VOLUM OMAGIAL - Facultatea de Ştiinţe ale Naturii şi Ştiinţe Agricole

Changes in bacterial abundance and biomass... / Ovidius University Annals, Biology-Ecology Series 14: 139-145 (2010)
Sample collection was done using improvised
piston corers (20 mL syringes with the forepart
detached, but with the gradation intact). From each
sample, the surficial 5 cm 3 (corresponding to a depth
of 17.5 mm) was taken for analysis.
Each sample was suspended in 5 ml of buffered
formaline (4% final concentration) [7, 8, 9, 10]. The
formaldehyde solution acts as a fixative, killing the
microorganisms and preventing contamination and
cell deformation. The labeled tubes containing the
subsamples were preserved by refrigeration at +4°C.
Cell separation. Dislodgement of bacteria
attached to sand grains is an important step prior to
analysis. The procedure used was adapted, with some
modifications, from existing literature [11, 12, 13, 14,
15].
Sediment suspensions were diluted 5-fold,
incubated with Tween 80 (1 mg/mL final
concentration) for 15 minutes and vortexed at 2 400
r.p.m. for 5 minutes.
Direct counting of bacteria. Microorganisms
were visualised by epifluorescence microscopy, using
3,6-dimethylaminoacridinic chloride (acridine
orange) as a fluorochrome. This compound becomes
highly fluorescent by binding to the nucleic acids,
giving an orange-red fluorescence for single-stranded
nucleic acids (mostly RNA) and a green one for
double-stranded acids (DNA) [16]. Acridine orange
stains both living and dead cells [17, 18].
The technique employed was an adapted and
simplified version of the protocols used by other
authors [3, 8, 10, 11, 19, 20]. 1 ml was collected from
each suspension and incubated for 5 minutes with 1
ml acridine orange (5 µg/mL final concentration).
The resulting solution was filtered through a 0.45 µm
Millipore filtering membrane, using a syringe and a
Millipore holder. Filtered membranes were
previously stained with Sudan Black, in order to
reduce background fluorescence.
Each filter was washed with 50-60 ml of distilled
water, placed on a glass slide and examined using a
Hund Wetzlar H 600 AFL 50 microscope, at an
500× enlargement. An eyepiece grid micrometer was
employed.
For each filter, 15-20 grids were randomly
chosen (from different areas of the membrane, except
for its margins), photographed with a digital camera
and visualised with MBF ImageJ for Microscopy
140
software (http://www.macbiophotonics.ca/downloads.
htm.) [21].
Fluorescent cells in each grid were counted
manually. Fluorescent anorganic particles and
obviously eukaryotic structures (by size and
morphology) were excluded. In case sediment
particles masked bacterial cells, any bacteria found
on the surface of such particles were counted twice
[7, 8, 17, 22]. The mean bacterial density was
calculated for each sample according to the following
formula:
N = n ×Af / Ag × V / v
where:
N = mean bacterial number per cm 3 of
sediment;
n = mean bacterial number per grid for each
subsample;
Ag = grid area;
Af = filter area;
v = volume of the filtered sediment
suspension;
V = volume of the total sediment suspension
containing 1 cm 3 of sediment.
Bacterial biomass estimation. All the
microorganisms observed were classified into three
morphological categories: cocci, bacilli (including
coccobacilli and vibrios) and filamentous bacteria
(those having a length more than five times greater
than the width) [3]. Cell dimensions (diameter,
respectively length and width) were measured using
the grid micrometer.
Biovolume was determined for each cell
according to the formula [8, 16]:
V = (π/ 4) d 2 (l – d / 3)
where:
l = cell length;
d = cell width/diameter.
For cocci, the formula becomes:
V = πd 3 / 6
To determine dry biomass based on the
biovolume, several authors proposed different
conversion factors. In the present study, the following
formula was used [23]:
md = 435 × V 0,86
where:
md = dry biomass (fg);