Mar 22, 2023

Public workspaceDAPI-Based Polyphosphate Estimation with Extraction Sufficiency Validation: A Method for Quantifying Polyphosphate from Microalgae Samples

DOI
dx.doi.org/10.17504/protocols.io.ewov1ox87lr2/v1
  • 1Dalhousie University
Ying-Yu Hu
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DOI: dx.doi.org/10.17504/protocols.io.ewov1ox87lr2/v1
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2023. DAPI-Based Polyphosphate Estimation with Extraction Sufficiency Validation: A Method for Quantifying Polyphosphate from Microalgae Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1ox87lr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 18, 2023
Last Modified: March 22, 2023
Protocol Integer ID: 77245
Keywords: DAPI, polyphosphate, microtiter plate, microplate, microalgae, fluorescence
Funders Acknowledgement:
CBIOMES
Grant ID: 549937
SCOPE-Gradients
Grant ID: 723789
Abstract
The utilization of DAPI-based fluorometric estimation for polyphosphate (polyP) analysis from microalgae has become increasingly prevalent in field samples since its publication by Martin P. et al. This technique involves evaluating the fluorescence of DAPI-stained samples in quartz cuvettes using a spectrofluorometer. To reduce the consumption of reagent, time, and labor while minimizing DAPI photobleaching, we have adapted this method to a 96-well black microtiter plate with a black film-covered lid. Additionally, the calculation method has been modified to account for matrix effects in microplates.
Testing the number of treatment rounds necessary to extract all polyP is crucial. However, even when collecting samples from the same field location or cultivation condition, there can be high variability in treatment rounds among replicates, leading to significant background fluorescence and rendering the polyP from the sample undetectable. This challenge is especially prominent when measuring polyP from field samples. Limited sample availability and insufficient polyP extraction, combined with high background fluorescence, make the laborious measurement unpredictable and hinder accurate polyP measurement. This obstacle is a significant hurdle in polyP measurement.
In our assay, we overcome the challenge by validating the sufficiency of extraction for each sample and then measuring the polyP values.
To conduct the assay, roughly 400 uL RNase, 400 uL DNase, and 700 uL proteinase are required for four samples.
CITATION
Martin, Patrick & Van Mooy, Benjamin. Fluorometric Quantification of Polyphosphate in Environmental Plankton Samples: Extraction Protocols, Matrix Effects, and Nucleic Acid Interference. Applied and Environmental Microbiology.
LINK
http://doi.org/10.1128/AEM.02592-12


Guidelines

  1. Extracted polyphosphate must be measured on the same day. Polyphosphate loss has been observed if the extraction is processed days after.
  2. The polyphosphate standard aliquot can only be thawed and used once. Do not refrozen and thawed multiple times.

Materials
Chemicals

ReagentTris Buffer 1M pH 7.0Fisher ScientificCatalog #BP1756-500
ReagentSodium phosphate glass type 45Sigma AldrichCatalog #S4379-500MG
ReagentProteinase-K Fisher ScientificCatalog #BP1700-500
ReagentRNase A: 500 U/mL; RNase T1: 20000 U/mL Fisher ScientificCatalog #AM2288
ReagentTURBO DNase 2 U/uLFisher ScientificCatalog #AM2239
ReagentDAPI: 4′6-Diamidino-2-phenylindole dihydrochlorideFisher ScientificCatalog #D1306


Sample collection
Sample collection
Filter microalgae in liquid media onto GFF or PC filters, using gentle vacuum pressure (5 inches Hg).
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU

Rinse sample with filtered artificial seawater (no nutrients)
Place sample filters in cryogenic vials
Filter blank media (without cells) through GFF or PC filter as blank.
Flash freeze filters and stored at Temperature-20 °C

Freeze dry before measurement.
Equipment
FreeZone® 2.5 L Benchtop Freeze Dryers
NAME
Labconco®
BRAND
700202000
SKU

Preparation of reagents
Preparation of reagents
Tris buffer Concentration20 Mass Percent Ph7.0
Note
Budget:
About 250 mL per four samples

In a 1 L volumetric flask, top Amount20 mL Concentration1 Mass Percent Ph7.0 Tris buffer to 1 L with MilliQ
Store at TemperatureRoom temperature
PolyP primary standard stock
Weigh one glass pellet of polyP (45) and write down the weight.

Equipment
Microbalance
NAME
Cubis series
TYPE
Sartorius
BRAND
MSE6.6S-000-DM
SKU

Transfer the pellet into a 100 mL graduated cylinder.
Dilute to 100 mL with Tris Concentration20 Mass Percent Ph7.0
Aliquot primary stock into 10~50 uL per microtube with Stepper and store at Temperature-20 °C
PolyP secondary standard stock
If the pellet is far more than 10 mg, dilute primary to secondary to bring down the concentration before preparing working standard
Proteinase K Concentration20 Mass Percent
Add Amount25 mL MilliQ directly into the original package of Proteinase K, vortex to mix
Aliquot 700 uL to microtubes and keep frozen at Temperature-20 °C
DAPI primary stock Concentration14.3 Mass Percent
Add Amount2 mL MilliQ directly into the original package and keep frozen at Temperature-20 °C
Preliminary extraction efficiency test
Preliminary extraction efficiency test
Prepare boiling bath.

Equipment
VWR® Advanced Hot Plates
NAME
VWR
BRAND
97042-658
SKU

Equipment
Hollow Polypropylene (PP) Ball Bath Covers, 20 mm
NAME
Cole-Parmer
BRAND
UZ-06821-04
SKU

Equipment
Tube rack
NAME
Simport MultiRack™
BRAND
CA48648-606
SKU

Prepare Temperature37 °C incubator/shaker.

Transfer sample into glass centrifuge tube
Equipment
Disposable Glass Screw-Cap Centrifuge Tubes
NAME
10 mL
TYPE
Corning®
BRAND
99502-10
SKU

Label centrifuge tube for different samples, place one Pasteur pipet into the tube for transferring extract from the same sample
Label 15 mL Falcon tube from 1 to 15 for each one sample.
Add Amount4 mL Tris buffer Concentration20 Mass Percent Ph7.0 , vortex and then sonicate.
Equipment
Specific Pipette Tips 5mL
NAME
Thermo Scientific™ Finntip™
BRAND
21-377-304
SKU

15s
Keep in boiling bath.

Note
Make sure the tube rack is in the middle of the boiling bath and covered with PP balls. Tris solution in the tube should be boiling during the 5 minutes' incubation.

5m
Sonicate
15s
Vortex and then transfer extract to 15 mL Falcon tube, according to the extract number.
Note
Do not push filter to the bottom. Use Pasteur pipet, gently lift the filter upwards, and then transfer as much extract as possible. Gently press the extract out of the filter.

Equipment
Disposable Soda-Lime Glass Pasteur Pipets
NAME
5 3/4"
TYPE
Fisherbrand
BRAND
13-678-6A
SKU

Repeat Step 17 to Step 20 until complete 15 times' extraction in total.
Centrifuge the extract
Centrifigation3200 rpm, Room temperature, 00:05:00
5m
Use forward pipetting, load black microtitre plate with Amount200 µL supernatant from the extract (one well for one extract, no need to load replicates).
Tris buffer Concentration20 Mass Percent Ph7.0 is used as blank.

Equipment
96-Well Black Microplates
NAME
Polystyrene
TYPE
Greiner Bio-One
BRAND
655076
SKU


Prepare DAPI working solution Concentration100 Mass Percent
Dilute Amount12.6 µL of Concentration14.3 Mass Percent DAPI stock with Amount1800 µL MilliQ in a foil wrapped microtube and vortex.
In the dimmed room with only red light bulb on add Amount24 µL Concentration100 Mass Percent DAPI to each sample in the plate.
Adhere black film on the top of a microplate lid and cover the plate with this lid.
Equipment
Black Vinyl Films for Fluorescence and Photoprotection
NAME
VWR
BRAND
89087-692
SKU

Shake at room temperature for Duration00:07:00
7m
Read fluorescence: excitation at 410 nm and emission at 550 nm
Equipment
Varioskan LUX Multimode Microplate Reader
NAME
Thermo Fisher
BRAND
VL0L00D0
SKU

Plot fluorescence intensity versus number of extraction.
The number of extract (N) is the stationary point where the fluorescence of stained extract stops decreasing or the derivative of the fluorescence after that point is close to zero.

If , proceed to extract five additional times. And then measure the stained extract following the previous steps.
Combine Extraction 1 to Extraction N into a falcon tube.
Note
Try to transfer all solution including debris from each tube.
If the total volume is over 50 mL, use a beaker instead.

Sample codeNV(Tris) per extract (mL)


Enzyme treated extract
Enzyme treated extract
Well mix 1~N extract, transfer 12 mL into 15 mL falcon tube, centrifuge Centrifigation3200 rpm, Room temperature, 00:05:00
5m
Transfer Amount1.8 mL supernatant to a 2 mL tube (Set S).
Note
Sample is triplicated into S1a, S1b and S1c; S2a, S2b, S2c...etc.

Centrifuge extract "N+1" Centrifigation3200 rpm, Room temperature, 00:05:00
Note
Blank is duplicated into B1a and B1b; B2a and B2b... etc.

5m
Transfer Amount1.5 mL supernatant into a 2 mL tube (Set B).
In Set S, add Amount18 µL RNase and Amount18 µL DNase
Note
RNase tends to leave residue in the tip. However one package has only 1 mL RNase, it will be a waste to use reverse pipetting. After dispensing RNase into the vial, use the same tip to draw the solution and gently dispense it back into the solution for about three time, so that there is no residue remaining in the tip. Replace a new tip for the next vial.

Note
Require ~400 uL RNase and ~400 uL DNase.

In Set B, add Amount15 µL RNase and Amount15 µL DNase
Incubate at Temperature37 °C , shake continuously
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU

Note
Start the timer when temperature reaches Temperature37 °C


10m
Thaw proteinase (~700uL)

In Set S, add Amount36 µL Proteinase
In Set B, add Amount30 µL Proteinase
Incubate at Temperature37 °C , shake continuously.
Note
Start the timer when temperature reaches Temperature37 °C


30m
Enzyme treated standard amended extract
Enzyme treated standard amended extract
Prepare PolyP working standard [PO3]~Concentration7.6 Mass Percent
Based on the actual concentration of PolyP (45) primary or secondary standard stock, dilute a certain volume of stock with Tris buffer Concentration20 Mass Percent Ph7.0
For a final concentration Concentration7.6 Mass Percent
Note
Total volume = 160 X N (ul), where N = sample number

Note
FW(45Na2O.55P2O5)=10600
Mol of PO3 per mol of PolyP (45) = 110


Transfer Amount840 µL of enzyme treated extract (1~N) into 2 mL tubes (Set A).
Note
Forward pipetting, aspire and dispense for three times to mix.


Add Amount160 µL Concentration7.6 Mass Percent polyP working standard to Amount840 µL of enzyme treated extract, vortex.
Prepare DAPI working solution Concentration100 Mass Percent

Dilute Amount12.6 µL of Concentration14.3 Mass Percent DAPI stock with Amount1800 µL MilliQ in a foil wrapped microtube and vortex.
Load microtiter plate
Load microtiter plate
7m
7m
Load Amount200 µL blanks (B: N+1), samples (S: 1~N) and amended samples (A: Amended 1~N) to the microplate. Organize samples as shown in the following scheme:


Note
Reverse pipetting


In a dimmed room with only red bulb on, add Amount24 µL DAPI working solution Concentration100 Mass Percent to each sample in the microplate except for those labelled with (UN).
Note
Forward pipetting


Adhere black film on the top of a microplate lid and cover the plate with this lid.
Shake at room temperature for Duration00:07:00
7m
Shake duration: 1 min
Shaking type: continuous
Shaking speed and force: 600 rpm/High
Fluorescence: excitation at 410 nm and emission at 550 nm
Measurement time: 300 ms
Excitation bandwidth: 5 nm
Calculation
Calculation


Citations
Martin, Patrick & Van Mooy, Benjamin. Fluorometric Quantification of Polyphosphate in Environmental Plankton Samples: Extraction Protocols, Matrix Effects, and Nucleic Acid Interference
http://doi.org/10.1128/AEM.02592-12