|Sampling protocol||Monthly sampling protocol at coastal stations
|Step by step Powerpoint presentation
by Fabrice at Barcelona meeting
|Measurement protocols||Measurement assignement to each group
|Lugol stock solution|
|Flow cytometry protocol||Marie et al. 1999 PDF file
Presentation by D. Marie (Power point)
|Heterotrophic flagellates stained with DAPI|
|HPLC for pigments (including sampling)|
|Nucleic acid extraction (Massana)|
| TSA-FISH protocol (including sampling)
List of probes to be applied (Excel file)
Electron microscopy (Laure Guillou)
|Nucleic acid extraction|
|SSCP C.Tebbe protocol (Power point)|
March 2001: A new update of the protocol for the monthly survey of year 2 has been done in (download summary Excel file). Please note the following points:
July 2000: The protocols have been updated in to take into accounts the conclusion which were reached during the startup meeting in Roscoff. In particular, please note that:
Below, the (strongly) recommended protocols for coastal sites are listed in yellow boxes. Other supplementary protocols have been added that can be useful.
Download table as Excel file
|Parameter||Method||Sample prefiltration||Replicates||Sample volume (ml)||Sample processing||Sample Preservation||Sample analysis||Sample analysis done by||Remark|
|Chlorophyll a||Fluorometry||-20 °C||Locally|
|Phytoplankton composition||Microscopy||2||50||Lugol 0.25%||Store in the dark at RT||Sedimentation and observation with inverted microscope (volume to sediment : 20-100mL)||Locally|
|Picoplankton taxonomy||TEM of whole mounts||< 3 um||3||200||1 fixed with osmic acid
1 fixed with 0.25 % glutaraldehyde and 1% acidic Lugol.
1 fixed with 1% glutaraldehyde in 0.2 M cacodylat.
See Wenche protocol for details on fixation
|Store in the dark at 4° C||EM protocol by Wenche||Wenche||Samples should not be accumulated but sent immediately to Wenche.|
|Heterotrophic eukaryotes abundance||Epifluorescence micorscopy||Total||90||Fix 90 ml of seawater with 10 ml ice-cold glutaraldehyde 10% (1% final) then filter (see protocol by Ramon)||as filters at -20 °C||See protocol by Ramon||Locally|
|Picoplankton abundance (autotrophs)||Flow cytometry||Total||1.5||1% paraformaldehyde+ 0.1 % glutar||in cryovials at - 80°C||Unstained on flow cytometer||Locally||See Marie et al 99|
|<3 um||1.5||1% paraformaldehyde+ 0.1 % glutar||in cryovials at - 80°C||Unstained on flow cytometer||Locally||See Marie et al 99|
|Bacteria abundance||Flow cytometry||Total||1.5||1% paraformaldehyde+ 0.1 % glutar||in cryovials at - 80°C||SYBR Green stained||Locally||See Marie et al 99|
|Pigments||HPLC||Total||1000||Filter on 25 mm||as filters at -80°C||HPLC protocol||Mikel||See Mikel protocol|
|< 3 um||1000||Filter on 25 mm|
|> 3 um||1000||Filter on 47 mm|
|FISH for eukaryotes||FISH-TSA||< 3 um||3 filters||200||See detailed protocol by Fabrice. It is critical that 1 - fixation with PF does not exceed one hour and 2 - filters be always kept humid during filtration and dehydration||as filters at -80°C in individual plastic box||FISH Protocol||Fabrice||See FISH-TSA protocol|
|FISH for prokaryotes||FISH-TSA||< 3 um||2 tubes||50||glutaraldehyde (0.1% v/v) final concentration for 10 min at 4 oC||as 50 mL flacon tubes at -20°C||FISH Protocol||Dave|
|Picoplankton diversity||DGGE eucaryotes||< 3 um||5000||Sample collected on Sterivex 0.2 um Millipore unit (see Rmaon protocol)||as Sterivex filters at -80°C||DGGE protocol||Ramon||A single DNA sample will be collected. After DNA extraction, the DNA will be split between the different groups|
|DGGE procaryotes||DGGE protocol||Dave|
|Quantitative PCR||Fluorescent PCR||Isabelle Roscoff|
|DNA chips||DNA chips||Klaus|
|Remark||This is the volume for a single sample||When mentionned -80°C samples must be stored in a deep freezer, not a regular freezer||All frozen samples are to be sent on dry ice|
For all measurements on picoplankton population, samples will need to be pre-filtered through 3 um. The best way will be to use in line fitler filter (25 or 47 mm) with a peristaltic pump at low rate. This will ensure sample homegeneity
Filter used: 47 mm polycarbonate 3 um
No fixed protocol will be used for library building (in particular choice of DNA extraction, , in order to maximize the diversity of recovered sequences. However we agreed on a set of primers and enzymes for screening, as well as on some recommendations. See the library protocol below.
|Sample storage||Sampling protocol||Analysis protocol|
|Clone library||5000||-80°C||R. Massana protocol (recommended)
D. Scanlan protocol
|Library building and analysis|
Each starting culture will be duplicated. Starting cultures are done in 50 ml Falcon flasks.
Parameters to be varied (not all combinations though):
All cultures will be started by individual partners from water filtered once or twice through 3 um or 0.6 um filter (protocol available on PICODIV Web site). The following elements will be varied (of course not all combinations will be tested at the same time).
150 uEin m-2 s-1
10 uEin m-2 s-1
Once cultures have started to grow up, they will be followed weekly by flow cytometry and optical microscopy and will be purified by one the following approach to obtain unialgal cultures. This part is of the responsibility of the individual partners.
- Fix 90 ml of seawater with 10 ml ice-cold glutaraldehyde 10% (1% final concentration)
- Store samples at 4°C until processing the samples (before 24 hours)
Preparation of filters
- Place a 0.8 µm (25 mm ø, cellulose acetate) filter on the base of the filtration system and wet it with sterile water. Put on top of it a 0.6 µm (25 mm ø, polycarbonate) filter.
- Add 20 ml of fixed seawater on the tower (the volume depends on the abundance; check it and adjust to convenience).
- Filtrate the sample until the seawater volume is reduced to 5 ml.
- Add 50 µl of DAPI working solution (5 µg/ml, final concentration) and stain for 5-10 min.
- Put one drop of low fluorescent oil on one slide (Cargille, Nikon), place the filter on the drop and wait until the filter became transparent.
- Add another oil drop on top of the filter and put the cover slide.
- Keep the slides in special plastic boxes frozen at -20°C
Counting flagellates on the epifluorescence microscope
- Observe the slides with UV excitation: pico and nanoflagellates appear as round cells with nucleus (a brighter area). Sometimes the flagella is also visualized.
- Immediately, change to blue light (485nm excitation, 530 nm emission wave-length, and 505 nm dichroic mirror) to discriminate between colorless cells from cells with plasts and pigments. This makes the possibility to separate heterotrophic from phototrophic nanoflagellates.
- Counts of each group of nanoflagellates (phototrophic and heterotrophic) are made separately, enumerating the nanoflagellates through transects (5 - 10 mm). For each sample is recommended to count at least 50-100 cells. Hence, we will make the necessary number of transects (3 transects of 5 or 10 mm are enough).
Dye working solution (DAPI)
- Add 1 ml of filtered seawater (0.2 µm) to the 10 mg DAPI vial.
- Add the 10 mg/ml of DAPI to 19 ml of sterile filtered seawater (0.2 µm). We will have a concentration of 0.5 mg/ml de DAPI.
- Filter this solution through 0.2 µm (Swinex and sterile syringes), and put it in a clean 20 ml vial. THIS IS THE WORKING DAPI SOLUTION
- Take 20 criovials and add to each one1 ml of DAPI working solution. Keep the criovials frozen (-20°C) until its use.
New procedure for samples (as of May 2001)
==> Use dark glass bottles if possible
Bottle 1: Osmium fixation
Bottle 2: 1% glutaraldehyde in 0.2 M cacodylat (final concentrations)
Bottle 3: 0.25 % glutaraldehyde and 1% acidic Lugol (final concentrations)
|Remarks from Mikel based on the analysis from the first Roscoff samples (May 2000)
Collecting biomass for marine DNA extraction:
Nucleic acid extraction
Use 1 ng of whole microbial DNA as template for Polymerase Chain Reaction (PCR) amplification of bacterial or eukaryal SSU rDNA. The reactions contain 200 µM of each of the deoxynucleoside triphosphates, 0.3 µM of each of the primers, 1.5 mM MgCl2, 1x PCR-buffer and 1 Unit of Taq DNA Polymerase in a final volume of 50 µl.
Eukarya: Eukaryal specific forward primer EukA (Medlin et al. 1988) and universal reverse primer 516r (ACC AGA CTT GCC CTC C) with a 40 bp GC-clamp, which amplify a 560 bp DNA fragment of eukaryal 18S rDNA.
Initial denaturation at 94°C for 5 min; 10 touchdown cycles of denaturation (at 94°C for 1 min), annealing (at 65 to 55°C for 1 min, decreasing 1°C each cycle) and extension (at 72°C for 3 min); 20 standard cycles of denaturation (at 94°C for 1 min), annealing (at 55°C for 1 min) and extension (at 72°C for 3 min), and a final extension at 72°C for 5 min.
PCR product check
Four µl of the PCR product are verified by electrophoresis on a 0.8% agarose gel stained with ethidium bromide, and the DNA yield of the PCR is quantified loading a standard in the same gel (Low DNA Mass Ladder, GIBCO BRL).
Denaturing Gradient Gel Electrophoresis (DGGE) is performed with the DGGE-2000 system (C.B.S. Scientific Company) as described in Muyzer et al. (1997).
A 6% polyacrylamide gel is casted by mixing two stock solutions of acrylamide (37.5:1 acrylamide: bisacrylamide) containing different amounts of DNA denaturant agents: 40 and 80% for bacterial PCR products and 45 and 65% for eukaryal PCR products (100% denaturant agent is defined as 7 M urea and 40% deionized formamide). The reproducibility of the gradient is obtained by using a two chambers gradient maker and controlling the flow of the acrylamide into the plates with a peristaltic pump at around 5 ml min-1. The gradient is overlaid with nondenaturant acrylamide in order to obtain well polymerized slots. The gel is casted several hours before loading.
800 ng of PCR product (typically 40 µl of the PCR product) are loaded in the gel slots with a Hamilton syringe for each sample (a maximum of 18 samples per gel). The gel is run at 100 V and 60°C for 16 h in 1x TAE buffer (40 mM Tris base [pH 7.4], 20 mM sodium acetate, 1 mM EDTA).
We used the DNA stains GelStar (FMC BioProducts) or SYBRGold (Molecular Probes). One of the glass plates is detached from the gel and 15 ml of the stain solution (15 ml of 1x TAE with 3 µl of the stain stock solution) are added covering the whole gel. The gel is stained for 30 minutes in the dark, rinsed with a large volume (around 500 ml) of 1x TAE buffer, removed from the glassplate and transferred carefully to a UV transparent gel scoop (Sigma). The gel is visualized with U.V. in the Fluor-S MultiImager (Bio-Rad) with the Multi-Analyst software (Bio-Rad). High-resolution images (1312 x 1034 pixels, 12-bits dynamic range) are saved as computer files (4.6 Mb).
Quantitative analysis of DGGE fingerprints
(from Amann 1995 and modified from Schönhuber et al. 1997, Adapted by Biegala and Simon for bacteria. Updated nov 00 for eukaryotes)
A. Cell preparation
A1. Fix the cells
A3. Storage and alteration of cell wall
B. In situ hybridization
B3. Rinse briefly
B5. TSA reaction (Tyramide Signal Amplification)
B7. Rinse briefly
B6. DAPI labelling
B8. Mount slide for epifluorescence observations
Stock solutions for FISH of eukaryotes
10% paraformaldehyde fixative (PFA10) **MADE FRESHLY**Stored at -20°C then at 4°C
Hybridization buffer (0.01% SDS, 50% to 36% Formamide according to probes)
Note: it should have the same Tm as the hybridization buffer even though there is no formamide and the equivalent stringent power is created by increasing NaCl concentration (see corresponding tables).
CitiFluor (AF3 mountant solution, Univ. Of Canterbury)
Stock solution aliquoted and kept at 4°C. Working solution = unchanged work solution.
Recommended method is CTAB.
Table 1: Primers and enzymes for clone libraries
Purifying PCR products
Products (500 ng) were electrophoresed on an agarose gel (1%) in the presence of Crystal Violet (add 0.5 - 1m l of a 2 mg/ml stock soln. Per ml gel) without Ethidium bromide. This allows visualisation of the DNA band under normal light. Bands were cut out and purified from the agarose with easypure" from Biozym.
Cloning of PCR products
Purified PCR products were cloned using the TOPO XL zero background kit from Invitrogen. With this kit a standard reaction produces 500 - 1000 clones with an efficiency >95%. Therefore it is possible to generate more than 10,000 - 20,000 18S clones from 500 ml filtered seawater.
Selection of clones
Analysis of clones
Filter 4 - 10 litres of seawater onto a 0.2 um pore size Gelman Supor-200 47 mm diameter filter on a vacuum of approx. 5-8 in Hg. Use of a 0.45 um pore size Gelman Supor-450 47mm diameter filter improves rate of filtration significantly and is fine for both Synechococcus and Prochlorococcus. The Supor filters are advantageous because they have:
Protocol I: Mainly as set out in Kerkoff and Ward (1993). AEM 59:1303-1309
Yield: 1 - 4 ug DNA depending on site and season.
Protocol II: Mainly as set out in Gordan and Giovannoni (1996). AEM: 62(4): 1171-1177.
We did this test during MIDAS workshop II in the Alicante solar salterns in May 1999. If you want to see a brief description of the salterns and the workshop you can check the MIDAS web page : http://www.icm.csic.es/bio/projects/midas
What we did was to have four different groups of people use their respective protocols for extraction of DNA in several ponds along the salinity gradient, and then we run a DGGE. The most laborious protocol was Ramon's and the easiest the one from Bergen, where they directly PCR from 1 ml of sample. I enclose the picture for half of the ponds (the other half shows the same). The agreement between methods is striking, while the differences between ponds are substantial.
The lanes are (starting from the left):
25X SET buffer:(250 ml)
Saline EtOH fixative (freshly prepared for every experiment!)
5X SET buffer:(25 ml)