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International Journal of Marine Science 2013, Vol.3, No.37, 295-305
http://ijms.sophiapublisher.com
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specimens of each sponge species. Thin, transverse
sections of sponge tissue were obtained using sharp
scalpel blade and observed under Hirox (model:
MX-2010Z) video microscope attached to computer.
Light microscopy
Prior to the washing of each sponge species in
distilled water, squashed suspension of sponge tissue
was subjected to microscopic slide and observed
under POLYVAR (REICHERT-JUNG) compound
microscope to understand the structural array of
spongin and spicules.
Electron microscopy
Scanning Electronic Microscopy was done according
to the method of Bozzola and Russell (1999). Briefly,
distilled water cleaned sponge tissues were dissected
with thin scalpel blade and the sections were mounted
over the stubs with double-sided carbon tape and were
fixed with a 4% aqueous Osmium tetroxide vapours
for 2 hr. A thin layer of Platinum (palladium) coat was
then applied over the samples using an automated
sputter coater (JEOL JFC-1600) for about 4 min. Then
the samples were scanned under Scanning Electron
Microscope (Model: JOEL-JSM 5600) at various
magnifications.
1.4 Macromolecular analysis
Each specimen of collected sponge species was
washed under running tap water for two to three times
and then washed finally with 0.1M phosphate buffer
(pH 7.5). For protein estimation, each experimental
sponge tissue was chopped and minced using sharp
dissection scissors and homogenized (10% w/v) in
ice-cold 0.1M phosphate buffer (pH 7.5) using
Heidolph DIAX 900 homogenizer. The homogenates
were centrifuged at 500 g for 10 min and the
supernatant was further recentrifuged at 5000 g for 10
min using refrigerated centrifuge (Kubota; Model: 6930).
The resultant supernatant of sponge homogenates
were used to estimate the total protein content by
spectrophotometer (SpectraMAX Plus, Molecular
Devices; Model: S/NP02512) assisted with software,
SoftMax Pro ver. 5.0. following the method of
Bradford (1976), using bovine serum albumin (BSA)
as standard.
Total carbohydrate content was measured using the
amended phenol-sulfuric acid method of Taylor
(1995), whereas the total lipid content was measured
by using a mixture of chloroform:methanol (1:2)
according to the previous method of Brooks et al
(1998).
For carbohydrate estimation, 1 g of each test sponge
tissue was homogenized with distilled water and the
homogenate was taken into a test tube, in which, 100
µL of 8% phenol was added. 1 mL of concentrated
sulfuric acid was then added rapidly, the steam of acid
being directed against the liquid surface rather than
against the side of the test tube in order to obtain good
mixing. The tubes were thoroughly mixed and later
they were placed in a water bath for 10 to 20 minutes
at room temperature to obtain yellow orange colored
complex. The color was found to be stable for several
hours and readings may also be taken later,
accordingly. The absorbance was measured at 490 nm
for hexoses and 480 nm for pentoses by using
UV-Visible Spectrophotometer (Molecular Device,
USA; Softmax pro 5.0). Reference standard curve
constructed for the particular sugar were used to
determine the amounts of carbohydrate present in each
sponge species. All solutions were prepared in
triplicate to minimize errors resulting from any
accidental contamination.
For lipid analysis, 1 g of each experimental sponge
tissue was homogenized with 10 mL distilled water
and the resultant pulp was transferred to a conical
flask containing 30 mL of chloroform-methanol
mixture and mixed well. The extraction was left
overnight at room temperature in a dark place. After
this, equal volumes (20 mL) of chloroform and
distilled water were added and the suspension was
gently mixed by vortexing and subjected to
centrifugation, to obtain three layers. The methanol
layer was discarded and the lower layer of chloroform
containing all the lipids was carefully collected free of
interphase, by sucking out with a fine capillary tube.
The organic layer was carefully evaporated by leaving
the extraction in warm water (around 50
℃
) with a
flow steam of nitrogen gas on the surface. As some
lipids get polymerized or decomposed on exposure to
light, heat and oxygen; the sample was covered with a
dark paper to protect from light. When the solution
was free of organic solvents, the total lipid content
was determined and the results are expressed in terms
of weight in micrograms of total lipid per gram dry
tissue.