Biochemical identification of unknown bacteria - Lab Report Example

LAB REPORT Biochemical identification of unknown bacteria Unknown ID 001 Nerjes Alsalman 3/18/09 Biochemical identification of unknown bacteria RESULTS: Morphological characteristics: Nutrient Agar Slant: The tube in 37oC: The amount of growth was slight, white in color, and had effuse form of growth. The tube in 25oC: The amount of growth was slight, white in color, and had Echinulate form of growth. Nutrient Agar plate: The colony characteristics on the plate looked like convex and round. Nutrient Broth: The surface and subsurface growth was flocculent, and the amount of sediments was granular. Fluid thioglycollate medium: The bacterium was facultative. The growth of bacteria was almost everywhere which means that the organism can grow aerobically or anaerobically depending on the culture conditions. Nutrient gelatin: Gelatin was found to be liquefied after incubation indicating gelatinase activity Physiological characteristics (fermentation and oxidative tests): Standard results CONTROLS O/F Durham Citrate Mixed acid V-P Oxidase Catalase NO3 NG FTM   A/An Dex Man Lac                 Pseudomonas aeruginosa +/- -/- -/- -/- + - - + + - + A Escherichia coli +/+ +/+ +/+ +/+ - + - - + + - F Enterobacter aerogenes +/+ +/+ +/+ +/+ + - + - + + - F Staphylococcus aureus -/- +/- +/- +/- - + - - + +/- + F - Results obtained for unknown 1 Sample O/F Durham Citrate Mixed acid V-P Oxidase Catalase NO3 NG FTM  001 A/An Dex Man Lac                 #001 +/+ +/- +/+ +/- - + + - + + + F Discussions Experiments were performed to identify and characterize given unknown bacterial culture. Different biochemical analyses were carried out to distinguish the closely related bacterial species. Following are the results for each experiment. 1) Nutrient agar slant: Given culture was streak on the nutrient agar slant and incubated at two different temperature 25°C and 37°C respectively. After 24h of incubation, slants were taken out from incubator and growth was observed. Growth on tube incubated at 37°C was found to be effuse and the growth was extensive while in case of tube incubated at 25°C growth was found to be lesser compared to previous tube and echinulated in nature. There was no pigmentation observed and growth was found to be whitish in color and translucent in nature. Results indicated that given unknown bacteria is either Escherichia coli, Enterobacter aerogenes or Staphylococcus aureus ,being human /animal inhabitant The optimum temperature for growth of these bacteria is 37°C (i.e. human body temperature) while in case of Pseudomonas aerogenosa being free living as well as human inhabitant, it can grow at 25°C as well as 37°C . Similarly it also produces bluish green pigment and grows as mucoid colony. 2) Nutrient agar: Given culture was streak on to the nutrient agar plate using quadrant streak technique and incubated at 37°C for 24 h. After incubation colonies were found to be convex, circular and having entire margin. Optically colonies were found to be translucent and without any pigmentation. This observation again ruled out the possibility of Pseudomonas aeroginosa. Similarly colony of Staphylococcus aureus is opaque, slightly elevated, appears yellowish white. While translucent, convex, round and glossy appearance of colony indicates presence of E.coli having fundamental characteristic of this kind, but needs to be investigated further for further confirmation. 3) Nutrient broth: To investigate growth pattern of given culture in liquid media, nutrient broth was inoculated and incubated at 37°C for 24h. After incubation growth was observed visually. Flocculent growth was observed having cloudy appearance while shaking. Sediments were found to be granular. 4) Fluid thioglycollate medium: To investigate oxygen requirement of given unknown organism thioglycollate medium was used. Thioglycollate and cystine (major constituent of media) provides reducing environment and higher viscosity restricts oxygen penetration in to the tube. Thus, the growth of organism can be monitored at different level as per their oxygen requirement. Here growth was observed at intermittent level and at top of the tube indicating presence of aerobic organism can also grow at lower oxygen concentration (facultatative anaerobes). E. coli and Staphylococcus aurous are aerobic in nature but can grow at lower oxygen concentration and thus possibilities of ether of them are high. 5) Nutrient gelatin: For detection of gelatinase activity, culture was grown on to gelatin agar plate. After incubation liquefaction of gelatin was observed indicating gelatinase producing organism. 6) O/F Glucose media: O/F glucose media is similar to glucose fermentative broth but having more concentration of glucose. This is mainly used to detect utilization of glucose by given organism in two different way 1) fermentative utilization and 2) respirator utilization. In case of respiratory utilization one can visualize acid production and thus color change of indicator dye at top of the tube while in case of fermentative utilization, there is excessive acid production leading to complete color change through out the media. After incubation complete color change was observed indicating glucose fermentation. Out of three predictive organisms E. coli and Enterobacter aerogenes are know to be glucose fermentor and thus eliminates possibility of Staphylococcus aureus. Now there are two possibilities for given organism 1) E coli and 2) Enterobacter aerogenes and both belonging to coliform group of organisms and to differentiate between them few more experiments were carried out. 7) Sugar fermentation with Duraham tubes: For further investigation to differentiate two organisms, sugar fermentation with gas production was monitored using duraham tubes. Three sugars were taken namely lactose, dextrose and manitol. All three sugars was found to be fermented with gas production reconfirms the presence of ether E. coli or Enterobacter aerogenes as Staphylococcus aureus fermented all these sugar without gas production. 8) MR-VP test (methyl red voges Proskaur Test): This test is mainly performed to distinguish two near by colifom bacteria and in this case E.coli and Enterobacter aerogens. Here, glucose fermentation and production of acetoin were monitored in two tubes parallel incubated. After incubation for 48 h it was observed that test for mixed acid production was positive while VP test was negative clearly indicating the presence of E.coli and eliminated possibility of Enterobacter aerogenes since, E.coli ferments glucose and produces the acid while Enterobacter aerogenes does not produce acid but ferments the glucose via production of butanol which subsequently is converted to butanidiol and thus forms acetoin via reaction with Berrites’s reagent. 9) Citrate test: To further validate and confirm the presence of E. coli citrate utilization test was performed. Simino citrate agar was used to monitored citrate utilization and in positive case color of tube become deep blue due to generation of ammonia from ammonium salt. Rise in pH also leads to color change of pH indicator bromothymol blue. The test was found to be negative confirming the presence of E coli. 10) Nitrate utilization, Catalse and Oxidase test: Final confirmatory test for E. coli were performed. These include nitrate utilization, catalse and oxidase. Results were found to be Negative for nitrate utilization and oxidase while it was found to be positive for catalase, confirming the presence of E coli in given unknown sample. Conclusion: Identification of given sample was carried out using different morphological and biochemical investigations. Standard chart was used for various tests for comparison of results. Nutrient slant and nutrient agar plate used for colony morphology studies where organism found to be grown as white, translusion, convex, and without any pigmentation. These results have indicated presence of E coli or Enterobacter aerogenes. Subsequent investigations with sugar fermentation experiments have further validated the previous observations. Here organism was found to be fermentative and production of gas and acid was observed in O/F glucose media and sugar fermentation with durham tubes experiments. Both this experiments have eliminated possibility of Pseudomonas aeruginosa (sugar fermentation negative) and Staphylococcus aureus (gas production negative). Differentiation between of E coli and Enterobacter aerogenes was carried out using mixed acid production and VP test where mixed acid test was found to be positive and VP test was negative clearly confirming the presence of E.coli( positive for mixed acid and negative for VP) and also eliminating the possibility of Enterobacter aerogenes (Mixed acid negative and VP positive).The confirmative test for E.coli was carried out using Citrate utilization, Nitrate utilization, Catalase activity and Oxidase activity. The results for these tests were also found to be in accordance with standard E. coli positive test, confirming the unknown sample as E. coli. References: 1) Microbiology – Pelzar, Reid and Chan. Tata McGrawHill Publishing company Limited, 1996. 2) Bergeys manual of determinative bacteriology- David Hendricks Bergey, John G. Holt, Lippincott Williams & Wilkins, 1994 3) Prescotts Principles of Microbiology –Joanney wille, Chris wolverton, and Linda Sherwood. McGraw-Hill Higher Education, 2008 Read More