The Physical And Microbiological Investigation Of Some Local Beverage Drinks
[A CASE STUDY OF ILORIN METROPOLIS, KWARA STATE]

3.5.1 Purification and maintenance of microbial isolates
The bacteria isolates were transferred onto fresh nutrient agar medium and incubated at 370C for 24 hours. Pure colonies of bacteria were maintained on slopes of nutrient agar (NA) slant and stored in a refrigerator at 80C. They were brought out when needed for studies.  
3.5.2 Characterization and identification of the isolates
Standard inoculums were prepared from the preserved stock culture by taking a loopful of the isolates and aseptically inoculated onto sterile nutrient agar (NA) plates. The plates were incubated at 280C for 24 hours. The characterization of the isolates were performed, by employing Gram staining reaction and other biochemical tests such as: Oxidase test, Catalase, Citrate test, Urease test, Coagulase test, TSI (Triple Sugar Iron agar) test, MIO (Motility Indole Ornithine) test and Methyl Red and Voges Proskauer test as described by Bailey and Scott’s Diagnostic Microbiology 13thdition (2014).

3.5.3 Gram staining technique
The organisms isolated were gram stained and examined using oil immersion objective to differentiate gram positive from gram negative organism. Smears of isolated organisms were made from each representative organism.
Procedure: Smear of the isolated organisms was made from each representative colony and allowed to air dried and then heat fixed gently and laid on staining rack. They were stained with crystal violet for 60 seconds and rinsedwith water and excess water drained off .They were flooded with Gram’s iodine and allowed to stain for 60 seconds and then rinsed with water. The smears were decolourised or differentiated briefly with acetone and washed off with distilled water immediately and finally counterstained with safranine for 1 minute and wash off thoroughly with distilled water.The back of the slides were wiped with damped cotton wool. The stained smears were then allowed to air dried and added 1 drop immersion oil to each slide and examined using x100 objective lens.
Result:Gram positive cocci appears deep purple and round in shapes
Gram negative cocci appear pinkish red in colour but round in shapes.
A gram positive bacillus appears purple and rod like in shapes.
A gram negative bacillus appears pinkish red and rod in shapes.
Gram negative or positive coco-bacilli appears short rod joined in 2 or 3 almost appears   cocci shapes but not.
3.6   Biochemical Tests for Identification of Bacteria Isolate
Catalase Test:
A small amount of cultured colonies was picked from culture using another edge of sterile glass slide, this was inserted into a solution of hydrogen peroxide (H202) on slide and observed for production of gas which indicates a positive reaction but if there is no bubbles of gas it indicates negative reaction, Staphylococcus aureus NCTC 6571 was used as positive control.
Indole Test:Isolate was inoculated in a bottle containing 3ml of sterile peptone water and incubated overnight at 370C. The indole was tested by adding 0.5ml of (Kovac’s reagent)Para-dimethylaminobenzaldehyde with gentle shake. It was observed for 5 minutes. Red ring surface layer indicates indole positive test while no red ring surface layer indicates a negative test, Escherichia coli ATCC 25922 as used as positive control.
OxidaseTest: A piece of filter paper was placed in a clean Petridis and drops of freshly prepared N,N,N,N-Tetra methyl-p-phenylenediamine (oxidase reagent) was added, A piece of glass rod was used to collect the isolate and the isolate was smeared  on  the  filter  paper. It was observed after 10 seconds – deep or dark purple indicates a positive oxidase. No colour change indicates a negative oxidase test, Pseudomonas aeruginosa ATCC 27853 was used as a positive control.
Citrate Test: Simmons’s citrate agar slant was prepared according to manufacturer’s instruction and autoclaved at temperature of 121°C for 15 minutes. Isolates was stabbed and streak on the slope using the wire loop. The tubes were incubated at 30°C for 18-24hours after which is it is observed. A bright blue colour indicates a positive citrate test while a green colour (no colour change) indicates negative citrate test.
Sugar Fermentation Test: Fermentation test was carried out using 3g of the following sugars: glucose, lactose, sucrose Maltose Mannose and fructose in medium which comprises of 1.0% of peptone and 1.0% NaCl respectively.  Three (3) drops of 0.1% of phenol red which serves as indicator were added to the medium and 10mls were discharge into test tube and sterilised at1100C for 15mins. After sterilization, it was inoculated with isolate from subculture and Durham tubes were inverted position in each of the tubes for detection of gas production. Acid production from orange (Alkaline) to yellow (Acid) in the fluid. Gas production was indicated by the presence of air space at the bottom of the inverted Durham tubes the results were recorded.
3.7    Statistical Analysis of Data.
This section presents the data analysis, testing of hypotheses and interpretation of results. All the data collected from the laboratory experiment are presented and interpreted. The analysis was carried out with the use of Statistic Package for Social Science (SPSS). The hypotheses were tested at 0.05 level of significance. The statistical tools used in analyzing the data were Analysis of Variance (ANOVA) and Duncan test.