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With the help of sterile petri dishes, one (1) ml of sterile water was added into the each petri dish. A U shaped glass rod was placed in each of the petri dish. Sterile slides were placed on each of the U shape glass rod in the petri dishes. With the help of a scalpel blade the already prepared SDA was cut into cubes. Using a sterile wire loop, four days to 1 week fungal growth was collected and smeared by the four sides of the SDA. A sterile cover slip was collected using a well flamed forcept and placed on the inoculum in the petri dish. The plate was then covered and kept at room temperature for 4 to 7days as growth was observed before examination.

Staining with lactophenol blue

A drop of lactophenol (LP) was placed on a clean microscopic slide The cover slip from the culture was gently removed using a sterile forcept and placed in the drop of lactophenol (LP). A drop of LP was dropped on the slide from the old culture as the media cultured on the slide was gently removed. Sterile cover slips was placed on the slide containing a drop of lactophenol blue and observe microscopically. It was first viewed at X10 to focus the lens well then X40 to get a clearer view.

CHAPTER FOUR 3.0 RESULT

The growth of different genera showed different cultural characteristics on the agar (table 1). The different colors use for identification of the fungi was also observed (Figure: 1- 4). These fungal isolate belong to the genera of Penicillium, Aspergillus, Fusarium and Mucor. (Table 2) The most common genus isolated was Aspergillus (Table 2). Some species of the dominant genus are Aspergillus flavus, A. fumigatus and A. niger. These different species of Aspergillus were

isolated from different samples tested along with other genera which were also identified.

TABLE 1

MORPHORLOGY APPEARANCE OF THE FUNGAL ISOLATES

COLOR SHAPE ELEVATION SIZE

SAMPLE A Black

Grey-Green White White-Pink Gray green

Irregular Oval Irregular Oval Oval

Elevated Elevated Elevated Elevated Flat

1-9cm in diameter 40-50mm 20um 64-70mmd 2-3um SAMPLE B Yellow-green

White White-Pink Black

Oval Irregular Oval Irregular

Elevated Elevated Elevated Elevated

3-6um 20um 64-70mm 1-9cm in diameter SAMPLE C Yellow-green

Grey-green

Oval Oval

Elevated Not elevated

3-6um 2-3um

Gray-green Oval Elevated 40-50mm

SAMPLE D Yellow-green Oval Elevated 3-6um

TABLE 2

THE IDENTIFIED FUNGI

GENUS SPECIE CORN FLOUR

SAMPLE A SOYBEAN

FOUR SAMPLE B

MEASURED DANO SAMPLE C

SACHET DANO SAMPLE D

Aspergilus bn A. flavus + + + +

A. niger + + _ _

A. fumigatus + _ + _

Penecillium P. digitatum + _ + _

Mucor M. hiemalis + + _ _

Fusarium F. Oxysporum + + _ _

THE MACROBILOGICAL VIEW OF THE FUNGI ISOLATE

FIG.1 FIG.2

Aspergillus fumigatus Aspergillus niger

FIG.3 FIG.4 Penecillum Sp Mucor Sp

MICROSCOPIC VIEW OF THE FUNGI ISOLATE

FIG.5 FIG.6 Aspergillus flavus Aspergillus niger

Fig.7 fig.9

Penicillium roqueforti Fusarium oxysporum

Fig.10 fig.11

Mucor hiemalis Aspergillus fumigatus

CHAPTER FIVE 5.0 DISCUSSION

From the research conducted, on corn flour, soybean flour and powder milk (measured and sachet), many genera and species of fungi were isolated and identified and they include Pspergillus, Penicillium, Fusarium and the Mucor specie. These food products were possibly contaminated by either the biotic or the abiotic factors which appeared to be one of the major factors that support fungal growth in food products. (Hill and Waller, 1999) As all the samples had different specie of the fungi isolated. The corn flour was more contaminated than other samples cultured because they were placed in exposed containers. Also storage facilities such as sacks, polythene bags and natural fibre, which are air-tight being used by the traders in the market (Personal observation) for storage of all the varieties might have encouraged the fungal growth. This is because they can lead to continuous increase in humidity and temperature of the food product, which consequently favours fungal growth as reported by Ahmad (2003). The most common fungus causing spoilage of food product is the Aspergillus flavus which was

identified from the samples collected which showed the highest occurrence in this research (Table 2). Moreover, food products can encounter fungal infestation by influences from outside environment, such as insect’s infestation, wound and presence of foreign matter such as sand, dust and debris among others. Thus some of the identified fungal species could have come from any of these sources. Similarly, the constant exposure of food products to the outside environment at the time of sales could have aided in deposition of the fungal spores on them.

Therefore, spores can germinate on the food products when temperature and humidity triggers the growth processes. Damage by insects has also been known to provide entry points for fungal infection (Dennis, 2002) and aid in their rapid spread. Hence, presence of insects may under certain critical circumstances be quite essential for establishment of infection. While several fungal species cause spoilage of food products worldwide, it is aiso noteworthy that presence of this known organisms isolated from this samples, are known to produce different mycotoxins like the ochratoxin, neurotoxin and aflatoxin. This mycotoxins may cause serious mycotoxicoses in man and in animal if produced in these food products tested. Example Penicillium roquefortine is known to produces neurotoxin. The extent to which neurotoxin affect nerve function depends on the toxicity of the substance either by ingestion or by inhalation depending on the individual age and immune status. This toxin can have long lasting effect by causing neurons to malfunction or by disrupting interneuron communication which may eventually lead to paralysis or death. The ochratoxin is a naturally occurring foodborne mycotoxin found in a wide variety of agricultural products that can be produced by several fungal specie and genera like Penicillium or the Aspergillus specie. Ochratoxin (OTA) causes nephrotoxicity and renal tumors in different animal species. However health effect has linked OTA exposure with human disease known as Balkan endemic nephropathy (BEN) and chronic intestinal nephropathy (CIN)

as well as other renal diseases. The generally most common and deadly mycotoxin produce is the aflatoxin known to be produce by aspergillus species. Aspergillus infections have grown in importance in the last years. However, most of the studies have focused on Aspergillus fumigatus, the most prevalent species in the genus. In certain locales and hospitals, Aspergillus flavus is more common in air than A. fumigatus, for unclear reasons (Guinea et al., 2005). After A. fumigatus, A. flavus is the second leading cause of invasive aspergillosis and it is the most common cause of superficial and systemic infection. Common clinical syndromes associated with A. flavus include chronic granulomatous sinusitis, keratitis, and cutaneous aspergillosis, wound infections, osteomyelitis following trauma, liver disease and even deep or systemic aspergillosis. Outbreaks associated with A. flavus appear to be associated with single or closely related strains, in contrast to those associated with A. fumigatus. In addition, A. flavus produces aflatoxins, the most toxic and potent hepatocarcinogenic natural compounds ever characterized which is very deadly because it is a byoroduct that can cause serious damage to the DNA.

Prolong exposure to aflatoxin, cell accumulate DNA mutations and thus are at risk of developing into cancer cells. Aflatoxins are known to be heat stable therefore heating of food products conterminated with aflatoxin cannot be destroyed by heat. This known mycotoxins that can be produce by this fungi species gets into the body either by inhalation of the spores, ingestion or cuts from the skin and this mycoses are more severe in immunocompromised individual. The spores of this fungi are in the air making it very easy for them to invade exposed food products or during the administration of immunosuppressive drugs use during organs transplanting thereby giving them easy access to cause infection as can be encouraged some environmental factors.