Apr 04, 2022

Public workspaceInorganic polyphosphate from microalgae: A DAPI-based estimation in microtiter plate V.6

This protocol is a draft, published without a DOI.
  • 1Dalhousie University
Ying-Yu Hu
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Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2022. Inorganic polyphosphate from microalgae: A DAPI-based estimation in microtiter plate . protocols.io https://protocols.io/view/inorganic-polyphosphate-from-microalgae-a-dapi-bas-b64brgsnVersion created by Ying-Yu Hu
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: April 04, 2022
Last Modified: April 04, 2022
Protocol Integer ID: 60259
Keywords: DAPI, polyphosphate, microtiter plate, microplate, microalgae, fluorescence
Abstract
The DAPI-based fluorometric estimation of polyphosphate from microalgae has been widely used in field samples since the method was published by Martin P. et al., where fluorescence of DAPI-stained samples is analyzed in quartz cuvettes by spectrofluorometer. In order to minimize the photobleaching of DAPI and reduce the consumption of reagent, time and labor, we have now scaled this method to 96-well black microtiter plate. Regarding to the matrix effects in microplate, the calculation has been modified accordingly.
Our method permits processing nine samples by using only 250 uL of extracted sample, 500 uL of RNase, 500 uL of DNase, 1000 uL of proteinase and <2000 uL of DAPI (100 uM). A lid with black film can protect all DAPI-stained samples from photobleaching.
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. Total particulate phosphorus (TPP) measurement is recommended prior to the extraction of polyphosphate. The level of TPP helps to estimate the volume of extraction solution.
  2. Different species or different sample locations (for field samples) require different numbers of extraction. A preliminary extraction efficiency test helps to obtain optimized number of extraction for extracting the most amount of polyphosphate with the least number of extraction.
  3. Extracted polyphosphate must be measured on the same day. Polyphosphate loss has been observed if the extraction is processed days after.
  4. 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

Budget of enzyme for every nine samples
  • RNase A (AM2288): half package
  • DNase (AM2239): one package
  • Proteinase-K: two tubes of aliquot (about 600 uL/tube)


Sample collection
Sample collection
Filter microalgae in liquid media onto precombusted GFF 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 seawater
Place sample filters in cryogenic vials
Filter blank media (without cells) through precombusted GFF 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 mM Ph7.0
Note
Budget:
About 400 mL per nine samples

In a 1 L volumetric flask, top Amount20 mL Concentration1 M Ph7.0 Tris buffer to 1 L with MilliQ
Filter through Rapid-flow and store at TemperatureRoom temperature
Note
If Tris buffer is to be used right away, this step is not necessary.

Equipment
Sterile Disposable Filter Units with PES Membrane
NAME
Thermo Scientific™ Nalgene™ Rapid-Flow™
BRAND
5964520
SKU

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 mM 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 mg/ml
Add Amount25 mL MilliQ directly into the original package of Proteinase K, vortex to mix
Aliquot 600 uL to microtubes (around 45 microtubes) and keep frozen at Temperature-20 °C
DAPI primary stock Concentration14.3 mM
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

Transfer sample into glass centrifuge tube.

Equipment
Disposable Glass Screw-Cap Centrifuge Tubes
NAME
10 mL
TYPE
Corning®
BRAND
99502-10
SKU

If the sample has less than 3 ug total particulate phosphate, use Amount2 mL Tris Buffer Concentration20 mM Ph7.0 for each extraction.
Otherwise, use Amount4 mL Tris Buffer Concentration20 mM Ph7.0 for each extraction.
Add Amount2 mL or Amount4 mL Tris buffer Concentration20 mM 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.
5m
Sonicate
15s
Vortex and then transfer extract to a 20 mL scintillation vial, label the vial with number of extraction.

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

Equipment
VWR® Vials, Borosilicate Glass, with Phenolic Screw Cap
NAME
22.18 mL
TYPE
VWR
BRAND
66012-044
SKU
https://ca.vwr.com/store/product/en/4618574/vwr-vials-borosilicate-glass-with-phenolic-screw-cap
LINK
24-400 cap: VWR 89076-764
SPECIFICATIONS

Go to Repeat until total extract number reaches 10.
Transfer 2 mL of extract to a 2 mL microtube.
Centrifigation13300 rpm, Room temperature, 00:05:00
Load black microtitre plate with Amount250 µL extract (triplicate).
Tris buffer Concentration20 mM Ph7.0 is used as blank.

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

Expected result





Prepare DAPI working solution Concentration100 uM
Dilute Amount2.1 µL of Concentration14.3 mM DAPI stock with Amount300 µL MilliQ in a foil wrapped microtube and vortex.
Volume of total 100 uM DAPI = 30 X (10 X 3 X N + 3), where N is the number of culture samples tested.
In the dimmed room with only red light bulb on, by using either stepper or pipette, add Amount30 µL Concentration100 uM DAPI to each sample in the plate.

Equipment
Finntip™ Stepper Pipette Tips
NAME
500 uL
TYPE
Thermo Scientific™
BRAND
9404170
SKU

Equipment
Finnpipette Stepper Pipette
NAME
Thermo Scientific™
BRAND
4540000
SKU

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
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.
Extraction of polyphosphate from samples
Extraction of polyphosphate from samples
Prepare boiling bath.
45m
Prepare Temperature37 °C incubator.

Transfer samples into glass centrifuge tubes.
Add same amount of Tris buffer Concentration20 mM Ph7.0 as preliminary test, vortex and then sonicate
15s
Place vials in boiling bath
5m
Sonicate
15s
Vortex and then remove extract to a 50 mL Falcon tube, and then Go to until total extract reaches N+1 .
Keep using the same pasteur pipet



Equipment
Falcon® Centrifuge Tubes
NAME
Polypropylene, Sterile, 50 mL
TYPE
Corning®
BRAND
352070
SKU

Combine extract 1~N into the same Falcon tube, keep extract N+1 in the centrifuge tube.
Main setup

Enzyme treated extract
Enzyme treated extract
Centrifuge the mixture of 1~N extractCentrifigation3200 rpm, Room temperature, 00:05:00
Equipment
General-purpose benchtop centrifuge
NAME
IEC CENTRA CL2
TYPE
Thermo
BRAND
00427 0F
SKU

Transfer Amount4 mL supernatant to a scintillation vial, add Amount40 µL RNase and Amount40 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.

Incubate at Temperature37 °C , shake continuously
Equipment
SHAKING INCUBATOR
NAME
71L
TYPE
Corning® LSE™
BRAND
6753
SKU

10m
Add Amount80 µL Proteinase
Incubate at Temperature37 °C , shake continuously.
30m
Enzyme treated N+1 extract
Enzyme treated N+1 extract
Centrifuge extract "N+1" (in the centrifuge tube) Centrifigation3200 rpm, Room temperature, 00:05:00
Transfer Amount1.5 mL supernatant into a 2 mL tube, add Amount15 µL RNase and Amount15 µL DNase
Incubate at Temperature37 °C , shake continuously
Note
Thaw proteinase during the 10-minute incubation.

10m
Add Amount30 µL Proteinase
Incubate at Temperature37 °C , shake continuously
30m
Enzyme treated standard amended extract
Enzyme treated standard amended extract

Prepare DAPI working solution Concentration100 uM
Dilute Amount2.1 µL of Concentration14.3 mM DAPI stock with Amount300 µL MilliQ in a foil wrapped microtube and vortex.
Total volume = 30 X 63 (ul) for one microplate
12.6 ul Concentration14.3 mM DAPI stock with Amount1800 µL MilliQ
Prepare PolyP working standard Concentration7.6 uM
Based on the actual concentration of PolyP (45) primary or secondary standard stock, dilute a certain volume of stock with Tris buffer Concentration20 mM Ph7.0
For a final concentration Concentration7.6 uM
Total volume = 320 X N (ul)
N = sample number

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



Load each enzyme treated sample Amount250 µL (no need to have replicates) to microplate.
In a dimmed room with only red bulb on, add Amount30 µL DAPI working solution Concentration100 uM to each sample in the microplate
Adhere black film on the top of a microplate lid and cover the plate with this lid.
Shake at room temperature.
7m
Read fluorescence: excitation at 410 nm and emission at 550 nm
Transfer Amount1680 µL of enzyme treated extract into a scintillation vial.
Note
Reverse pipetting

Note
If fluorescence of the enzyme treated samples is higher than 5, dilute samples into 50% or more with Tris buffer until the estimated fluorescence is lower than 5 (this dilution factor must be added into the final calculation).


Add Amount320 µL Concentration7.6 uM polyP working standard to Amount1680 µL of enzyme treated extract, vortex.
Load microtiter plate
Load microtiter plate
Load Amount250 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

Note
If samples have been diluted in Go togo to step #54 , load diluted samples in this step


In a dimmed room with only red bulb on, add Amount30 µL DAPI working solution Concentration100 uM to each sample in the microplate except for those labelled with UN.
B: N+1
S: 1~N
A: Amended sample

Note
Use stepper to dispense DAPI is faster but show bigger deviation.

Adhere black film on the top of a microplate lid and cover the plate with this lid.
Shake at room temperature.
7m
Read fluorescence: excitation at 410 nm and emission at 550 nm
Calculation
Calculation

Definition of symbol














Note
If samples have been diluted in Go togo to step #54 , add dilution factor into the 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