2.0MATERIALS AND METHODS
2.1.1 Glass wares
include conical flasks, pipettes, measuring cylinders, glass spreader, beakers,
test tubes, McCartney bottles, glass slides.
incubator, refrigerator, light microscope, spectrophotometer, weighing balance,
hot plate, hot air oven.
include crystal violet, safranin, 70% ethanol, Gram’s iodine, hydrogen
peroxide, immersion oil.
include nutrient broth, nutrient agar, mannitol salt agar, normal saline,
Mueller-Hintonagar, DNase agar. Other materials are inoculating loop,
inoculating needle, Petri dishes, cryovials, cotton wool, and aluminium foil.
2.2.1 Sterilization of Materials
of the glasses ware used in the research were sterilized in a hot air oven at
160°C for more than two hours before use. The following disinfecting procedures
were carried out during this research: inoculating loops and the needles were
flamed till red hot in a Bunsen flame before use.
spreaders were also dipped in 70% ethanol and passed through a Bunsen flame
before use. The necks of conical flasks were flamed regularly while pouring
agar into Petri dishes to prevent contamination of the media. Glass slides were
cleaned with 70% ethanol to ensure they are grease free and the work bench was
swabbed with 70% ethanol solution before and after work
2.2.2Preparation and Sterilization
used were prepared by weighing an appropriate amount of the media powder and
dissolving it in an appropriate volume of water in a conical flask according to
the manufacturer’s instruction and then homogenized on a hot plate.
solid media, the media was autoclaved at 121°C and 15psi for 15 minutes and
then allowed to cool before pouring into Petri dishes. The media was allowed to
set before inverting the Petri dishes to prevent the condensate from dropping
to the agar surface. For liquid media, the media were first dispensed in
appropriate volumes into test tubes and corked with a cotton plug before being
placed in a container and autoclaved at 121°C and 15psifor 15 minutes.
wastewater samples used in this research were collected from the following
halls ofresidence in Obafemi Awolowo University, Ile-Ife :Fajuyi Hall (7.5181°
N, 4.5183° E).
water samples were collected in sterile reagent bottles from gutters of the
above mentioned hall of residence in
Obafemi Awolowo University and taken immediately to the laboratory for
processing. A total of samples were collected between September 2017 and November
2.2.5 Isolation of Staphylococci
10-fold dilutions were carried out for each wastewater sample in 9ml of sterile
normalsaline solution. Out of the 10-1, 10-2and sometimes
10-3 dilutions, 0.1 ml of the diluents were plated out onmannitol
salt agar plates using the spread plate technique and incubated at 37°C for 48
or yellow coloured, dome shape colonies were picked into nutrient broth tubes
frommannitol salt agar plates with colony counts of between 30 and 300, and
incubated at 37°Cfor 18-24 hours. The broth culture were then streaked on
nutrient agar plates and incubated at37°C for 18-24 hours. The recovered pure
isolates were then stored in cryovials and stored inthe fridge at 4°C prior to
further biochemical tests.
staining is one of the most important procedures in microbiology for
bacterialidentification and taxonomic division. It was developed by Hans
Christian Gram in 1884. Itseparates bacteria into Gram positive and Gram
negative bacteria based on their cell wallcomposition.
positive bacteria appear purple under the microscope as they retain thecrystal
violet stain (primary stain) because of the thick peptidoglycan layer of their
cell wall,while Gram negative bacteria appear pink, they lose the crystal
violet stain and absorb thesafranin stain (counter stain) because their cell
wall has only a thin peptidoglycan layer.
drop of distilled water was placed on a clean grease free slide with a dropper.
Two or threecolonies from the pure bacterial culture on nutrient agar were then
picked using a inoculatingloop and used to make a smear on the slide. The smear
was allowed to air dry and was heatfixed by passing it over a flame. The slide
was then flooded with crystal violet for 60 secondsand rinsed gently under
The slide was flooded with Gram’s iodine for
30seconds and then rinsed off in a flow of tap water. Thereafter, the slide was
then floodedwith ethanol for 10-15 seconds and rinsed off, after which the
slide was flooded with safraninfor 45 seconds and rinsed under running water.
The slide was then allowed to dry and a dropof immersion oil was placed on the
slide after which it was viewed under oil immersion(×100 magnification) using a
enzyme catalase is produced by microorganisms that live in oxygenated
environments toneutralize toxic forms of oxygen metabolites,H2O2.
Catalase breaks down hydrogen peroxideH2O2 into water and
oxygen resulting in bubble formation.
drop of hydrogen peroxide was placed on a clean and sterile grease- free slide,
a few bacteria colonieswere then transferred to the slide using a wire loop.
The rapid evolution of bubbles indicatesa positive test.
DNase test detects the ability of an organism to produce deoxyribonuclease
which is anenzyme that cleaves DNA.
loopful from an 18-24 hour old culture on nutrient agar was streaked on DNase
agar andincubated at 37°C for 18-24 hours. After incubation, the plates were
flooded with 1N HCland left for about 4 minutes. The presence of a clear zone
around the line of streak indicates apositive test while the absence of a clear
zone is indicative of a negative result.