Microbial Contamination Of Vended Fruit

METHODOLOGY

3.1. Study Area
This study was conducted in Applied Microbiology Laboratory Unit, Ebonyi State University, Abakaliki while the samples were collected from different fruit vendors in Abakpa Main Market, Abakaliki, Ebonyi State. Abakpa Main Market, Abakaliki, also known as “meat market” is the largest market in Ebonyi State with different people selling different items like foodstuffs, fruits, vegetables, wears and other exciting goods. A great number of traders there are involved in fruit selling. And most of them are sliced or processed because most of their customers may not be able to afford or have time to process the fruits properly.

3.2. Sample collection
A total of seventeen (17) vended fruit samples consisting of carrot, cucumber, tiger nuts, sliced watermelon and pineapple were collected on duplicates from three different fruit vendors and put into a white polyethene bags to differentiate them based on the vendors they were bought from. 

3.3. Materials and Reagents used
The materials and reagents used during the course of this research include: weighing balance, beakers, conical flasks, autoclave, petri-dishes, 70% ethanol, non-absorbent cotton wool, aluminum foil, test tubes, wire loops, incubators, microscope, blender, nutrient agar, potato dextrose agar, mannitol salt agar, salmonella-shigella agar, macConkey agar, peptone water and distilled water.

3.4. Methods
3.4.1 Media preparation
The different media which included nutrient agar, potato dextrose agar, mannitol salt agar, macConkey agar and salmonella-shigella agar; and peptone water were prepared according to the manufacturer’s instruction.

3.4.2 Isolation of micro-organisms from the vended fruit samples
About 10g of each of the fruit sample was weighed and homogenised in 90ml of sterile distilled water using an electric blender. Then, ten-fold dilutions of the homogenates were made with sterilized peptone water; after that 1ml of the 10-4 dilutions of the homogenates were dispensed into the petri-dishes that were labelled based on the agar used by pour plate method and allowed to gel. After gelling, the petri-dishes that contained mannitol salt agar, nutrient agar, macConkey agar and salmonella-shigella agar were incubated at 37°C for 24hours while the petri-dishes that contained potato dextrose agar were incubated at 25°C for 3days.
    
The nutrient agar, macConkey agar, mannitol salt agar and salmonella-shigella agar were used to check for total bacterial count, total coliform count, presence of Staphylococcus aureus, Salmonella and Shigella spp respectively.
 
At the end of the incubation period, the plates were brought out of the incubators and the colonies were counted using a colony counter device and each count was expressed in colony forming unit per ml (CFU mlˉ). 

3.5. Isolation of the cultured micro-organisms  
The distinct colonies on nutrient agar and potato dextrose agar were carefully examined using microscope for their morphological characteristics like colour. Then these colonies were sub-cultured on nutrient agar using streaking method and were incubated at 37°C for 24hours.

3.6. Identification of Isolates
Gram staining and other biochemical tests were carried out based on the method of Cheesbrough (2004). The biochemical tests performed here included catalase test, oxidase test, indole test and coagulase test.

3.7 Biochemical tests
3.7.1 Catalase test
The discrete colonies of each of the isolates were collected with a wooden stick and emulsified in a drop of hydrogen perioxide (H2O2). Bubbles of gas indicated a positive result according to Cheesbrough (2000). 

3.7.2 Indole test
Here a little portion of each of the isolates is inoculated into 5ml of sterilised prepared peptone water which is contained in different test tubes using a wire loop. And then, the test tubes containing the organisms were left to incubate at 37°C for 48hours. After incubation period, 3-4drops of indole reagent known as Kovac’s reagent was added and shook gently. A positive result gave a red surface layer after 10minutes while a negative result gave no red surface layer after 10minutes according to Cheesbrough (2000).

3.7.3 Oxidase test
A piece of filter paper was placed in a clean petri dish and 2-3drops of freshly prepared oxidase reagent was added. With the aid of a wooden stick, discrete colonies of the isolates were collected separately and smeared on the filter paper. A positive result gave a purple-blue colouration after 10seconds while a negative result gave no such colour after 10 seconds according to Cheesbrough (2000). 

3.7.4 Coagulase test
A drop of distilled water was placed on each end of a slide and a colony of the test organism was emulsified in each of the drops to form a thick suspension. Then a loopful of plasma was added to one of the suspensions and swirled gently. A positive result showed clumping after 10 secconds while a negative result showed no clumping after 10 seconds according to Cheesbrough (2000).

3.8 Gram staining 
A thin smear of the isolates were made on different slides with the aid of a wire loop and left to dry and after they were heat fixed and allowed to cool. Then the different smears were covered with crystal violet stain for 30-60 seconds and rapidly washed off with clean water. Then the smears were covered with Lugol’s iodine for 30-60 seconds and rapidly washed off with clean water. The smears were decolourised rapidly with alcohol and washed out immediately with clean water. Then the smears were covered with safaranine for 30-60 seconds and washed immediately with clean water. The stained smears were then allowed to air-dry. After drying, a few drops of oil immersion were dropped on the stained smears and viewed with the aid of a microscope (×100 oil Objective lens) to check for the microscopic properties of the organisms like the Gram reaction, morphology (Cheesbrough, 2006). 

For the fungal isolate, a drop of lactophenol cotton blue stain was dropped in the centre of a clean slide. And then a fragment of the fungus was collected with the aid of a wire loop and placed in the drop of the stain and teased gently and covered with a coverslip. The coverslip was not pushed down or tapped to avoid the dislodging of the conidia from the conidiophores. Then the stained isolate was viewed under the microscope with ×10 and ×40 objective lens for its morphological characteristics (Cheesbrough, 2006)

3.9 Identification of fungal isolate
3.9.1 Lactophenol cotton Blue test
For the fungal isolate, a drop of lactophenol cotton blue stain was dropped in the centre of a clean slide. And then a fragment of the fungus was collected with the aid of a wireloop and place in the drop of the satin and teased gently and covered with a coverslip. The coverslip was not pushed down or tapped to avoid the dislodging of the conidia from the conidiophores. Then the stained isolate was viewed under microscope with X10 and X40 objective lens for its morphological characteristics (Cheesbrough, 2006)