THE ROLE OF GOVERNMENT IN SOLVING HOUSING PROBLEMS IN ANAMBRA STATE

11 CHAPTER THREE MATERIALS AND METHOD 3.1 Materials The materials used are; sample containers, spatula, cotton wool, masking tape, Nutrient Agar, Potatoes Dextrose Agar (PDA) measuring cylinder, bottle wate rs, weighing balance, autoclave, incubator, petri dish, syringe, Pasteur pipettes, Bunsen burner, conical flash, test tubes, microscope slides, beakers, microscope, wire loop, distilled water. 3.2 Study Area Calabar is a major city in Cross River State in Southern Nigeria as well as being the state headquarters with a population ranging between 466,800 and 1 million, it is located at about 32m (105pt) above sea level, density of 910/km 2 (2,400/sq m), area of 406 square diameters (157/sq m) with coordinates of 4 O 57’ 0" N and 8 O 19’ 30" E. Calabar features a tropical monsoon climate with a lengthy wet season spamming ten months and a short - day season covering the remaining two months. The harmattan, which significantly influences weather in West Africa, is noticeably less pronounced in the city. Temperature are relatively constant throughout the year, with average temperatures of about 25 to 28 degree Celsius. The city economy is based on tourism as it described as the tourism capital of Nigeria. 3.3 Collection of Samples One bottles each of ten different brands available at various retail outlets at these sites were randomly purchased using a simple random sampling technique. The selected bottles before purchase were inspected for good condition of the cap and the protective seal. The brand name, dates of manufacture and expiry, batch number, NAFDAC registration number, the process of water purification employed, and place of manufacture we re documented. Samples were transported in good condition to the Microbiology Laboratory in Cross River University of Technology for microbial analysis. All samples were stored at 4 O C before analysis. 3.4 Culture Media The culture media used include nutrie nt agar to determine the total viable bacterial count, Potatoes Dextrose Agar. Culture media were all prepared according to the respective manufacturers specification and sterilized in an autoclaved at 121 O C at 15 psi for 15 minutes. 3.5 Sample Preparation Ten - fold serial dilution was carried out for each of the sample (10 samples), 9ml of distilled water was measured into each of the 10 test tubes of the first samples. Then 1ml of the bottle water was 12 measured using a sterile syringe and added to the first test tube of the first product, 1ml was measured again from the first test tube into the next test tube and thus, to the others. This dilution procedure was carried out for all the test tubes containing the water samples, the diluted sample were labelled accordingly for easy identification. 3.6 Enumeration and isolation of bacteria A 1ml aliquot of the 9 th and 10 th sample test tube water was inoculated aseptically on each of the sterilized agar using pour - plate method. The plates were incubated for 24hours at 37 O C, after which visible colonies were counted and expressed in CFU/mL district colonies were selected and purified by repeated streaking on nutrient agar plates. Pure cultures were preserved on agar slant at 5 O C. 3.7 Characterization and identificati on of isolates Morphological characterization The colony of the pure culture of each bacterial isolate was observes for morphological features using Berger’s manual of determinative bacteriology, cell safe was determined under x100 objective of the light m icroscope and biochemical test. 3.7.1 Identification of indicator organisms An overnight Nutrient agar culture of each isolate from each water sample was taken through Gram’s stain and biochemical tests for the identification of each of the organisms. The biochemical tests carried out on the Gram - negative organisms included MRVP, citrate utilization, indole, growth in Triple Sugar Iron agar, and catalase. The growth characteristic of the Gram - positive cocci on Mannitol Salt agar was observed and the cocci w ere tested for catalase and coagulase production. The Gram - positive rods were observed for sporulation characteristics. Maintenance of strains Distinct colonies of each isolate from the samples were stored in nutrient agar at 4°C and cryo - preserved in glyc erol - nutrient broth in a deep freezer. Microbial examination of water samples Total coliform was detected by membrane filtration technique (0.45 μm, Pall Corporation, Washington). 100ml samples were filtered through membrane filters and filters were transferred to petri plates containing M - endo agar with the grid side upward then incubated at 37 O C fo r 24hrs. The colonies on membrane filter were counted. The samples were analyzed for heterotrophic bacteria count by spread plate technique and coliforms by using the standard five - tube most probable number (MPN) method by spreading 0.1ml original water sa mple and 0.1ml of 10 times diluted water sample on plate count agar separately and aseptically. Both plates were incubated at 37 O C for 24hrs all 13 colonies were enumerated and mean was taken and all heterotroph were identified by various biochemical techniqu es. For the detection of fungus, potato dextrose agar (PDA) media was used and was identified by staining with lactophenol cotton blue. Forty - two bacterial isolates were selected randomly for antibiotic sensitivity testing against eight antibiotics using s tandard methods. 3.4.2 Biochemical tests and identification of isolates The following biochemical tests were carried out to determine the characteristics of isolated bacteria. 3.7.1 Gram stain A heat fixed smear of 18 - 24 hours old culture was prepared on a clean microscopic slide. The smear was stained with crystal violet for 30 - 60 second, after which the stain rinsed off with distilled water. Logol’s iodine was added to intensify stain for 60 seconds and rinsed with distilled water. 90% ethanol was added t o the slide as a decolorizer for 10 - 15 seconds and rinsed off under a running tap. The smear was again covered with safranin and allowed to act for 30 seconds, then rinsed again with water. It was air - dry and then observed under X100 oil immersion objectiv e binoculum microscope. 3.7.2 Catalase Test This is to determine if the bacterial can produce catalase enzyme. 3ml of 3% of hydrogen peroxide were transferred into a sterile slide and a colony from the plate were picked with clean tooth pick into the slide immediately discharged of bubbles shows positive while no bubble shows negative. 3.7.3 Oxidase Test This test is performed to determine if the bacteria produce oxidase enzyme using the test oxidase reagent (PL.390) from most diagnostic (Nesto, Wirral, UK) in accordance with the manufactures published protocol. A well - isolated pure colony was placed on a filter paper using a sterile wire loop. A drop of test oxidase reagent (Kovac’s reagent B) was added and mixed. After 10 - 20 seconds, the filter was observe d for a colour change with oxidase positive isolates producing a purple colour being taken as presumptive aeromonas and pseudomonas isolates. Oxidase negative colonies were colorless and were presumptively considered to be Escherichia coli 3.7.4 Indole Tes t Indole test using kovac’s reagent determine whether the microbe produce indole from amino acid tryptophan. A loopful of organism was inoculated in test tube containing the 3ml of peptone water and sealed with cotton wool and foil paper. It was then incu bated for 48 hours at 34 O C after 48 hours of incubation, drops of kovac’s reagent were drafted into each tube using a sterile syringe, shaken gently and observation recorded immediately. Pink coloured ring after addition of appropriate reagent 14 indicates po sitive result while no colour change even after the appropriate reagents indicate negative results. 3.7.5 Citrate utilization This test determines the ability of bacteria to use citrate as a sole carbon source with water. About 15.8g of Simmons citrates agar was weighed on a Mettle Toledo balance and dissolved into distilled water and sterilized at 121 O C for 15 minutes allow to cool to about 45 O C before 3ml was then measured and poured into each test tube using a sterile syringe and allowed to solidify. A sterilized wire loop was used to pick an inoculum from the subculture plate and inoculated on freshly citrate agar by stabbing/streaking, and then was incubated for 24 hours. The test tubes were observed for colour change with citrate positive isolates pr oducing a blue colour while the negative produces a green colour. 3.7.6 Methyl red test This test is performed to determine the organisms that have the ability to carry out mixed acid fermentative when supplied with glucose. The pH indicator methyl red det ects a pH change to the acid range as a result of acidic end - products such as lactic, acetic and formic acid. A loopful of organisms was inoculated in a test tube containing the 3ml of peptone water and was sealed with cotton wool and foil paper. It was th en incubated for 48 hours at 37 O C, after 48 hours of incubation, the tubes were removed from the incubator and 0.5 drops of methyl red reagent were dropped in each of the tubes and observed immediately for colour change. Results were then recorded. 3.7.7 V oges - Proskauer Test This test detects the presence of action ( acetylmethlcarbinol ) and 2,3 - butanediol substance with are formed during the butanediol fermentation of glucose. A 2.72g weight of Glucose broth buffer, was dissolved in 160mls of distilled wate r. The medium was properly dissolved by heating it over the Bunsen flame. Approximately, 4ml of the glucose broth was measured into sterilized test tubes. The tubes were sealed with aluminum foil and sterilized at 121 O C in an autoclave for 15 minutes, then removed and allowed to cool. A sterile wire loop was used to pick the organism from the slant, and it was inoculated into the glucose broth. The tubes were sealed with aluminum foil and inoculated at 37 O C for 48hours after which 0.5ml of alpha - napthol sol ution and 0.5ml of 40% potassium hydroxide solution were sequentially added using a sterile pipette. The tubes were observed for colour changes. 3.7.8 Motility Test Motility test is used to determine if an unknown organism is motile or not. Motile organism s possess locomotive organelles such as cilia, flagella or pseudopodia. A semi - solid medium is used to carry out 15 this test. Approximately, 6.7g of nutrient agar was weighed and dissolved in 240mls of distilled water and it was flamed over the Bunsen burner (i.e., for proper dissolution). 6ml of the agar was poured into 40 test tubes. Then, the tubes were sealed with aluminum foil paper, and the agar was sterilized in an autoclave. After 15 minutes of steriliz ationa wire loop was used to pick the organism fr om the slant and inoculated in the tube, making a single stab down the center of the tube to about half the depth of the medium. The tubes were then sealed again with aluminum foil and incubated at 37 O C. after incubation, the tubes were examined at interva ls, e.g., after 6 hours and 1 and 2 days. Non - motile bacterial generally give growths that are confirmed to the stab line, have sharply defined margins and leave the surrounding medium clearly transparent. Motile bacteria typically give diffused, hazy grow ths that spread through the medium rendering it slightly opaque. 3.7.9 Triple Sugar Iron Test This is a test named for its ability to test microorganism’s ability to ferment sugars (lactose, sucrose and glucose) and to produce hydrogen sulphide. It is ofte n used in the selective differentiation of enteric bacteria. About 14.26g of triple sugar iron agar was dissolved in 240mls of distilled water and heated over the Bunsen flame, 3ml of the agar was measured into each test tubes, and the tubes were sealed wi th aluminum foil paper and sterilized in an autoclave. After sterilization (15 minutes), the tubes were removed and kept in a slant position until the medium solidified. A sterile wire loop was used to pick the organism from the slant and smeared on the su rface of the agar. The wire loop was again sterilized and used to stab the butt gently. The tubes were sealed with aluminum foil paper. The tubes were incubated at 37 O C for 24 hours, and thereafter the slant and butt were observed for colour changes, prese nce of cracks and bubbles evidence for gas production. 16 CHAPTER FOUR RESULT 4.1 Total Heterotrophic bacteria count in CFU/mL for all ten samples s/n Sample code Total heterotrophic bacteria count (x10 9 CFU/mL ) 1 C/W 2.10 x 10 9 2 U/W 4.0 x10 9 3 Sw/W 1.60 x 10 9 4 Mn/w 9.4 x 10 9 5 W/W 5.4 x 10 9 6 L/W TNC 7 E/W 1.27 x 10 9 8 A/W 1.92 x 10 9 9 Nes/W 1.51 x 10 9 10 Sp/W 1.21 x 10 9 Table 4.1 Shows the total heterotrophic bacteria count that ranges from 9.4 x 10 9. Sample Mn/W shows the heterotrophic bacteria count of 9.4 x10 9 andSp/W shows the lowest Heterotrophic bacteria count of 1.21 x 10 9 4.2 Biochemical characteristics of the Isolates A total of 32 isolates were obtained and 6(18.8%) produced Alkaline Slant, 23(71.9%) produced Acid Butt, 9(28.1%) produced Acid Slant while 8(81.3.0%) were Alkaline Butt; H 2 S (Hydrogen Sulfide) were (90.1%) negative while the remaining (9.4%) positive. Gas which produced 21(65.6%) were negative, (34.4%) were positive. Methyl Red 24(75%) were positive, 8(25%) were negative; Indole 32(100%) were negative; Citrate 32(100%) were positive. Oxidase produced 21(65.6%) positive, 11(34.4%) negative; and Catalase produced (96.9%) positive, 1(3.1%) were negative. Gram staining (71.9%) were positive, 9(28.1%) produced negative 4.3 Frequency and percentage occurrence of Bacterial species from different Water Samples The frequency and percentages of occurrences of the isolates for different bottled water are presented in t able 3 belo w, CRUTECH water from the result obtained had t hree isolates (3) of Bacillus spp with the percentage of 9.37%. Unical water had four (4) isolates of which three (3) are Staphylococcus spp having the percentage of 9.37% and one (1) Bacillus spp with percentage of 3.13%. Swam water had 17 four (4) isolates, two of Pseudomonas spp having the percentage value of 6.25%, (1) Staphylococcus spp value of 3.12% and (1) Bacillus spp with the percentage of 3.125%. Also, five (5) isolates were obtained from Mangero water of which Bacillus spp was three (3) being 9.38%, t wo (2) isolates from Pseudomonas spp with the percentage of 6.25%. Wynny water had (4) isolates in which Staphylococcus spp was ( 1 ) with the percentage of 3.12% and B acillu s spp was 3 isolates with the percentage of 9.37%. Lasien Water had 4 isolates in which Aeromonas spp was two (2) with the percentage of 6.25%, Pseudomonas spp with 1 isolate with the percentage value of 3.13% and Bacillus spp having ( 1 ) isolate with the percentage of 3.13%. Eva Water had two (2) isolates (1) Staphylococcus spp with 3.13% and (1) Bacillus spp with 3.13% respectively. Two isolates were obtained from Aquafina water in which (1) was Aeromonas spp in the percentage of 3.13% a nd (1) Bacillus spp in the percentage of 3.13%. Nestle water had two (2) isolates which is Staphylococcus spp with the percentage of 6.25%. The result of the isolate obtain from Spring water shows two isolates which (1) was Pseudomonas spp with 3.13% and B acillus spp with 3.13% respectively ( ref table 2 ). Total frequency and occurrence of different bacterial isolates in the sample Bacillus spp had the highest frequency of occurrence of 46.9 % followed by Staphylococcus spp which is 2 5.0 %, Pseudomonas spp being 21.9 %, and Aeromonas spp which had 9.37% as presented in the table below ( ref table 3 ) 18 T ABLE 1: CULTURAL, MORPHOLOGICAL AND BIOCHEMICAL CHARACTERISTICS OF ISOLATES Isolates code Media Edges Elevation Colour Texture Gram staining Catalase Oxidase Citrate Indole Methyl red Slant TSI Butt TSI Voges Proskauer H 2 S Gas Motility Form Suspected Organism C/W1 N/A Entire Raised Creamy Rough + + + + - + A A - - - + Short rod Bacillus spp C/W2 N/A Entire Raised Creamy Rough + + + + - + A A - - - + Shot rod Bacillus spp C/W3 N/A Entire Raised Creamy Rough + + + + - + A A - - - + Shot rod Bacillus spp U/W1 N/A Round Pulvinate Milky Smooth + + - + - + A A + - - - Cocci Staphylococcus spp U/W2 N/A Round Pulvinate Milky Smooth + + - + - + A A + - - - Cocci Staphylococcus spp U/W3 N/A Round Pulvinate Milky Smooth + + - + - + A A + - - - Cocci Staphylococcus spp U/W4 N/A Entire Flat Creamy Dry + + + + - - A A - - + + Shot rod Bacillus spp Sw/W1 N/A Round Pulvinate Milky Smooth + + - + - + A A + - - - Cocci Staphylococcus spp SW/W2 N/A Rough Flat Creamy Dry + + + + - + A A - - - + Short rod Bacillus spp Sw/W3 N/A Curled Raised Milky Smooth - + + + - - K K - - + - Long rod Pseudomonas spp Sw/W4 N/A Curled Raised Milky Smooth - + + + - - K K - - + - Long rod Pseudomonas spp Mn/W1 N/A Entire pulvinate Creamy Smooth + + + + - + A A - - - + Shot rod Bacillus spp Mn/W2 N/A Entire Raised Creamy Moist + + + + - + A A - - - + Shot rod Bacillus spp Mn/W3 N/A Curled Raised Milky Smooth - + + + - - K K - - + - Long rod Pseudomonas spp Mn/W4 N/A Entire Raised Creamy moist + + + + - - A A - - + + Shot rod Bacillus spp Mn/W5 N/A Curled Raised Milky Smooth - + + + - - K K - - + - Long rod Pseudomonas spp W/W1 N/A Curled Raised Creamy Dry + + + + - + A A - - - + Shot rod Bacillus spp W/W2 N/A Round Pulvinate Milky Smooth + + - + - + A A + - - - Cocci Staphylococcus spp 19 W/W3 N/A Entire Raised Creamy Rough + + + + - + A A - - - + Shot rod Bacillus spp W/W4 N/A Entire Raised Creamy Rough + + + + - + A A - - - + Short rod Bacillus spp L/W1 N/A Entire Raised Milky Rough - + + + - + A K + + + - Cocco - bacilli Aeromonas spp L/W2 N/A Entire Raised Milky Rough - + + + - + A K + + + - Cocco - bacilli Aeromonas spp L/W3 N/A Entire Raised Creamy Rough + + + + - + A A - - - + Shot rod Bacillus spp L/W4 N/A Curled Raised Milky Smooth - + + + - - K K - - + - Long rod Pseudomonas spp E/W N/A Round Pulvinate Milky Smooth + + + + - + A A + - - - Cocci Staphylococcus spp E/W N/A Entire Raised Creamy Rough + + + + - + A A - - - + Short rod Bacillus spp A/W N/A Entire Raised Milky Rough - + + + - + A K + + + - Cocco - bacilli Aeromonas spp A/W N/A Entire Raised Creamy Rough + + + + - + A A - -