TITLE: Lipids in microalgae: Quantitation by acid-dichromate method in microtiter plate
AUTHORS: Yingyu  Hu, Zoe  V Finkel

[DESCRIPTION]
This is a protocol for quantitating total lipids in microalgae. 

The acid-dichromate method is widely used to perform colorimetric analysis of extracted lipids. Here we present a protocol using 96-well microtiter plate for safe and efficient sample handling with high throughput. Only 500 ul of 0.15% acid-dichromate is required for each sample, which greatly reduces the amount of corrosive and toxic reagent.  

In addition, comparing with the absorbance at 440 nm, the absorbance at 348 nm yields five-time higher sensitivity in lipids quantitation.

Total lipids in the samples should not excess 100 ug. Accurate quantitation can be achieved with as little as 20 ug. A working detection limit is about 5 ug. 
 


[BEFORE_START]
Pre-combust 12 ml amber vials (one for each sample/sample blank/standard/standard blank) and glass vials (for MilliQ water, concentrated sulphuric acid, acid-dichromate reagent).

Rinse vial caps with 95% ethanol and dry.

Rinse serological pipet (0.5 and 5 mL) with chloroform. The pipets are used to measure and transfer extract,  concentrated sulphuric acid and acid-dichromate reagent.

Rinse syringe with chloroform and dry.


[GUIDELINES]
Lipids of microalgae is extracted by Folch solvent. The extract is dissolved in chloroform and stored at -80 degree ULT freezer.
 

Estimate the mass of extracted lipids as 10~30% of total cell mass. 

The quantitative range of this method is 0~80 ug.


[STEPS]
SECTION: Preparation of Standard
3. Prepare  glyceryl tripalmitate (GTP) primary standard solution (around 1 mg/ml)
SECTION: Preparation of Standard
3.1. Place frozen GTP in vacuum desiccator with lose cap until it is warmed to  Room temperature before making primary standard solution

SECTION: Preparation of Standard
3.2. Weigh around 1 mg GTP, take note of the actual weight. 

SECTION: Preparation of Standard
3.3. Dissolve GTP by 1 mL chloroform in amber vial, gently vortex. 

SECTION: Preparation of Standard
4. Prepare working standards:
SECTION: Preparation of Standard
4.1. 5 ug/vial:
In two 12 ml amber vials, add 5 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
SECTION: Preparation of Standard
4.2. 10 ug/vial:
In two 12 ml amber vials, add  10 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
SECTION: Preparation of Standard
4.3. 20 ug/vial:
In two 12 ml amber vials, add  20 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
SECTION: Preparation of Standard
4.4. 40 ug/vial:
In two 12 ml amber vials, add  40 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
SECTION: Preparation of Standard
4.5. 80 ug/vial
In two 12 ml amber vials, add  80 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
SECTION: Preparation of Standard
5. Dry working standards at  Room temperature  under N2 gas stream (<2 psi).

SECTION: Preparation of acid-dichromate reagent
6. Estimate the total volume of potassium dichromate required:
Number of standards and standard blanks: 12
Number of samples and sample blanks: N

V=0.5x(N+12) ml
SECTION: Preparation of acid-dichromate reagent
7. Transfer concentrated sulphuric acid to a glass vial for temporary storage
SECTION: Preparation of acid-dichromate reagent
8. Weigh a glass vial, and tare the balance
SECTION: Preparation of acid-dichromate reagent
9. Use 5 ml serological pipet to measure and transfer concentrated sulphuric acid to this vial. The volume of sulphuric acid is several milliliter more than estimated in  . Write down the weight of sulphuric acid.

SECTION: Preparation of acid-dichromate reagent
10. The weight of dichromate required for the 0.15% (w/w) acid-dichromate reagent equals the weight of sulphuric acid multiplied by (0.15/99.85).
SECTION: Preparation of acid-dichromate reagent
11. Weigh dichromate and dissolve it into concentrated sulphuric acid. Cap the vial and vortex gently.
SECTION: Reaction of lipids and acid-dichromate reagent
12. Allow frozen extract warm up to Room temperature .
SECTION: Reaction of lipids and acid-dichromate reagent
13. Label two 12 mL vials with “+ Blank” and “- Blank”.
"-Blank" is 0 ug GTP.
"+Blank" is the reference of absorbance.
SECTION: Reaction of lipids and acid-dichromate reagent
14. Prepare boiling water bath on hot plate, place a vial rack in the water bath
SECTION: Reaction of lipids and acid-dichromate reagent
15. Add 0.5 mL of acid-dichromate reagent to each vial (standards, +Blank and –Blank, samples and sample blanks). Cap and vortex right after. 

 

SECTION: Reaction of lipids and acid-dichromate reagent
16. Keep reaction vials in boiling water for 15 min.

SECTION: Reaction of lipids and acid-dichromate reagent
17. Cool vials to Room temperature in the fumehood
SECTION: Reaction of lipids and acid-dichromate reagent
18. Prepare 0.2 g/ml sodium sulphite solution
Weigh  0.2 g sodium sulphite in a 2 ml microtube.
Add 1 mL MilliQ water into the tube.
Vortex
SECTION: Reaction of lipids and acid-dichromate reagent
19. Add 1.125 mL MilliQ (1 mL + 125 uL by pipet) to each vial. Cap immediately and vortex.
SECTION: Reaction of lipids and acid-dichromate reagent
20. Cool vials to room temperature.
SECTION: Reaction of lipids and acid-dichromate reagent
21. Add 25 µL  0.2 g/ml sodium sulphite solution to the “+Blank” vial. Vortex.
SECTION: Colorimetric analysis
22. Vortex each vial and load 250 µL reactant into microtiter plate.
 

SECTION: Colorimetric analysis
23. Read absorbance at 348 nm
SECTION: Calculation
24. Subtract absorbance of "+Blank" from the absorbance of standards.
SECTION: Calculation
25. Plot the resulted absorbance versus mass of GTP (ug).  
 

 

SECTION: Calculation
26. Subtract absorbance of "+Blank" from the absorbance of samples and sample blanks.
SECTION: Calculation
27. Calculate the mass of lipids by using the standard curve and the resulted absorbance.
SECTION: Calculation
28. Subtract the lipids mass of the blank filter from the lipids mass of the samples.
SECTION: Calculation
29. Convert the resulted mass to total extracted lipids based on the fraction of extract used in the reaction.
SECTION: Preparation of Standard
4.6. 100 ug/vial
In two 12 ml amber vials, add  100 µL GTP primary standard to each vial. Cap the vial to avoid contamination.
SECTION: Note
1. If the biomass is unknown, process one of the replicates first, and then decide the dilution factor and the amount that can be used for phospholipids. 

SECTION: Note
2. If the result from Step 1 shows the lipids collected is lower than low-limit-of-detection, consider combine replicates as one sample.