Site Loader
Rock Street, San Francisco

CHAPTER
THREE

3.0       MATERIALS
AND METHODS

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

3.1       Sampling Location

The
cocoa Theobroma cacao plant parts,
the husk, root, leaves and bark were collected from Federal University of
Agriculture, Abeokuta, Ogun State. The fish samples were collected from Badagry
and Alimosho Local Government Areas of Lagos State.

 

3.2       Collection of Fish

One
hundred and twenty adult African mud catfish Clarias gariepinus were collected in all. Sixty were from Sejiro, Adeola,
Wusu, Awhansu and Gandonu farms in Badagry Local Government Area of Lagos
State. Another sixty were collected from Lakunle, Latia, Rashida, Raymond and
Pius farms in Alimosho Local Government Area of Lagos State. They were
collected in sterile jerry cans and transported to the Veterinary Microbiology
laboratory, Federal University of Agriculture, Abeokuta for length and weight
measurement and microbiological analyses.

 

3.3       Determination of Fish Length and Weight

i.                   
Fish weight: Each fish ample was placed on
weighing balance (name and model of the balance) and measured in duplicate and
mean value was found.

ii.                 
Fish length: Standard length was measured
using graduated meter rule from head to tail in duplicate and mean was
estimated.

 

 

 

3.4       Selection of Meicinal Plant and Extracts
Preparation           

3.4.1    Husk of Theobroma
cacao (cocoa)

Some
pieces of cocoa, Theobroma cacao
(cocoa) pods were collected, opened up and the seeds removed, the husks were
then washed thoroughly and allowed to air-dry after which these were chopped
with knife. About 40g was weighed with an electronic weighing balance. This was
done in triplicates and soaked in 250ml ethanol for extraction and were
concentrated using rotary flask evaporator and preserved at 4°C in airtight
bottle until further use.

 

3.4.2    Leaves of Theobroma cacao (cocoa)

Some
leaves of cocoa, Theobroma cacao
(cocoa) plant were collected, washed thoroughly and allowed to air-dry after
which these were chopped with knife. About 40g was weighed with an electronic
weighing balance. This was done in triplicates and soaked in 250ml ethanol for
extraction and were concentrated using rotary flask evaporator and preserved at
4°C in airtight bottle until further use.

 

3.4.3    Bark of Theobroma
cacao (cocoa)

Some
pieces of cocoa, Theobroma cacao
(cocoa) barks were chopped from the cocoa plant, collected, and washed
thoroughly and allowed to air-dry after which these were chopped with knife.
About 40g was weighed with an electronic weighing balance. This was done in
triplicates and soaked in 250ml ethanol for extraction and were concentrated
using rotary flask evaporator and preserved at 4°C in airtight bottle until
further use.

 

 

 

3.4.4    Root of Theobroma
cacao (cocoa)

Some
pieces of cocoa, Theobroma cacao
(cocoa) root were collected, and then washed thoroughly and allowed to air-dry
after which these were chopped with knife. About 40g was weighed with an
electronic weighing balance. This was done in triplicates and soaked in 250ml
ethanol for extraction and were concentrated using rotary flask evaporator and
preserved at 4°C in airtight bottle until further use.

 

3.5
      Microbiological analyses 

3.5.1
   Total Bacterial Count

Estimation
of total bacteria count in water and fish samples was done according to method
of Miles and Mistra described by Hedges (2002).

i.
Total bacteria count in water: One ml of the  water sample was aseptically and thoroughly
mixed with 9 ml sterile distilled water 
and 1 ml of this diluted water was then 
serially diluted in sterile tubes containing 9 ml of sterile water to
make 1/102, 1/103, 1/104, 1/105,
1/106, and 1/107 respectively. One mililitre from 1/106
dilution was mixed with warm prepared Nutrient agar, gently mixed and allowed
to solidify. The plates were incubated at 370C for 24 hours.  Colonies of bacteria found were counted and
estimated accordingly.

ii. Total bacteria count in fish: One g of the skin, gill and
intestinal sample was aseptically and thoroughly homogenized with 9 ml sterile
distilled water and 1 ml of this diluted homogenate was then serially diluted
in sterile tubes containing 9 ml of sterile water to make 1/102,
1/103, 1/104, 1/105, 1/106, and
1/107 respectively. One mililitre from 1/106 dilution was
mixed with warm prepared Nutrient agar, gently mixed and allowed to solidify.
The plates were incubated at 370C for 24 hours.  Colonies of bacteria found were counted and
estimated accordingly.

3.5.2 Culture, Isolation and Identification of
Bacteria

i.
Culture and isolation of bacteria: Isolates obtained from
the Nutrient were sub-cultured on Nutrient agar and MacConkey agar were
incubated at 37oC for 24 hours to observe the growth. Isolated
colonies were further sub-cultured to obtain pure isolate.

ii. Colonial
examination and microscopic identification of bacteria isolates: The
colonial morphology and characteristics such as size, shape, colour,
consistency, elevation and edges were examine according to Cowan and Steel (1993).
Microscopic examination was performed by gram staining to examine the cell
morphology.

iii. Biochemical Identification:  The following biochemical tests were performed
to further characterise the bacteria isolates according to Cowan and Steel
(1993).

(1)Sugar fermentation Test: Sugar
containing medium was inoculated with the pure test isolates and incubated
at 37OC for 18-24hours. The production of acid and gas as a result
of fermentation was shown by changes in the colouration of the medium and gas
production with the following sugar solution containing 1% Andrade indicator;
glucose, lactose, maltose, mannitol, and sucrose.

(2)Oxidase Test:
Production of cytochrome oxidase by certain bacteria that can catalyse the
transport of electron between electron donor-bacteria and redox dye tetramethyl
paraphenylene diamine, reducing it to deep purple colouration was done as
follows. A piece of filter paper was soaked with prepared oxidase reagent (1%
freshly prepared tetramethyl-p-phenylene diamine dihydrochlomide) and a pure
colony of the bacteria was smeared on it. Purple colouration of the colony
indicates positive result within few seconds; no colour changes indicate
negative reaction.

(3) Catalase Test:
Loopful of pure inoculums was dipped into 3% Hydrogen peroxide. Bubble
production indicates positive test while no bubble indicate negative result.

(4) Urease Test:
Ability of different isolates to break urea by production of urease
enzyme.  Pure colony of the organism was
inoculated into urea medium and incubated for 24 hrs at 37oC to
observe red pink colour changes which indicate a positive test.

(5) Indole test: This
test was done to demonstrate ability of the isolates to decompose amino acid
tryptophan to indole. Presence of indole was tested for its reaction with
p-dimethyl amino benzaldehyde by production of pink ring.

(6) Citrate utilization Test: This
test was done to demonstrate the ability of the organism to utilize citrate as
its only source of carbon. Pure colony of the isolate was inoculated into
Simmon-citrate agar medium which contain sodium citrate, an ammonium salt and
an indicator bromothymol blue, then incubated at 37oC for 24 hrs.
Blue colouration indicates positive test while original green indicates
negative test.

(7) Methyl Red test: This
test was performed to detect the production of acid from glucose which lowers
the pH of the medium, resulting in colour changes. A few drops of methyl red
were added to overnight glucose phosphate broth with the resultant red
colouration indicating a positive reaction.

 

3.6       In-Vitro
Antibacterial Susceptibility Test of Plant Extracts and Synthetic Antibiotics

3.6.1    Preparation of Parts of Theobroma cacao (cocoa) Extracts

Some
parts of cocoa parts used in the preparation of the plant extracts are the
leaves, roots, barks and the husks. Each of these parts was freshly collected,
washed thoroughly and allowed to air-dry after which these were chopped with
knife. About 40g was weighed with an electronic weighing balance. This was done
in triplicates and soaked in 250ml ethanol for extraction and were concentrated
using rotary flask evaporator and preserved at 4°C in airtight bottle until
further use.

 

3.6.2       
Antibacterial Activity of T. cacao through Disk Diffusion Method

Filter
disc diffusion method was used for testing of the medicinal plant extracts
against some bacterial fish pathogens. Whatman No. 1 filter paper disc (5 mm
diameter) was impregnated with crude plant extracts (5 mg disc) and then placed
on nutrient agar which has been inoculated with bacterial fish pathogens. The
sterile disc impregnated with water (solvent) was used as a negative control.
All plates were incubated at 37°C for 24 hrs after which the zone of inhibition
was measured and recorded in millimetre (mm) diameter. Replicates of each
experiment were carried out thrice and the mean was calculated for both the
test and control.

 

3.6.3    In-Vitro
Antimicrobial Susceptibility Test of Synthetic Antibiotics

Pure bacterial isolates of 0.5McFarlan was spread on Mueller-Hinton Agar and allowed to dry at room
temperature. Different antibiotics were
tested against the bacteria isolates by the Kirby Bauer disc diffusion method
on Mueller Hinton agar according to Bauer et
al (1996). The following antibiotic discs used were; Ampicillin (10µg), Cotrimoxazole
(25µg/5µg), Amoxycillin (10µg), Penicillin (10µg), Streptomycin (10µg),
Cefuroxime (30µg), Gentamicin (10µg), Ciprofloxacin (5µg), Tetracycline
(30µg) and Ofloxacin (10µg). Each of these
synthetic antibiotics discs
mentioned above was placed at about 20mm distance from each other on the
inoculated agar and incubated at 370C for 18 to 24 hours. The
inhibition zones were measured using graduated metre ruler to determine the
diameter of the inhibition zones and interpreted as sensitive, intermediate and
resistant, according to CLSI guidelines (CLSI, 2014).

 

 

 

3.7       Phytochemical Screening of Extracts

The
screening was carried out to check for the presence or absence of active
secondary metabolites or different constituents such as tannins, flavonoids,
phenols and alkaloids. These were performed as follows:

i. Tannin test: Half
of 1g of powdered part was boiled in 20ml distilled water for few minutes with
addition of about 3 drops of 5% FeCl3. A positive result was recorded
if a brownish–green or blue colouration was developed (Sofowora, 1993).

ii. Flavonoids test: One
gram of the powdered part is added with 10ml ethyl acetate and subjected to
heat over a steam bath (40-50°C) for about 5mins and the filtrate was treated
with 1ml diluted ammonia. A positive was indicated by yellow colouration
(Sofowora, 1993).

iii. Phenols test:
Extract was mixed with few drops of diluted Folin Ciocalteu reagent and aqueous
sodium carbonate solution. The mixture was allowed to stand for 10mins and
formation of grey colouration indicated the positive result of phenol groups
(Sofowora, 1993).

v. Alkaloids test:
Few drops of diluted HCl and 0.5ml Wagner’s reagent were used. A brown flocculent
precipitate indicated the presence of alkaloid (Sofowora, 1993).

 

3.8       Minimum
Inhibitory Concentrations (MIC) and Minimum Bactericidal Concentrations (MBC)
of Antimicrobials

3.8.1    MIC of Synthetic Antibiotics and Plant Extracts

Standard broth
micro-dilution method was used to determine the MIC of the bacteria isolates
obtained from disc diffusion test. All the bacteria isolates were tested against the following antibiotics
dilution ranges from 0.5–64 µg/mL; Ampicillin, Gentamycin, Ciprofloxacin,
Cefuroxime, Tetracycline, amoxicillin, penicillin, streptomycin, ofloxacin and
cotrimoxazole. Each antibiotic was serially diluted in 1% peptone of 100µl according to their respective ranges and equal volume of 100µl of overnight broth culture of 0.5 MacFarland; was added to all the
dilution ranges from well 1 to well 10 and incubated at 37°C in ambient air for
24hours. Turbid wells were indicated to have growth while clear wells were
identified to have no growth after incubation. The MIC of each antibiotic was
noted as the highest dilution showing no growth.

Procedures
above were repeated using the variously prepared T. cacao parts on bacterial isolates to determine the MIC of plant
extracts.

 

3.8.2    MBC
of Synthetic Antibiotics and Plant Extracts

The
MBC is defined as the lowest dilution of antibiotic that kills the bacteria
isolates. Sub-culturing was made from two fold dilutions before each well
showing the MIC onto Nutrient agar plates. Following overnight incubation at
37°C in ambient air for 24hours, the plates were examined for growth of
bacteria colonies. Lack of growth indicates that the antibiotic is having
bacteriacidal potential, while growth indicates bacteriostatic activity.

Procedures
above were repeated using the variously prepared T. cacao parts on bacterial isolates to determine the MBC of plant
extracts.

 

3.9       Analysis of Data

Data obtained from bacteriological analysis such
as the plate count and inhibition diameters of sensitivity assay were
statistically analyzed using Analysis of Variance (ANOVA) and Duncan Multiple
Range Test (DMRT) to determine the significant differences between means.
However, bacterial characterization and identification, phytochemical
analysisas well as data obtained from the MIC and MBC data were presented in
tables. 

Post Author: admin

x

Hi!
I'm Jeremy!

Would you like to get a custom essay? How about receiving a customized one?

Check it out