Identification of the Bacteria Pseudomonas Aeruginosa - Lab Report Example

ABSRACT: The bacteria that are present in the given sample must be identified for many reasons. There are several traditional and modern methods thatwe can use to identify, characterize the bacteria organisms. They are physical characteristics, biochemical tests, serological tests, Nucleic acid analysis methods, staining techniques fro the identification. The bacterial physiology, Gram’s staining method, Microscopic examination, colony morphology, cellular morphology; biochemical testing and DNA sequencing are the methods that will help us to identify the micro organisms that are present in the given sample. The micro organisms are ubiquitous in nature. The presence of some of these micro organisms in the food and in human body will lead to health effects. Hence the identification and characterization of these micro organisms is of vital importance. These micro organisms can be identified based on some special characteristics exhibited by them. The biochemical testing method is designed to identify the ability of the micro organisms to produce some specific metabolites. Most of the micro organisms invade the respiratory tract, genitourinary tract and gastrointestinal tract. Here the above mentioned methods were used to identify the given unknown micro organism. The unknown micro organism was found to be Pseudomonas aeruginosa. INTRODUCTION: The identification of the bacteria in the given sample can be done by gram’s staining, biochemical and physiological tests. The colony morphology is the first step in the identification process. The size, shape and the gram’s reaction will give us an idea about the bacteria. Based on the Gram’s stain, we can classify the bacteria as gram positive or gram negative. After the classification the biochemical tests are done. The basic biochemical tests include: Indole production test, Methyl Red test, Voges Prosker Test, Citrate test, Catalase test, oxidase test, Urease test, Triple sugar test, Motility staining test and Morphology identification. These tests will give us, the idea of what type of bacteria it is. (Todar 2008). The identification of the physiological characteristics by anaerobic test and motility test will also give us better idea regarding the nature of the bacterium. Then using the Bergey’s Manual of systematic Bacteriology Volume 1, we can identify the bacterium. (Shimeld and Rodgers 1999), (Harley 2004). AIM: To characterize and identify the given bacterium by studied staining and biochemical test methods. MATERIALS AND METHOD: Bacterial Characterization and Identification: 1. Colony Morphology: Colony morphology is an important tool in the identification of the bacterial cultures. The bacteria are classified based on their size, forms, cellular morphologies and Gram reactions. The colony morphology is the same for a given micro organisms and 2. Cellular morphology by Gram staining: The bacterial cells are translucent in nature and hence staining of the cells is required for identifying the gram positive and those that take the color of the counter stain are called gram negative. Here the primary dye is crystal violet and the secondary dye is safranin O or basic fuchsin. 3. Biochemical tests: a. Catalase test: This test is used to identify the micro organisms which produce the enzyme catalase. The catalase enzyme converts the hydrogen peroxide into water and oxygen. If the catalase enzyme is produced by the bacteria, when a drop of hydrogen peroxide is added to the medium, the bubbles are found to have formed indicating the positive reaction. (Brown 2010). b. Oxidase test: The oxidase test is used to differentiate the pseudomonadacease and Enterobacteriaceae families of bacteria based on the production of the enzyme cytochrome oxidase. This enzyme transfer the electrons from the donor molecule to oxygen, this oxygen evolved turn blue or purple within 15 seconds. This is a positive test. c. Antibiotic Sensitivity test: This test aims to find the susceptibility of the bacteria for a variety of antibiotics. The antibiotics containing discs were placed on the medium containing the P.aeruginosa and the antibiotic sensitivity tests were performed. (Brown 2010). 4. Physiological Characteristics: a. Anaerobic growth: The bacterium is made to grow under anaerobic condition. b. Motility test: This test is used to determine the ability of the bacteria to determine the migration level from the inoculation site. The ability of the bacteria to move from the site of inoculation is found out by the appearance of cloudiness in the media. (Harley 2004). c. Salinity test: Some bacteria depend upon the amount of salinity present in the water for growth. More the salinity, the lesser is the growth. The unknown sample is dropped in the medium containing the increased salt concentration. d. Multiple test system: This is used to conduct five biochemical tests in a single strip. Based on the results obtained we can easily identify the bacteria RESULTS AND DISCUSSION: From the colony morphology studies, it was found that the bacterium is a rod shaped bacteria. Some were also found as single colonies and some as chains. Thus it belongs to the rod shaped bacterium. When looked into the cellular morphology, the cells were translucent in nature and it required Gram’s staining. The result was Gram negative, pink color cells were found on the microscopic view. Thus it was confirmed that they are rod shaped gram negative bacteria belonging to the class of Gamma proteobacteria. Then further tests were conducted to check for the family, whether it belongs to the Enterobacteriaceae or Pseudomonaceae. (Bergey and Holt 1994) .After that, the biochemical tests were done to identify the bacteria. The bacteria showed the results as follows: Table 1: Gram’s Staining test. Name of the Test Result Gram’s Staining Negative The table 1 shows the result of the gram’s staining. The bacteria were found to be gram negative. Table 2: Biochemical test. Name of the Test Result Catalase test Positive Oxidase test Positive The table 2 shows the results of the biochemical tests. The bacterium was found to be positive for the catalase test and oxidase test. Table 3: Physiological Test. Name of the Test Result Anaerobic test No Growth Motility test Positive From the table 4, the bacterium was found to be anaerobic and have motility in the medium. This was confirmed by the appearance of the cloudy nature in the medium. Table 4: Antibiotic sensitivity test Name of the Test Result Zone of growth Teracyline Positive none Streptomycin Negative 0.9 cm Vancomycine Positive none Penicilline Positive none Choramphenico Positive none Ampicillin Positive none The table 4 gives the results of the antibiotics sensitivity test. It was found from the graph that the bacterium was found to be resistant for a variety of antibiotics except Streptomycin. Picture 1: Standard oxi-ferm Test tube II results column. Picture 2: The oxi-ferm Test tube II results: Biocode : 30303 Based on the oxi-ferm test tube results, the results correspond to the results of the Pseudomonas aeruginosa. This is confirmed by the biocode 22303 which is similar to the one that we received by the experiments. The confirmative tests such as catalase, oxidase, urease, triple sugar tests are as per the BBL Oxi/Ferm Tube II. Thus the given bacteria was identified to be fermentative,oxidase positive, catalase positive, triple sugar negative, urease negative, methyl red and Voges Prosker negative from the results. Graph 1: Effect of salinity concentration on the growth of unknown bacteria: From the indole production test, the presence of red color on the surface of the alcohol layer of the broth indicated that they have the tryptophanase enzyme. The methyl red test tube was yellow in color indicating a negative test. The citrate test was positive as we can find the change of the color from green to blue. It was also found that the unknown bacteria were not able to ferment glucose and lactose and sucrose properly. Thus the triple sugar test was negative. In the catalase test, when hydrogen peroxide was added to the medium, they were broken down into oxygen and bubbles were found on the medium indicating a positive test. The oxidase test was also found to be positive. From the results it was concluded that the unknown bacterium was pseudomonaceae. The bacteria had the motility in the motility test. This indicates that the bacterium has flagella or other motility organs for their movement in the medium during their growth. Similarly as the salinity is increased the bacterium was not able to grow. The growth rate decreased as the salinity increased. Similarly the urease test was also negative. The bacteria were not able to reduce the urea into ammonia. The solution remained in the yellow color indicating the negative reaction. CONCLUSION: When all these results were compared with the Bergey’s Manual of systematic Bacteriology Volume 1, the bacteria were confirmed to be Pseudomonas aeruginosa. P.aeruginosa is a gram negative bacterium having a flagella and motility showing positive test results for catalase, oxidase and negative for urease, methyl red and voges proscker test. The triple sugar iron test containing lactose, sucrose and glucose as the carbon source was also tested which indicated that P.aeruginosa cannot utilize them for the metabolism. Thus the unknown bacterium given was confirmed as Pseudomonas aeruginosa. (Bergey and Holt 1994). Pseudomonas aeruginosa is a gram-negative bacterium belonging to the Gamma Proteobacteria class of bacteria. They are rod shaped bacteria belonging to the family of Pseudomonadaceae. P.aeruginosa when isolated from soil sample or water sample they produce small, rough colony of cells. (Todar 2008).Whereas if isolated from the clinical samples, they form smooth colonies either with fried –egg appearance or with mucoid appearance. The P.aeruginosa produces two types of pigments pyoverdin and pyocyanin. The first pigment is a fluorescent pigment and the latter is a blue pigment, which will be produced at large concentrations if the medium contains low-iron concentration. (Pommervile, 2009).The pyocyanin is found to have the virulence factor with them. (Brown 2010). P.aeruginosa is resistant to most of the micro organisms. They are very sensitive to most of the antibiotics such as fluoroquinolones, gentamicin and imipenem. The main reason for the development of the antibiotic resistance is due to its natural habitat of living in association with the actinomyces, bacillus and molds. References: Pommerville, JC 2009, Alcamo’s Fundamentals of microbiology: body systems, Jones & Bartlett Learning. Bergey, DH and Holt, JG 1994, Bergeys manual of determinative bacteriology, Lippincott Williams & Wilkins Shimeld, LA and Rodgers, AT 1999, Essentials of diagnostic microbiology, Cengage learning. Todar, K 2008, Online textbook of bacteriology, viewed on September 10, 2009 http://www.textbookofbacteriology.net/index.html Brown, AE 2010, Bensons Microbiological Applications: Laboratory Manual in General Microbiology, Mc Graw Hill Co. Harley, JP 2004, Laboratory Exercises in Microbiology, McGraw Hill. Read More