METHODOLOGY
Collection of Samples
In Ilesha, Osun State, a total of five (5) tomato samples were collected. All of
the samples were taken aseptically in sterile universal containers and
transported to the laboratory for examination right away.
Media Preparation
Nutrient agar, mannitol salt agar, eosin methylene blue agar, MacConkey
agar, salmonella-shigella agar, and peptone water were all made according
to the manufacturer's instructions.
Isolation of Bacterial from the Tomatoes Samples
Each tomato sample was weighed and homogenized in 90 mL of sterile
distilled water, then blended in a sterile blender, and 1 mL of the
homogenate was constituted in 9 mL of sterile peptone water. Then, using
the pour plate technique, 0.1 mL of the last two dilutions (10 -4 and 10-6) were
inoculated in triplicate on properly prepared media. The plates were then
incubated for 24 hours at 37°C.
The plates were checked after incubation to see if there were any separate
colonies. The colony counter was used to count colonies, which were
quantified as colony forming units per gram (CFU/g) of sample homogenate.
On nutritional agar, a total aerobic count was done, and Escherichia coli were
counted on Eosin methylene blue agar.
According to Oranusi and Olorunfemi, Mannitol salt agar and MacConkey
agar were used to count Staphlococcus aureus and non-E. coli coliforms,
respectively, while Salmonella Shigella agar was used to count Salmonella
after 24 hours of pre-enrichment of sample homogenate in Selenite-Flouch
(2011). Discrete colonies on various media were separated and purified
through repeated subculturing on the same media. For further
characterization, pure colonies were kept on agar slants at 4 oC.
Purification and Maintenance of Isolates
Each distinct colony on a petri dish was transferred into plates containing
newly prepared nutritional agar using a sterile inoculating loop and cultured
at 37°C for 24-48 hours. The colonial morphologies (Cultural traits) of the
isolates were documented after incubation and compared to descriptive
features in Holt et al (1994). The isolates were then conserved on nutrient
agar slants and kept at 4 degrees Celsius in the fridge.
Biochemical characterization and Identification of Isolates
The bacteria isolates were identified using the method developed by Oranusi
et al. (2004). Catalase, mrthyl red, oxidase, citrate utilization, and coagulase
and indole assays were among the biochemical tests utilized to further
describe the bacteria. The gram-negative isolates were also subjected to an
oxidase test to determine whether they were oxidase positive or negative.
The identities of coliforms and bacteria were then confirmed using the
Bergy's Manual for Determinative Bacteriology's identification procedures
(Holt et al., 1994).
Identification of Isolates
Cheesbrough's method was used to perform Gram staining and other
biochemical assays (2006). Each bacterial isolate's colonial appearance on
the plate was inspected and classified using the following criteria: color,
shape, edge, elevation, surface, and opacity (Olutiola et al., 2000). Catalase,
oxidase, indole, and coagulase tests were among the biochemical tests
performed here.
Biochemical Characterization
Gram Staining
Gram staining was employed in this investigation to distinguish Gram
positive and Gram-negative bacterial isolates based on their capacity to
preserve the primary (Gram positive bacterial) or secondary (Gram negative
bacterial) stain (gram negative bacterial). A smear of the test isolate was
emulsified in a drop of sterile distilled water on a clean glass slide, forming a
smooth suspension, air dried, heat fixed by passing the inoculated glass slide
through the Bunsen burner flame for about 2-3 times, stained with the
primary stain, crystal violet for 60 seconds, and then rinsed off in slow
running tap water. It was then decolorized for about 5 seconds with 70%
ethanol, followed by a heavy rinse with water to avoid decolorization.
Finally, the smear was counter stained with safranin for 60 seconds, rinsed
with water, and allowed to air dry before a drop of immersion oil was applied
to the smear and viewed under a microscope with oil immersion lenses. Cells
of isolates that held the purple color of the primary stain, crystal violet, were
labeled as gram-positive bacteria, whereas those that were unable to retain
the color of the primary stain but stained with the pink color of the counter
stain were labeled as gram-negative bacteria (Fawole and Oso, 2001).
Catalase test
In a test tube, 2-3 mL of hydrogen peroxide was purified. A colony of test
organisms was obtained and immersed in hydrogen peroxide solution using a
sterilized wooden or glass rod. The presence of bubbles indicated the
presence of oxygen. The organism was catalase positive if bubbles were
formed; if bubbles were not created, the organism was catalase negative.
Coagulase test
The test was carried out according to Barrow and Gelthan's instructions
(1993). 0.1 mL of an 18–24-hour old broth culture of the tested organism was
added to 0.5 mL of a 1:10 dilution of human plasma in saline, incubated at
37°C for 2-24 hours, and coagulation was assessed. A favorable outcome
was suggested by the creation of a distinct clot.
Citrate test
In 100 mL of distilled water, 2.4 g of citrate agar was dissolved. 10 mL citrate
medium was dispensed, covered, sterilized, and allowed to cool in a tilted
posture in test tubes. The tubes were injected once over the surface with the
24-hour-old culture organisms. The transition from green to blue indicates
that the citrate has been used (Cheesbrough, 2006).
Indole test
The test bacterial colony was injected in tryptophan broth and cultured for
24-28 hours at 37°C. Then Kovac's reagent (0.5 mL) was added. A positive
result was obtained. After adding the reagent, a pink tinted rink appeared.
Negative test: no color change after the addition of the reagent.
Methyl red (MR) test
5 mm glucose phosphate broth (1 g glucose, 0.5 percent KH2PO4, 0.5
percent peptone, and 100 mL distilled water) was poured and sterilized into
clean test tubes. After inoculating the tubes with the isolated test organisms
and incubating them at 37°C for 48 hours, a few drops of methyl red solution
were given to each test, and the color change was noted. A good reaction is
indicated by the color red (Olutiola et al., 2000).
Voges-proskaeur (VP) test
In clean test tubes, 5 mm of glucose phosphate broth (1 g glucose, 0.5
percent KH2PO4, 0.5 percent peptone, and 100 mL distilled water) was
poured and sterilized. The test organisms were then put into the tubes,
which were then incubated at 37 °C for 48 hours. Following incubation, 6
percent -naphthtol and 6 percent sodium hydroxide were added to around 1
mL of the isolated organisms' broth culture. Within 30 minutes, a bright red
coloration signals a favorable reaction (Olutiola et al., 2000).