Discussion
Materials and Methods
An unknown broth culture labeled # 17 was supplied by the lab instructor to identify the bacteria. We prepared a SOP by incorporating laboratory techniques that were considered as determinative tests in the dichotomous keys (appendix B and appendix C). The first two steps we performed to form the basis of identification were Gram’s staining and plate streaking of the sample. The supplied broth culture was streaked out simultaneously on one of each Trypticase Soy Agar (TSA), Mannitol Salt Agar (MSA) and MacConkey Agar (MAC) plate. The streaking was performed using the quadrant streak method described in the lab manual (Leboffe & Pierce, 2010). After observing the colony morphology and growth pattern, we further
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We arranged the subsequent determinative biochemical tests after the dichotomous keys for definite identification of the bacteria present in broth sample. As the initial screening tests identified the unknown bacterial broth as a mixture of gram-positive cocci and gram-negative bacilli, we organized two different test regimes to follow the dichotomous keys. Figure 1 Flow diagram of biochemical tests for Gram-positive cocci
Figure-1 illustrated the test result and the direction of tests that we accomplished to set up the SOP of gram-positive cocci. The catalase test was vital because it categorized the gram-positive cocci into two major groups. We evaluated the test results with the standards of media reactions described in Difco manual (Zimbro, et al., 2009). Most tests of SOP supported the identity of gram-positive coccus as Micrococcus luteus. However, glucose test did not support the dichotomous key, and it was fermentative. For this, we performed more biochemical tests of oxidase, nitrate reduction, and cultured further the pure culture on BPEY tellurite Agar. These later tests reinforced the identification of Micrococcus luteus that was shown in figure
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The test results as evaluated with standard media reactions suggested the most reasonable identity of gram-negative bacilli as Citrobacter freundii. The oxidase test was vital as it categorized the gram-negative bacteria into two major groups. The tests scheduled in SOP supported the definitive identification of gram negative bacillus. Some of the tests provided more implications than others. Since all the tests were consistent with the dichotomous key, we did not perform further tests for species characterization of the bacillus. A photograph of isolated gram-negative bacillus was presented in figure
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
One bacterium was gram negative. It underwent four different tests. These tests were the EMB test (Eosin Mehylene Blue), the Sulfur Indole Motility (SIM) test, the Urease test, and the Simmon’s Citrate Utilization test. The EMB test checks for a bacteria’s ability to ferment lactose. This test is accomplished by placing the bacteria on Eosin Methylene Blue agar. The agar is selective for gram negative bacteria and those bacteria that can ferment lactose will have colored growth, usually a metallic green sheen.
Streak plate technique was used to isolate pure culture for each bacteria (2). The Gram stain was used to determine Gram reaction and morphology of each bacteria (2) Selective and differential media such as, salt agar, MacConkey agar and blood agar were used for bacterial identification (2). Gelatin deeps were inoculated to detect production of gelatinase (2). Starch Agar plate were inoculated to detect amylase (2). Ocular reticle used to determine bacteria size (2). Motility deeps were inoculated to detect motility on bacteria (2). Thioglycollate broth used to determine oxygen requirements (2). Carbohydrate fermentation
The purpose of this study is to identify an unknown bacterium from a mixed culture, by conducting different biochemical tests. Bacteria are an integral part of our ecosystem. 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. These biochemical tests are process of elimination that relies on the bacteria’s ability to breakdown certain kinds of food sources, their respiratory abilities and other biochemical conditions found in nature.
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.
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
Phenotypic methods of classifying microorganisms describe the diversity of bacterial species by naming and grouping organisms based on similarities. The differences between Bacteria, Archaea and Eukaryotes are basic. Bacteria can function and reproduce as single cells but often combine into multicellular colonies. Bacteria are also surrounded by a cell wall. Archaea differ from bacteria in their genetics and biochemistry. Their cell membranes are made with different material than bacteria. Just like bacteria, archaea are also single cell and are surrounded by a cell wall. Eukaryotes, unlike bacteria and archaea, contain a nucleus. And like bacteria and archaea, eukaryotes have a cell wall. The Gram stain is a system used to characterize bacteria based on the structural characteristics of their cell walls. A Gram-positive cell will stain purple if cell walls are thick and a Gram-negative cell wall appears pink. Most bacteria can be classified as belonging to one of four groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, and Gram-negative bacilli) (Phenotypic analysis. (n.d.).
mutans was problematic due to its difference with Bergey’s Manual result for the catalase test. However, after comparing it with a peers results, it seems very possible that the strain we are working with varies from the strain used in Bergey’s. Bacteria possess the ability to develop varying phenotypes within the same species due to frequent mutation and horizontal gene transfer. Therefore, it is possible that the results obtained in our lab may vary from those provided in Bergey’s Manual. Arriving to the conclusion that the Gram negative bacteria was Klebsiella pneumoniae was much more direct. Using Bergey’s Flowchart for identification, the bacteria shared the test results and had a similar shape and
This pathogen, Streptococcus pneumoniae, is a gram-positive coccus that is long shaped and usually seen in groups of pairs (Todar, 2008-2012). This pathogen ranges from o.5-1.25 micrometers, which is pretty small in size (Todar, 2008-2012). It “lacks catalase and ferments glucose into lactic acid” (Todar, 2008-2012). To grow this bacterium in the lab the best way to do it would be to grow it on a blood agar at 37 degrees Celsius and produces a green zone arou...
Music and Art are two important factors in a society. They are apart of a neighborhood's History. They show how a community has lived, and what was important to the people and how they lived. The Art and Music during certain time periods can show how that community has grown and how it developed. There were many important artist and musician that played a big role in how Detroit, Michigan grew. They also had a big impact on the society of Detroit. Till this day those Artist and Musicians still have an impact on Detroit.
Southern hospitality is the best in the world. People that live in the South are very nice and are always willing to help another person in any way they can. If someone is from out of town and needs directions to a certain place southerners will make sure he or she knows how to get there before he or she leaves them. Southerners are very polite. Every time we pass someone on the rode, we are going to wave at him or her. Towns in the South have fewer people and everyone knows everyone. The people in the South are nicer than anywhere else in the United States.
When the three of us decided to use Texas as our micro-culture, I thought it was a great idea. I am not a Texan, since by definition to be a Texan, you must have been born in Texas, no exceptions (http://www.texas-best.com), but do consider myself an honorary Texan.
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.
Royal Melendy writes about a rising social culture taking place at the turn of the twentieth century. He depicts this culture as the ambiance emitted in early Chicago saloons. “Saloons served many roles for the working-class during this period of American history, and were labeled as the poor man’s social clubs” (summary of saloon culture, pg. 76).
Leboffe, M. J., & Pierce, B. E. (2010). Microbiology: Laboratory Theory and Application, Third Edition 3rd Edition (3rd Ed.). Morton Publishing