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Margaret E Gibson July 20, 2009 Microbiology Dr. Metera Lab Report 3: Labs 7 and 8- Metabolism and Biochemical Tests Abstract This experiment focused on metabolism and biochemical tests. The goal of performing these tests was to differentiate microbes from one another and to compare how metabolic and biochemical processes differ from species to species.

The tests performed include: the Fermentation of Sugars Test (sucrose, glucose, and lactose), the Urease Test, the Fermentation of Lactose Test, the Sulfide Indole Mobility (SIM) Test, the Nitrate Reduction Test, the Protein Hydrolysis Test, the Catalase Test, and the Cytochrome Oxidase Test. The microbes that were tested during this lab were: Escherichia coli, Bacillus cereus, the unknown, Proteus vulgaris, Staphylococcus epidermis, Enterobacter aerogenes, the control, and Pseudomonas fluorescens.

The microbes tested during these various tests were looking for which would: reduce sulfur/produce sulfate, produce indole, or possess motility, reduce nitrate, and contain protease, catalase and oxidaase. Introduction The purpose of these labs was to observe various metabolic processes by determining the pH of certain bacteria, determining if the bacteria was urease positive or negative, determining which bacteria ferment which sugar(s) during fermentation, and determining if bacteria are lactose fermenters and non-lactose fermenters.

Metabolic processes can also be observed by determining if bacteria reduce sulfur/produce sulfate, produce indole, or possess motility, determining which bacteria are able to reduce nitrate, determining if bacteria contain protease, determining if bacteria contain catalase, and determining if bacteria contain oxidase. The tests performed to determine these metabolic processes include: the Fermentation of Sugars Test (sucrose, glucose, and lactose), the Urease Test, the Fermentation of Lactose Test, the Sulfide Indole Mobility (SIM) Test, the

Nitrate Reduction Test, the Protein Hydrolysis Test, the Catalase Test, and the Cytochrome Oxidase Test. The bacteria tested include: Escherichia coli, Bacillus cereus, the unknown, Proteus vulgaris, Staphylococcus epidermis, Enterobacter aerogenes, the control, and Pseudomonas fluorescens. The different types of microbes studied in this experiment include: Escherichia coli, Bacillus cereus, Proteus vulgaris, Staphylococcus epidermis, Enterobacter aerogenes, and Pseudomonas fluorescens.

Escherichia coli is mainly found in animal feces and comprises their intestines as well (US Food and Drug Administration). Bacillus cereus is a known medium of food poisoning and causes vomiting and abdominal cramps (Todar). Proteus vulgaris is connected with food spoilage of meat, poultry, and seafood and may cause diarrhea in infants (Schenectady Country Community College). Staphylococcus epidermis often infects hospital patients with weak immune systems in catheter wounds (European Bioinformatics Institute).

Enterobacter aerogenes is the source of numerous infections such as bacteremia, lower respiratory tract infections, skin and soft tissue infections, urinary tract infections (UTIs), endocarditis, intra-abdominal infections, septic arthritis, osteomyelitis, and ophthalmic infections (E Medicine). Pseudomonas fluorescens are able to grow in various conditions such as soil, water, and plant habitats (European Bioinformatics Institute). Several hypotheses arise during this experiment due to the many subjects being tested. However, since there are numerous tests being performed, a more general hypothesis can be ascertained.

The hypothesis for all tests in both Lab 7 and Lab 8 is that the outcome of the tests will produce the desired results in order to differentiate various species of bacteria from one another and to reveal certain characteristics of metabolic and biochemical processes. Materials and Methods Lab 7 For Part A of Lab 7, label Escherichia coli, Proteus vulgaris, the unknown, and Enterobacter aerogenes on a blue (sucrose), a green (glucose), and a red (lactose) tube. Then, using aseptic technique, inoculate each bacteria into each color tube by sticking the inoculating loop to the bottom of the tube and twirling it, then pulling it straight out.

Record the results. For Part B, label the tubes Escherichia coli, Proteus vulgaris, unknown, and Enterobacter aerogenes. Using aseptic technique, inoculate each tube with the corresponding bacteria by streaking the surface of the agar slant. Record the results. For Part C, label Staphylococcus epidermis, Proteus vulgaris, and Escherichia coli on the Petri plate with the MacConkey agar. Using aseptic technique, inoculate the labeled parts of the plate. Record the results. Lab 8 For Part A of Lab 8, label each tube Enterobacter aerogenes, Staphylococcus epidermis, and Proteus vulgaris.

Using aseptic technique, “stab” the inoculating loop ? of the way to the bottom of the tube and then pull it straight out to inoculate each tube with the corresponding bacteria. Record the results. For Part B, label each tube Enterobacter aerogenes and “control. ” Using aseptic technique, inoculate each Tryptic Nitrate tube by sticking the inoculating loop to the bottom of the tube and twirling it, then pulling it straight out. Then, add ten drops of sulfanilic acid anddemehtyl-1-napthylamine. If a red color develops after this step, record the record the results. If not, add zinc dust to the tube and vortex it.

Record the results. For Part C, label Enterobacter aerogenes and Bacillus cereus on the milk agar plate. Using aseptic technique, inoculate the plate with the corresponding bacteria. Record the results. For Part D, put a few drops of water on the slide and then inoculate it with Bacillus cereus. Next, add one drop of hydrogen peroxide to the sample. Record the results. For Part E, use a sterile swab to transfer the cells from Enterobacter aerogenes and Pseudomonas fluorescens to a disk. Use a new swab for each sample. Add one drop of water to each disk. Record the results. Results Lab7: Part A [pic] |[pic] | |Figure 1 |Figure 2 | |Figure 1 is the unknown for sucrose. As shown, it had an orange |Figure 2 is Escherichia coli for sucrose. As shown, it was | |ring at the top that fades to yellow at the bottom, was cloudy |orange throughout, had darker solution inside the tube than out, | |all the way through, and had no bubbles. |was very cloudy at the bottom, and had no bubbles. |[pic] |[pic] | |Figure 3 |Figure 4 | |Figure 3 is Enetrobacter aerogenes for sucrose. As shown, it was|Figure 4 is Bacillus cereus for sucrose. As shown, it had a dark| |yellow and cloudy throughout, and had no bubbles. |orange ring at the top and was light orange, it was cloudy at the| | |bottom, and had no bubbles. |[pic] |[pic] | | | | |Figure 5 |Figure 6 | | | | |Figure 5 is Enterobacter aerogenes for glucose.

As shown, it was|Figure 6 is the unknown for glucose. As shown, it had an orange | |all yellow and cloudy (++), and had no bubbles. |ring at the top, was yellow and cloudy (++) throughout, and had | | |no bubbles. |[pic] |[pic] | | | | |Figure 7 |Figure 8 | | | | |Figure 7 is Escherichia coli for glucose. As shown, it was |Figure 8 is Bacillus cereus for glucose.

As shown, it was orange| |yellow, cloudy at the top, and had no bubbles. |throughout and had no bubbles. | |[pic] |[pic] | | | | |Figure 9 |Figure 10 | | | | |Figure 9 is the unknown for lactose.

As shown, it was uniformly |Figure 10 is Enterobacter aerogenes for lactose. As shown, it | |light red and cloudy (+), and had no bubbles. |was light orange and cloudy (++), had a red ring at the top, and | | |had no bubbles. |[pic] |[pic] | | | | |Figure 11 |Figure 12 | | | | |Figure 11 is Escherichia coli for lactose. As shown, it was |Figure 12 is Bacillus cereus for lactose.

As shown, it was red | |yellow, cloudy at the top, and had bubbles. |throughout and had no bubbles. | Lab 7: Part B |[pic] |[pic] | |Figure 13 |Figure 14 | |Figure 13 is the unknown. As shown, it had a red streak of red |Figure 14 is Enterobacter aerogenes. As shown, it had faint | |colonies (+++) and remained the same color. |cloudy colonies (+) and remained the same color. |[pic] |[pic] | |Figure 15 |Figure 16 | |Figure 15 is Escherichia coli. As shown, it had faint cloudy |Figure 16 is Proteus vulgaris. As shown, it was bright pink | |colonies (+) and remained the same color. |throughout, orange at the bottom, and experienced a change in | | |color. | Lab 7: Part C pic] Figure 17 Figure 17 is Staphylococcus epidermis, Proteus vulgaris, and Escherichia coli. As shown, the Staphylococcus epidermis showed no growth, the Pseudomonas vulgaris showed substantial growth (+++), and the Escherichia coli showed substantial growth (+++) and turned pink. Lab 8: Part A |[pic] |[pic] | |Figure 18 |Figure 19 | |Figure 19 is Enterobacter aerogenes.

As shown, it showed |Figure 20 is Staphylococcus epidermis. As shown, it showed no | |substantial growth (+++). |growth. | |[pic] | | |Figure 20 | | |Figure 21 is Proteus vulgaris. As shown, it showed substantial | | |growth (+++), turned black, and exhibited a red ring at the top. | Lab 8: Part B |[pic] |[pic] | |Figure 21 |Figure 22 | |Figure 22 is Enterobacter aerogenes. As shown, it was red ? of |Figure 23 is the control. As shown, it was red ? of the way | |the way through separated by black at the bottom. |through separated by black at the bottom. | Lab 8: Part C [pic] Figure 23

Figure 24 is Enterobacter aerogenes and Bacillus cereus. As shown, Bacillus cereus exhibited a lot of growth (++++). Lab 8: Part D [pic] Figure 24 Figure 25 is Bacillus cereus. As shown, it formed bubbles. Lab 8: Part E [pic] Figure 25 Figure 26 is Enterobacter aerogenes and Pseudomonas fluorescens. As shown, the Pseudomonas fluroescens turned purple. Discussion The results of this experiment prove that the hypothesis was correct: the expected results were obtained and therefore made it possible to differentiate various species of bacteria from one another and to reveal certain characteristics of metabolic and biochemical processes.

For example, in the Fermentation of Sugars test, the unknown’s pH was slightly alkaline and no carbon dioxide gas was given off (Figures 1, 6, and 9). The Escherichia coli had a pH around neutral for all three of the sugars and there were bubbles in the Durham tube for glucose, so the bacteria produced carbon dioxide gas during fermentation (Figures 2, 7, and 11). The Enterobacter aerogenes had a slightly acidic pH and no carbon dioxide gas was given off (Figures 3, 5, and 10).

The Bacillus cereus had a slightly alkaline pH and no carbon dioxide gas was given off (Figures 4, 8, and 12). In the Detection of Urease test, the unknown remained the same color, so it was urease negative (Figure 13). The Enterobacter aerogenes remained the same color, so it was urease negative (Figure 14). The Escherichia coli remained the same color, so it was also urease negative (Figure 15). The Proteus vulgaris turned red, meaning it became alkaline with the production of ammonia, so it was urease positive (Figure 16).

In the MacConkey Agar test, the Staphylococcus epidermis exhibited no growth, meaning it is Gram positive, and it does not ferment lactose (Figure 17). The Proteus vulgaris exhibited growth, so it is Gram negative, and it does not ferment lactose (Figure 17). The Escherichia coli exhibited growth, so it is Gram negative, and it turned red, so it ferments lactose (Figure 17). In the Sulfur Indole Motility test (SIM), Enterobacter aerogenes exhibited growth above the inoculation line, so it is motile (Figure 18). The Staphylococcus epidermis did not exhibit any growth, so it is not motile (Figure 19).

The Proteus vulgaris exhibited growth above the inoculation line, turned black, and showed a red ring at the top of the solution, so it is motile, a phosphorus reducer, and an indole producer (Figure 20). In the Nitrate Reduction test, the Enterobacter aerogenes turned red, so the nitrate was not reduced by nitrate reductase, meaning it was nitrate reductase negative (Figure 21). The control also turned red, so the nitrate was not reduced by nitrate reductase, meaning it was also nitrate reductase negative (Figure 22).

In the Protein Hydrolysis test, the Enterobacter aerogenes did not exhibit any growth, so it was protease negative (Figure 23). The Bacillus cereus exhibited a lot of growth and turned the milk agar clear, so it was protease positive (Figure 23). In the Catalase test, the Bacillus cereus bubbled, so it is catalase positive (Figure 24). In the Cytochrome Oxidase test, the Enterbacter aerogenes did not change color, so it is cytochromoe oxidase negative (Figure 25). The Pseudomonas fluorescens turned purple, so it is oxidase positive (Figure 25).

As expected in all laboratory experiments, this one had the possibility of human error. Mistakes could have been made by failing to sterilize the inoculating loop correctly, which would result in possible contamination of the sample. Another error could have been possibly occurred by mislabeling the plates according to species, which would produce invalid results. Finally, failing to inoculate the SIM tubes ? of the way to the bottom of the tube would result in the inability to observe whether or not the species is motile or not. Although this experiment went rather smoothly, there is always an opportunity for mprovement. An example of how this experiment could be made better is by testing more of the same microbes in each test. In Labs 7 and 8, many of the microbes used in the tests were not consistently present in each one. If the same bacteria were used, it would aid greatly in differentiating the same bacteria from one another and observing how metabolic and biochemical processes differ from species to species. This experiment and its results are important to the scientific community because they ultimately serve as a basis for further study of the subject.

By learning basic metabolism and biochemical tests used to differentiate microscopic organisms from one another, researchers can then develop more advanced and more specific tests that can further distinguish microbial species from each other. This will aid in discovering new microbes and different ways microbes react to certain factors. By doing so, researchers will have a better idea of how to distinguish helpful, potentially life-saving microbes from pathogenic or harmful ones. References

US Food and Drug Administration. Escherichia Coli. 5 Oct. 2006. . . Todar, Kenneth. Bacillus Cereus Food Poisoning. 2006. . . Schenectady County Community College. Proteus Vulgaris, P. Mirabilis.. . . European Bioinformatics Institute . Staphylococcus Epidermis Can Cause Infections in Wounds. 2006-2007. . . E Medicine . Excerpt from Enterobacter Infections. 1996-2006. . . European Bioinformatics Institute . Pseudomonas Fluorescens Is Being Researched as a Biological Control Organism. 2006-2007. . .

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