meth_1.16 Water Sampling from Brisbane River Estuary

Contributor(s): Christian Rinke (email) and Apoorva (email).

Citation:

AM Project ID: AM0005

Please note, the below protocol includes all aspects of the Brisbane River program, sampling, flow cytometry, and DNA extraction.

A. Sampling SOP (every two months) 

  1. At each of the sampling site (Shorncliffe = SH, Eagle Farm = EF, Indooroopilly = IN), collect ~35 L of subsurface water off the deck/pontoon using smaller containers and transfer to the 35 L container.
  2. Label the container and wheel it back to the car, where it is transported into the vehicle using a wooden plank/crates.
  3. Collect 250 mL unfiltered sample (in triplicates) from each sampling site and transfer into green containers supplied by ALS Environment testing for checking chemical parameters (Nitrates/Nitrates). Label the bottles with name, date and time of sampling.
  4. Collect 60 mL unfiltered sample (in triplicates) from each sampling sites and into purple containers supplied by ALS Environment testing for checking chemical parameters (Ammonium, Phosphates). Label the bottles with name, date and time of sampling.
  5. Collect 60 mL of field filtered (0.45 µm) sample in triplicates, from each sampling sites, into red containers supplied by ALS Environment testing for checking chemical parameters (Silicates). Label the bottles with name, date and time of sampling
  6. Record physical parameters of surface waters (10 cm deep) with Horiba probe (temperature, pH, dissolved oxygen, salinity, TDS, GPS for location). Take observations in triplicates, and record them onto table (below) and backup the observations with pictures of the readings.
  7. Store the green, purple and red bottles in eskys filled with frozen ice packs. At the end of sampling, carry the quote, sample list and hand over the bottles to ALS Environment Testing Centre, Blyth Street, Stafford, Brisbane. Also email them a list of samples as well as temperature and pH from each sampling site.
  8. Transport ~35 L of the water sample (SH, EF and IN) to the lab at ACE, Level 5, Building 76, UQ St Lucia using trolleys. 


 B. Physico-Chemical Parameters 

  1. Using the Horiba, record the readings (temperature, pH, dissolved oxygen, and GPS for location) in triplicates from each sampling site (SH, EF and IN) on the contextual metadata table.
  2. Record the values for dissolved silicates, nitrates, nitrites, ammonium, phosphates as obtained from ALS Environmental Testing onto the contextual metadata. 

C. Sample processing by filtration (Filtermaster 2001, designed and fabricated by Dr Rinke, ACE) 

  1. Prepare a filtration setup for amplicon samples. This includes three PVC lines connected to each of the 35L sample containers to their respective 0.22 µm Sterivex filters through a peristaltic pump.
  2. Keeping rotation less than 100 rpm, filter X volume of samples using the 0.22 µm Sterivex filter.
  3. Seal the Sterivex filters using filter caps and store them in UV-ed 50 mL falcon tubes and store at -20 °C until further processing.
  4. For metagenomics samples, the filtration setup is prepared such that water from 2 x 35 L containers (SH and EF at first, then IF) is drawn simultaneously into 2 x filtration towers containing 8 µm pre-filters, with the help of a peristaltic pump set at 100 rpm. The 2 x filtration towers are, in turn 6 x 0.22 µm Sterivex lines (3x for each sample) and the sample is filtered out into 6 x 10 L containers. Filter the X L of the sample through EACH of the Sterivex filters. Note: The filtermaster 2001 will allocate different volumes to each Sterivex filter. Therefore, it is important to watch the volume flowing through each filter. Once the first filter reaches the required sample volume, stop the flow to this filter using a flow control clip tubing roller, this will allocate the flow to the remaining filters and speed up filtration. You can now remove this filter.
  5. Seal the 0.22 µm Sterivex filters using filter caps, and store the Sterivex and the 8 µm prefilter for respective samples in UV-ed 50 mL falcon tubes and store at -20 °C until further processing.
  6. For microbial enumeration, prepare 2 % glutaraldehyde stocks of 5 x whole water samples, 5 x 8 µm filtered samples, 5 x 0.22 µm filtered samples from each of the sampling site. Flash freeze the stocks in liquid nitrogen and store at -80 °C.

Fig: Part of filtermaster 2001. The blue house is connected to the 8um prefilter tower, with the three lines connected to 0.22um Sterivex filters. 

 

 D. DNA extraction by Phenol-Chloroform 

  1. Defrost the filter and seal the bottom of the 0.22 µm Sterivex filter using a Sterivex cap.
  2. Fill the filter with 1.8 mL Lysis buffer and add 18 µL of lysozyme (100mg/mL) to the Sterivex filter. Cap the top of the filter.
  3. Incubate in a beaker of water for 1 h in a shaking incubator at 37 °C.
  4. Add 200 µL of Proteinase K (2 mg/mL) and incubate in a shaking 100 rpm incubator for 1 h at 55 °C.
  5. Recover the lysate into 4 x 2 mL tubes such that 600 µL was collected into 2 tubes from each Sterivex.
  6. Add equal volumes of Phenol:Chloroform:Isoamyl alcohol (25:24:1) and invert to mix.
  7. Spin at max 16,000 g for 10 min, recover aqueous phase to a new tube.
  8. Add equal volumes of Chloroform:Isoamyl alcohol (24:1) and invert to mix.
  9. Spin at max 16,000 g for 10 min, recover aqueous phase to a new tube.
  10. Precipitate DNA with 600 µL of isopropanol and let sit for 15 mins at room temperature (RT).
  11. Spin at max 20,000 g for 30 min at RT, discard supernatant.
  12. Add 500 µL of 70 % EtOH, flick tube to mix.
  13. Spin at max 20,000 g for 10 min at RT, discard supernatant.
  14. Air dry till EtOH is evaporated.
  15. Resuspend the pellet in 25-50 µL of sterile water and incubate overnight at 4 °C.
  16. Quantify DNA with Qubit and store at 20 °C. Record the results in the contextual metadata. 

 

E. Sample submission for Sequencing

  1. Submit x µL of samples from each sampling site (SH, EF & IN) for metagenomics sequencing (~10 Gb)
  2. Submit y µL of samples from each sampling site for amplicon sequencing
  3. Record results in the contextual metadata. 

 

F. Microbial Enumeration of samples (Cytoflex protocol communicated by Dr Martin Ostrowski, UTS) 

Note: limit of size for autotrophs is 100 μm and TE buffer is preferred to PBS for clearer cytograms for flow cytometry.

Reagents 

Tris-EDTA (TE) Buffer

  1. Purchase molecular grade TE buffer – 93283 Sigma Aldrich 500 mL ~$100 
  2. Filter through a 0.2 μm syringe filter to ensure a clear solution.

 SYBR Green I Nucleic Acid Gel Stain 10,000X concentrate in DMSO – Invitrogen S7563 ~$429 for 500 μL *Need a 5×10-5 final concentration of SYBR Green in sample for staining (Marie et al., 1999, Brussaard et al., 2004, Seymour et al., 2005, Patten et al., 2008).

  1. Make a working stock of a 1:500 dilution of SYBR Green in TE Buffer. To do this, add 10 μL to 4990 μL TE Buffer in a 15 mL centrifuge tube. Aliquot 500 μL into Eppendorf tubes and freeze until needed. One 500 μL tube will be enough to analyse 1 plate of samples. 

 

Reagents Preparation 

  1. Thaw one of the 500 μL aliquots of the 1:500 diluted SYBR Green working stock.
  2. Transfer the 500 μL to a 50 mL centrifuge tube and add 15.5 mL TE Buffer. This will further dilute the SYBR Green working stock by 1:32 (which will yield a final dilution of 1:20,000 once added to the plate).

Note: this is enough for one full plate of samples. 

Number of Wells

SYBR Green Working Stock

TE Buffer

96

500 μL

15.5 mL 

72

375 μL

11.625 mL 

48

250 μL

7.75 mL 

36

187.5 μL

5.815 mL 

24

125 μL

3.875 mL 

12

62.5 μL

1.94 mL 

6

31.25 μL

0.97 mL 

  1. Filter sterilize the SYBR Green working stock using a syringe and disposable 0.2 μm filter
  2. If you are simultaneously analysing stained and unstained fractions of the samples (to determine background levels), filter sterilize an extra 16 mL TE buffer using a syringe and disposable 0.2 μm filter. 

 

Sample Preparation Procedure 

  1. Set up flow cytometer.
  2. Turn on heat block to 37 °C. 
  3. Collect samples from liquid nitrogen or -80 °C freezer.
  4. Thaw samples at 37 °C on heat block for approx. 2-3 mins.
  5. Viruses, bacteria and autotrophs need to be analysed on separate plate.
  6. Prepare Autotroph plate first and run while you prepare the virus, then prepare the bacteria plate while the virus plate is running. 

 

Autotrophs

  1. Prepare size beads by diluting 1 drop beads into 300 μL MQ.
  2. Add MQ to wells B1-B2 and B10, B11, B12. 3. Add 200 μL Yellow Green Beads (YB) to well B3.
  3. Add size beads to wells B4-B9 in reverse order of size (2.0, 1.0, 0.5, 0.2, 0.1, 0.02 μm).
  4. Add 200 μL neat sample to well remaining in the 96 well plate. (e.g., x1 CTD per row, therefore 8 samples per row).
  5. Add MQ in two wells at the end of samples in each row as blanks. 

 

Viruses

To set up virus unstained plate: 

  1. Add 160 μL of TE Buffer to each well.
  2. Add 40 μL sample to each well.
  3. Cover plate with foil.
  4. Incubate on the heat block at 80 °C for 10 mins.
  5. Add TE in two wells at the end of samples in each row as blanks. 

To set up virus stained plate: 

  1. Add 160 μL of prepared SYBR Green I and TE Buffer diluted solution to each well.
  2. Add 40 μL sample to each well of a u-bottom 96 well plate.
  3. Cover plate with foil.
  4. Incubate on the heat block at 80 °C for 10 mins. 
  5. Add TE with SYBR in two wells at the end of samples in each row as blanks. 

 

Bacteria

  1. Add 160 μL of prepared SYBR Green I and TE Buffer (1:20,000) diluted solution to each well.
  2. Add 40 μL sample to each well of a u-bottom 96 well plate.
  3. Cover plate with foil.
  4. Add TE with SYBR in two wells at the end of samples in each row as blanks.

Record all the values in contextual metadata file.