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So the ladder serves as another positive control. The electrophoresis was run at 100V and not more than 120-150mA for 1 hour 10 minutes. In the end of the process, the gel picture was taken under UV light using gel documentation system. The bands were read and compared with the bands at DNA ladder results.
3.2.2 Determination of the climatic factors influencing the survival and population of
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based on the manufacturer‟s instruction. Using a weighing balance, 12g of nutrient agar powder was weighed and poured into a conical flask. After, 250 ml of distilled water was poured into the flask and stirred. This was sterilized using autoclave at 121°C for 15 minutes. After cooling, the media was poured into Petri dishes sterilized using hot air oven at 160°C for 90 minutes. This was then allowed to solidify.
One milliliter of each water sample was spread into separate nutrient agar plates and incubated at 37°C for 24 – 48 hours. Each colony was observed on a plate and checked for its purity. The impure colony was subcultured by transferring it to nutrient broth and then streaking into nutrient agar plates for pure colony growth. Bacterial colonies were further purified and preserved on nutrient agar slants at 4°C for identification.
3.3.1.2 Identification of bacteria isolates
The bacteria isolates were identified using colony cultural characteristics, colony morphology and biochemical characterization. The cultural characteristics of bacterial isolates such as colony color, colony elevation, colony margin and colony surface were inspected visually in a plate.
Morphological characteristics of bacterial isolates such as spore stain, gram staining, shape and motility tests were determined by light microscope when viewed using ×40 objective lens (Cheesbrough, 2006).
3.3.1.2.1 Gram staining procedure
A sterile wire loop was used to put a drop of normal saline at the center of grease – free slide. A portion of colony was picked and emulsified in the drop and allowed to air dry before heat fixing. Crystal violet was applied for 1 minute on the smear; it was later replaced with Lugol‟s iodine for another one minute before rinsing with distilled water. The slides were later flooded with 95% ethanol (decolouriser) for few seconds until the slides‟ appearance became free of violet stain. Slides were then rinsed with water and Safranin stain applied for 1 minute. This was followed by rinsing and air drying before being observed microscopically under × 100 objective lens in oil immersion. The bacterial cell that retained the purple color against the counter stain background indicated a Gram positive organism while the Gram negative organisms stained red.
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3.3.1.2.2 Biochemical characteristics of the bacteria isolates
Biochemical characteristics of isolates, such as catalase test, coagulase test, indole test, motility test, oxidase test, and sugar fermentation test were carried out according to Cheesbrough (2006).
3.3.1.2.2.1 Catalase test
In catalase test, a drop of 3% hydrogen perioxide was put on a grease free slide. Portion of the colony of the test organism was picked and emulsified in it using a wire loop. This was examined for gas bubble which indicates catalase positive and the absence of gas bubbles indicates catalase negative. The test was used to differentiate between Staphylococcus species. The gas bubbles observed in the reaction was due to the breakdown of hydrogen peroxide to oxygen and water by enzymes called catalase.
3.3.1.2.2.2 Coagulase test
A drop of physiological saline was put on a clean glass slide, followed by making smear of 24 hours old isolate of test organism. Then a drop of human serum was added into it to make a suspension. Clumping indicates positive result which implies the ability of the test organism to produce coagulase, an enzyme that coagulate blood plasma while in a negative result no clumping was observed (Cheesbrough, 2006). This test is used to differentiate pathogenic Staphylococcus aureus from non-pathogenic staphylococci.
3.3.1.2.2.3 Indole Test
This test was done as described by Cheesbrough (2006); Miller and Wright (1982). It was carried out to determine the organisms that breakdown amino acid tryptophan into indole. Peptone water of 1.5g weight was dispensed into 250ml capacity conical flask. 100ml of distilled water was gradually added and shaken. This was finally dispensed into the test tubes and corked for sterilization in the autoclave at 121°C for 15minutes. A speck of each isolate was inoculated into 5ml of sterile peptone water and was incubated at 37°C for 48 hours. About 4 drops of Kovac‟s reagent was added into each tube and gently shaken. Positive test was indicated by a red colour that occured immediately at upper part of the test tube.
50 3.3.1.2.2.4 Motility test
The isolate was cultured in sterile peptone water medium in the test tube and incubated for 24 hours at 37°C. A drop of the suspension was placed on a cover slip and glass (concave glass slide) with plasticin made circular on it was inverted on the cover slip. This was carefully inverted and then observed for motility under ×40 objective lens.
3.3.1.2.2.5 Oxidase Test
This is used to determine the presence of oxidase enzyme. Filter papers were dipped into a freshly prepared oxidase reagent and allowed to dry for a little while. With a wire loop, a colony of the test organism from the culture plate was removed and smeared on the filter paper. A purple colour change of the organism immediately it is smeared on the filter paper indicated a positive result while purple colour change after 10-20 seconds indicated a negative result.
3.3.1.2.2.6 Sugar fermentation (lactose, maltose and fructose) test
This test is used to determine the ability of the organism to ferment a specific carbohydrate with or without the production of gas. Bromothymol blue was used as an indicator in the media.
Fermentation of the carbohydrate produces acids, causing the media to change from blue to yellow. Inverted tubes called Durham tube, traps some of the gas the organism produces, allowing production to be seen (if it ferments, gas will be produced). Each of the test tubes containing the carbohydrate medium was inoculated with the test organisms and incubated at optimum temperature for 24 hours. When the media turned yellow (fermentation had occurred) and gas is produced, it indicated positive result and when it remained blue (no fermentation occurred) which indicated negative result.
3.3.1.2 Isolation of fungi from the breeding sites of mosquito 3.3.1.2.1 Preparation of Media
The media used was Sabouraud Dextrose Agar (SDA) for the isolation of fungal organisms. The media were prepared according to the manufacturer's instruction by weighing 65g of the powder and dissolving it in 1000ml of water. The solution was homogenized using the magnetic stirrer
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and sterilization at 121oC for 15minutes was done using an autoclave. The prepared media was allowed to cool and poured into petri dishes.
3.3.1.2.2 Innoculation of samples on the plate.
The water samples were inoculated on the prepared SDA by streaking. This was incubated aerobically at 25oC for 3 days. Respective colonies formed were sub-cultured and incubated severally to get pure culture. After that, the plates were observed for colony morphology.
3.3.1.2.3 The slide culture technique
The slide culture technique according to Wijedasa, and Liyanapathirana (2012) was also employed. The SDA medium in the Petri dish was cut into square blocks, 1cm × 1cm with a sterile blade. An agar square was placed at center of the sterile slide on a sterile V shaped bent glass rod in a sterile Petri dish. A wire-tip of the culture was inoculated into the mid-point of each of the four edges of the agar square. The cover slip was lifted by means of sterile forceps and placed over the surface of the inoculated agar block. About 10ml of sterile distilled water was poured in the bottom of the Petri dish and the dish covered. The slide culture plates were incubated at room temperature for 3 -5 days. The growth was examined after 3 or 5 days. When sufficient growth occurred, stained preparation was made. The cover slip was removed and the agar block discarded without disturbing the rectangles of growth on the slide and cover slip. A drop of lacto-phenol blue was immediately added and the second sterile cover slip gently placed.
Similarly, the growth on the cover slip was stained and placed on the second sterile slide. The two slides preparations were examined under the low (×10) and high (×40) power of the microscope. Observed features such as the hyphae and spore heads were recorded.
3.3.1.2.4 Identification of the fungal isolates
Both macroscopic and microscopic identification were carried out based on the criteria described by St-Germain and Summerbell (1996) and Kidd et al., (2014). Macroscopically, the culture plates were observed from the third day for colonial morphological characteristics of the isolates.
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Features observed were pigmentation, texture (dry, powdery, velvety, gelatinous) and colour of reverse of the plates. Microscopically, the features below were sought for after slide culture included: hyphae (septate or non-septate), mycelium (hyaline or coloured), type of asexual spores (sporangiospores, conidia, arthrospores or blastospores), appearance of conidia (shape, size (micro or macroconidia), colour, smooth or rough, one, two or many celled), characteristics of asexual spore-head, appearance of sporangiophores or conidiophores (simple or branched, and if branched, the type of branching), size and shape of the collumella at the tip of the sporangiophore, size and shape of the vesicle at the tip of the conidiophores, appearance of conidiophores (single or in bundle), presence of sexual spores (oospores, zygospores or ascospores) and Presence of special structures or spores (stolons, rhizoids, chlamydospores).
3.2.4.2.5 Biochemical test for yeasts identification
The suspected yeast-like organisms were identified by performing germ tube test as described by Sandven (1990). Three (3) drops of fresh pooled human serum were dispensed into 12 x 75 mm labeled test tubes using a Pasteur pipette. A colony of the isolate was picked with a sterile wooden applicator and emulsified in the serum. The stick was discarded in a discard container.
The suspension was incubated at 37oC for 2 to 4 hours. A drop of the suspension was placed on a clean microscope slide. A clean cover glass was placed over the suspension and then examined with a microscope using the low power objective. High power objective was used to confirm the presence or absence of germ tubes. Controls were read and results recorded.
3.3.2 Determination of physicochemical characteristics of the various breeding habitats