Bioscience Methods 2017, Vol.8, No.2, 18-30
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For fungi, the Potato Dextrose Agar was also poured into the petridishes containing the 10
-2
dilution of the snacks
sample in triplicate, the plates were gently rocked and allowed to cool and solidify before taping the plates. The
plates were observed for 7 days for fungi growth.
1.7.4 Enumeration of microbial colonies
Colony counting was carried out visually by counting the number of visible colonies that appeared on the plates.
Calculation of colony forming unit (CFU) per ml for the bacteria and the spore forming unit (SFU) per ml for the
fungi was based on the formula:
1.7.5 Identification of bacteria and fungi
The identification of bacteria and fungi was based on the cultural characteristics (shape, colour of the pigment,
opacity, elevation, edge of colony, consistency and surface), staining reaction (gram staining and lacto phenol
cotton blue staining), and the biochemical tests (nitrate reduction, indole production, urease, coagulation and sugar
fermentation) as reported by Cowan and Steel, (2002) (Famurewa et al., 2009).
1.7.6 Cultural characteristics of the bacteria
The bacterial isolates were streaked for single colonies, incubated at 37°C for 24 hours and observed directly on
Agar plates to note the shape, size, colour, of pigment, opacity, elevation surface and edge of the colony.
1.7.7 Gram staining for bacteria
The gram staining steps include, the preparation of a heat fixed smear from an 18-24 hour culture, which was
stained with crystal violet for one minute before the solution was poured off and rinsed off with gram iodine
solution, the iodine was allowed to react for one minute then poured out and the slides washed with 95% alcohol
until no more violet comes out from the slides. The slides were rinsed under a gentle running tap water and
counter stained with safranin for 1 minute, the slides were blotted dry and examined with microscope
(LXSZ-107BN: England) under oil immersion lens to observe the morphology of the cells.
1.7.8 Fungi staining
Few drops of lactophenol cotton blue solution were placed on slides, the inoculating needle was flamed and
swirled for 20 seconds, after which, it was used to transfer fungus onto the slides, emulsified and the organisms
covered with a cover slip. The prepared slides were viewed under microscope (LXSZ-107BN: England) and the
fungi were observed.
1.7.9 Spore staining
The ability of bacteria isolates to produce endospores was determined. A heat smear was prepared from an 18-24
hours culture; malachite green solution was added and steamed for 5-10 minutes. The slides were washed with
water, blotted dry and examined under the microscope (LXSZ-107BN: England) with an oil immersion objective
lens; spores were stained green while bacteria cells were stained red.
1.8 Biochemical test
1.8.1 Catalase test
The tested organisms were inoculated onto Nutrient Agar plates and incubated at 37°C for 24 hours. 3% volume
concentration of hydrogen peroxide was placed on a colony, emulsified and examined for oxygen bubbles which
indicate the presence of catalase.
1.8.2 Coagulase test
An 18-24 hours culture was used for this test. A loop full of normal saline was placed on each section of a slide
and was emulsified with a small amount of the 18-24 hours old culture until a homogenous suspension was
obtained.
