International Journal of Aquaculture, 2016, Vol.6, No.10, 1
-
9
7
aerobically at 21
o
C for 6 h. The viability of bacterial isolates was evaluated by pipetting 100 µL of the mixture,
serially diluted and plated onto CMC agar at 0 h and 6 h. After 2- day incubation, the number of bacterial colonies
on each plate was enumerated.
3.5 Survival in stomach environment
A simulated stomach juice was prepared according to a modified protocol of Geraylou et al. (2014). In brief,
pepsin (3 mg. mL
-1
) was diluted in normal saline solution (NSS; 0.85%), and pH was adjusted to 4.53 with 1 N
hydrochloric acids (HCL). Fresh culture of cellulose - degrading bacteria was harvested by centrifugation, and the
bacterial cells was washed and suspended into PBS (~ 1.0 x 10
8
CFU.mL
-1
). Thereafter, 100 μL of this suspension
was inoculated into duplicate of 10 mL stomach simulation, and incubated for 3 h at 21ºC. Then, viable cells of
the tested bacteria were monitored by plating the bacterial mixture onto CMC - agar at 0 h and after 3 h
incubation.
3.6 Survival in intestinal condition
A simulated intestinal juice was prepared by mixing these chemical compounds (125 m.mol
-1
NaCl, 7 m.mol
-1
KCl, 45 m.mol
-1
NaHCO
3
, 0.3% bile salt and 3 g.L
-1
trypsin) in sterile distilled water. Subsequently, 100 μL of
fresh bacterial culture of cellulose – degrading bacteria (~ 1.0 x 10
8
CFU.mL
-1
) was inoculated into duplicates of
10 mL intestinal simulation; followed by incubation for 4 h at 21ºC. Then, cell viability was monitored by plating
of the mixture on CMC agar at 0 h and after 4 h incubation.
3.7 Toxicity assay
A total of 30 juvenile hybrid abalones (0.47±0.13g) were divided into 3 experimental groups in a small - scale in
vivo experiment. Each group had 2 rearing tanks and each tank had 5 juvenile abalones. The abalone were fed
with 1.5% body weight.day
-1
with commercial pellet impregnated with either
Stenotrophomonas
sp or
Bacillus
sp
at a cell concentration of ~1.0 x 10
9
CFU.g
-1
. The animals were reared in 10 L filtered seawater (32 ppt) for 14
days, and the rearing water was replaced every three days. During the experiment, dead abalone was recorded on
daily basis.
3.8 Data analysis
Data obtained were statistically analyzed using either independent Sample t - test or one-way analyses of variance
(ANOVA), followed by tukey comparison test.
Acknowledgement
This work was supported by the Institute for Marine and Antarctic Studies (IMAS), an institute of the University of Tasmania. The
author appreciates Nick Savva for providing abalone used for this study.
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