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International Journal of Marine Science 2013, Vol.3, No.40, 319-332
http://ijms.sophiapublisher.com
328
locations in two ponds (pond 1 and pond 2; sampling
points denoted as A, B and C in each pond) and two
locations in the source water (creek 1 and creek 2) as
shown in Figure 2.
A Niskin water sampler was used to collect water
samples from ponds and the source water (creek)
between 0800 and 0900 h. The samples were
transferred aseptically into sterile BOD bottles. The
sediment samples were collected with the help of a
hand held acrylic core (400 mm long, 40 mm dia) and
were transferred immediately into new sterile
polythene bags. All samples were transferred
immediately to an ice box and transported to the
laboratory for analysis. From 30-doc onwards, for
enumeration of bacteria in the hepatopancreas, five
shrimps from each pond were collected at every
fortnight from feed-check trays. Collected shrimp
were transferred immediately to polythene bags
containing seawater which was filled with oxygen gas
prior to packing. Samples were processed for
microbiological analysis within 3-4 h after collection.
3.3 Physico-chemical analysis
Environmental parameters such as temperature,
salinity and pH were measured with the help of
centigrade thermometer, hand-held refractometer
(Atago, Japan) and calibrated pH meter (Lab-India,
PHAN) respectively. The oxidation-reduction
potential (Eh) was measured with the help of
microprocessor-based analyzer (Lab-India, model
PHAN) fitted with an
Orion
combination
platinum/Ag/AgCl electrode. Dissolved oxygen (DO)
content was estimated by Winkler’s method (Parsons
et al., 1984). For the estimation of particulate organic
carbon (POC), a known volume of water sample was
filtered under vacuum (1/3 atmospheric pressure)
through a Whatman GF/F filter (47 mm diameter; 1.2
µm pore size) which was previously ignited in a
muffle furnace at 450
℃
for 3 h. Two mL of sodium
sulphate solution (4.5% v/v) was added separately to
remove interference of chloride ions. Filters were then
dried and stored in an aluminum foil until further
analysis. The material retained on the filter paper
was oxidized by acid-chromate solution and the
reduction in optical density (OD) was measured
spectrophotometrically (spectrophotometer model
UV-1201; Shimadzu, Japan). Standardization was
carried out using glucose as the carbon source. The
POC has been expressed as g C m
-3
. Organic matter in
the sediment was estimated following the method
described by El Wakeel and Riley (1956).
3.4 Bacterial quantification
Total heterotrophic bacteria (THB) were estimated
using half-strength nutrient agar (NA; HiMedia, India)
as preliminary tests showed highest retrievability on
50% NA. Total
Vibrio
like organisms (TVLO) were
enumerated on
Vibrio
-selective medium- thiosulphate
citrate bile-salt sucrose agar (TCBS; HiMedia, India).
Luminescent bacteria (LB) were quantified using
half-strength NA supplemented with 1% glycerol. An
inoculum of 100
μ
L pond water was directly spread
plated onto respective media. For isolation of vibrios
in sediment, 10 g of wet sample was diluted in 90 mL
of 50% sterile seawater (SSW). Appropriate dilutions
were carried out and 100
μ
L were spread plated
onto media.
For quantification of bacteria in the hepatopancreas,
live shrimps were surface-disinfected by swabbing
with 75% alcohol. Hepatopancreas from shrimps were
aseptically removed, pooled and weighed. They were
then washed twice with 50% sterile seawater (SSW) to
remove loosely adhering particles and homogenized.
A known quantity of hepatopancreas was suspended in
50% SSW (w/v) and serially diluted (up to 10
-7
) and
plated onto TCBS and NA media. The LB plates were
stored in the dark and colonies were counted after
18-24 h of incubation at room temperature. After
spread-plating, TCBS and NA plates were incubated at
room temperature for 48-96 h and counts in
source/rearing water, sediment and hepatopancreas of
shrimps were expressed as CFU mL
-1
, CFU g
-1
dry
weight and CFU g
-1
wet weight respectively.
3.5 Bacterial isolation and identification
To analyze the composition of the TVLO in each
sample, 1 to 3 numerically dominant colonies from
each plate containing about 20-200 colonies were
selected based on their morphological appearance and
were further purified on NA plates. Identification of
Vibrio
spp
.
was based on their morphological,
physiological (growth, temperature and NaCl
tolerance or requirements) and biochemical
characteristics (oxidase, catalase, glucose utilization,
nitrate reduction, indole production, Voges-Proskauer
test, methyl red, citrate, arginine dihydrolase,