Processes 2018, 6,38
set of samples; the residue represented a relatively small proportion of total cellular carbohydrates.
Thus, different amounts of biomass were tested for each species to identify a quantity that would place
all measurements within the standard curve. One milligram dry weight was found appropriate for
E. coli and A. acidocaldarius, and 0.5 mg dry weight was used for Synechococcus 7002 (i.e., organisms
with higher carbohydrate content require less biomass for the assay).
Additionally, the procedure outlined in Del Don et al. [48] prescribes washing the glycogen pellet
with methanol until the pellet is white. However, in the current work, it was observed that glycogen
pellets from cyanobacterial samples remained slightly blue after three successive methanol washes,
most likely due to photosynthetic pigments. The anthrone assay was tested on glycogen extracts
from Synechococcus 7002 samples after one, two, or three washes. The carbohydrate content was not
significantly different among the three treatments (p > 0.05 for all pair-wise T-tests), indicating that a
single wash is sufficient (data not shown).
5. DNA
5.1. Literature Review
DNA represents a small but important component of cellular biomass, and its content changes
with specific growth rate. For example, slower-growing cells contain more DNA on a cell mass
basis than fast-growing cells [4,17]. De Mey et al. [50] provided a summary and comparison of
methods for quantifying DNA and RNA. De Mey et al. tested different absorbance, colorimetric,
and fluorescence-based assays on purified DNA solutions and reported accuracy and sensitivity
for bacteria of differing GC contents [50]. UV absorbance is precise but requires a pure sample
(for example, from kit extraction) to minimize interference from RNA and protein. Orcinol can
be used to quantify DNA colorimetrically, but has differing sensitivities for different nucleic acids
and is not as precise for mixtures of DNA and RNA. The diphenylamine assay [51] is a commonly
used method but has lower sensitivity for low GC content and is not as precise as other mentioned
methods [50]. The diphenylamine assay also seems to be sensitive to the purity and preparation of the
reagents [18]. Fluorescence methods for DNA detection are becoming popular [50]. Hoechst 33258 is a
DNA-intercalating dye and is reported to be biased toward AT content [50]. However, cell lysate can
be used due to low affinity of the dye for protein and RNA. Thiazole orange is another dye with good
precision, but it requires a pure sample and is biased toward GC content [50]. Additional fluorescent
dyes that require pure sample for good quantification include PicoGreen and RiboGreen [52].
Considerations when selecting a DNA quantification method include the purity of the sample,
interfering substances, and bias toward nucleotide content. Based on these considerations, the Hoechst
fluorescent assay was selected for the current study. It is more quantitative than extraction kits and is
safer and more precise than the classic diphenylamine method. It is recognized that AT nucleotide
bias and the DNA standard used will influence the resulting estimation. Downs and Wilfinger [53]
developed and validated an alkali lysis procedure with subsequent Hoechst quantification of DNA
using rat pituitary cells. Downs and Wilfinger showed equivalent accuracy but greater precision than
the diphenylamine assay [53].
5.2. Procedure (After Downs and Wilfinger, 1983)
5.2.1. Reagents
•
Cell pellet (0.4–1 mg dry biomass, fresh or frozen).
•
Alkali extraction solution: 1 N NH 4 OH, 0.2% Triton X-100.
•
Assay buffer: 100 mM NaCl, 10 mM EDTA, 10 mM Tris, pH 7.0 with HCl.
•
Standard buffer: 100 mM NaCl, 10 mM EDTA, 10 mM Tris, pH 7.0 with HCl, 0.025 N NH 4 OH,
0.005% Triton X-100.
160
set of samples; the residue represented a relatively small proportion of total cellular carbohydrates.
Thus, different amounts of biomass were tested for each species to identify a quantity that would place
all measurements within the standard curve. One milligram dry weight was found appropriate for
E. coli and A. acidocaldarius, and 0.5 mg dry weight was used for Synechococcus 7002 (i.e., organisms
with higher carbohydrate content require less biomass for the assay).
Additionally, the procedure outlined in Del Don et al. [48] prescribes washing the glycogen pellet
with methanol until the pellet is white. However, in the current work, it was observed that glycogen
pellets from cyanobacterial samples remained slightly blue after three successive methanol washes,
most likely due to photosynthetic pigments. The anthrone assay was tested on glycogen extracts
from Synechococcus 7002 samples after one, two, or three washes. The carbohydrate content was not
significantly different among the three treatments (p > 0.05 for all pair-wise T-tests), indicating that a
single wash is sufficient (data not shown).
5. DNA
5.1. Literature Review
DNA represents a small but important component of cellular biomass, and its content changes
with specific growth rate. For example, slower-growing cells contain more DNA on a cell mass
basis than fast-growing cells [4,17]. De Mey et al. [50] provided a summary and comparison of
methods for quantifying DNA and RNA. De Mey et al. tested different absorbance, colorimetric,
and fluorescence-based assays on purified DNA solutions and reported accuracy and sensitivity
for bacteria of differing GC contents [50]. UV absorbance is precise but requires a pure sample
(for example, from kit extraction) to minimize interference from RNA and protein. Orcinol can
be used to quantify DNA colorimetrically, but has differing sensitivities for different nucleic acids
and is not as precise for mixtures of DNA and RNA. The diphenylamine assay [51] is a commonly
used method but has lower sensitivity for low GC content and is not as precise as other mentioned
methods [50]. The diphenylamine assay also seems to be sensitive to the purity and preparation of the
reagents [18]. Fluorescence methods for DNA detection are becoming popular [50]. Hoechst 33258 is a
DNA-intercalating dye and is reported to be biased toward AT content [50]. However, cell lysate can
be used due to low affinity of the dye for protein and RNA. Thiazole orange is another dye with good
precision, but it requires a pure sample and is biased toward GC content [50]. Additional fluorescent
dyes that require pure sample for good quantification include PicoGreen and RiboGreen [52].
Considerations when selecting a DNA quantification method include the purity of the sample,
interfering substances, and bias toward nucleotide content. Based on these considerations, the Hoechst
fluorescent assay was selected for the current study. It is more quantitative than extraction kits and is
safer and more precise than the classic diphenylamine method. It is recognized that AT nucleotide
bias and the DNA standard used will influence the resulting estimation. Downs and Wilfinger [53]
developed and validated an alkali lysis procedure with subsequent Hoechst quantification of DNA
using rat pituitary cells. Downs and Wilfinger showed equivalent accuracy but greater precision than
the diphenylamine assay [53].
5.2. Procedure (After Downs and Wilfinger, 1983)
5.2.1. Reagents
•
Cell pellet (0.4–1 mg dry biomass, fresh or frozen).
•
Alkali extraction solution: 1 N NH 4 OH, 0.2% Triton X-100.
•
Assay buffer: 100 mM NaCl, 10 mM EDTA, 10 mM Tris, pH 7.0 with HCl.
•
Standard buffer: 100 mM NaCl, 10 mM EDTA, 10 mM Tris, pH 7.0 with HCl, 0.025 N NH 4 OH,
0.005% Triton X-100.
160