The Use of Biochemical Techniques in Identifying Unknown Bacteria #4 Report

Intro Microbiology Title: The Use of Biochemical Techniques in Identifying Unknown Bacteria #4 Abstract: Unknown Bacterial Identification is widely used in laboratories throughout the world, for a variety of purposes. Most common use of Bacterial Identification is in outbreaks, particularly in food distribution settings. Before resolving the outbreak, identifying the specific type of Bacteria is necessary to eliminate contamination and proper distribution of food. Within the laboratory, well known traditional biochemical techniques were used, such as Urea Hydrolysis, Citrate utilization, Nitrate Reduction, FTM, Methylene Red (MR) indication, and Vogues-Proskauer (VP) test, for purposes of identifying the unknown bacteria. The test results of led to aerobic bacteria with negative Urea, Nitrate, MR, and positive Citrate. According to the findings, the unknown bacteria #4 was likely Gram Negative Rod-shaped Alcaligenes. Introduction: Identification of Bacteria pays huge dividends within the field of Microbiology by having the ability to detect harmful strains and prevent outbreaks of these strains. Within the advancement of technology, there are many various techniques when experimenting with an unknown bacterium. Traditional Biochemical and Molecular techniques are used within lab settings to investigate the presence of a specific bacteria. These techniques are still used in modern lab settings and often provide conclusive data to an unknown organism. These methods can also prevent false data, which means tests should be accounted for and done numerous times and efficiently. In this experiment, Gram staining was done to identify the bacteria as gram positive or gram negative. This technique plays fundamental importance within Microbiology by identifying bacteria when an outbreak, such as in the food industry occurs. Gram staining is a necessary practice while doing so, to distinguish whether the bacteria is Gram Positive or Gram-negative by identifying its resulting final color. Not only will the color of the bacteria after performing the gram stain be a distinguishable factor but bacteria are transparent, making it difficult to see under a microscope without being stained. The experiment included determining whether the unknown bacteria strain #4 was either a gram-positive or gram-negative by its color change after gram staining. The purpose of the operation was to use traditional biochemical methods to characterize the unknown bacteria sample 4. Among the various types of tests to perform on an unknown bacteria, Molecular methods are willing to provide numerous organisms in the presence of a single study. Sufficiency within the molecular method includes adequate sensitivity and selectiveness and can overall improve on culture-based strategies within a short time frame. (Liyanapathirana 2017) Before there was DNA sequencing, molecular analysis allowed molecular geneticists to experiment reactions within the body and how it responds to various microbial disease. A biochemical test can be used to identify chemicals that are particularly unique to individual species. The test is represented as a classic and least expensive method to examine an organism. The biochemical technique is useful when identifying types of microbes while using a manual to find key distinctions, traditionally by incorporating test tubes and plates to analyze bacteria. Throughout the years, the technique has allowed for a reduction in costs while having adequate, efficient results and reproducibility with a more significant number of tests. (Parker 2018) The purpose of the exercise was to use traditional biochemical techniques to identify unknown organism #4. Methods: Gram Staining: After sterilizing the inoculating loop, a pure culture was dabbed, taken, and smeared onto the slide, while being careful not to clump the sample. Once the pure culture was on the slide, it was heat fixed with a lighter for 5 seconds. The first step included an application of the first (primary) stain crystal violet to the slide directly for approximately 30 seconds. After the 30 seconds of the solution, the slide was then rinsed lightly with distilled water. Following the primary stain, the second step included Iodine solution, which served as the mordant and applied for 60 seconds. Following saturation and another rinse of water, the third stain was an ethyl alcohol wash; this was used and left on the surface of the slide for 12 seconds. Water was applied once again to rinse the alcohol wash. Lastly, the application of the safranin which was utilized as the counterstain had been applied for 60 seconds. A rinse of the slide was then done. Finally, the use of the safranin which was used as the counterstain had been applied for 60 seconds. The last rinse of water had been done to eliminate the remainder of any stain. After the experiment, the slide was then viewed under a Bright field microscope at 1000x magnification in determining the color displayed was reddish/pink to decide on bacteria distinction. The suggested that the bacteria were, in fact, gram-negative bacteria whereas a gram-positive would have appeared purple. Urea: The primary culture tube was shaken to suspend bacteria in reassuring and performing inoculation of a culture accurately. After sterilization of the inoculation loop, a sample was taken and placed within the Urea test tube and incubated for 48 hours. A positive result would be the color pink, representing a strong urease production. If the sample were negative with no pink color, no urea hydrolysis would have taken place. Citrate: Once a sample of bacteria had been taken with a sterile inoculation needle, a BBL Simmon's Citrate Agar Slant tube was then inoculated with a slant and stab technique. The test tube had then been incubated for 48 hours at 37oC. The test had been done to examine if bacteria can or can't use citrate to grow. Nitrate: The Nitrate test consisted of the growing of unknown bacteria #4 in a culture test tube with nitrate broth that had contained 0.5% potassium nitrate (KNO3) and Durham tubes. Bacteria #4 was inoculated from the culture by a sterile loop and tapped slightly within the inside of the test tube to ensure the sample was appropriately attained. The incubation period consisted of 48 hours at approximately 37OC. After incubation, the test tube was examined for specific growth or absence of growth. A positive result within the Nitrate test would have occurred by the presence of gas bubbles, identifying that the unknown bacteria had reduced nitrate to a gaseous end product. Negative results within the procedure would have been present by no formation of gas bubbles, meaning the organism may have had the formation of nitrite or possibly other nongaseous end products. Approximately 0.5mL of the nitrate test reagent had been mixed within the culture tube for the appearance of a possible color change. If the color pink or red had occurred would have been the confirmation of a positive test, providing evidence the medium is negative. The confirmation of a negative test result would entail the addition of 5 to 10 drops of zinc powder while gently swirling the tube. The actual color change will determine the final result. The appearance of color red within five to ten minutes indicates the presence of nitrate, while no color change resulted in no existence of nitrate within the sample. FTM: The test involved a result of determining unknown bacteria #4 and if the microorganism required oxygen for growth. The aerotolerance of the bacteria would have ultimately determined if the unknown organism was aerobic, meaning it needs or requires oxygen for sustainability. When determining the oxygen capacity or necessity for oxygen use of the unknown, three individual tests were implemented. The three tests consisted of MR-VP, Citrate slant, and Nitrate with Durham tube to assure the presence of oxygen requirements for the unknown bacteria. Various results of these given procedures would justify the findings of the unknown sample #4. Methyl Red Test: The Methyl Red test consisted of using a tube of BBL MR-VP broth, which had been inoculated with a sterile loop containing unknown bacterium #4. As well as the many other tests, it had been incubated for 48 hours at a temperature of 37oc. Following incubation, one-third of bacterial culture #4 was transferred into a separate test tube. Next, five drops of methyl red indicator were added. Observations after the addition were taken to indicate if bacteria #4 had the capability of fermenting glucose, lowering the pH of the medium. Vogues-Proskauer Test: A divisor of approximately one-third of the methyl red test had been taken into a separate test tube. Next, fifteen drops of Barritt's solution A and five drops of solution B was added to each culture, shaking the test tube to incorporate aeration. Reactions within the test tubes would occur immediately or within a 20-minute time frame. Positive results of the test would involve the color red. The test was ultimately focused upon the unknown bacterium having the capability of fermenting glucose, as well as the production of acetylmethylcarbinol. Results: Figure 1a: Picture of Alcaligenes cells consecutive to Gram Staining Viewed at 400x Magnification through a Brightfield Microscope. Figure 2a: Picture of Alcaligenes cells consecutive to Gram Staining Viewed at 1000x Magnification Oil Immersion lens through a Brightfield Microscope. Many steps were done in order to find the differential nature of bacteria between grampositive and gram-negative cells. Differential staining is necessary for determining the type of bacteria because of how they differ chemically, physically, as well as their reaction to the staining procedure. Shown in Figures 1 and 2 at different magnification is unknown organism #4. The unknown bacterial cells following gram staining were observed having a display of pink/red color and rod-shape, with clear indication of individual cells. Table 1: Tests and Procedures in Identifying Unknown Bacteria #4 Test Purpose Expected Observed Gram Stain The test was used to identify the specific type of peptidoglycan present among bacteria depending on final stain color. Gram positive bacteria appear purple in color while having a layered peptidoglycan. Gram negative bacteria appear pink in color while having a thin layer of pseudopeptidoglycan. Unknown organism #4 appeared to be pink through microscope representing the bacteria as gram negative. Urea Hydrolysis The Urea Hydrolysis test would indicate the presence of urease enzyme by representing the color pink. If urease positive the color of the sample would display pink. If the organism was urease negative it would display yellow. Unknown organism #4 appeared negative for urease enzyme with no pink color. Simmon’s Citrate The Simmon’s citrate test indicates if organism can utilize citrate by displaying a blue color. If citrate positive test tube will display a blue color. If the organism is citrate negative it will display a green color. Unknown organism #4 test tube appeared blue in color which means it had broken down citrate for utilization. Nitrate Reduction Nitrate test was utilized in determining if the unknown organism is capable of reducing Nitrate. After incubation, complete reduction should be present while partial reduction can be tested using other reagents. Unknown organism #4 was negative by displaying a brown color that went away, with initial bubble no longer present. +Zinc was negative by having a hot pink color. Methyl Red Test The methyl red procedure was used to determine the type of fermentation used by unknown organism. (Mixed acid fermentation or 2,3- Butanediol) Positive results would indicate the presence of gas displaying a bubble in the Durham tube, +Reagents A&B being red, +zinc having no change in color. Negative results would indicate no presence of gas bubble, +Reagents A&B with no change in color, +zinc displaying red in color. A positive test result would be indicated by the color Red representing mixed acid fermentation & negative for 2,3Butanediol. Negative results would be indicated by no color change for no mixed acid fermentation. Figure 1b: Photographs of Test Results procedures. Unknown organism #4 was negative by displaying the color yellow with an orange rim. Organism did not represent any presence of mixed acid fermentation. Figure 2b: Flow chart Identifying Unknown Bacteria #4. The traditional Biochemical techniques used to identify Unknown Bacteria #4 are shown in Figure 2b (above). Evidence shows that a gram-negative bacterium had been present following the gram staining procedure. After Gram staining, unknown bacteria appeared as Rod-shaped through magnification, as shown in figure 1a and b. Positive results after Citrate test represented the utilization of citrate being shown a blue color. Negative results with Urea hydrolysis, Nitrate reduction, FTM, and Methyl Red contributed to the findings of unknown organism #4. Following the observation of all procedures taken place, using Bergey’s Manual of Determinative Bacteriology 9th Edition led to the outcome of Unknown bacteria #4 most likely the Proteobacteria Alcaligenes. Discussion: Biochemical and Molecular techniques have advanced within microbiology and are used within a wide variety of laboratories throughout the world when identifying various microorganisms. Gram staining led to the observation that unknown bacteria #4 was most likely Alcaligenes, which is a non-endospore forming bacteria. When given an unknown bacterium, it was necessary to gram stain by differentiating whether or not a gram-negative or gram-positive bacteria was present. The critical distinction after carrying out the gram staining procedure is accurately obtaining the presence of thick or thin peptidoglycan. Because the bacteria following gram staining displayed a pink color of representation, it is considered a gram-negative with a thin layer of peptidoglycan. If the organism had been gram-positive bacteria and had a thick layer of peptidoglycan, it would display the color purple. Another distinction found within the gram staining procedure was the rod shape of the bacterial cells. Various other tests such as Simmon's citrate procedure had been done to examine the utilization of citrate and ability for unknown to break down citrate. It was a positive result following incubation presenting a blue color within the citrate test tube. The Methyl Red test was an indication of what type of fermentation was taken place with unknown organism #4. Although previous tests had led to the conclusion of the possible bacteria it was, further testing would reassure accuracy towards results making sure all findings fall within the range. Conclusive to incubation, unknown bacteria presented a yellow color with orange rim showing no presence of mixed acid fermentation. After the MR test was observed as a negative result, it was a possibility that the bacteria could be Enterobacter. Urea Hydrolysis was done to indicate the organism had a urease enzyme by displaying the color pink. Once incubation was complete, no pink color was present. The test resulted in negative urea hydrolysis with no urease enzyme present within the bacteria. Accuracy among unknown identification was done by using a minimum of five biochemical techniques. Later evidence showed Gram Stain, FTM, and Nitrate reduction identified the bacteria among the other procedures. Early findings suggested that after all procedures were complete, it was a possibility that unknown bacteria #4 may have been Enterobacter. After no confirmation towards the gram-negative bacteria being Enterobacter, it was later discovered unknown #4 might have been Alcaligenes. Evidence that represented the gram-negative bacteria as rod shape and aerobic and no sign of nitrate reduction ultimately led to the conclusion the unknown was Alcaligenes following confirmation. After identifying the shape and color of the bacteria, another distinction was to find if the organism was aerobic or facultative aerobic. Identifying one from the other would give a closer identification of the bacteria. The Fluid Thioglycollate Medium (FTM) procedure had been used by observing the growth of bacteria within a specific part of the tube. Following incubation, evidence showed that the bacteria was obligate aerobic with growth towards the surface of the medium as shown in figure 1b. Following FTM, according to Bergey’s Manual, two options of identifying unknown bacteria as Pseudomonas or Alcaligenes were present, knowing these were the only aerobic organisms given. Test results following Nitrate reduction had ultimately provided evidence that the unknown bacteria were gram negative Alcaligenes. Nitrate Reduction tested to see if nitrate was reduced after incubation. There was no evidence to support the reduction in nitrate with no bubble formation and the color pink (Figure 1b). If the unknown bacteria had a positive result following the nitrate test, then it would support evidence that the unknown bacteria were Pseudomonas, because this organism would be expected to adequately reduced nitrate. Alcaligenes bacteria was the identification of unknown bacteria #4 among the phylum of Proteobacteria and genus of gram-negative, aerobic, rod-shaped and motile. Proteobacteria are a phylum that was discovered by the scientist Carl Woese during the 1980’s by using scientific methods of nucleotide sequence homology. (Parker) Proteobacteria have various classifications such as alpha, beta, gamma, delta, and epsilonproteobacteria. Among these classes falls specific separation in orders, families, genera, and species. This bacterium is most commonly found within water and soil and falls within the class of Betaproteobacteria. Alcaligenes derives from the family of gram-negative non-spore forming bacteria in the order of Burkholderiales. The well-known genera of Burkholderiales consist of the order Achromobacter, Alcaligenes, and Bordetella. (Gottschling)