Analysis of Bacterial Identification - Assignment Example

Analysis of Bacterial Identification CONCLUSIONS & DISCUSSION After identifying your unknown as closely as possible and with reference to Bergy’s Manual FULLY discuss your findings, by answering the questions on the following page. 1. Summarise each of the tests or assays used in the identification process, breaking them down according to a) Primary and b) Secondary tests. The basis of bacterial identification is rooted in taxonomy. Taxonomy is concerned with cataloging bacterial genus and species. Genus is a group of relates species and species is a group of related strains. Schemes for bacterial identification include bacterial physiology. The basis lies in the fact that not all characteristics will be present in all bacteria. Different bacteria will have different physiology. Physiological methods of speciation of bacteria include morphological and metabolic characteristics, the former is called primary tests and the latter is termed as secondary tests. In the present exercise, the primary tests used after bacterial culture were the pigment production by the given organisms followed by microscopic examination for the cellular shape, size, arrangements and motility. The given organisms produced yellow pigments and were non-motile, spherical in shape and were arranged in clusters of four to six. Next, Gram staining was done to see if the organisms were gram positive or negative. The given organisms were gram positive as indicated by their purple coloration. The secondary tests done were to see if the organisms were aerobic, i.e. if they required oxygen for survival and growth. Also several nutrients were checked to see if the bacteria could substitute the normally used glucose for nutrition with any other saccharide. These were lactose, sucrose, and citrate utilization tests. Possession of the enzyme nitrate reductase by the bacteria was tested by performing the nitrate reductase test. In this test nitrates are reduced to nitrites which react with reagent A and B to give red dye. The ability of the bacteria to synthesize enzymes to fight oxidative end products (mostly lethal super oxide radicals), namely catalase and oxidase, were also done. Enteric bacteria usually produce stable acid and turn methyl red to yellow. Methyl red test was done to see the capability of the organism to convert saccharides into acids. This acid is the end product of glucose metabolism and was tested using the oxidation/fermentation tests. The enzyme gelatinase, a protein digesting enzyme, if produced by the bacteria can liquefy solid gelatin. This test turned out to be negative (Washington, W. 2006) 2. Which a) primary and b) secondary test characteristics were the most significant for correct identification of your organism? The most important primary test was the identification of the colony color imparted by the pigments produced by the given bacteria. Not many bacterial species produce pigments and if they do, they have different colors. The organism in this exercise produced yellow pigments and hinted that they could be from the luteus species; (luteus means yellow in Latin). In fact this characteristic alone was enough in the present study to identify the bacteria, because the other two pigment producing bacteria synthesized red and green pigments, quite different from yellow. The most important secondary test proved to be the optimal growth of the organism at 30 degrees celcius instead of 37 degrees on nutrient agar. This test not only showed that the organisms thrive at otherwise sub-optimal temperature of 30 degrees, but also that they are aerobic too; because they grow and divide in presence of oxygen in the incubator. The test ruled out S. marcesens, the only other bacteria thriving at 30 degrees, since the latter are gram negative single rods against the given organisms which are gram positive spherical cells. 3. Did all the observed characteristics agree with those in Bergey’s Manual for your unknown bacterium? Discuss any that did not and give possible explanations that might account for this: No, all the observed characteristics did not agree with those in Bergey’s Manual for the given unknown bacteria. The oxidation/fermentation test result did not agree with that mentioned in the Bergey’s Manual. The colors in both tubes should have had changed to yellow from greenish-blue but instead remained unchanged indicating a negative oxidation/fermentation tests. The Bergey’s Manual mentions this test positive for M. Luteus, while I got it negative. Sugar digesting bacteria degrade glucose either fermentatively or oxidatively, i.e. either not using or using oxygen respectively. The end products of fermentation and oxidation are mixed acids that can be detected by routine fermentation/oxidation test medium. In the present exercise, the bacteria failed to give this test positive because the bacteria might be producing extremely weak acids from the oxidative degradation of glucose. The second reason could be that more sensitive oxidation-fermentation medium would have been required for their detection, sensitive enough to detect weaker and smaller amount of acids. The third reason could be that the mixed acids produced by the given bacteria were further broken down to more neutral products like CO2, water or O2. The fourth reason could be that the given species of bacteria is a slower-growing species and needs an incubation period of 3 days or longer (Hugh, 1953). 4. Recommend some NON-CULTURE based tests that could be carried out to further confirm the identity of your or any unknown microorganism. Taxonomy, nowadays generally uses molecular biology (genetic) approaches for bacterial identification. Included in such approaches are real time polymerase chain reaction (RT-PCR) and micro array. RT-PCR relies on primer sequences designed to facilitate bacterial identification, while micro array uses species-specific oligonucleotide probes designed for specific regions of bacterial genes. Mass spectroscopy can also be used for species and strain identification (Premier Biosoft International) RT-PCR utilizes a pair of primers, which are complementary to a defined sequence on each of the two strands of the cDNA of the bacteria. cDNA is made from the DNA. These primers are then extended by a DNA polymerase and a copy of the strand is made after each cycle, leading to logarithmic amplification. In short, after useful selection of primer, if one gets several copies of DNA, then the bacteria has been identified accurately. This method is not so expensive and is fast. Tuberculosis in several countries is being identified by this process now. In microarray, short oligonucleotides, called probes, are immobilised on a cassette and short sections of the bacterial gene sample is hybridized with these probe. If one gets the hybridization, then the bactewria has been identifed accurately tracing back to the probe. This method is very expensive and cannot be used for general purposes. Mass spectroscopy with or without gas chromatography can be used to identify bacterial proteins or other metabolites, which can then be traced back to a species. For example muramic acid, found in the cell walls of most bacteria, is readily detected in the spinal fluids of patients with pneumococcal meningitis (15) or synovial fluids from septic arthritis (Fox, 2006) 5. Fully discuss, with examples of relevant situations, the significance of accurately identifying bacteria: Accurate identification of bacteria is not only important in medicine, but is equally important for industry and environment. If the Gram staining alone is done correctly, it can provide valuable data, since it can provide elucidative clues about the source of microbial contamination in a company or a hospital or a laboratory. For example, if microbial identification of isolates reveals Gram-positive cocci, the source of contamination could be derived from humans. If microbial identification of isolates reveals Gram-positive rods, the source of contamination could be derived from dust or strains resistant to disinfectants, like wise Gram-negative rods points the contaminant to be water or moisture (Pelczar 1993). Accurate identification of bacteria is of utmost important in medicine. A clinician should have accurate bacterial identification to render an immediate presumptive diagnosis based on which he/she can make more rational decision about antimicrobial therapy. For example, both Salmonella paratyphi A and S. paratyphi B causes gastroenteritis, but the former also causes septicemia, while the latter also causes paratyphoid fever. Now if the microbiologist gets a sample to confirm the species of Salmonella, she first does the glucose test. Both produce acid and gas. She then does the Lactose test. Both are not fermented. She then does the mannitol test. Both are fermented. She then does the xylose test. One produces H2S while the other does not (Pelczar 1993). What if she, for some reasons, does not notice the gas and misinterprets her results! In this case the clinician needs accurate species to prescribe the medicine; otherwise it could have drastic effects on the prognosis of the patient. Certain bacterial diseases like bronchopneumonia, meningitis, and urinary tract infection require accurate identification and a ‘good guess’ is not to be made. It can be fatal. Another reason for accurate identification is to avoid the use of broad spectrum antibiotics by the doctors where laboratory facilities are not available. A clinician may prescribe combination of 3 to 5 antibiotics and wait for the response. The problem though is that this use of unnecessary antibiotics is creating drug resistance amongst the organisms and this is making general public more sick instead of improving their health. Pharmaceutical companies require clean rooms for production and packaging their products. From quality assurance point of view, they need to identify bacteria from final product if any, they have to demonstrate that certain specific bacteria is absent in sterile product or water, they have to confirm certain test organisms in validation process. When it comes to quality control of fermentation stocks in biotechnology companies, there is little chance for errors. Accurate bacterial identification is expected by Food and Drug Administration (FDA). This helps the authority to determine a certain flora for a specific site as normal or abnormal and accordingly evaluate the effectiveness of cleaning or to troubleshoot any source of contamination in case of epidemics. A bacterial species normal in downtown Miami may not be so normal in Arizona. References used: 1. Fox, A. (2006). Mass spectroscopy for species or strain identification after culture or without culture: past, present, and future. Journal of Clinical Microbiology, 44(8), 2677-2680 2. Hugh, R. and Leifson, E. (1953). The taxonomic significance of fermentative versus oxidative metabolism of the carbohydrates by various gram-negative bacilli. Journal of Bacteriology, 66, 24-26. 3. Pelczar, M., Chan E., Krieg,N. (1993). Microbiology Concepts and applications. New York. McGraw-Hill, Inc. 4. Premier Biosoft International. Identifying and distinguishing bacterial strains using real time PCR and micro arrays. Retrieved from http://www.premierbiosoft.com/tech_notes/bac-id.html 5. Winn, W., Allen, S., Janda, W., Koneman, E., Procop, G., Schreckenberger, P., Woods, G. (2006). Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. Philadelphia. Lippincott Williams & Wilkins. Read More