International Journal of Aquaculture, 2017, Vol.7, No.18, 122-127
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However, the MIC of the present study revealed that 500 µg/ml was the least concentration that prevented the
growth of bacteria after 24 hours incubation except
A. hydrophila
and
S. iniae
who had 1000 µg/ml for aqueous
and ethanolic extracts respectively while
S. aureus
recorded 250 µg/ml in ethanolic extracts of soursop leaves.
The results were in agreement with the report of Oyedeji et al. (2015), Haro et al. (2014) and Bello et al. (2013).
4 Materials and Methods
4.1 Plant collection
The soursop (
A. muricata
) leaves used in the study was obtained in Teaching and Research Farm, Ondo State
University of Science and Technology, Okitipupa and was identified by Dr D.O. Aworinde in the Department of
Biological Sciences, Ondo State University of Science and Technology, Okitipupa.
4.2 Preparation and extraction of soursop leaves
Soursop leaves was extracted as described by Ajaiyeoba and Fadare (2006). The fresh leaves soursop were
air-dried for 2 weeks and grounded with a hammer mill, 100 g of the powder of the plant leaves were immersed in
300 ml of water, ethanol and methanol for 72 hours and 48 hours respectively. The plant leaves were rightly
blended with water, ethanol and methanol, filtered using a sterile muslin cloth after which the extracts was
acquired, air-dried and store at 25°C until further study.
4.3 Source of test organisms
Microorganisms isolated from
Clarias gariepinus
juveniles were
Aeromonas hydrophila
,
Streptococcus sp.
,
Bacillus subtilis
and
Staphylococcus aureus
. The isolation and characterization of bacteria using bio-chemical test
was carried out at Microbiology Laboratory, Faculty of Science, University of Ibadan, Nigeria.
Aspergillus flavus
was collected from the laboratory stock of the Department of Microbiology, Ondo State University of Science and
Technology, Okitipupa. The pure cultures were labeled, sub-cultured on nutrient agar slants and nutrient broth(s)
and potato dextrose agar (PDA), kept in the refrigerator at 4ºC until further study.
4.4 Counting of microorganisms
The tissues of
C. gariepinus
were individually softened and put into sterile bottles containing sterilized distilled
water and homogenized (Shalaby et al., 2006). Serial dilution was adopted and 1ml each from 10-1 to 10-6
dilution factors were dispersed into Petri dishes that were correctly labeled and molten sterilized medium was
poured aseptically into Petri dish. The plates were twisted kindly for even distribution of inocula and allowed to
set/gel and then incubated at 37ºC for 24 hours. The organisms grew into visible different colonies after 24 hours.
Total viable counts and enterobacteriacea counts were ascertained, the result were expressed in log
10
CFU/g.
4.5 Antimicrobial assay
A cup plate diffusion assay as described by (Bello et al., 2013) was used. Pre-poured indicator [pathogen (4 mm
depth)] was overlaid with a 10 ml soft agar (0.7%) lawn of indicator culture (thus generating a potential mat for
the indicating of bacteria). Wells of 10 mm diameter were cut into these agar plates using cork borer and 0.1 ml of
the plant extracts was placed into each well (Bello et al., 2013). Distilled water was used as negative control while
antibiotics, chloramphenicol (10 mg/mL and 20 mg/mL) were used as positive control. The Petri dish was
assessed for diameter of zones of inhibition which was scored positive, if the width of the clear zone was 10 mm
or longer. The diameter of zones of the inhibition was taken to be proportional to the logarithm of the
antimicrobial compounds in soursop leaves (Maria et al., 1994).
4.6 Minimum inhibitory concentration
Double dilution of 2000 µg/ml of soursop leaves was made in 2 ml volume of broth to 15.63 µg/ml. One row of
the test was inoculated with 0.02 ml of 1 in 100 dilution of the overnight broth culture of the organism (Stokes and
Ridgeway, 1980). The test was incubated at 37ºC for 24 hour aerobically. The minimum inhibitory concentration
was the lowest concentration that prevented the growth of bacterial after 24 hour incubation (Osoba, 1979).
