air dried for a few days The slides can be kept in closed boxes for some time Preparing microsope slides for observation First of all cell concentrations in cultures should be high enough for photography Otherwise a centrifuge should be used to concentrate the cells Three drops of 20 22 and 25 ul are transferred onto an agarose coated slide by using a micropipette 0 1 0 2 ml Each drop is covered immediately with a cover
108. r sources of ignition 3 Extreme risk of explosion by shock friction fire or other sources lof ignition Forms very sensitive explosive metallic compounds Heating may cause an explosion 4 5 6 Explosive with or without contact with air 7 May cause fire 8 Contact with combustible material may cause fire 9 Explosive when mixed with combustible material Flammable Highly Flammable Extremely Flammable Reacts violently with water Contact with water liberates extremely flammable gases Explosive when mixed with oxidizing substances Spontaneously flammable in air In use may form flammable explosive vapour air mixture May form explosive peroxides Harmful by inhalation Harmful in contact with skin Harmful if swallowed Toxic by inhalation Toxic in contact with skin Toxic if swallowed Very Toxic by inhalation Very Toxic in contact with skin Very Toxic if swallowed Contact with water liberates toxic gas Can become highly flammable in use Contact with acids liberates toxic gas Contact with acids liberates very toxic gas Danger of cumulative effects Causes burns Combination of particular risks 14 15 ases 15 29 20 21 20 21 22 Harmful by inhalation and in contact with skin Harmful by inhalation in contact with skin and if swallowed 20 22 Harmful by inhalation and if swallowed 21 22 Harmful in contact with skin a
109. rded data The Rotor Gene Software Before starting the Rotor Gene Software you have to switch on the cycler After starting the Rotor Gene program the main window new experiment will appear Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 71 6 Processing of nucleic acids tm Browse sen te Select the template on which to base your experiment then click New PM Use Experiment Wizard to Configure Experiment F Show this Screen when Rotor Gene Opens i A d S B z Exp Info Progress Profile Temperature Samples Arrange Des gu gt n m us Vi New Open Save Start Pause Stop Help bait Figure 17 Rotor Gene Software toolbar Before starting the run set up your experimental parameters using the toolbar Exp Info settings The name of the operator and the name of the experiment has to be inserted Also the reaction volume the detection channel and the kind of rotor has to be chosen Profile The temperature program for the run has to be set up You can choose among the options HOLD CYCYLING and MELT HOLD Choose a temperature which will be hold over a certain time period DENATURE special option for the first denaturing step Cycling Choose max 5 following temperatures and time periods Denaturing Annealing Elongation Set the time point of fluorescence detection The fluorescence detection will take place directly after the elongation step
110. removed discarded and the pellet is resuspended in 1 5 ml fresh medium All steps are carried out under sterile conditions clean bench The evaluation is done based on the turbidity due to bacterial growth in the inoculated wells Spot tests are done regarding single wells as control It has to be considered that used substrates partially cause a turbidity independent from bacterial growth OO OO OOO0000000009 DOOOOOOOOOOOO Zellfreie Kontrolle EOOO OOOO COO0O FOOOOOOOOOOOO SOOOOOOOOOOOO FOOODOOOOOOOOO St mme Substrate gt Figure 25 Scheme for the inoculation of microplates with respect to testing various substrates Recording of growth curves The most important parameter in the context of growth experiments is the growth yield regarding specific substrates This circumstance is reflected by individual dry weights and cell counts Growth rates of bacteria cultures are determined by turbidity measurements optical density OD using the Ratio XR Turbidimeter HACH Germany To convert the OD to cell counts a conversion factor has to be determined Therefore a culture exhibiting a strong turbidity is analysed in respect to its cell concentration and then serially diluted The OD of the dilutions is noted and the respective cell counts calculated cells OD Equation to calculate growth rates In x In xo t ty u growth rates h x OD current date Xo OD at the beginning of exponent
111. rmful danger of serious damage to health by prolonged lexposure if swallowed 48 23 Toxic danger of serious damage to health by prolonged exposure through inhalation 48 23 24 Toxic danger of serious damage to health by prolonged exposure through inhalation and in contact with skin 48 23 24 25 Toxic danger of serious damage to health by prolonged exposure through inhalation in contact with skin and if swallowed 51 53 Toxic to aquatic organisms may cause long term adverse effects in the aquatic environment 52 53 Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment 68 20 Harmful possible risk of irreversible effects through inhalation 68 20 21 Harmful possible risk of irreversible effects through inhalation and in contact with skin 68 20 21 22 Harmful possible risk of irreversible effects through inhalation in contact with skin and if swallowed 68 2 1 22 Harmful possible risk of irreversible effects in contact with skin and if swallowed 68 22 Harmful possible risk of irreversible effects if swallowed 37 Wear suitable gloves 38 In case of insufficient ventilation wear suitable respiratory lequipment 39 Wear eye face protection 40 To clean the floor and all objects contaminated by this material use to be specified by the manufacturer 41 In case of fire and or explosion do not breathe fumes Carl von Ossietzky Universitat Oldenburg 12
112. rned on otherwise the microcalorimeter will start to heat The tempertature in the internal waterbath of the microcalorimeter keeps it temperature because it is connected to the thermostate next to the microcalorimeter which can cool down as well as heat Always pay attention that the external waterbath contains enough water Because the microcalorimeter itself can only heat the temperature of the external water bath must be Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 90 9 Unrelated experimental procedures lower than in the internal one To adjust the appropriate temperature of the external as well as the internal water bath have a look at the temperature regulator unit Fig 1 in front of the microcalorimeter Now the microcalorimeter and then the computer can be turned on The password is agcyp Recording data By choosing the calorimetry software Digitam for windows four red lights will appear on the front panel of the microcalorimeter Choose Control and Initiate experiment Fill in the parameters for each channel Channel 1 2 3 or 4 for each measuring cylinder Amplifier setting 300uW 1000uW or 3000u W usually 1000uW is enough Operator enter your name Results name Use the right mouse button and choose Browse file and enter a name for the data file For example jutta001 3 the first number is the number of the run and the second one the number o
113. running length of the four approaches results in the uncovering of the unknown sequence Finally the sequence will be transmitted into a internet data bank e g EMBL RDP and compared with known sequences Material Equipment Thermocycler LiCor DNA Sequencing System 4200 Chemicals and reagents DYEnamic Direct Cycle Sequencing Kit Amersham Sequencing primer IRD marked Stock solutions TBE buffer 10 x Tris Base 890 mM 108 g Boric Acid 890 mM 55 g Na EDTA x 2H 0 20mM 744 g H 0 dest ad 1 l Working solution Gel solution dest H O 14 5 ml Urea NF 12 6 g 10x TBE buffer 3 6 ml Long Ranger 50 gel solution 4 5 ml Procedure Sequencing reaction The sequencing reaction is carried out via DYEnamic Direct Cycle Sequencing Kit Amersham For one single sample you will need four 0 2 ml tubes in order to pipette all four nucleotides G A T C The DNA sample volume 2 ul will be transferred in each of the four tubes Afterward you prepare the master mix for each sample you need 1 ul of ddNTP s 2 ul of IRD labelled primer One aliquot of the master mix contains a volume of 3 ul and will be transferred into your sample tube Finally the tubes will be put into the cycler and the you can start the run The program depends upon your primer Sequencing program Temp C Time min Cycles n 95 5 1 95 0 5 0 5 30 72 1 4 co The annealing temperature depends upon the melting temperature Ty of t
114. s Phenol chloroform extraction pH ethanol precipitation take up DNA in TE buffer DNase treatment of the RNA samples Phenol chloroform extraction pH 4 0 ethanol precipitation take up RNA in TE buffer Rapid test for quantifying DNA 1 ul of sample is pipetted on an EtBr plate Set up an 1 5 agarose gel 1 x TAE buffer and boil up the solution using a microwave After cooling down 60 C 15 ul ethidium bromide 10 mg ml are added per 100 ml agarose The agarose is poured into petri dishes Additionally 1 ul of herring sperm is added in varying concentrations e g 10 30 50 100 150 ng ul Using a transilluminator the DNA content can be estimated based on the standard DNA extraction from sediments with FastDNA Spin Kit Compared to the phenol chloroform extraction this is a fast but expensive method A further advantage of a kit is that filters are used for extraction so that the application of toxic substances such as phenol and chloroform can be minimized Besides the chemicals columns and tubes that are included in the kit four sterile 1 5 ml Eppicaps as well as acetate and ethanol are needed Unless otherwise noted an Eppendorf centrifuge is used by 13 000rpm transfer 0 5 g sediment 918 ul Sodium Phosphate Puffer 122 ul MT Buffer 40 ul Poly A solution and 20 ul sodiumpyrophosphate solution to a Lysing Matrix E Tube shake the tube slightly for 30 sec and wait two minutes before starting the cell lysis
115. s carried out by using the DNA fluorescence dye PicoGreen as well as the fluorescence reader The calculation is carried out via a calibration line resulting from defined concentrations of DNA 1 Pipette the following DNA standards TE buffer N PicoGreen 1 200 diluted in TE DNA concentration pH 7 5 DNA stock solution buffer pH 7 5 100 ng ul 99 ul lul 100 ul 50 ng ul 99 ul lul 100 ul 25 ng ul 99 ul lul 100 ul 0 ng ul 100 ul Oul 100 ul 2 Pipette the DNA samples TE buffer DNA sample PicoGreen 1 200 diluted in pH 7 5 TE buffer pH 7 5 99 ul lul 100 ul gt Pipette Pico Green PicoGreen not until measurement gt Incubation of 5 min in the reader 1 Turn on the microtiterwellreader 2 Turn on the computer 3 Start program FLUOstar Optima 4 Push the button Test Protocols gt Test name Reinhard Versuch gt Settings gt Basic parameters Test name Reinhard Versuch Microplate Nunc F96 Microwell 96 Positioning delay 0 1 Number of kinetic windows 1 Number of cycles 3 Measurement start time 0 0 Number of flashes per cycle 20 Cycle time variable depends on the number of samples x fluorescence intensity Number of multichromatics 1 Excitation filter 485 nm Emission filter 520 nm Gain variable depends on the highest DNA concentration Start 1 Stop 1 Pause before cycle 0 a Layout Record the adjustment of your samples and standards on the plate and push the button check timing 5
116. slip 18x18 mm Normally the liquid part of the cell suspension is soaked into the agarose while the bacterial cells are arranged on the top In some cases it is necessary to prevent the cell suspension from being evaporated A paraffin solution is used to seal all sides of the cover slip by using a warm spatula Don t forget to take some nice pictures of your cells It is worth it See also How to get the perfect photomicrograph of Heribert Cypionka http www icbm de pmbio lehre ws0708 ringvi digitalemikrofotos pdf Determination of gram type Gram differentiation Bartholomew 1962 Gregersen 1978 Gram negative bacteria do not retain the initial crystal violet stain They are decolourised by the organic solvent and hence show a pink red counterstain Gram positive bacteria instead retain the violet dye This difference basically lies in the cell wall structure of the bacteria a Gram staining 1 Plate freshly grown cells not older than 24 h onto a clean object slide or cover slip and allow the film to air dry Fix the dried film by passing it quickly through the Bunsen flame heat fixation do not pyrolyse the cells Two reference strains one gram negative and one gram positive strain are treated the same way Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 40 5 Microscopy and cell counts 2 Stain the fixed cells for up to 1 min with Huckers ammonium oxalate cry
117. stal violet reagent 3 Drain the staining solution Wash off briefly with water for 5 sec Drain 4 Flood the slide with Gram s Iodine solution for ca 1 minute Wash off with water Drain 5 Drain staining solution Wash off briefly with water for 5 sec Remove excess water 6 Counterstain 3 x 30 sec each in 3 drops of n propanol 7 Wash off with water for 5 sec 8 Check quality using a microscope 9 Flood slide with safranin solution and allow to counterstain for 30 seconds Drain counterstain solution and wash off with water Remove excess water Dry 10 Find dry and stained bacteria under 10x 40x lenses Then examine the bacteria using oil immersion but no cover glass 11 Compare your results with the gram test of gram negative and gram positive reference strains b KOH solution test Put some drops of KOH solution 3 onto a clean microscopic slide Add cell material colonies or cell pellets by using an inoculating loop and mix for 5 to 10 seconds Carefully pull back the platine loop Slimy filaments indicate gram negative cells In comparison treating gram positive cells with KOH does not end up in the formation of slimy threads Flagella staining Ryu 1937 Based on a bacterial cell suspension a microscopic specimen is prepared As soon as the majourity of cells is fixed staining solution is added to the preparation After allowing the staining to take place 5 15 minutes the flagella should be visible
118. store at 20 C Reagents PTN Puffer 120mM 16 02 g l Na gt HPO 0 86 g l NaH PO 11 2 g l Tris HCl 6 6 g l Tris NaPO 125 mM Base 1 46 g l NaCl adjust to pH 8 with HC filter sterilize autoclave Tris 25 mMNaCl pH8 1x TE 10 mM Tris HCl 1 mM EDTA pH 8 20 SDS 20 g SDS in 100 ml H2O prepare with sterilized H2O in baked glassware 30 PEG 1 6 M 150 g polyethylene glycol 6000 46 76 g NaCl in RNAse free water 500 NaCl pecipitation ml final volume Prepare in baked glassware first dissolve PEG in solution microwave adjust to final volume autoclave EB Buffer 10 mM Tris pH 8 5 prepare with RNAse free water filter sterilize autoclave Better take from a fresh QiaQuick Kit Agarose gelelectrophoresis DNA fragments of 200 50 000 bp are separated by agarose gelelectrophoresis due to their size Negatively charged DNA molecules move through an agarose matrix with an electric field Shorter molecules move faster and migrate further than longer ones The movement decelerates logarithmically according to the number of base pairs After the electrophoresis the separated fragments are stained with ethidium bromide This is an intercalating fluorescent dye which is mutagen and carcinogen so that gloves and a lab coat are required Preparation of agarose gels The gels are prepared in Plexiglas trays which have only two opposite side walls so that the gel is in contact with the electric field Before preparation the open sides
119. strates of anaerobic isolates eles ceseceseeeeeesecnseceseeeseeees 78 Recording of growth curves a ana sn savnasedinas dees enee aaee a E a a aa e oaaae 79 Determination of B glucosidase activity ueseseessessessensennsensnesnenensonnnnnnennonsennnnnnnennnnennnnnn 80 Determination of B glucosaminidase activity useesensessensensensnnsnesnensennnenennennennennennennnnennnnn 8l Determination of leucine aminopeptidase activity e sseesesseeeeeseesesresresrtstesrssresrestsresseertsseesreses 82 Determination of B glucosaminidase activity in microtiterplates sseeneene 83 Determination of ATP Bergmeyer 1983 2u 20022442202s0nesnnennnnnnnnnnnesnnesnnesnnenennnnnnnenne nen 83 9 Unrelated experimental procedures sssssssssssssssnsssssnnsnsssssnnnnsnsnsssnnnsnssnnnnssnsnsnssnnnnsse 85 Determination of methane concentrations via gas chromatography uersersneessesnseenseennnenn 86 Phage dislodgement and extraction from sediment samples Danovaro et al 2001 87 Microcalorimetry soe een aaa a a a s a datshatadonet 88 Determination of physicochemical gradients ccccsscccssscsesesesesesesesesesesecceesesesesesescscececeeeeeees 90 Determination of the oxygen profile by a needle profile nersersneesseenseensnesneenneenneennnnnn 90 Molecular diffusion coefficients for various ions and gases in aqueous solutions 9 Fick s law of diffusi
120. t a final concentration of 1 as an antioxidant The staining solution can be kept at 4 C for several weeks do not freeze Fix Mix 63ml destilled water 30ml Methanol 2ml GDA Glutardialdehyde 25 5ml Tween 80 0 5 The solution is filtered 0 2um Nalgene Total cell counting A minimum of 30 squares must be counted each with 30 50 cells a Determination of cell number ml Prilwasser cell number ml y A a V y mean cell number per square A filtration area 314 16 mm d 20 mm a square area 0 01 mm A a total cell numbers of the squares y A a number of cells per filter V filtration volume ml b Determination of cell number g sediment cell number g y A a Manalysed Manalysed analysed sediment weight g weight g dilution factor c Counting tool Count Them Examine specimens 40 x magnification largest field of vision document by digital fotography and Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 43 5 Microscopy and cell counts quantify using the counting tool Count them introduction by H Cypionka Total cell counts of water samples Hobbie et al 1977 2 5 ml of a water sample are mixed with 200 ul of a particle free acridine orange solution 0 1 and stained for 2 minutes Next the solution is filtered through a Nuclepore filter pore size 0 2 um and pre stained using Irgalan b
121. tection of nitrite Solutions A 1 65 g sulfanilic acid is dissolved in 375 ml of hot water 125 ml glacial acetic acid is added B 0 5 g a naphtylamine is suspended in 100 ml of water and 125 ml glacial acetic acid is added The mixture is stirred until a clear solution is gained The solution is filled up with water to a final volume of 500 ml Prevent skin contact and spilling Use a pipettor and rinse used vessels pipettes thoroughly Procedure 0 5 ml of sample is mixed with 0 5 ml of solution A and 2 5 ml of solution B The extinction is measured after 10 minutes at 530 nm Calibration curves are generated based on KNO using standards with concentrations ranging from 0 to 100 uM The principle behind the assay is depicted in Figure 2 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 22 2 Analysis of nitrogen compounds Detection of nitrate Goltermann Solutions A 1 M HCI B 1 M NaOH C Reducing agent C1 0 039 g CuSO 5 H O in 100 ml of distilled H O C2 0 12 g hydrazine sulphate N H H2SO0 in 25 ml of distilled H O 5 ml of solution C1 and 25 ml of solution C2 are mixed and filled up with distilled H O to a final volume of 50 ml The completed reducing agent and solution C are not maintainable and have to be prepared daily Procedure 10 ml of sample centrifuged particle and sulphide free if necessary remove sulphide using CO are blended with 0 25 ml NaOH
122. thods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 70 6 Processing of nucleic acids Solution Volume ul Temp C Be Cycles 10 x buffer 2mM MgCl 2 5 96 4 1 dNTPs 2 5mM each 2 94 N BSA 20mg mL 0 25 55 1 40 MgCl 10mM 0 75 PCR HO dest 74 72 2 Fl detection 357f 10pmol uL 0 5 7 10 l 907R 10pmol uL 0 5 25 l l SYBRGreen I 1 400 1 NEB Taq Polymerase 5U uL 0 1 DNA Template 10 Final volume 25 The thermo cycler Thermal cycling is performed using a Rotor Gene RG 3000 four channel multiplexing system Corbett Research Sydney Australia The cycler operates with a computer using the software Rotor Gene For detail see Rotor Gene User Manual The cycler provides a 37 72 well dual channel system We will use the 36 well rotor using 36 flat cap tubes 0 2 mL The rotor has to be completely loaded no empty positions The Rotor Gene cycler has 2 detection units Channel 1 detects at 510 nm and channel 2 at 555 nm Therefore you can run a multiplex detection using 2 dye solutions at the same time Once the tube passes the detection unit it will be excited caused by high energy flashes The resulting emission light will be recorded by a photomultiplier The data will be transferred to the PC and represent graphically Fluorescence Figure 16 Left hand side Rotor Gene RG 3000 four channel multiplexing system Right hand side Graphical presentation of reco
123. tion Appl Environ Microbiol 69 5 2928 2935 Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 96 Solorzan L 1969 Determination of ammonia in natural waters by phenolhypochlorite method Limnol Oceanogr 14 5 799 amp Steenbergen C L M amp Korthals H J 1982 Distribution of phototrophic microorganisms in the anaerobic and microaerophilic strata of Lake Vechten the Netherlands Pigment analysis and role in primary production Limnol Oceanogr 27 5 883 895 Tabatabai M A 1974 A Rapid Method for Determination of Sulfate in Water Samples Environmental Letters 7 3 237 243 Tschech A amp Pfennig N 1984 Growth yield increase linked to caffeate reduction in Acetobacterium woodii Arch Microbiol 137 2 163 167 Tucker F L amp Bartholomew J W 1962 Variations in gram staining results caused by air moisture Stain Technol 37 3 157 162 Widdel F 1980 Anaerober Abbau von Fetts uren und Benzoes ure durch neu isolierte Arten Sulfat reduzierender Bakterien Technical report Universit t G ttingen Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg
124. tion Position water pump temperature regulator unit measururg cup nections to external water citeulator Peitier elements Fig 2 Thermal Activity Monitor m metal heat sink electrical calibration resistor Figure 29 Thermal acitivity monitor Figure 30 Measuring cylinder The microcalorimeter Thermal Activity Monitor 2277 is an ultrasensitive instrument that measures the heat flow in uW It consists of four measuring cylinders Fig 1 that are surrounded by a water bath with a constant temperature isotherm Each cylinder is divided into two channels the left one for the sample and the right one for the reference Fig 2 When heat is produced in the sample the temperature difference is conversed into an electricity flow by the surrounding Peltier elements Fig 2 The temperature increase of the internal water bath is compensated by an external thermostate next to the microcalorimeter In this case the microcalorimeter is used to monitor growth experiments Heat is produced when a substrate is degraded due to microbial activity This is plotted in a heat production versus time diagram on a computer With the data set the energy yield can be calculated 1Joule 1 Watt Second Vice versa the energy yield can be predicted if the reactions in the degradation processes of the substrate are known Running the microcalorimeter First of all the thermostate on the left side of the microcalorimeter must be tu
125. together the Gel Sandwich length 33cm thickness 0 25 mm Wear gloves and a lab coat while working acrylamide is carcinogenic The glass plates are marked in order to identify the interior and outerior plates Spill the plates using SDS 25 ddH 0 and ethanol 98 You have to remove ethanol completely because it constricts the polymerization of acrylamide Afterwards put the glass plates in the brackets of the equipment Casting the gel Prepare the gel solution not until casting the gel under the flume hood Mix the chemicals using the protocol in the beaker on a magnet stirrer Add TEMED 20 ul and APS 200 ul to start the polymerization of the gel and Pipette the gel through a 0 2 um filter between the horizontal glass plates using a 50 ml syringe Avoid the formation of air bubbles by knocking gently on the glass plates Afterwards attach the precomb into the gel The complete polymerization time lasts 1 5 hours Install the Gel sandwich After polymerization is done you have to pull out the precomb and clean the upper border of the gel Clean the glass plates in the scope of the detector using ethanol Install the gel Sandwich into the sequencer and Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 65 6 Processing of nucleic acids mount the upper and lower buffer chamber Fill both chambers with 1 x TBE buffer ca 1 1 up to the check mark and flush the gel slote with TBE
126. ul PBS H O 3 ul tyramide DMF at 46 C 20 min in the dark 21 Dab filter on blotting paper and wash in 10 50 ml ddH gt O 1 5 min RT dark 22 Wash in 10 50 ml 96 ethanol 1 5 min RT dark To keep the background fluorescence low it is important to wash the filters in large volumes of water and ethanol 23 Let air dry dark 24 Counter stain with DAPI Put filter section on glass slide Stain with 10 ul DAPI solution Incubate min 30 min in dark Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 51 5 Microscopy and cell counts Table 9 Frequently used CARD FISH probes Probe Target Sequence 5 3 of probe FA Reference Agrol Rhizobium sp HEISE eae PEELA unknown S et al 2006 EUB338 Bacteria SETO o COTAHH 55 Amann et al 1990 NON33 None negative ACT CCT ACG GGA GGC g d ntrel AGC 55 Wallner et al 1993 Desulfobacter CAC AGG ATG TCA AAC DSB985 D bacula CCAG 20 Manz et al 1998 GTG CTC CCC CGC CAA Arch915 Archaea TTC CT 55 Stahl amp Amann 1991 Cren537 Crenarchaeota ER ae oe 10 Teira et al 2004 DSV698 Most Desulfovibrio GTT CCT CCA GAT ATC 40 Manz et al 1998 spp TAC GG Mu mann et al 2005 formamide concentrations in hybridisation buffer Solutions Lysozyme solution 10 mg ml Prepare buffer 1ml 0 5M EDTA 1ml IM Tris HCl and 8 ml ddH O Add 100mg Lysozyme when solution is needed Phosphate buffered saline PBS pH 7 6 10
127. urg 18 1 Media preparation Oxic medium and agar plates Oxic medium The components of the medium Table 7 are weighed into 400 ml distilled H gt O using a 1 litre cylinder and dissolved under stirring magnetic stirrer The provided solutions are added using a sterile graduated pipette or one way syringes cannulae Table 7 Oxic medium for the cultivation of marine aerobes Substances Net weight per litre Sterile solutions Supply per litre NaCl 24 32 g Trace elements solution SL 10 1 ml MgCl 6H O 10g Tungsten Selenite solution 1 ml CaCl 2 H O 15 KBr 0 84 M 1 ml KCl 0 66 g H BO 0 4 M 1ml NaSO 4g SrCl 0 15 M 1 ml HEPES 2 38 g NaF 0 07 M 1 ml Glucose 5g The medium used for the plates is filled up to 700 ml using distilled H2O The medium used for the liquid cultures is filled up to 1 1 The media is transferred into 1 1 Schott bottles The pH of the medium is adjusted to 7 2 7 4 using NaOH The media is autoclaved for 25 min at 121 C After cooling down the following substances are added Ammonium Phosphate solution 10 ml NaHCO3 solution 0 2 g in 10 ml H O 10 Vitamins solution 5 times concentrated 2 ml The pH of the medium is checked and if necessary adjusted under sterile solutions Casting the plates The agar solution 4 gt 12 g in 300 ml distilled H O at least 5 times washed is autoclaved for 15 min at 121 C and kept liquid using a water bath 90 C The medium is preheated at 60 C using a
128. v Afterwards samples are sonicated for 15 minutes at 35 C in the ultrasonic bath To remove detrital and inorganic particles the samples are centrifuged for 1 minute with 2 000 rpm 500 1 000 ul of the supernatant are filtrated on a black 0 2 um polycarbonate filter and stained with SybrGreen I In case of sediment samples the supernatant is diluted 1 100 with TAE buffer before filtration The filter is triply flushed in each case with 1 ml TAE buffer and transferred to a microscopic slide 6 ul staining and mounting solution are dropped in the centre of a cover slip 18 mm x 18 mm The cover slip is brought to the object slide with the staining solution towards the filter To distribute the staining solution equally on the filter you can carefully press with tweezers onto the cover slip After an incubation time of 15 to 30 minutes at 4 C refrigerator the total cell count is determined via epifluorescence microscopy The storage of specimens at 20 C allows the determination of the cell count also a few months after staining Alternativ Take 1cm sediment by a 2ml syringe and extrude it into a sterile 15ml centrifuge tube Roth containing 9 ml Fix Mix samples can be stored 4 C 2weeks Mix it and put 1 ml of this mixture into an 2ml Eppendorf tube Treat the samples 15 min 35 C with ultrasonication and mix again Dilute 5 100ul of this solution the supernatant in 10 ml TAE Vaccumfilter this solution on 0 2um polycarbonatef
129. x PBS 1x PBS 1 litre 1 litre NaCl 80 g 88 KCl 2g 0 2 g Na HPO 7 HO 26 8 g 2 17g KH PO 2 48 0 2 10 Blocking reagent Blocking reagent is dissolved in Maleic acid buffer to a final concentration of 10 w v with shaking and heating either on a heating block or in a microwave oven Adjust the Maleic acid buffer to pH 7 5 Then dissolve 10g of Blocking reagent in 100 ml of Maleic acid buffer This stock solution is autoclaved and stored at 20 C Amplification buffer 2 ml PBS 20x 0 4 ml Blocking Reagent 10 16 ml NaCl 5M Add sterile H O to a final volume of 40 ml Add 4g dextrane sulphate heat 40 60 C Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 52 5 Microscopy and cell counts 1 M Tris HCl pH 8 0 Dissolve 121g Tris base in 800 ml H20 Adjust to desired pH with concentrated HCl Mix and add H O to 1 litre 0 5M EDTA pH 8 0 Dissolve 186 1 g Na EDTA 2H 0 in 700 ml H O Adjust pH to 8 0 with 10 M NaOH ca 50 ml Add H30 to 1 litre Maleic acid buffer 100 mM Maleic acid 150 mM NaCl pH7 5 20 C Hybridization buffer 2 ml 10 ml NaCl 5 M 360 ul 1800 ul Tris HCl 1 M 40 ul 200 ul Dextrane sulphate 0 28 lg Formamide xul xul MQ water x ul xul Blocking reagent 200 ul 1000 ul SDS 10 2ul 10 ul Keep the mixtures in a water bath 48 60 C until the dextrane sulphate dissolves Washing buffer 50 ml NaCl 5 M xul Tris HC1 1 M
130. xed cells that are microscopically examined The selectivity of staining is based on the particular organisation of the ribosomal RNA Due to comparative sequence analyses it is possible to identify scopes on the small subunit of the ribosomal RNA 16S or 18S rRNA which are consistent on distinct phylogenetic levels class up to domain Gene sections with a length of 18 base pairs provide the target sequence for the oligonucleotides labelled with a fluorescence dye e g Cy3 Primarily the cells of pure cultures or bacterial communities from environmental samples will be fixed on a microscope slide Afterwards cells are penetrated and entered by the oligonucleotide probe which hybridizes with the ribosomal RNA target sequence In addition it is necessary to establish particular conditions for every single probe After the hybridization non specific probes are washed off and cells are labelled using a conventional dye like DAPI Finally samples are analysed by epifluorescence microscopy The option of different microscopic filters enables the discrimination of non specifically DAPI and specifically FISH labelled cells Anyway some problems could occur Frequently cells from natural environmental samples possess a lower amount of ribosomes and therefore feature a lower degree of fluorescence Also interfering compounds of the sampling site that are characterized by their own fluorescence e g sediment or precipitations could have an disturbing e
131. zing plug A bottling i tube magnetic stirrer Figure 1 Glass vessel for preparing anaerobic medium Widdel 1980 Oxic media After autoclaving the lateral screw caps are leak proofed closed and the rubber stopper is removed from the connecting piece for gas inflow Now air can only enter the vessel through the cotton filter To add the supplementary solutions to the cooled down medium the vessel is connected to a N gas inflow 5 kPa The vessel with the completed medium remains connected to the nitrogen influx because the excess pressure is needed for bottling The completed medium is bottled to sterile containers Anoxic media After autoclaving the head space above the medium is flushed with N CO 80 20 v v immediately The screw caps are leak proof closed and the medium is cooled down while being stirred under N CO 80 20 v v 5 kPa exposure The supplement solutions are aseptically added to the cooled down medium The pH value of the medium is adjusted if necessary to 7 2 7 4 using sterile 1 M NaCO or 1 M HCI The completed medium is bottled to sterile containers Methods Script AG Cypionka ICBM Carl von Ossietzky Universit t Oldenburg 14 1 Media preparation Mineral base medium for many anaerobic bacteria Cypionka and Pfennig 1986 Table 1 Mineral base medium for many anaerobic bacteria F gt fresh water B gt brackwater M gt marine B M F B M Substance MW g l

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