International Journal of Aquaculture, 2017, Vol.7, No.23, 143-158
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Quantitative determination of exo-enzymes production (amylase, cellulase, lipase and protease) were carried out
after the standard methods portrayed by Bernfeld (1955), Denison and Koehn (1977), Bier (1955) and Walter
(1984), respectively, and a detailed description for which has been presented by Bairagi et al. (2002). Quantitative
assay of phytase and xylanase activities were measured following Yanke et al. (1999) and Bailey (1992),
respectively. Protein content in the enzyme sample was measured after Lowry et al. (1951) and unit (U) activity
has been presented.
2.7 Fish pathogens and culture maintenance
Six fish pathogenic strains
Aeromonas hydrophila
MTCC-1739 (AH),
Aeromonas salmonicida
MTCC-1945 (AS),
Aeromonas sobria
MTCC-3613 (AB),
Bacillus mycoides
MTCC-7538 (BM),
Pseudomonas putida
MTCC-1072
(PP) and
Pseudomonas fluorescens
MTCC-103 (PF) were obtained from the Microbial Type Culture Collection,
Chandigarh, India. In addition,
Aeromonas veronii
(AV) and
Pseudomonas
sp. (P) were isolated from diseased
fish. The fishes were suffering from pale gills, bloated appearance, skin ulcerations and hemorrhage. Experimental
onset of disease by intraperitoneal injection to
O. niloticus
confirmed pathogenicity of the isolated strains.
Pathogenic strains used in the study were maintained in the laboratory (4°C) on TSA (HiMedia, Mumbai, India)
slants. Stock cultures were stored at -20°C in 0.9% NaCl with 20% glycerol in tryptone soya broth (TSB) to
provide consistent inoculums during the study (Sugita et al., 1998).
2.8 Assay for pathogen inhibitory activity and Co-culture test
Inhibitory activity of the isolated strains against the said eight fish pathogens was primarily noticed through
‘cross-streaking’ (Madigan et al., 1997).
Co-cultured activity of selected bacterial and yeast isolates were tested with previously isolated ten autochthonous
fish gut bacterial isolates, e.g.
Bacillus subtilis
(JX292128),
Bacillus atrophaeus
(HM246635),
Bacillus pumilus
(KF454036),
Bacillus flexus
(KF454035),
Bacillus subtilis
(HM352551),
Bacillus methylitrophicus
(KF559344),
Bacillus subtilis
subsp.
Spizizenii
(KF559346),
Enterobacter hormaechei
(KF559347),
Bacillus amyloliquefaciens
(KF623209) and
Bacillus sonorensis
(KF623291). At first, the isolates from
O. niloticus
were streaked and grown
(30°C, 24 h) on nutrient agar plate. Afterward, autochthonous fish gut isolates (pure cultures) were streaked on the
same plate at a 90°angle with the growth line of the strains from
O. niloticus
keeping a hairline gap (0.1 mm).
Following incubation (30°C, 24 h), growth of microbiota was checked with previously streaked bacteria and
disappearance of the gap indicated compatibility of the yeast and bacteria strains with the autochthonous fish gut
bacteria.
2.9 Growth on fish mucus
Fish gut mucus was collected from live
O. niloticus
and thereafter processed following Dutta and Ghosh (2015).
Growth on mucus was determined at 30°C by counting the number of bacterial cells with a Petroff-Hausser
counting chamber at 24 h, 48 h and 72 h intervals. OD at 600 nm was taken after 24 h, 48 h and 72 h cultures.
Sterilized un-inoculated mucus was served as the control.
2.10 Bile tolerance
Bile tolerance of the selected gut isolates was evaluated through determination of minimum inhibitory
concentration (MIC). Crude bile juice (pH 5.6) was collected from dissected gall bladder in aseptic condition,
sterilized by passing through filter papers (0.8 μm and 0.22 μm pore) (HiMedia, Mumbai, India) and stored at
-20°C until use. Cultures grown in TSB (30°C, 24 h) were centrifuged (10,000 g, 10 min, 4°C) and microbial
suspensions were prepared in PBS. Sterile PBS (control) or sterile PBS supplemented with 5-100% (v/v) fish bile
juice was inoculated (10
7
CFU mL
-1
) with the microbial suspension. Following incubation (1.5 h, 30°C), the
microbial samples were serially diluted in sterile PBS and viable counts were determined on TSA media plates.
2.11 Bio-safety evaluation
Bio-safety evaluation of two selected isolates was carried out through in vivo studies conducted in 350 mL glass
aquaria using 30 healthy
O. niloticus
fingerlings (Average body weight: 35±5.1 g). The fishes were acclimatized
