Identifying an Unknown Microbial Organism
Introduction
The purpose of identifying an unknown microbial agent is so that the five “I’s” of Microbiology, which are, Inoculation, Incubation, Isolation, Inspection and Identification can be practiced. Providing an unknown microbial agent tests the ability of ones skill of the above techniques and to accurately obtain the correct results and compare these results to biochemical test results that are already established. (Forrest & Elliott, 2012, p.111)
Test used include the Gram Stain, the Streak Plate and Biochemical test such as the MR-VP, SIM, Phenylalanine Deaminase, Simmon’s Citrate along with the Carbohydrate Fermentation and Urease tests.
Biochemical Test of Importance
Several Biochemical test were performed, however, only three will be three will be discussed due to these test being the most significant in the identification of the microbial agent. These test helped narrow down the options of which organism the results matched with. The tests, which will be further dissected, are the SIM, Simmon’s Citrate and the Urease tests.
First, the SIM test consisted of Sulfur, Indole, and Motility; however, the focus will be narrowed to the Sulfur portion of the test only due to it being the most significant. Two methods used in this portion of testing were direct testing on the specimen and culture/Isolation. During the SIM testing, aseptic technique was cautiously used. An inoculation needle was used to obtain culture. Afterwards, the needle was taken and stabbed into the SIM media. The media was then stored at 37 degrees Celsius for 24-48 hours. (Forrest & Elliott, 2012, p.114)
The Sulfur portion of the SIM test consisted of the enzymes Thiosulfate reducta...
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Cowan, M. K., & Talaro, K. P. (2009). Infectious Diseases Affecting the Respiratory System. In Microbiology: A systems approach (3rd ed., pp. 645-54). Boston, MA: McGraw-Hill.
Forrest, S., & Elliott, M. (2012). Lab 16 Identification: Biochemical Tests. In Microbiology: An Introduction to Laboratory Procedures & Techniques (pp. 111-116). El Dorado, KS: McGraw-Hill Companies, Inc.
Forrest, S., & Elliott, M. (2012). Lab 20 Identification of an Unknown Bacteria/ Published Results. In Microbiology: An Introduction to Laboratory Procedures & Techniques (pp. 135-138). El Dorado, KS: McGraw-Hill Companies, Inc.
PODSCHUN, R., & ULLMANN, U. (1998). Klebsiella spp. as Nosocomial Pathogens: Epidemiology, Taxonomy, Typing Methods, and Pathogenicity Factors. Clinical Microbiology Reviews. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC88898/
The isolate possesses some enzymes required for hydrolytic reactions. Hydrolytic enzymes found to be secreted from the bacterium, are -amylase, casein, and PYRase. In the starch hydrolysis and casein tests, there was a zone of clearing around the bacterium, which was indicative of the secreted enzymes necessary to break down starch and casein. In the PYR test, the presence of PYRase was detected by a color change to red on the PYR disc after the addition of the PYR reagent (p-dimethylaminocinnamaldehyde). Hydrolytic enzymes for which the EI tested negative were urease, gelatinase, and DNAse. In the Urea Hydrolysis test, it was observed that the urea broth did not have a color change, indicating that there was no urease secreted to break down urea in the broth. Similarly, there was no gelatinase present to break down gelatin in the Gelatin Hydrolysis test, so the nutrient gelatin remained solid. It was concluded that the EI does not possess DNase because there was no clearing zone around the bacteria, indicating that DNA had not been
After 5 days of growth each slant was tested using the gram staining technique to confirm the complete isolation of the bacteria. Both isolations were completely successful. Then each sample of bacteria was subjected to a series of tests for identification.
In this lab project, the microbiology students were given 2 unknown bacteria in a mixed broth each broth being numbered. The goal of this project is to determine the species of bacteria in the broth. They had to separate and isolate the bacteria from the mixed broth and ran numerous tests to identify the unknown bacteria. The significance of identifying an unknown bacteria is in a clinical setting. Determining the exact bacteria in order to prescribe the right treatment for the patient. This project is significant for a microbiology students because it gives necessary skills to them for future careers relating to clinical and research work.
They can be found anywhere and identifying them becomes crucial to understanding their characteristics and their effects on other living things, especially humans. Biochemical testing helps us identify the microorganism present with great accuracy. The tests used in this experiment are rudimentary but are fundamental starting points for tests used in medical labs and helps students attain a better understanding of how tests are conducted in a real lab setting. The first step in this process is to use gram-staining technique to narrow down the unknown bacteria into one of the two big domains; gram-negative and gram-positive. Once the gram type is identified, biochemical tests are conducted to narrow down the specific bacterial species.
After the end of the experiment the unknown 10 sample was Staphylococcus epidermidis. Came to this conclusion by first beginning with a Gram Stain test. By doing this test it would be easier to determine which route to take on the man made flow chart. Gram positive and gram negative bacteria have a set of different tests to help determine the unknown bacterium. Based on the different tests that were conducted in lab during the semester it was determined that the blood agar, MSA, and catalase test are used for gram positive bacteria while Macconkey, EMB, TSI, and citrate tests are used for gram negative bacteria. The results of the gram stain test were cocci and purple. This indicated that the unknown bacteria were gram positive. The gram stain test eliminated Escherichia coli, Klebsiella pneumonia, Salmonella enterica, and Yersinia enterocolitica as choices because these bacteria are gram negative. Next a Blood Agar plate was used because in order to do a MSA or a Catalase test there needs to be a colony of the bacteria. The result of the Blood Agar plate was nonhemolytic. This indicated that there was no lysis of red blood cells. By looking at the plate there was no change in the medium. Next an MSA test was done and the results showed that there was growth but no color change. This illustrates that the unkown bacteria could tolerate high salt concentration but not ferment mannitol. The MSA plate eliminated Streptococcus pneumonia and Streptococcus pyogenes as choices since the bacteria can’t grow in high salt concentration. Staphylococcus aureus could be eliminated because not only did the unknown bacteria grow but also it didn’t change color to yellow. Lastly a Catalase test was done by taking a colony from the Blood Agar plate...
I also inoculated a tryptic soy broth (TSB), a nutrient gelatin deep, a motility agar deep, a fluid thioglycollate medium (FTM) tube, and a TSA plate with my unknown culture. All of these inoculated media were incubated until the next class period (about 48 hours). Then when I came to class most of my inoculated tubes and my streak plate appeared to have growth. The next step I took was making a gram stain to determine the gram reaction and cellular morphology of my unknown. I used my working slant to do this, after careful examination of the gram stain, I learned that my unknown was a gram-positive bacterium. I then preceded by making a negative stain to see the size of the cells of my unknown bacteria. The cell shape was cocci and the cells occurred in clusters of tetrads. After discovering that my unknown bacteria was gram-positive cocci, I turned to page 207 of the lab manual to narrow down my options, there was only four out of the gram-positive list that were
The eighteenth exercise of the laboratory manual titled Unknown Identification and Bergey’s Manual is an experiment to identify an unknown bacterium. In this exercise, a student must randomly choose a numbered bacterium available to the class. The keys in Appendix H, located on the last pages of the book, are the major helpful tools in this exercise because it provides completed steps of tests that needs to be performed in order to distinguish certain bacteria. This means that in this exercise, various types of tests and techniques must be performed to identify the chosen unknown bacterium. The unknown bacterium that I selected was number thirty-nine in which I discovered as the Bacillus megaterium after conducting several tests.
electrophoresis. The way the PCR method works is by first mixing a solution containing the
Bacteria play a large role in our health, the environment, and most aspects of life. They can be used in beneficial ways, such as decomposing wastes, enhancing fertilizer for crops, and breaking down of substances that our bodies cannot. However, many bacteria can also be very harmful by causing disease. Understanding how to identify bacteria has numerous applications and is incredibly important for anyone planning to enter the medical field or begin a career in research. Having the background knowledge of identifying an unknown bacteria may one day aid healthcare professionals diagnose their patient with a particular bacterial infection or help researchers determine various clinical, agricultural, and numerous other uses for bacteria.
The purpose of this investigation was to identify an unknown bacterium. “At any time there are millions of bacteria living around, on, or inside us” (The Plague). Bacterium can’t be identified by merely looking at it. Many bacteria have the similar appearances in growth. “In most cases, detection is based on the reaction of an enzyme with a certain substrate” (Sigma-Aldrich). Identification is usually based on the results of the bacterium’s cells metabolic capacities.
By using aseptic technique, which consisted of using gloves and wiping area with disinfectant, we were then able to inspect the growth on the three dishes, without removing the lids. We compared what we saw to samples we looked at and took notes on the week prior. A small live culture of the unknown bacteria was then made from a petri dish of our choosing. The colony had to be isolated and fairly large. In a 1.5 ml micro centrifuge tube 400 µl of sterile water were mixed with about half of our selected colony, which was transferred by using the broad end of a sterile toothpick and taking from our petri dish.
I identified the genus and species of an unknown bacterial culture, #16, and I applied the following knowledge of morphologic, cultural and metabolic characteristics of the unknown microorganism according to the laboratory manual as well as my class notes and power point print outs. I was given an incubated agar slant labeled #16 and a rack of different tests to either examine or perform myself; the tests are as follows: Gram Stain; Nutrient Gelatin Test; Carbohydrate Fermentation; Dextrose, Lactose and Sucrose; IMVIC tests; Citrate, Indole, Mythel-Red and Vogues Proskauer test; as well as a Urease and TSI Test. Materials and Methods/Results Upon receiving the Microorganism (M.O.) #16, I prepared a slide by cleaning and drying it. Then, using a bottle of water I placed a sterile drop of water on the slide and used an inoculating loop, flame sterilized, I took a small sample of the unknown growth in my agar slant and smeared it onto the slide in a dime sized circle and then heat fixed it for ten minutes.
Due to contamination, the Micro-ID Listeria unit could not be used in the identification of the Listeria species used to inoculate it. This result may have something to do with the contamination visualized on our blood agar plate and oxidase test.
Person, A. & Mintz, M., (2006), Anatomy and Physiology of the Respiratory Tract, Disorders of the Respiratory Tract, pp. 11-17, New Jersey: Human Press Inc.
Fischbach, Frances, A Manual of Laboratory & Diagnostic Tests, 4th ed., J. B. Lippincott Company, Philadelphia