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| Biochemical tests | Summary of the procedures | References |
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| Catalase test | The bacterial isolates were grown on the YEMA medium and mixed with 3% hydrogen peroxide. Appearance of bubbles indicated catalase-positive isolates | [15, 39–42] |
| Oxidase test | The test involved streaking actively growing bacterial cells on a filter paper containing drops of 1% Kovac’s oxidase reagent. Oxidase enzyme-producing isolates changed the blue dye from dark purple to black within 5–10 seconds |
| Methylene blue test | Methylene blue 0.1% (v/v) was added to the YEMA medium. The isolates were then streaked on the medium and incubated at 30°C for 48 hours before analyzing the growth of the isolates |
| Citrate utilization test | Simmon’s citrate agar medium was prepared, and the test isolates were streaked onto it. Then, it was incubated at 30°C for 24 hours to observe the growth. A blue coloration indicated a positive test, while citrate-negative isolates did not change the color of the medium and remained green |
| Urease test | The urea agar medium (Christensen) incorporated with phenol red as pH indicator was inoculated with test organism and incubated at 30°C for 24–48 hrs. Urease enzyme production was observed by the color change of the medium from yellow to pink |
| Gelatinase activity | The fresh cultures were inoculated in test tubes containing 10 ml yeast mannitol broth comprising 2% gelatin powder. Then, test tubes were incubated for 48 hrs at 28 ± °C |
| Then, the test tubes containing culture broth were subjected to low temperature in the refrigerator (4°C) for 30 mins. After incubation at a low temperature, the culture broth was removed and observed for the presence of the gelatinase enzyme. The isolates that produced gelatinase enzyme (positive isolates) remained liquefied, whereas the negative isolates remained solid due to the presence of not degraded gelatin |
| Starch hydrolysis test | The isolates were cultured on yeast mannitol Agar medium supplemented with 0.2% (w/v) starch powder. This medium was inoculated with bacterial isolates and incubated at 30°C for 24 hours. After incubation, 5 ml of iodine solution (0.340 grams iodine and 0.660 grams potassium iodide in 100 mm distilled water) was poured on the plates. The formation of a blue-black color due to the starch iodine complex and the observation of a clear zone around the colonies indicated that the isolates were starch degrading due to the production of amylase enzyme by the cells |
| Triple sugar iron agar test | The fresh isolates were transferred and streaked onto triple sugar iron agar (TSI) slants. After 48 hours of incubation, the colors of the butt and slant were analyzed for each isolate |
| Sugar fermentation test | The sugar fermentation test was performed in peptone water supplemented with phenol red indicator solution (0.01%). Eight different sugars (lactose, glycerol, galactose, maltose, fructose, sorbitol, sucrose, and glucose) with a final concentration of 1% were used in the experiment |
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