This method is based on Brendel et al. (2000), Kumar & Turner (2015), and Greer et al. (2018) to develop a simple and rapid procedure for cellulose quantification using ground plant material.
1. Places a few leaves in 20 mL polyethylene vial with 4-5 metal beads for grinding and cap appropriately. Grind leaves using a cryogenic bead grinder.
2. Take 2 mL round-bottomed microcentrifuge tube and using a pin or sharp tweezers make two holes in the lid.
3. Weigh out ~20 mg of ground plant material into a 2 mL round-bottomed microcentrifuge tube.
4. Add 400 µL of acetic acid (80%; v/v, if using glacial acetic acid prep to this concentration beforehand) and 40 µL of concentrated nitric acid (69%; v/v) to the sample and leave with lids open for 15 minutes.
5. Cap tube tightly and move to heating block, start with heating block at room temperature and set temperature to 120⁰C. NOTE: it is important to start with the heating block at room temperature to prevent overpressure inside the microcentrifuge tubes and potential spillage of the acid.
6. Once heating block reaches 120⁰C boil samples for 30 minutes tapping the top of each tube every 5 minutes to prevent condensation building up on the lid.
7. Move tubes to rack to cool.
8. Wash the sample as follows:
Add the required reagent and invert to mix using parafilm to cover holes in lid. Centrifuge for 5 minutes at 7000 rpm and discard supernatant.
a. 1 x 1000 µL ethanol, to remove extraction breakdown products,
b. 1 x 1000 µL deionized water, to remove traces of nitric acid,
c. 1 x 1000 µL ethanol,
d. 1 x 1000 µL acetone.
9. Cover rack with samples with aluminium foil and leave to dry in fume hood overnight. Figure 1 displays some examples of cellulose after the digested and washed products have dried.
10. Make a 67% sulphuric acid solution by slowly adding 335 mL concentrated sulphuric acid to 165 mL milli-Q water and allow solution to cool. NOTE: Always add acid to water and never the other way around.
11. Add 1 mL of the 67% sulphuric acid solution to the dried cellulose, only add acid to the number of samples you plan to process that day.
12. Shake at room temperature for 1 hour until no material can be seen.
13. Prepare the glucose standards as outlined in Kumar & Turner (2015). These standards should have concentrations of 0, 10, 20, 40, 60, 80 and 100 µg/mL of glucose.
14. In a new 15 mL screw capped tube add 500 µL of milli-Q water and add 20 µL of a sample to the water. Additionally, aliquot 500 µL of each glucose standard into a 2 mL screw capped tube.
15. Prepare 100 mL of a 0.3% anthrone in concentrated sulphuric acid solution over ice (stability ~ 3 hours). Mix anthrone into sulphuric acid using a glass stirring rod.
16. Add 1 mL of the anthrone solution to each test sample and standard slowly down the side of the microcentrifuge tube to prevent mixing of samples as much as possible. Cap tightly and mix via inversion. NOTE: tubes will get very hot, wear appropriate PPE.
17. Place in boiling water bath for five minutes and cool on ice. CRITICAL: It is important not to exceed the five minute timeframe when boiling the samples as they will lose their blue/green colouring and go an opaque white which is no longer viable for measurement.
18. Place 200 µL of sample in microplate and record OD620.
19. Generate a regression curve using the glucose standard readings and determine glucose concentration of each sample.
20. Use equation 1 to determine the concentration of cellulose in the ground plant material.
Equation 1: Cellulose (% of ground plant material) = glucose (µg/mL) / (weight of samples (µg) x 100 x 50 (to get total amount in 1 mL))