Fifteen samples of plain cakes were collected from eatries and parties from Jos metropolis
The test Microorganisms were Isolated from the samples of cakes gotten from eateries and parties in Jos metropolis
Isolation of spoilage microorganisms of plain cake was carried out using the method described by Ayanda., et al. (2013). The samples were soaked in sterile water for 30 minutes to dissolve. Serial dilution of the sample were made to 108. 1ml of each dilution was innuculated on each labeled plate and 20ml of nutrient agar was poured into plates and incubated at 37° for 24 to 48 hours.
This technique is used to differentiate gram positive bacteria from gram negative bacteria based on their cell wall composition. For gram positive, their cell wall was purple because they retain the primary stain which is crystal violet, while gram negative bacteria were stained pink, because they retain counter stain. The procedure is as follows: A smear was prepared using 18-24hours old culture. One looped of sterile distilled water was transferred on clean grease free glass slide and colony from 24hours old culture was transferred and emulsified on it to make thin preparation. The smear was air dried and the edge of the glass slide was passed over Bunsen flame for about three  times to heat fix the smear. Primary stain (crystal violet) was added to the smear and left for about 60seconds. The stained smear was rapidly washed with distilled water, the smear grams iodine solution was added for another 60 seconds and washed with distilled water, then the smear was decolourised with 95% alcohol for 5seconds and washed with water. Finally, the smear was counterstained with saffranin dye for 60seconds and washed with distilled water. The smear was then observed under x 100 oil immersion objective lens of the microscope.
Biochemical test was performed for identification purposes and confirmatory tests and the organisms used for inoculation of the test media was sourced from fresh cultures. Specific biochemical tests performed include:- catalase test, carbohydrate fermentation test, urease test, indole production test, citrate utilization test, mortility, oxidase test and coagulase test (Cheesbrough, 2009).
This test detects the presence of enzyme coagulase. A smear of 24 hours old culture of the organism under study was made on a slide, some drops of blood were added to the smear and left to stand for about 1 hours. As positive reaction to this test is indicated by clotting and coagulation of the blood plasma (suggesting that the enzymes coagulase was produced by the organism). However, absence of clotting or coagulation is a negative reaction.
This test detects the presence of cytochrome C oxidase that is able to reduce oxygen. A smear is made on a slide, a piece of filter paper soaked with a few drops of oxidase reagent strip, was placed on the smear for 4 minutes. A positive reaction the oxidase test is indicated by a deep purple colour while the other colours or no change in colour is indicated of a negative reaction.
This test detects the presence of enzymes catalase, which contents hydrogen peroxide to water and oxygen. It is used to differentiate bacteria species based on their positive or negative reaction with hydrogen peroxide solution. A smear was made on a slide and 3 drops of 3% hydrogen peroxide solution was added to it. Production of effervescent oxygen bubbles indicated reaction.
2H202 + Catalase 2H20202
This test detects the enzyme that splits Urea to Ammonia and Carbondioxide in bacterial. It is used to distinguish Proteus spp, Klebsiella spp, which produce urease, also E.coli that do not produce urease. When Urea is added to the smear preparation of the organism to be identified, evolution of CO2 is a positive reaction while a negative reaction results if there is no evolution of carbondioxide gas.
This test is used on the identification of enteric bacteria. Enterobacter spp are positive to this test, while Escherichia coli will give a negative reaction. The test is used to check the ability of organisms to utilize citrate as a sole carbon source. The isolate was inoculated aseptically using a sterile wire loop to streak on simmon’s citrate Agar medium. It was then incubated for 48hours. After incubation at 38°c, a blue colouration on the medium indicates a positive reaction, while no change in the colour of the medium, indicates a negative reaction to citrate test.
This was carried out to identify the isolate to species level. The production of turbidity and gas from carbohydrates (lactose, glucose, sucrose, manitol) was used to identify isolates to species level.
This test is performed to distinquish the motile organisms from the non-motile ones. Isolates was inoculated aseptically using a sterile wire loop to stab the test organism in nutrient broth. It was then incubated for 48 hours. Diffuse hazy growth that spreads throughout the medium, making it look opaque indicate motility and when growth is confine to the line of inoculation it indicate non- motility.
The morphological and cultural characteristics with special reference to the sporulation of the fungi isolates was used to identify them. Where the plates were observed daily for growth rate of each of the cultural isolates. The colour and morphology of the colonies was recorded. A wet mount of each fungus was prepared by suspending a loopful of isolate in a few drops of lacto-phenol cotton blue solution on a microscope slide and then cover with a slip then viewed under x40 magnification.
Three species of Bacteria were isolated; Bacillus laterosporus, Staphylococcus aureus and Staphylococcus epidermidis. Bacillus laterosporus – an aerobic filamentous, spore bearing gram positive rod. Staphylococcus aureus and Staphylococcus epidermidis are non-motile gram positive cocci that are catalase positive, where Staphylococcus aureus is coagulase positive, Staphylococcus epidermidis is coagulase negative. See table 3.
Two genus were identified (Zygomycetes and Deuteromycetes) and they are common contaminant on various substrates like grains, fruits etc. the general cultural and microscopic characteristics of the species are showed in table 4, adapted from fungi Atlass (Samson., et al. 2004).
It was observed for bacteria species, that Bacillus laterosporus had the highest occurrence of 62.5%, where Staphylococcus epidermidis and Staphylococcus aureus had 25% and 12.5% respectively. Rhizopus stolonifer had 29.41%, Aspergillus fumigatus 23.53% where Aspergillus flavus, Aspergillus niger, Penicillius citrinum and Penicillium chrysogenum had 11.76% respectively for fungi species. Table 5 showed the result.
+ = Positive
- = Negative
|Cultural characteristics on Czapek agar||Microscope examination of solid culture||Probable identify|
|Blue, green colour, wooly surfaces growth and the reverse is yellow||Short conidiophores, smooth-walled and conical shaped terminal vesicles.||Aspergillus fumigatus|
|White fluffy growth of colonies with elevated mycelia that turned black after 36 hours||Single celled spore and flattened conidia in chains developing at the end of the sterigma, arising from the terminal bud of the septate hyphae.||Aspergillus niger|
|Colonies usually consist of a dense yellow green conidiophores becoming dark yellow-green||Conidia heads typically radiates, later splitting into loose columns conidia globose to subglobose phialides borne directly on the vesicle.||Aspergillus flavus|
|Whitish colony becoming grayish-brown and brown black sporangia, often over 20mm high smooth or slightly rough walled stolons and blackish brown at maturity.||Columella is brown, spores are short ellipsoidal, zygospores brownish-black, sporangiospores irregular in shape often polygonal or avoid.||Rhizopus stolonifer|
|Growing restrictedly within 7days 1-5cm in diameter consisting of a dense felt of conidiophores appearing leathery blue green reverse normally yellow||It has septate, branching fungal mycelia. Its conidiophores are one stage branched smooth walled terminating in a whorl of 6 – 10 phialides, phialides, flask shaped, conidia produced in columns, globose to sub globose.||Penicillium citrinum|
|Rapid growth, lobed margin, pale green blue changing to darker green shades, odour is fruity usually velvety and flat||Septate hyphae, conidiophores simple or branched.||Penicillium chrysogenum|
|Organism Isolated||No of Occurrence||% Frequency|
Frequency of occurrence = No of a specie isolated x 100
The total no of microorganism (Bacteria or fungi)
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