Processes 2018, 6,38
phenol sulfuric acid assay [40]. In the L-cysteine assay, pentose, heptose, and deoxy sugars contribute
to absorbance, and absorbance stability varies among different carbohydrates. Pentoses also contribute
to signal in the anthrone assay, but the absorbance fades rapidly and presents minimal interference.
Different hexoses may also produce differential responses in the anthrone assay; for example, mannose
produces 55% percent of the measured absorbance intensity of glucose [43]. Minimizing interference
from pentoses is a key consideration when selecting assays to avoid measuring nucleotide bases twice
in both nucleic acid and carbohydrate assays.
Glycogen is the most common form of carbohydrate storage for bacteria [44]. Glycogen content
can indicate cellular responses to changing nutrient conditions; for instance, E. coli and Synechococcus
7002 have both been found to increase glycogen storage during nitrogen limitation [45,46]. Glycogen
can be precipitated from cells with KOH, but alkalinity causes some degradation of glycogen.
An alternative method using sodium sulfate to adsorb and co-precipitate glycogen has been developed
for mosquitoes [47] and adapted to bacterial samples [48] and was selected for the current study.
The anthrone assay was selected for quantification of hexoses due to minimal pentose interference.
The method employs sulfuric acid to hydrolyze polysaccharides to glucose monomers. In the
presence of anthrone, glucose monomers are converted to hydroxyaldehydes and dehydrated to
hydroxymethylfurfurals [49], which form blue-green colored complexes with anthrone. The current
study tested the hexose quantification assay on cell pellets, glycogen extracts, and the residue remaining
after the glycogen extraction process. The sum of the glycogen extract and residue measurements
was compared with the total cell pellet measurement to verify recovery of all cellular carbohydrates.
Differentiation between glycogen and other cellular carbohydrates, such as cell wall sugars, can
provide useful parameters for metabolic models.
4.2. Procedure (After Del Don et al., 1994)
4.2.1. Reagents
•
Cell pellet (0.5–1 mg dry biomass, fresh or frozen, washed with carbon-free media).
•
Anthrone reagent: (per reaction) mix 10 mg anthrone and 250 µL fresh absolute ethanol (anthrone
will partially dissolve), add 75% sulfuric acid to a final volume of 5 mL, and stir until anthrone is
completely dissolved [18]. Prepare fresh daily (within 24 h of use) and store at 4 ◦ C.
•
2% sodium sulfate (w/v).
•
Methanol.
•
Glucose standards (prepare from fresh 1 mg/mL glucose solution). A linear response was observed
using 10–250 µg/mL standards (e.g., 10, 50, 90, 130, 170, 210, 250 µg/mL). The limit of detection
with anthrone has been previously reported as 5 µg/mL [48].
4.2.2. Quantification of Glycogen
(1) Re-suspend cell pellet in 200 µL 2% sodium sulfate in 2-mL Eppendorf tube.
(2) Seal tube with parafilm to prevent cap from popping open and heat for 10 min at 70 ◦ C
(VWR analog heat block).
(3) Add 1 mL methanol, and vortex in two 10-s rounds to co-precipitate sodium sulfate and glycogen.
(4) Centrifuge for 15 s at 10,000 rpm to pellet the precipitate (Eppendorf 5415D microcentrifuge) and
decant the supernatant.
(5) Wash the precipitate with 1 mL methanol (add methanol, vortex, centrifuge, and decant).
(6) Re-suspend the pellet in 1 mL water, transfer to a clean glass test tube, and place on ice to chill.
(7) Add 5 mL ice-cold anthrone reagent (mixing is unnecessary).
(8) Chill on ice for 5 min, vortex briefly to homogenize the solution, and incubate in a boiling water
bath for 10 min.
158
phenol sulfuric acid assay [40]. In the L-cysteine assay, pentose, heptose, and deoxy sugars contribute
to absorbance, and absorbance stability varies among different carbohydrates. Pentoses also contribute
to signal in the anthrone assay, but the absorbance fades rapidly and presents minimal interference.
Different hexoses may also produce differential responses in the anthrone assay; for example, mannose
produces 55% percent of the measured absorbance intensity of glucose [43]. Minimizing interference
from pentoses is a key consideration when selecting assays to avoid measuring nucleotide bases twice
in both nucleic acid and carbohydrate assays.
Glycogen is the most common form of carbohydrate storage for bacteria [44]. Glycogen content
can indicate cellular responses to changing nutrient conditions; for instance, E. coli and Synechococcus
7002 have both been found to increase glycogen storage during nitrogen limitation [45,46]. Glycogen
can be precipitated from cells with KOH, but alkalinity causes some degradation of glycogen.
An alternative method using sodium sulfate to adsorb and co-precipitate glycogen has been developed
for mosquitoes [47] and adapted to bacterial samples [48] and was selected for the current study.
The anthrone assay was selected for quantification of hexoses due to minimal pentose interference.
The method employs sulfuric acid to hydrolyze polysaccharides to glucose monomers. In the
presence of anthrone, glucose monomers are converted to hydroxyaldehydes and dehydrated to
hydroxymethylfurfurals [49], which form blue-green colored complexes with anthrone. The current
study tested the hexose quantification assay on cell pellets, glycogen extracts, and the residue remaining
after the glycogen extraction process. The sum of the glycogen extract and residue measurements
was compared with the total cell pellet measurement to verify recovery of all cellular carbohydrates.
Differentiation between glycogen and other cellular carbohydrates, such as cell wall sugars, can
provide useful parameters for metabolic models.
4.2. Procedure (After Del Don et al., 1994)
4.2.1. Reagents
•
Cell pellet (0.5–1 mg dry biomass, fresh or frozen, washed with carbon-free media).
•
Anthrone reagent: (per reaction) mix 10 mg anthrone and 250 µL fresh absolute ethanol (anthrone
will partially dissolve), add 75% sulfuric acid to a final volume of 5 mL, and stir until anthrone is
completely dissolved [18]. Prepare fresh daily (within 24 h of use) and store at 4 ◦ C.
•
2% sodium sulfate (w/v).
•
Methanol.
•
Glucose standards (prepare from fresh 1 mg/mL glucose solution). A linear response was observed
using 10–250 µg/mL standards (e.g., 10, 50, 90, 130, 170, 210, 250 µg/mL). The limit of detection
with anthrone has been previously reported as 5 µg/mL [48].
4.2.2. Quantification of Glycogen
(1) Re-suspend cell pellet in 200 µL 2% sodium sulfate in 2-mL Eppendorf tube.
(2) Seal tube with parafilm to prevent cap from popping open and heat for 10 min at 70 ◦ C
(VWR analog heat block).
(3) Add 1 mL methanol, and vortex in two 10-s rounds to co-precipitate sodium sulfate and glycogen.
(4) Centrifuge for 15 s at 10,000 rpm to pellet the precipitate (Eppendorf 5415D microcentrifuge) and
decant the supernatant.
(5) Wash the precipitate with 1 mL methanol (add methanol, vortex, centrifuge, and decant).
(6) Re-suspend the pellet in 1 mL water, transfer to a clean glass test tube, and place on ice to chill.
(7) Add 5 mL ice-cold anthrone reagent (mixing is unnecessary).
(8) Chill on ice for 5 min, vortex briefly to homogenize the solution, and incubate in a boiling water
bath for 10 min.
158