TITLE: Inorganic polyphosphate from microalgae: A DAPI-based estimation in microtiter plate 
AUTHORS: Yingyu  Hu, Zoe  V Finkel

[DESCRIPTION]
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.
 



[GUIDELINES]

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


[STEPS]
SECTION: Preparation of reagents
7. Tris buffer  20 mM pH 7.0 
 

SECTION: Preparation of reagents
7.1. In a 1 L volumetric flask, top 20 mL 1 M pH 7.0 Tris buffer to 1 L with MilliQ
SECTION: Preparation of reagents
7.2. Filter through Rapid-flow and store at Room temperature
 

 

SECTION: Preparation of reagents
8. PolyP primary standard stock
SECTION: Preparation of reagents
8.1. Weigh one glass pellet of polyP (45) and write down the weight.

 

SECTION: Preparation of reagents
8.2. Transfer the pellet into a 100 mL graduated cylinder.
SECTION: Preparation of reagents
8.3. Dilute to 100 mL with Tris 20 mM pH 7.0 
SECTION: Preparation of reagents
8.4. Aliquot primary stock into 10~50 uL per microtube with Stepper and store at -20 °C
SECTION: Preparation of reagents
9. 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
SECTION: Preparation of reagents
10. Proteinase K 20 mg/ml 
SECTION: Preparation of reagents
10.1. Add 25 mL MilliQ directly into the original package of Proteinase K, vortex to mix
SECTION: Preparation of reagents
10.2. Aliquot 600 uL to microtubes (around 45 microtubes) and keep frozen at -20 °C
SECTION: Preparation of reagents
11. DAPI primary stock 14.3 mM  
Add 2 mL MilliQ directly into the original package and keep frozen at -20 °C
SECTION: Preliminary extraction efficiency test
12. Prepare boiling bath.

 

 

 

SECTION: Preliminary extraction efficiency test
13. Transfer sample into glass centrifuge tube.

 

SECTION: Preliminary extraction efficiency test
14. If the sample has less than 3 ug total particulate phosphate, use 2 mL Tris Buffer 20 mM pH 7.0 for each extraction.
Otherwise, use 4 mL Tris Buffer 20 mM pH 7.0  for each extraction.
SECTION: Preliminary extraction efficiency test
15. Add 2 mL or 4 mL Tris buffer 20 mM pH 7.0 , vortex and then sonicate.
 

SECTION: Preliminary extraction efficiency test
16. Keep in boiling bath. 
SECTION: Preliminary extraction efficiency test
17. Sonicate
SECTION: Preliminary extraction efficiency test
18. Vortex and then transfer extract to a 20 mL scintillation vial, label the vial with number of extraction.

 

 

SECTION: Preliminary extraction efficiency test
19.   
SECTION: Preliminary extraction efficiency test
20. Transfer 2 mL of extract to a 2 mL microtube. 
13300 rpm, 5 min
SECTION: Preliminary extraction efficiency test
21. Load black microtitre plate with 250 µL extract (triplicate).
Tris buffer 20 mM pH 7.0 is used as blank.

 

 



SECTION: Preliminary extraction efficiency test
22. Prepare DAPI working solution 100 uM
Dilute 2.1 µL of 14.3 mM DAPI stock with 300 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.
SECTION: Preliminary extraction efficiency test
23.  In the dimmed room with only red light bulb on, by using either stepper or pipette, add 30 µL 100 uM DAPI to each sample in the plate.

 

 

SECTION: Preliminary extraction efficiency test
24. Adhere black film on the top of a microplate lid and cover the plate with this lid. 

 

SECTION: Preliminary extraction efficiency test
25. Shake at room temperature
SECTION: Preliminary extraction efficiency test
26. Read fluorescence: excitation at 410 nm and emission at 550 nm

 

SECTION: Preliminary extraction efficiency test
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.
SECTION: Extraction of polyphosphate from samples
28. Prepare boiling bath.
SECTION: Extraction of polyphosphate from samples
30. Transfer samples into glass centrifuge tubes.
SECTION: Extraction of polyphosphate from samples
31. Add same amount of Tris buffer 20 mM pH 7.0 as preliminary test, vortex and then sonicate
SECTION: Extraction of polyphosphate from samples
32. Place vials in boiling bath
SECTION: Extraction of polyphosphate from samples
33. Sonicate
SECTION: Extraction of polyphosphate from samples
34. Vortex and then remove extract to a 50 mL Falcon tube, and then  . 
Keep using the same pasteur pipet



 

SECTION: Extraction of polyphosphate from samples
35. Combine extract 1~N into the same Falcon tube, keep extract N+1 in the centrifuge tube.
SECTION: Enzyme treated extract
37. Centrifuge the mixture of 1~N extract3200 rpm, 5 min 
 

SECTION: Enzyme treated extract
38. Transfer 4 mL supernatant to a scintillation vial, add 40 µL RNase and 40 µL DNase
SECTION: Enzyme treated extract
39. Incubate at 37 °C, shake continuously
 

SECTION: Enzyme treated extract
40. Add 80 µL Proteinase
SECTION: Enzyme treated extract
41. Incubate at 37 °C, shake continuously.
SECTION: Enzyme treated standard amended extract
47. Prepare PolyP working standard 7.6 uM 
Based on the actual concentration of PolyP (45) primary or secondary standard stock, dilute a certain volume of stock with Tris buffer 20 mM pH 7.0
For a final concentration  7.6 uM
Total volume = 320 X N (ul)
N = sample number
FW=101.9617
SECTION: Enzyme treated standard amended extract
48. Transfer 1680 µL of enzyme treated extract into a scintillation vial.
SECTION: Enzyme treated standard amended extract
49. Add 320 µL  7.6 uM  polyP working standard to 1680 µL of enzyme treated extract, vortex.
SECTION: Enzyme treated N+1 extract
42. Centrifuge extract "N+1" (in the centrifuge tube) 3200 rpm, 5 min 
SECTION: Enzyme treated N+1 extract
43. Transfer  1.5 mL supernatant into a 2 mL tube, add 15 µL RNase and 15 µL DNase
SECTION: Enzyme treated N+1 extract
44. Incubate at 37 °C, shake continuously
 

SECTION: Enzyme treated N+1 extract
45. Add 30 µL Proteinase
SECTION: Enzyme treated N+1 extract
46. Incubate at 37 °C, shake continuously
SECTION: Load microtiter plate
50. Load 250 mL samples to the microplate. Organize samples as shown in the following scheme:
  

  

SECTION: Load microtiter plate
51. Prepare DAPI working solution 100 uM
Dilute 2.1 µL of 14.3 mM DAPI stock with 300 µL MilliQ in a foil wrapped microtube and vortex.
Total volume = 30 X 54 (ul) for one microplate
12.6 ul  14.3 mM DAPI stock with 1800 µL MilliQ
SECTION: Load microtiter plate
52. In a dimmed room with only red bulb on, add 30 µL DAPI working solution 100 uM to each sample in the microplate except for those labelled with UN.
       

 

SECTION: Load microtiter plate
53. Adhere black film on the top of a microplate lid and cover the plate with this lid. 
SECTION: Load microtiter plate
54. Shake at room temperature.
SECTION: Load microtiter plate
55. Read fluorescence: excitation at 410 nm and emission at 550 nm
SECTION: Calculation
56. 
Definition of symbol

  

SECTION: Calculation
57. 
  
SECTION: Calculation
58. 
     

SECTION: Extraction of polyphosphate from samples
29. Prepare 37 °C incubator.

SECTION: Calculation
59.    
   

SECTION: Calculation
60.    

SECTION: Calculation
61.   

SECTION: Extraction of polyphosphate from samples
36.    

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

SECTION: Sample collection
2. Rinse sample with filtered seawater
SECTION: Sample collection
3. Place sample filters in cryogenic vials
SECTION: Sample collection
4. Filter blank media (without cells) through precombusted GFF filter as blank.
SECTION: Sample collection
5. Flash freeze filters and stored at -20 °C 

SECTION: Sample collection
6. Freeze dry before measurement.