Inorganic polyphosphate from microalgae: A DAPI-based estimation in microtiter plate

Ying-Yu Hu, Zoe V. Finkel

Published: 2022-01-21 DOI: 10.17504/protocols.io.b3xkqpkw

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 http://doi.org/10.1128/AEM.02592-12

Before start

Steps

Sample collection

Filter microalgae in liquid media onto precombusted GFF filters, using gentle vacuum pressure (5 inches Hg).

Equipment

Value	Label
Filter forceps	NAME
blunt end, stainless steel	TYPE
Millipore	BRAND
XX6200006P	SKU
http://www.emdmillipore.com/	LINK

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 -20°C

Freeze dry before measurement.

Equipment

Value	Label
FreeZone® 2.5 L Benchtop Freeze Dryers	NAME
Labconco®	BRAND
700202000	SKU

Preparation of reagents

Tris buffer 20mM 7.0

Note

Budget:About 400 mL per nine samples

7.1.

In a 1 L volumetric flask, top 20mL 1M 7.0 Tris buffer to 1 L with MilliQ

7.2.

Filter through Rapid-flow and store at Room temperature

Note

If Tris buffer is to be used right away, this step is not necessary.

Equipment

Value	Label
Sterile Disposable Filter Units with PES Membrane	NAME
Thermo Scientific™ Nalgene™ Rapid-Flow™	BRAND
5964520	SKU
http://www.fishersci.com/us/en/home.html	LINK

PolyP primary standard stock

8.1.

Weigh one glass pellet of polyP (45) and write down the weight.

Equipment

Value	Label
Microbalance	NAME
Cubis series	TYPE
Sartorius	BRAND
MSE6.6S-000-DM	SKU
http://www.fishersci.com/us/en/home.html	LINK

8.2.

Transfer the pellet into a 100 mL graduated cylinder.

8.3.

Dilute to 100 mL with Tris 20mM 7.0

8.4.

Aliquot primary stock into 10~50 uL per microtube with Stepper and store at -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

10.

Proteinase K 20mg/ml

10.1.

Add 25mL MilliQ directly into the original package of Proteinase K, vortex to mix

10.2.

Aliquot 600 uL to microtubes (around 45 microtubes) and keep frozen at -20°C

11.

DAPI primary stock 14.3mM

Add 2mL MilliQ directly into the original package and keep frozen at -20°C

Preliminary extraction efficiency test

12.

Prepare boiling bath.

Equipment

Value	Label
VWR® Advanced Hot Plates	NAME
VWR	BRAND
97042-658	SKU

Equipment

Value	Label
Hollow Polypropylene (PP) Ball Bath Covers, 20 mm	NAME
Cole-Parmer	BRAND
UZ-06821-04	SKU
http://www.coleparmer.com	LINK

Equipment

Value	Label
Tube rack	NAME
Simport MultiRack™	BRAND
CA48648-606	SKU

13.

Transfer sample into glass centrifuge tube.

Equipment

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

14.

If the sample has less than 3 ug total particulate phosphate, use 2mL Tris Buffer 20mM 7.0 for each extraction.

Otherwise, use 4mL Tris Buffer 20mM 7.0 for each extraction.

15.

Add 2mL or 4mL Tris buffer 20mM 7.0 , vortex and then sonicate.

Equipment

Value	Label
Specific Pipette Tips 5mL	NAME
Thermo Scientific™ Finntip™	BRAND
21-377-304	SKU
http://www.lifetechnologies.com	LINK

16.

Keep in boiling bath.

17.

Sonicate

18.

Vortex and then transfer extract to a 20 mL scintillation vial, label the vial with number of extraction.

Equipment

Value	Label
Disposable Soda-Lime Glass Pasteur Pipets	NAME
5 3/4"	TYPE
Fisherbrand	BRAND
13-678-6A	SKU
http://www.fishersci.com/us/en/home.html	LINK

Equipment

Value	Label
VWR® Vials, Borosilicate Glass, with Phenolic Screw Cap	NAME
22.18 mL	TYPE
VWR	BRAND
66012-044	SKU
24-400 cap: VWR 89076-764	SPECIFICATIONS

19.

20.

Transfer 2 mL of extract to a 2 mL microtube.

13300rpm

21.

Load black microtitre plate with 250µL extract (triplicate).

Tris buffer 20mM 7.0 is used as blank.

Equipment

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

Citation

22.

Prepare DAPI working solution 100uM

Dilute 2.1µL of 14.3mM DAPI stock with 300µL 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.

23.

In the dimmed room with only red light bulb on, by using either stepper or pipette, add 30µL 100uM DAPI to each sample in the plate.

Equipment

Value	Label
Finntip™ Stepper Pipette Tips	NAME
500 uL	TYPE
Thermo Scientific™	BRAND
9404170	SKU
http://www.fishersci.com/us/en/home.html	LINK

Equipment

Value	Label
Finnpipette Stepper Pipette	NAME
Thermo Scientific™	BRAND
4540000	SKU
http://www.fishersci.com/us/en/home.html	LINK

24.

Adhere black film on the top of a microplate lid and cover the plate with this lid.

Equipment

Value	Label
Black Vinyl Films for Fluorescence and Photoprotection	NAME
VWR	BRAND
89087-692	SKU

25.

Shake at room temperature

26.

Read fluorescence: excitation at 410 nm and emission at 550 nm

Equipment

Value	Label
Varioskan LUX Multimode Microplate Reader	NAME
Thermo Fisher	BRAND
VL0L00D0	SKU

27.

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

28.

Prepare boiling bath.

29.

Prepare 37°C incubator.

30.

Transfer samples into glass centrifuge tubes.

31.

Add same amount of Tris buffer 20mM 7.0 as preliminary test, vortex and then sonicate

32.

Place vials in boiling bath

33.

Sonicate

34.

Vortex and then remove extract to a 50 mL Falcon tube, and then .

Keep using the same pasteur pipet

Equipment

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

35.

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

36.

Enzyme treated extract

37.

Centrifuge the mixture of 1~N extract3200rpm

Equipment

Value	Label
General-purpose benchtop centrifuge	NAME
IEC CENTRA CL2	TYPE
Thermo	BRAND
00427 0F	SKU

38.

Transfer 4mL supernatant to a scintillation vial, add 40µL RNase and 40µL 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.

39.

Incubate at 37°C, shake continuously

Equipment

Value	Label
SHAKING INCUBATOR	NAME
71L	TYPE
Corning® LSE™	BRAND
6753	SKU

40.

Add 80µL Proteinase

41.

Incubate at 37°C, shake continuously.

Enzyme treated N+1 extract

42.

Centrifuge extract "N+1" (in the centrifuge tube) 3200rpm

43.

Transfer 1.5mL supernatant into a 2 mL tube, add 15µL RNase and 15µL DNase

44.

Incubate at 37°C, shake continuously

Note

Thaw proteinase during the 10-minute incubation.

45.

Add 30µL Proteinase

46.

Incubate at 37°C, shake continuously

Enzyme treated standard amended extract

47.

Prepare PolyP working standard 7.6uM

Based on the actual concentration of PolyP (45) primary or secondary standard stock, dilute a certain volume of stock with Tris buffer 20mM 7.0

For a final concentration 7.6uM

Total volume = 320 X N (ul)

N = sample number

Note

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

48.

Transfer 1680µL of enzyme treated extract into a scintillation vial.

Note

Reverse pipetting

49.

Add 320µL 7.6uM polyP working standard to 1680µL of enzyme treated extract, vortex.

Load microtiter plate

50.

Load 250mL samples to the microplate. Organize samples as shown in the following scheme:

Note

Reverse pipetting

51.

Prepare DAPI working solution 100uM

Dilute 2.1µL of 14.3mM DAPI stock with 300µL MilliQ in a foil wrapped microtube and vortex.

Total volume = 30 X 54 (ul) for one microplate

12.6 ul 14.3mM DAPI stock with 1800µL MilliQ

52.

In a dimmed room with only red bulb on, add 30µL DAPI working solution 100uM to each sample in the microplate except for those labelled with UN .