complicating their measurement, and can only be assayed after processing for 10 s
with a weak output (10 w). The use (or lack thereof) of sonication and the output and
time should be uniform across every test. The length of Tolypothrix sp. in the
sample, which is sonicated as needed, is measured through a digital microscope
screen using a hemocytometer. If this is impossible, a micrometer should be used, or
the length should be measured based on the approximate length of the slide grid. The
unit of measure is cm/mL.
6.1.5 Chlorophyll a and b
There are many ways of extracting and assaying chlorophyll a and b, but an
extraction method was introduced by Burnison (1980) that uses dimethyl sulfoxide
(DMSO), which is generally efficient and easy to handle. Approximately 1–2 mL of
filtered culture medium and a glass fiber filter (either GF/F from Whatman, UK, or a
GF-75 from Advantec, Japan) 25 mm in diameter are used. Filter paper should be
lightly rounded and placed in a centrifuge tube of 1.5–2.0 mL. The DMSO is then
added and sonicated three times for 30 s. Once preserved, it may be measured later; it
may either be preserved at À20
C before the addition DMSO and sonication or it
may be completely extracted and preserved at 4
C. However, the preservation
method for all samples must be uniform. If the sample has been refrigerated, it
should be brought back up to room temperature and left to rest at 65
C for 10 min.
Centrifugal separation (i.e., at 12,000 rpm for 10 min) is then performed before the
removal of the filter and cell debris. The spectrum of the supernatant should be
measured with a spectrophotometer, and the concentration of chlorophyll a and b is
then calculated using the following equation (Wellburn 1994):
A λ ¼ absorbance in wavelength λ nm
ð
Þ À absorbance in wavelength 750 nm
ð
Þ :
Chl a ¼ 12:19 A 665 À 3:45 A 649
Chl b ¼ 21:99 A 649 À 5:32 A 665
where A is the absorbance in nanometers and Chl is chlorophyll. Additionally, it can
be assumed that the phaeophytization quotient (PQ level ¼ A 435 /A 415 ) is an index of
the stress.
6.1.6 Phycocyanin, Phycoerythrin, and Allophycocyanin
A simple method of extracting and assaying C-phycocyanin (C-PC),
C-phycoerythrin (C-PE), and allophycocyanin (APC) was developed by Patel
et al. (2005). Approximately 5–10 mL of culture medium must be centrifuged, and
the supernatant should be filtered by a 25 mm diameter glass fiber filter, either GF/F
6 Estimation Using the Microcosm N-System
47
with a weak output (10 w). The use (or lack thereof) of sonication and the output and
time should be uniform across every test. The length of Tolypothrix sp. in the
sample, which is sonicated as needed, is measured through a digital microscope
screen using a hemocytometer. If this is impossible, a micrometer should be used, or
the length should be measured based on the approximate length of the slide grid. The
unit of measure is cm/mL.
6.1.5 Chlorophyll a and b
There are many ways of extracting and assaying chlorophyll a and b, but an
extraction method was introduced by Burnison (1980) that uses dimethyl sulfoxide
(DMSO), which is generally efficient and easy to handle. Approximately 1–2 mL of
filtered culture medium and a glass fiber filter (either GF/F from Whatman, UK, or a
GF-75 from Advantec, Japan) 25 mm in diameter are used. Filter paper should be
lightly rounded and placed in a centrifuge tube of 1.5–2.0 mL. The DMSO is then
added and sonicated three times for 30 s. Once preserved, it may be measured later; it
may either be preserved at À20
C before the addition DMSO and sonication or it
may be completely extracted and preserved at 4
C. However, the preservation
method for all samples must be uniform. If the sample has been refrigerated, it
should be brought back up to room temperature and left to rest at 65
C for 10 min.
Centrifugal separation (i.e., at 12,000 rpm for 10 min) is then performed before the
removal of the filter and cell debris. The spectrum of the supernatant should be
measured with a spectrophotometer, and the concentration of chlorophyll a and b is
then calculated using the following equation (Wellburn 1994):
A λ ¼ absorbance in wavelength λ nm
ð
Þ À absorbance in wavelength 750 nm
ð
Þ :
Chl a ¼ 12:19 A 665 À 3:45 A 649
Chl b ¼ 21:99 A 649 À 5:32 A 665
where A is the absorbance in nanometers and Chl is chlorophyll. Additionally, it can
be assumed that the phaeophytization quotient (PQ level ¼ A 435 /A 415 ) is an index of
the stress.
6.1.6 Phycocyanin, Phycoerythrin, and Allophycocyanin
A simple method of extracting and assaying C-phycocyanin (C-PC),
C-phycoerythrin (C-PE), and allophycocyanin (APC) was developed by Patel
et al. (2005). Approximately 5–10 mL of culture medium must be centrifuged, and
the supernatant should be filtered by a 25 mm diameter glass fiber filter, either GF/F
6 Estimation Using the Microcosm N-System
47