3.

3 THE MEDIA PREPARATION

3.3.1 nutrient agar

The was prepared according to the manufacturer's instructions.

28 g of nutrient agar powder was dissolved in one liter of distilled water,The mixture was continuously
stirred while heating to ensure complete dissolution, followed by sterilization via autoclaving at 121°C
for 15 minutes. After cooling to approximately 45°C, the nutrient agar was poured into sterile petri
dishes and allowed to solidify (Forbes et al., 2022)

3.3.2 mannitol salt agar (MSA)

111 g of MSA powder was dissolved in 1000 mL of distilled water, the mixture was continuously stirred
while heating to ensure complete dissolution followed by sterilization Via autoclaving at 121°C for 15
minutes after cooling to 45°C the agar was poured into sterile petridishes and allowed to solidify (Patrick
et al., 2022).

3.3.3 Eosin methylene blue (EMBA)

Similarly, eosin methylene blue agar (EMBA) was prepared by dissolving 36 g of EMB powder in 1000 mL
of distilled water, followed by autoclaving, cooling, and pouring into plates (Patrick et al., 2022).

3.4 samples isolation and identification

3.4.1 samples Inoculation

,Bacterial samples were first inoculated onto nutrient agar plates and incubated at 35°C for 24 hours.
Subsequent subculturing was performed on selective media: EMBA to promote coliform growth and
inhibit Gram-positive bacteria, and MSA to select for staphylococci, with both incubated at 37°C for 24
hours (Forbes et al., 2022).

3.4.2 Gram staining

Gram staining was conducted by preparing a bacterial smear on a glass slide, air-drying, heat-fixing, and
sequentially applying crystal violet, iodine, decolorizer (95% ethanol), and safranin. The stained slide was
examined under a microscope using immersion oil and a 100x objective to determine cell morphology
and Gram reaction (Forbes et al., 2022).

BIOCHEMICAL TEST for Staphylococcus aureus

3.4. Catalase test

Procedure: A clean glass slide was first prepared, Using a sterile loop, a small amount of bacterial culture
was transferred onto the slide. A drop of 3% hydrogen peroxide (H₂O₂) was then added directly to the
bacterial sample. Immediate observation of bubble formation indicated a positive catalase test, while
the absence of bubbles signaled a negative (Bailey & Scott's, 2021).

3.4 Coagulase test

The coagulase test was conducted to identify the presence of coagulase enzyme, which is produced by
Staphylococcus aureus and causes plasma to clot.

Procedure: A small bacterial colony was transferred onto a drop of normal saline on a glass slide, a drop
of plasma was added to the bacterial suspension, and the mixture was gently stirred using a sterile loop.
The reaction was then monitored for clumping within 10 seconds, with clumping indicating a positive
coagulase test and no clumping signifying a negative result (Williams & Wilkins, 2021).

3.4 Urease test

Procedure: A sterile urease agar slant was inoculated with the test bacterium using a sterile wire
loop,the inoculated slant was incubated at 35–37°C for 24 hours. After incubation, the color of the
medium was observed. A positive result is indicated by a pink color change in the medium, while a
yellowish color signifies a negative result (Aryal et al., 2022).

3.4. BIOCHEMICAL TESTS FOR Escherichia coli

3.4. Indole Test

Procedure: A sterile test tube containing tryptone broth was inoculated with the test organism and
incubated at 37°C for 24–48 hours. After incubation, 5 drops of Kovac’s reagent were added to the tube,
which was gently shaken and allowed to stand for a few minutes. A positive result was indicated by the
formation of a red ring at the surface, while no color change denoted a negative result (Taylor and
Francis, 2020).

3.4. Methyl Red (MR) Test

Test Procedure: A sterile MR-VP broth was inoculated with the test organism and incubated at 37°C for
24 hours. Following incubation, 5 drops of methyl red indicator were added to the tube, which was
gently shaken to mix the contents. A red color signified a positive result (indicating acid production),
whereas a yellow color represented a negative result (Aryal et al., 2022).

3.4. Voges-Proskauer (VP) Test

Procedure: A sterile MR-VP broth was first inoculated with the test organism. The inoculated broth was
incubated at 37°C for 24 hours. After the incubation period, 0.6 mL of alpha-naphthol and 0.2 mL of 40%
potassium hydroxide (KOH) were added to the broth. The tube was then shaken vigorously and allowed
to stand undisturbed for 15–30 minutes. A positive result was indicated by the development of a red or
pink color, whereas no color change was interpreted as a negative result (Aryal et al., 2022).

3.4 Citrate Utilization Test

Procedure: A sterile Simmons citrate agar slant was streaked with the test organism using a sterile loop.
The inoculated slant was incubated at 37°C for 24 hours, after which the color of the medium was
observed. A positive result was indicated by a blue color change, while a negative result was noted if the
medium remained green (Forbes et al., 2022).

3.4 Motility Test

The motility test was conducted to assess bacterial motility by preparing a semi-solid agar medium in a
test tube

Procedure: A sterile inoculating needle containing the bacterial culture was used to stab the medium,
and the tube was incubated at 37°C for 24–48 hours. Post-incubation, the growth pattern was analyzed:
motile bacteria exhibited diffuse growth spreading from the stab line, causing cloudiness in the medium,
whereas non-motile bacteria showed growth restricted to the stab line (MacFaddin et al., 2021).

3.4 Urease Test

Procedure: A sterile urease agar slant was inoculated with the test bacterium using a sterile wire
loop,the inoculated slant was incubated at 35–37°C for 24 hours. After incubation, the color of the
medium was observed. A positive result is indicated by a pink color change in the medium, while a
yellowish color signifies a negative result (Aryal et al., 2022).
3.4 Preparation of McFarland Standard

The McFarland standard was prepared by first weighing 0.05 grams of barium chloride dihydrate
(BaCl₂·2H₂O), which was dissolved in 100 mL of 1% sulfuric acid (H₂SO₄). The solution was mixed
thoroughly until homogeneous, and its turbidity was verified using a spectrophotometer at a
wavelength of 625 nm to ensure an optical density (OD) between 0.08 and 0.13. The prepared standard
was then stored in a tightly sealed container and protected from light to maintain stability (Bailey &
Scott’s, 2022).

3.4 Standardization of the Inoculum

A 0.5 McFarland standard, either freshly prepared or sourced from a reliable provider, was used as a
reference. A bacterial suspension was created by transferring 3–5 isolated colonies of the test organism
into a test tube containing sterile saline. The suspension was vortexed vigorously to achieve
homogeneity, and its turbidity was visually compared against the 0.5 McFarland standard. Adjustments
to the suspension’s density were made by either adding more bacterial colonies or diluting with sterile
saline until the turbidity visually matched the standard (CLSI, 2021).

3.4 Preparation of Mueller Hinton Agar

Mueller Hinton agar was prepared by dissolving 38 grams of agar powder in 1 liter of distilled water. The
mixture was stirred until fully dissolved, brought to a boil, and autoclaved at 121°C for 15 minutes. After
cooling to 45–50°C, approximately 20–25 mL of the medium was poured into each sterile Petri dish
under aseptic conditions. The plates were allowed to solidify at room temperature before use (CLSI,
2023).

3. 4. ANTIBIOTICS SUSCEPTIBILITY TEST

The antibiotic susceptibility test was conducted using the Kirby-Bauer (disc diffusion) method. First, a
sterile cotton swab was dipped into the bacterial suspension and evenly streaked across the entire
surface of a Mueller-Hinton agar plate to create a uniform bacterial lawn. The plate was then allowed to
dry briefly to ensure proper absorption of the inoculum. Using sterile forceps, antibiotic-impregnated
discs were carefully placed onto the agar surface. Each disk was gently pressed to ensure full contact
with the agar, and they were spaced approximately 24 mm apart to avoid overlapping inhibition zones.
Once prepared, the plate was incubated at 35°C for 24 hours. After incubation, the plate was removed,
and the diameter of the zones of inhibition around each antibiotic disk was measured in millimeters
using a vanier caliper with all measurements recorded for analysis (Forbes et al., 2022).