reaction. DO NOT use less than a 1:10 dilution on acidic
hydrolysates as the excess acid can have an effect on the reaction. ALWAYS run a set of standards and a CVS with each
batch of samples.
15. Prepare the MBTH working solution right before adding to
samples: 1:1 (v/v) solution of 3 mg/mL MBTH and 1 mg/
mL DTT. Prepare enough of the MBTH working solution to
add 500 μL to each sample and standard in a batch. Keep
batches to 40 total tubes or less.
16. Do not exceed the incubation time. Do not allow the samples
and standards to sit around after addition of the NaOH or
MBTH working solution—they must be placed on the hot
block within a few minutes of adding these solutions.
17. Once the ferric solution has been added, remove the glass tubes
from the hot block, and carefully vortex to mix. Allow the
samples and standards to react with the ferric solution for
10–15 min while the samples cool to room temperature.
Tubes may remain open during this time. Allow them to cool
in a hood to control fumes.
18. Once mixed, place an aliquot of the samples and standards into
the appropriate cuvettes, and obtain an absorbance on the
spectrophotometer at 620 nm. Take an absorbance within an
hour after the final dilution step. Zero the spectrophotometer
on 18.2 MΩ water. DO NOT zero the spectrophotometer on
the 0 standard from the calibration standard set; this standard
should be included as a point on the calibration curve.
19. Be sure to include the 0 standard as a point in the calibration. When
building a linear calibration curve, DO NOT force the curve
through the origin. Remember to correct the sample carbohydrate
concentration for the amount of hydrolysate that was used in the
dilution (for a 1:10 in 0.5 mL, use 50 μL), and then get a total
Table 1
Suggested standard concentrations for the spectrophotometric method
Concentration of stock/working solution
(mg/mL)
Dilution stock + water
(μL)
Final concentration
(mg/mL)
0.25
0 + 500
0
0.25
20 + 480
0.010
0.25
30 + 470
0.015
0.25
50 + 450
0.025
0.25
75 + 425
0.0375
0.25
100 + 400
0.050
Total Carbohydrate Content Determination of Microalgal Biomass by Acid. . .
199
hydrolysates as the excess acid can have an effect on the reaction. ALWAYS run a set of standards and a CVS with each
batch of samples.
15. Prepare the MBTH working solution right before adding to
samples: 1:1 (v/v) solution of 3 mg/mL MBTH and 1 mg/
mL DTT. Prepare enough of the MBTH working solution to
add 500 μL to each sample and standard in a batch. Keep
batches to 40 total tubes or less.
16. Do not exceed the incubation time. Do not allow the samples
and standards to sit around after addition of the NaOH or
MBTH working solution—they must be placed on the hot
block within a few minutes of adding these solutions.
17. Once the ferric solution has been added, remove the glass tubes
from the hot block, and carefully vortex to mix. Allow the
samples and standards to react with the ferric solution for
10–15 min while the samples cool to room temperature.
Tubes may remain open during this time. Allow them to cool
in a hood to control fumes.
18. Once mixed, place an aliquot of the samples and standards into
the appropriate cuvettes, and obtain an absorbance on the
spectrophotometer at 620 nm. Take an absorbance within an
hour after the final dilution step. Zero the spectrophotometer
on 18.2 MΩ water. DO NOT zero the spectrophotometer on
the 0 standard from the calibration standard set; this standard
should be included as a point on the calibration curve.
19. Be sure to include the 0 standard as a point in the calibration. When
building a linear calibration curve, DO NOT force the curve
through the origin. Remember to correct the sample carbohydrate
concentration for the amount of hydrolysate that was used in the
dilution (for a 1:10 in 0.5 mL, use 50 μL), and then get a total
Table 1
Suggested standard concentrations for the spectrophotometric method
Concentration of stock/working solution
(mg/mL)
Dilution stock + water
(μL)
Final concentration
(mg/mL)
0.25
0 + 500
0
0.25
20 + 480
0.010
0.25
30 + 470
0.015
0.25
50 + 450
0.025
0.25
75 + 425
0.0375
0.25
100 + 400
0.050
Total Carbohydrate Content Determination of Microalgal Biomass by Acid. . .
199