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International Journal of Aquaculture, 2014, Vol.4, No.18 108
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112
http://ija.biopublisher.ca
109
systems has also been established (Molokwu and
Okpokwasili, 2002).
Information on the enzymatic activities of intestinal
bacteria from farmed
Clarias
gariepinus
is not
available. Assessment of the substrate degrading
ability of gut microflora is important in understanding
the nutrition and physiology of the host organism and
may help in formulating appropriate feeds (Tanu et al.,
2012). Therefore, the present study was undertaken to
establish the culturable bacteria associated with the
intestinal tract of farmed
Clarias
gariepinus
and their
degradative abilities.
1 Materials and Methods
1.1 Sample collection
The African catfish (
Clarias
gariepinus
) was obtained
from a private fish farm in Port Harcourt, Rivers State
of Nigeria.
1.2 Enumeration and isolation of culturable
intestinal bacteria
Fifty live fish with an average weight of approximately
25g were killed by physical destruction of the brain.
Before dissection, the fish were externally cleaned
with 70% ethanol. Pooled samples of 10 fish were
used for each replicate. From each pooled gut contents,
1.0g was taken aseptically and homogenized with
9.0ml sterile physiological saline. The homogenate
was serially diluted up to 10
-6
dilution. Then 0.1ml of
each dilution was plated in triplicate onto different
media using the spread plate method. The media
chosen were Nutrient agar (Oxoid), MacConkey agar
(BIOTECH), Thiosulphate citrate bile salt sucrose
(TCBS) agar (Oxoid), Salmonella-Shigella agar (Fluka),
Aeromonas medium with Ampicillin supplement
(Ryan) (Oxoid), Mannitol salt agar(Lab M), Pseudomonas
cetrimide agar (Oxoid) and de Man Rogosa and
Sharpe(MRS) agar (Oxoid).
1.3 Screening for amylase-producing strains
Bacterial isolates were screened for amylolytic
properties by starch hydrolysis test on starch agar
plate. The microbial isolates were streaked as a line on
the starch agar plate and plates were incubated at 37
o
C
for 24h. The plates were flooded with 1% prepared
iodine solution at the end of incubation. A clear zone
of hydrolysis surrounding the growth indicates
positive result while the presence of blue colour
around the growth indicates a negative result.
1.4 Screening of potent alkaline protease producing
strains
Bacterial isolates were screened for extracellular
protease production by streaking onto skim milk agar
plates. The plates were incubated at 37
o
C for 24h.
Protease production was demonstrated by the clearing
of opaque milk proteins in the area surrounding the
colony
1.5 Screening of lipolytic bacteria
The isolates were screened for lipolytic activity by
streaking on Egg Yolk Agar. The plates were
incubated at 37
o
C for 24-48h. The formation of a thin
iridescent layer overlying the colonies was considered
as positive result.
1.6 Screening for cellulolytic bacteria
Isolates were screened for cellulose activity by
streaking on Cellulose Agar. Plates were incubated for
24-48h at 37
o
C. At the end of the incubation period,
the plates were flooded with 1% congo red.
Appearance of clear zone around the colony showed
the presence of cellulase.
2 Results
2.1 Bacterial count and isolation
The total heterotrophic bacterial count was 3.8±0.02 x
10
8
cfu/g in the intestine of farmed
Clarias
gariepinus
.
A total of 18 bacterial isolates were identified
according to Holt et al. (1994). Bacteria of the genera
Bacillus
,
Staphylococcus
,
Vibrio
,
Aeromonas
,
Pseudomonas
,
Lactobacillus
,
Escherichia
,
Salmonella
,
Enterobacter
,
Micrococcus
and
Flavobacterium
were
isolated from the fish gut at different frequencies with
Bacillus
(22.2%) predominating (Table 1).
Table 1 Frequencies of isolation of the bacterial genera
Genus
Number of isolates
Bacillus
4 (22.22)
Staphylococcus
2 (11.11)
Vibrio
2 (11.11)
Aeromonas
2 (11.11)
Pseudomonas
2 (11.11)
Lactobacillus
1 (5.56)
Escherichia
1 (5.56)
Salmonella
1 (5.56)
Enterobacter
1 (5.56)
Micrococcus
1 (5.56)
Flavobacterium
1 (5.56)
Total
18 (100)
Note: Numbers in parentheses represent percentage frequencies