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Identification of Gram Negative unknown bacteria # 123444 labDaud Abdalla TA. Austin 04/07/2010Gram stain of Pseudomonas aeruginosa cellsAn abstract is an introduction to a formal research paper (some call it a thesis). It is a short-summary designed to present your main argument, topic you would like to address, and conclusions drawn from your research.AbstractThe main purpose of this experiment is to identify the given bacteria using comparative analysis of biochemical characteristics. Using aseptic technique the given sample was inoculated in different types of media and incubated for 24 hours and then stored in 4° C. Different types of bacteria produce different enzymes. Some bacteria may secrete same enzymes, so some specific tests were done to narrow down the possibilities. After performing various biochemical tests such as TSIA, H2S, Indole, Motility, MR, VP, Citrate, Gelatin, Urease, and Oxidase tests, the given unknown bacteria #12 was found out to be Pseudomonas anisms are mainly divided into two domains prokaryotes and eukaryotes. Prokaryotes do not have membrane bounded cells, they have simple internal structures, and they are generally smaller than eukaryotic cells. Prokaryotes are further divided into Bacteria and Archaea. Archaea are closely related to Eukarya. The domain bacteria contain an enormous variety of prokaryotes (Madigan et.al 2009). All know pathogenic prokaryotes fall under Bacteria but all bacteria are not pathogenic.Introduction: The opening paragraph of your paper will provide your readers with their initial impressions of your argument, your writing style, and the overall quality of your work. A vague, disorganized, error-filled, off-the-wall, or boring introduction will probably create a negative impression.Background/ IntroductionBacteria are very small, single cell microorganisms that normally found together in millions, and bacteria also is found in every habitat on Earth. They are a few micrometers in length and their morphology ranges from cocci (spherical) to bacilli (rod shaped) to and spirilla (curved walls). Bacteria are so widespread that it is possible only to make the most general statements about their life history and ecology. They may be found on the tops of mountains, the bottom of the deepest oceans, in the guts of animals, in the hot springs, and even in the frozen rocks and ice of Antarctica. It the year of 1676 bacteria were properly identified as microorganisms. Biochemical tests are the more definitive identification that the microbiologists have developed to differentiate even closely related organisms like bacteria, something too small for the naked eye to be seen. To identify the unknown bacterium which was one of the six organisms received, biochemical tests were used to differentiate these closely related organisms. Biochemical test help identify bacteria quickly and more accurately based on specific biochemical characteristics brought about by enzymatic reactions that are part of specific and well understood metabolic pathways. Organisms are mainly divided into two domains prokaryotes and eukaryotes. Prokaryotes do not have membrane bounded cells, they have simple internal structures, and they are generally smaller than eukaryotic cells. Prokaryotes are further divided into Bacteria and Archaea. Archaea are closely related to Eukarya. The domain bacteria contain an enormous variety of prokaryotes (Madigan et.al 2009). All know pathogenic prokaryotes fall under Bacteria but all bacteria are not pathogenic.Bacteria are divided into Gram Positive and Gram Negative considering the peptidoglycan layer. The Gram Positive bacteria have thicker layer of petidoglycan. This thicker peptidoglycan layer plus the interaction of Lipolic acid hold the primary stain strongly. So the gram positive bacteria are resistant to declolorization, but the Gram Negative bacteria are not because of thin peptidoglycan layer. The step in the identification of an unknown bacterial strain is Gramstaining. This procedure provides useful information about the shape and size of the cells, and distinguishes between Gram-Negative and Gram-Positive species. This distinction is essential for the decision as to which criteria should be used in the further identification of the strain (T.Gregersen 1978).The biochemical test that was used to identify the given unknown #12, among six possible unknown bacterias Escherichia coli, Pseudomonas aerogenosa, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter aerogenes , and Salmonella typhimurium were TSIA (Triple Sugar Iron Agar) which indicated a number of metabolic processes about a specific microorganism. The three sugars in TSIA differentiated bacteria on the basis of glucose fermentation, lactose fermentation, sucrose fermentation, and sulfur reduction. MR (Methyl Red) was used to detect organisms capable of performing a mixed acid fermentation and VP (Voges-Proskauer) was used to detect if organism capable of fermenting glucose could quickly convert their acid products to acetoin and 2,3-butanediol or not. SIM test was used for the determination of three bacterial activities: sulfur reduction, indole production, and motility. Citrate test helped see if bacteria could utilize citrate as the sole carbon source or not. Urease test tested for the presence of urease, similarly, oxidase tested for the presence of cytochrome oxidase, and gelatinase test tested for the presence of gelatinase. Materials and Methods/Experimental Procedures: The “Experimental Procedure” section of a formal lab report, also known as “Methods”, is the section where you tell your readers how you performed the experiment. Using your lab manual, handouts, and notes taken during the lab as a guide, describe in paragraph form the experimental procedure you followed. Be sure to include enough detail about the materials and methods you used so that someone else could repeat your experiment based on your description.Materials and Methods/Experimental ProceduresTo start with, the unknown bacteria # 12 broth was cultured in TSA plate (Trypticase Soy Agar) by performing T-streak so as to produce isolated colonies of bacteria from mixed culture. T streak was done by obtaining a sample with a sterile loop and dragging the loop back and forth across the agar surface starting at the edge, making sure that the loop was flamed after each ending and sterilized upon completion. The streaking technique also included incubation of streaked plate in an upturned position for 24 hours in the hot room temperature as it is a favorable condition for the most bacterial growth. Singled colonies were seen after the incubation of 24 hours. Aseptic technique was used every time while the media was inoculated with unknown gram negative bacteria so as to prevent any contamination of the culture, the sterile medium, or the surrounding. The inoculating instruments were sterilized prior to use by incinerating it in the Bunsen burner flame, holding it in an angle with the loop end pointing downward to prevent contamination of the media. Tube was flamed by passing the open end through the burner flame every time they were used to avoid any contaminations (again). Also, protective clothing were worn while handling the microbes which included lab coat, gloves, close-toed shoes, safety goggles, etc. Inoculating loop was mostly used to transfer the living bacteria, but in some of the tests like to see if the organism was motile or not, inoculating needle was used. It is important to cool the loop or the needle for few seconds so we will not kill a lot of bacteria. After completing the tests, the desk was wiped with a disinfectant Lysol as well. As required by the reaction process, chemicals and indicators such as methyl red, oxidase test reagents, kovac’s reagent, ampoule of Barritt’s A and Barritt’s B were used. Every test had its own purpose and own incubation time. Incubation time for some of them was initially none to 7 days for maximum.TSIATSIA is a solid differential media that give three test results: 1) Sugar utilization, 2) gas production and 3) H2S production. It contains the beef extracts, yeast extracts and peptones as a source of carbohydrates and sodium thiosulfate as a source of sulfur. The media is prepared from agar slant with a deep butt that provides both aerobic and anaerobic conditions. Bacteria produce acidic products when they ferment certain carbohydrates. The carbohydrate utilization tests are designed to detect the change in pH which would occur if fermentation of the given carbohydrate occurred. Acids lower the pH of the medium which will cause the pH indicator (phenol red) to turn yellow. The media remains red if the bacteria do not ferment carbohydrate. If gas is produced as a byproduct of fermentation, then bubbles or a splitting appears in the butt or the entire slant is pushed up from the bottom of the tube. If H2S is produced by the organism then a black precipitate is formed. The media was inoculated with the test organism using a heavy inoculum, stabbing the agar butt and then streaking the slant. The incubation time was 6hrs initially up to 24hrs. UreaUrea hydrolysis is a liquid medium used to test for the presence of the urea hydrolyzing enzyme, Urease. The enzyme hydrolyses urea to ammonia and carbon dioxide. It differentiates rapid urease-positive bacteria from slow urease-positive bacteria and Urease-negative bacteria. The media contains urea, peptone, potassium phosphate, glucose and phenol red. Phenol red is an indicator which turns yellow below 8.4 and pink above 8.4. Potassium phosphate is a mild buffer, which can resist alkalization of the medium from peptone metabolism. Color change to shocking pink indicates a positive test, but sometimes the incubation period might take up to eight days to see this result. MR/VPMethyl Red is used to determine if an organism is capable of performing a mixed acid fermentation. Methyl red is an indicator that is red at pH 4.4 and yellow at pH 6.2. A red color indicates a positive result (glucose can be converted into acidic end products) and a yellow color indicates a negative result (glucose is converted into neutral end products). The VP portion is used to determine if glucose can be converted to 2, 3-butanediol. Reagents 1 ampuale of Barritt’s A and B in VP oxidize the acetoin to diacetyl, which react with guanidine nuclei from peptone to produce red color indicating a positive test. No color or copper color indicates a negative test. Thus, MR and VP test was performed by the inoculating the loop. They both had an incubation period of 24 and 48 hours respectively. SIMSulfur Indole Motility (SIM) is a semi-solid media used to conduct three concurrent tests: 1) H2S production, 2) Indole production where Kovak’s reagent is used on inoculated, incubated culture, and 3) motility. The medium is semi solid that consists of casein and animal tissue (as a source of amino acid), iron containing compound, and sulfur in the form of sodium thiosulfate. Sulfur is reduced in the form of hydrogen sulfate gas in anaerobic respiration which combines with iron forming ferric sulfide, giving a black precipitate. Indole production is due to the presence of an enzyme called Tryptophanse (an enzyme that can hydrolyze tryptophan to pyruvate, ammonia and indole). Cloudiness in the medium outside the stab zone indicates motility. The media was inoculated with the test organism by inserting the needle only about two-thirds the depth of the agar. They were incubated for 24 hours in the hot room.Citrate Citrate test is used to identify if an organism is capable of utilizing citrate as a sole carbon source or not. Citrate medium contains sodium citrate as the only source of carbon and ammonium phosphate as the only source of nitrogen. Bromthymol blue dye (green at pH 6.9 and blue at pH 7.6) was used as an indicator in this medium. Thus, bacteria which possess citrate-permease can transport citrate in to the cell and produce pyruvate and show a color change from green to Prussian blue. This test was performed using an inoculating needle and streaking the slants with the test organisms. The result was observed after 48 hours of incubation. Sometimes it might even take a little longer time to see the change in color. Also, at times the change in color might not be evident, but if there is growth in the slant media then the test can be confirmed as a positive citrate test.Oxidase Oxidase test is used to detect the production of the enzyme cytochrome oxidase by Gram-negative bacteria .This test is performed using a chromogenic reducing agent that change or produce color as they become oxidized. A bacterial colony is transferred to a filter paper saturated with reagent and the oxidase test reagent is added directly over the colony. Within seconds, the reagent which act as an artificial electron donor, changes color i.e. turns black or dark blue if oxidized cytochrome is present. No change in color indicates the negative test end result. It is important that the test is read within one minute to ensure accurate results.Gelatinase Gelatinase test help us to determine microbial ability to produce gelatinases. Gelatinases comprise a family of extracellular enzymes produced and secreted by some microorganism to hydrolyze gelatin. This media is used to test if bacteria can digest the protein gelatin. To digest gelatin, the bacteria must make an enzyme called gelatinase. An inoculating needle is used to stab the gelatin. After incubating the inoculated media for at least 24 hrs, the tube is transferred to the refrigerator for an hour to verify the liquefaction is not because of temperature, but because of the Gelatinase formation. The tube should be completely chilled prior to observation. If the media is solid after refrigeration then the test is negative (the bacteria did not digest gelatin), and if the media is liquefied even after refrigeration, then the test result is positive (the bacteria is able to digest gelatin). If the media re-jells then it is either negative or is a slow gelatinase producer. Thus, the incubation time is 24 hours initially and then may have to be incubated up to 8 days because the production of gelatinase can be very slow.The results of experiment is shows the outcomes of your research and experiments. Good research paper includes not only textual explanation, but visual descriptions as well. Be sure to include graphs, raw collected data and statistics too. This is what the results section usually does. To make everything look more interesting, try to illustrate all or, at least, the most important of your findings. The results have to be described in detail, but not explained.ResultsGram Negative Results SheetMediumTSIAH2SIndoleMotilityGlucoseMRVPCitrateUreaseGelatinOxidaseResultsK/K-g-ve-ve-ve+A+g-ve-ve+ve-ve+ve+veTSIA was observed after the incubation period of 6 hours and it showed a red slant with red butt signifying no fermentation. The given bacterium was not fermented to any of the three carbohydrates but utilized peptone and amino acids that alkalinized the medium and turned it red. Both the slant and butt was appeared red because the peptone was used aerobically and anaerobically. After 24 hours of incubation, the result showed a red slant and red butt indicating no fermentation. Urease test showed a negative result because the first result was observed after 24 hours of incubation and was waited until 8 days to see if there appeared any color change in the broth. Thus, the given bacterium was unable to break down urea into ammonia. SIM was observed after 24 hours of incubation. The result showed no blackening of the medium indicates no sulfur reduction. For the Indole Test, 5 drops of Kovac’s reagent were added, and the result showed no color change giving a negative result for Indole test. This showed that the given bacteria could not hydrolyze tryptophan to pyruvate, ammonia and Indole. And for motility, the bacteria were nonmotile since it could not able to grow radiating outward from the stab line giving negative result. For MR test 5 drops of methyl red indicator was added in the medium after incubating it for 24 hours. No color changed indicating a negative MR test for the given sample. Thus, the bacteria were not able for mixed acid fermentation. VP test was done after the incubation period of 48 hours by adding 1 ampoule of Barritt’s A and 1 ampoule of Barritt’s B and waiting for an hour to see any change in color. No color change was observed showing that the bacteria was not able to convert glucose products to 2, 3-butanediol. Citrate test was observed after the incubation period of 48 hours and was found to be positive because the color changed from green to Prussian blue. This indicated that the bacteria contained citrate permease enzyme thereby, had an ability to convert citrate into pyruvate. The incubation period had to be at least 48 hours because no color change was observed prior to 48 hours of incubation. To perform oxidase test, bacterial colony was swabbed to a filter paper and chromogenic reducing agent was added in it. Immediate color change to black was appeared indicating a positive oxidase test. Therefore, the cytochrome c oxidase was present in the given unknown bacteria. Gelatin was performed initially after incubating the broth for 24 hours in the hot room. The media was then transferred to the cold room for an hour and it was seen that the gelatin was not solidify indicating a positive result. Thus, the gelatin was produced and gelatin hydrolysis was took place. Discussion/ Concliusion: In the discussion, you assess how the results answer to this question and discuss its relevance to the existing knowledge in the field.When writing a conclusion, you should try to answer a few questions, as succinctly as possible.You will have already answered some of these in your discussion, but the key is to leave some questions that another researcher can expand upon for their research project.Discussion/ ConcliusionAfter comparing all of the results to the standard chart of Gram Negative Unknown, my unknown sample # 12 was found to be Pseudomonas aerogenosa . Oxidase being positive was an important result which helped to eliminate the entire possible choices except Pseudomonas aerogenosa. Gelatin being positive helped to eliminating the other possible choices and just focus on Proteus mirabilis and Pseudomonas aerogenosa. Since, Motility result was negative for both Klebsiella pneumoniate and Pseudomonas aerogenosa, it was somewhat easier to exclude Proteus mirabilis. Citrate was positive for the entire unknown except Escherichia coli. Negative MR Test and negative urease test kept on giving an alternative between Enterobacter aerogenes and Pseudomonas aerogenosa but negative VP test for the entire unknown except Enterobacter aerogenes, it was excluded. Then finally, since TSIA test for gas production was seen negative, as it was positive for entire unknown. Colony morphology on TSA of Pseudomonas aerogenosa was small, beige and mucoid while other was white in color. Then finally, these entire features make a conclusion that the given unknown bacteria were none of other than Pseudomonas aerogenosa.Pseudomonas aerogenosa is a pathogenic gram-negative rod that belongs to the family Pseudomonadaceae. These pathogens are widespread in nature, inhabiting soil, water, plants, and animals (including humans). P. aerogenosa has become an important cause of infections such as pneumonia, urinary tract infections (UTIs), and bacteremia. Pseudomonal infections are complicated and can be life threatening. P. aerogenosa is opportunistic pathogens which rarely causes disease in healthy persons. The integrity of a physical barrier to infection such as skin, mucous membrane is lost or an underlying immune deficiency (eg, neutropenia, immunosuppression) is present in most cases of infections today.Citation of research paper is a must to avoid any PlagiarismMLA Style (Modern Language Association)APA Style (American Psychological Association)Work CitedLeboffe, Michael, and Burton E. Pierce. “Gram Stain.” Microbiology: Laboratory Theory and Application. 2nd Ed. Morton Publishing Company.2006.86-87. Print.Medigan, Michael T. et al. Brock biology of Microorganisms. 12th ed. California: Benjamin-Cummings, 2009. Print.Qarah, Samer. Pseudomonas aeruginosa Infections.Medscape’s continually updated clinical references,1994-2010. Web. 04 April. 2010.; ................
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