3.6.1 Presumptive Test: Measured amounts of 5ml single strength, 10ml double strength, MacConkey broth medium were sterilized in 20ml and 10ml bottles containing a Durham tube for indicating gas production. With sterile graduated Pipettes, the following amount of waste water were added. Into each of 5 bottles of 10ml double strength MacConkey broth were inoculated 10 ml of waste water. Into each of 5 bottles of 5ml single strength MacConkey broth were innoculated 1ml of waste water. Into each of 5 bottles of 5ml single strength MacConkey broth were inoculated 0.1ml of wastewater.
These were repeated for the respective wastewater samples. There after, the bottles were incubated at 37oC in a water bath and examined after 24hours for the presence or absence of acid and gas production. Those that showed acid and sufficient gas to fill the concavity of the top of the Durham tubes were considered to be contaminated or
presumptive positive as a result of the growth of Coliform bacilli. Any remaining negative bottles were reincubated for another 24hours for further checking, and if acid and gas develop, they too were regarded as being positive. Again, the negative bottles were subcultured unto fresh Nutrient Agar plates to check if there was other contaminants apart from Coliform bacilli.
3.6.2 Differential Coliform Test (EIJKMAN Test):
Here, Eijkman test is usually employed to ascertain whether the Coliform bacilli detected in the presumptive test were Escherichia coli. This test depended on the ability of E. coli to produce gas and turbidity when growing in Brilliant Green Bile Broth medium pre-warmed at 37oC. Here, all bottles considered presumptive positive were sub-cultured into Brilliant Green Bile Broth medium pre-warmed at 37oC. These were incubated at 44oC in a water bath and examined after 24hours for the presence of gas production and turbidity and for colour change.
For confirmatory test, all bottles showing gas production and turbidity were sub-cultured unto Eiosin Methylene Blue Agar. Peptone water broth were also inoculated and incubated at 44oC in a water bath and examined after 24 hours to check for indole production at 44oC.
For completed test, colonies developing were gram stained and were further inoculated into Indole test medium, methyl red test medium, voges-proskaur test medium, Citrate medium. Also, slopes were made on nutrient agar. All these were examined and the result were recorded respectively.
3.6.3 Identification of the Isolates
The isolates were identified using the following methods-
3.6.3.1 Macroscopic Examination
This was the visual examination of the appearance of the organisms isolated on the eiosin methylene blue agar media and nutrient agar media respectively. Such examination included colony morphology, size, colour, texture, whether smooth or rough surface, flat or raised and other features.
3.6.3.2 Gram Staining:
Gram staining method was most commonly used in microbiology since it differentiates bacteria into two classes- the gram positive organisms that stained dark purple and gram negative organisms that stained pale to dark red. To identify the cell type, and features of the isolates, the gram stain was therefore performed on each isolate.
Method
With a sterile wire loop, the isolates were smeared on a clean grease-free slide and fixed with mild heat, the slides were stained with 0.5% crystal violet solution for 1 minute. They were washed with water and flooded with Lugols lodine for 1minute followed by washing with water again. Each sample slide was rinsed or decolourised with acetone-alcohol solution. These were then washed with water. The slides were counter stained with neutral red for 2 minutes and washed with water. The back of the slides were wiped clean and were placed in a draining rack for the smears to air dry.
Each smear was examined microscopically using oil immersion, that is x 90 or x 100 objective.
Gram positive isolates stained dark purple, blue or violet while gram negative isolates stained pale to dark red.
3.6.3.3 Indole at 44oC
Using a sterile straight wire, 5ml of sterile Motility Indole Urea (MIU) medium was inoculated with a smooth colony of the test organisms. An indole paper strip was placed in the neck of the Motility Indole Urea tube above the medium and the stopper.
This was incubated at 37oC-44oC over night and examined for indole production by looking for a reddening of the lower part of the strip.
Reddening of strip showed positive test (indole was produced). No red colour showed negative test (No indole was produced).
3.6.3.4 Methyl Red Test:
A colony of the test organisms was inoculated in 0.5ml of sterile Glucose Phosphate Broth. After overnight incubation at 35-37oC, a drop of methyl red solution was added.
A positive methyl red test was shown by the appearance of a bright red colour, indicating acidity while a yellow or orange colour showed a negative test.
3.6.3.5 Voges-Praskaur test:
2ml of sterile glucose phosphate peptone water was inoculated with the isolates and incubated at 35-37oC for 48hours. A very small amount of creatine was added and mixed thoroughly. 3ml of sodium hydroxide reagent was added and shaken. The bottle cap was removed and this was left for 1 hour at room temperature. Pink-red colour showed positive test. No pink-red colour showed negative test.
3.6.3.6 Citrate Utilization Test:
Using a sterile straight wire, 3-4ml of sterile koser’s citrate medium was inoculated with a culture of the isolates. The inoculated broth was incubated at 35-37oC for up to 4 days, checking daily for growth.
Turbidity and blue colour showed positive test (Citrate was utilized). No growth showed negative test, (Citrate was not utilized).
3.6.3.7 Catalase Test:
A small portion of the colony was placed under test in a drop of hydrogen peroxide on a clean grease-free slide. Active bubbling or effervescence at the drop of hydrogen peroxode indicated positive test (Catalase was produced). No release of bubbles showed negative test (Catalase was not produced).
3.7 Determination of Organic Pollutants Using Gas Chromatrography-Mass