How to quantify residual cellulose

Before began:

Prepare 65% and 98% H2SO4

5% phenol (newly prepared), phenol was diluted with ddH2O

Steps:

1. 1mL sample was centrifuged at 5000rpm for 15min.
2. The pellet was suspended in distilled water and heat at 100 °С for 30 min to achieve cell lysis.
3. The residual cellulose was washed with distilled water 2 times through centrifugation at 500 rpm for 15 min and hydrolyzed into soluble sugar with 1 mL 65% H2SO4.
4. The hydrolysis sample was diluted 20 times and then soluble sugar were assayed by phenol-sulfuric acid method with glucose as standard (glucose standard was prepared with concentration range from 1-1600 µg/mL).
5. Mix three solutions in order: sample: 5% phenol: 98% H2SO4 = 150 µl : 150 µl : 750 µl. react for 30 min at room temperature.
6. Detect absorbance at 485 nm (492 nm is applicable).
7. Draw standard curve and calculate sugar content.