EMP1 Task 2 - Culturing Microbes LAB Report PDF

Preview

Summary

EMP1 Task 2 - CULTURING MICROBES LAB REPORT...

Description

Course: C453 EMP1 Task 2 - CULTURING MICROBES LAB REPORT Section I: Introduction Aseptic technique is a key laboratory skill in the field of microbiology. For carrying out procedures like moving cultures, inoculating media, seclusion of pure cultures, and executing microbiological tests, microbiologists use aseptic technique. To avoid adulteration of cultures from foreign bacteria inherent in the environment, precise aseptic technique is used. For example, microbes picked up from the researcher’s body, airborne microorganisms, the lab bench-top or other surfaces and microorganisms found on unsterilized glassware and equipment may possibly contaminate cultures, consequently interfering with the lab results. The risk of contamination can be eliminated greatly by using proper aseptic technique. Accurate aseptic technique helps to stop microbes used in the laboratory from accidentally being on the loose into the environment and/ or infecting people working in the laboratory. Section II: Materials and Methods Start activating yeast by dumping the packet of yeast into a disposable cup and then add 1/4 cup of warm water, shake it around, and let it sit for 10 minutes. Take 3 test tubes and label them as E. coli, Staph epidermidis and Saccharomyces cerevisiae (Yeast). We need E. Coli culture tube/via and S. Epidermidis culture via and activated yeast to grow the bacteria. Put them in nutrient broth found in bag number eight. Growing the cultures takes about 48 hours. Next, generate a streak plate of the three microorganisms. Use 2 nutrient agar test tubes, melt this agar by placing it in boiling water and then pour into 4 plates. Growing organisms on the streak plates will take about 72 hours. Once cultures and streak plates are ready we will move on to the actual Microbial Identification experiment. The first step before carrying out the Biochemical Testing for Microbial Identification—Methyl Red, Voges-Proskauer, and Catalase lab kit experiment was to put together the supplies. The supplies comprised of methyl red dye, Barrett's A and B reagents, MR-VP broths, empty test tubes, empty slides, hydrogen peroxide, the Staphylococcus epidermidis growth in test tube, and the Escherichia coli growth in test tube. Start by labeling the test tubes accordingly. Before executing the Methyl Red experiment, the contents in MR-VP broth test tube were split between the MR-VP broth test tubes and empty test tubes. The empty test tubes now containing half of the MR-VP solution were inoculated with S. epidermidis in test tube labelled Staph and E. Coli in other test tube labelled Coli. To conduct Methyl Red test on E. Coli, pour seven drops of methyl red reagent into the test tube labelled MR-VP containing E. Coli, then closely watch, you can see that the tube turned red. There was no incubation time needed to determine the results. Next to conduct Methyl Red test on S. Epidermidis, pour seven drops of methyl red reagent into the MR-VP tube containing S. Epidermidis, and then closely watch, you can see that the tube turned red. There was no incubation time needed to determine the results.

Start Voges-Proskauer test by labeling the test tubes accordingly. In the MR-VP broth test tubes containing half solution of MR-VP broth inoculated with S. epidermidis in test tube labelled Staph and E. Coli in other test tube labelled Coli. To conduct the test, pour twelve drops of Barrett's A reagent and four drops of Barrett's B reagent to the MR-VP test tube inoculated with E. Coli. Next, pour twelve drops of Barrett's A reagent and four drops of Barrett's B reagent to the MR-VP test tube inoculated with S. epidermidis. For this test you won’t get immediate results, wait for 30 minutes. At the end of 30 minutes you should get the same red color. But after 30 minutes you see that there is no color change, which means the test is negative. In the Catalase experiments E. Coli and S. epidermidis are used. Take two empty slides and label one as E Coli and other as Staph, next with a permanent marker make a circle target area. E. Coli and S. epidermidis were put on the appropriately labeled slides, then hydrogen peroxide was added as a reagent. Slides were viewed immediately to see if there was production of bubbles on the surface. We see bubbles, which are indicative of a positive result. In the Carbohydrate Fermentation experiment, S. epidermidis and Saccharomyces cerevisiae is used with the phenol red test tubes and small, empty Durham test tubes provided. Three sugars that we're testing will be glucose, fructose, and mannitol. Next, S. epidermidis and Saccharomyces cerevisiae were added to three test tubes, resulting in three phenol red test tubes containing S. epidermidis and three phenol red test tubes containing S. cerevisiae. Each of the three reagents, glucose, fructose, and mannitol, was added to each organism's separate test tubes, resulting one phenol red test tube of S. epidermidis containing fructose, second phenol red test tube of S. epidermidis containing glucose, and third phenol red test tube of S. epidermidis containing mannitol. The process was repeated for the three phenol red tubes that had S. cerevisiae resulting one phenol red test tube of S. cerevisiae containing fructose, second phenol red test tube of S. cerevisiae containing glucose, and third phenol red test tube of S. cerevisiae containing mannitol. Place small, empty Durham test tubes in each of the six phenol red test tubes. Incubate these for 24 hours to see if the solution had turned yellow or if any gas bubbles had formed. You can see that the solution has turned slight red in three phenol red test tubes containing S. epidermidis, so the test is negative. You can see that phenol red test tube containing S. cerevisiae with reagent fructose has turned slight yellow and there are gas bubbles, so the test is positive, whereas the rest two solutions with reagents glucose and mannitol has turned slight red, so the test is negative In the Motility experiment we use motility test agar. Label the test tubes accordingly. Place, E. Coli and S. epidermidis into separate motility test tubes. Take a pointed inoculation loop, minus the loop, flame it over the fire to sterilize it, and then gather some E. Coli from the plate containing E. Coli. Once the bacteria are on the inoculation loop, place the loop directly in a straight stabbing fashion, into the motility test tube, labeled E. Coli. Next gather some S. epidermidis from the plate containing S. epidermidis and place it into motility test tube labeled S. epidermidis. No reagents were used in this experiment. Wait 48 hours for incubation to see if bacteria have grown exactly where you placed it, or if it has moved.

Verification of Experiment

Steak Plates on 8/1/2021 (72 hours later Plated on 7/29/2021)

Methyl Red & Voges-Proskauer Experiments

Catalase Experiments

Carbohydrate Fermentation Experiments

Motility Experiments Section III: Results Methyl Red test on Escherichia Coli - When seven drops of methyl red reagent is added into the test tube containing E. Coli, the tube turned red. There was no incubation time needed to determine the results. Voges-Proskauer test on Escherichia Coli – When twelve drops of Barrett's A reagent and four drops of Barrett's B reagent is added to test tube inoculated with E. Coli. For this test you won’t get immediate results, wait for 30 minutes. At the end of 30 minutes you should get the same red color. But after 30 minutes you see that there is no color change, which means the test is negative. Catalase experiment on Escherichia Coli - Hydrogen peroxide was added as a reagent to the slide with E. Coli. Slides were viewed immediately to see if there was production of bubbles on the surface. We see bubbles, which are indicative of a positive result. Motility experiment on Escherichia Coli - Gather some E. Coli from the plate containing E. Coli. Once the bacteria are on the inoculation loop, place the loop directly in a straight stabbing fashion, into the motility test tube, labeled E. Coli. No reagents were used in this experiment. Wait 48 hours for incubation to see if bacteria have grown exactly where you placed it, or if it has moved. The E. Coli still looked the same after 48 hours of incubation, resulting in a negative test result. Bacteria, like E. Coli, are considered to have no flagella. Methyl Red test on Staphylococcus Epidermidis – When seven drops of methyl red reagent is added into the tube containing S. Epidermidis, the tube turned red. There was no incubation time needed to determine the results. Voges-Proskauer test on Staphylococcus Epidermidis – When twelve drops of Barrett's A reagent and four drops of Barrett's B reagent is added to test tube inoculated with E. Coli. For this test you won’t get immediate results, wait for 30 minutes. At the end of 30 minutes you should get the same red color. But after 30 minutes you see that there is no color change, which means the test is negative.

Catalase experiment on Staphylococcus Epidermidis - Hydrogen peroxide was added as a reagent to the slide with S. Epidermidis. Slides were viewed immediately to see if there was production of bubbles on the surface. We see bubbles, which are indicative of a positive result. Carbohydrate Fermentation experiment on Staphylococcus epidermidis – When each of the three reagents, glucose, fructose, and mannitol is added to three phenol red test tubes containing S. epidermidis and small, empty Durham test tubes placed in each of the three phenol red test tubes. Incubate these for 24 hours to see if the solution had turned yellow or if any gas bubbles had formed. We can see that the solution has turned slight red, so the test is negative. Motility experiment on Staphylococcus epidermidis - Gather some S. epidermidis from the plate containing S. epidermidis. Once the bacteria are on the inoculation loop, place the loop directly in a straight stabbing fashion, into the motility test tube, labeled S. epidermidis. No reagents were used in this experiment. Wait 48 hours for incubation to see if bacteria have grown exactly where you placed it, or if it has moved. The S. epidermidis still looked the same after 48 hours of incubation, resulting in a negative test result. Bacteria, like S. epidermidis, are considered to have no flagella. . Carbohydrate Fermentation experiment on Saccharomyces cerevisiae – When each of the three reagents, glucose, fructose, and mannitol is added to three phenol red test tubes containing S. cerevisiae and small, empty Durham test tubes placed in each of the three phenol red test tubes. Incubate these for 24 hours to see if the solution had turned yellow or if any gas bubbles had formed. You can see that the solution with reagent fructose has turned slight yellow and there are gas bubbles, so the test is positive, whereas the rest two solutions with reagents glucose and mannitol has turned slight red, so the test is negative. Section IV: Discussion To identify a bacteria, we use different biochemical characteristics of the bacteria. Based on their genetics, bacteria have different characteristics, and that determines what species and genes they belong to. Organisms that ferment glucose to glucose are encouraged to ferment the glucose which they can do that in several different pathways and we can use these differences to identify them. In the process of fermenting glucose, they're going to produce acids at the end. The methyl red test will tell us if that's true. After incubating for two to three days, add methyl red and see if color changes to red. If the color changes to red, that's MR positive, and this means that due to glucose fermentation the organism does produce mixed acids. Methyl Red test on Escherichia Coli - When seven drops of methyl red reagent is added into the test tube containing E. Coli, the tube turned red, that's means E. Coli is MR positive, and due to glucose fermentation the organism does produce mixed acids. Methyl Red test on Staphylococcus Epidermidis – When seven drops of methyl red reagent is added into the tube containing S. Epidermidis, the tube turned red, that's means S. Epidermidis is MR positive, and due to glucose fermentation the organism does produce mixed acids. The Voges-Proskauer test, tests for an alternate pathway. The Voges-Proskauer test is actually testing for the presence of butanediol. But you can't do it directly, so it's essentially detecting the presence of

acetoin. A color change to red means positive VP test, this indicates that due to glucose fermentation organism produces butanediol. Voges-Proskauer test on Escherichia Coli – When twelve drops of Barrett's A reagent and four drops of Barrett's B reagent is added to test tube inoculated with E. Coli. But after 30 minutes you see that there is no color change, which means the test is negative. This indicates that Escherichia Coli did not produce butanediol due to glucose fermentation. Voges-Proskauer test on Staphylococcus Epidermidis – When twelve drops of Barrett's A reagent and four drops of Barrett's B reagent is added to test tube inoculated with E. Coli. But after 30 minutes you see that there is no color change, which means the test is negative. This indicates that Staphylococcus Epidermidis did not produce butanediol due to glucose fermentation. We're observing to see if the organism produces catalase, in the catalase test. Organisms capable of living in an aerobic environment will produce catalase because hydrogen peroxide is produced as a byproduct, in using oxygen as part of metabolism. As hydrogen peroxide can be damaging to cells, so bacteria have to have a way to rid themselves of this hydrogen peroxide. The catalase converts hydrogen peroxide into oxygen and water, which is harmless. We should see bubbles when hydrogen peroxide is added as it converts hydrogen peroxide into water and oxygen. As oxygen is gas we will see bubbles when gas is released. So if we see bubbles, it’s indicative of a positive result. Catalase experiment on Escherichia Coli - Hydrogen peroxide was added as a reagent to the slide with E. Coli. Slides were viewed immediately to see if there was production of bubbles on the surface. We see bubbles, which are indicative of a positive result. So we can confirm the organism produces catalase. Catalase experiment on Staphylococcus Epidermidis - Hydrogen peroxide was added as a reagent to the slide with S. Epidermidis. Slides were viewed immediately to see if there was production of bubbles on the surface. We see bubbles, which are indicative of a positive result. So we can confirm the organism produces catalase. In Motility experiment we are trying to find if the organism is motile, whether the organism is capable of moving on its own or is it at the compulsion of the environment around it. The bacteria that are motile have flagella. Motility experiment on Escherichia Coli - Gather some E. Coli from the plate containing E. Coli. Once the bacteria are on the inoculation loop, place the loop directly in a straight stabbing fashion, into the motility test tube, labeled E. Coli. No reagents were used in this experiment. Wait 48 hours for incubation to see if bacteria have grown exactly where you placed it, or if it has moved. The E. Coli still looked the same after 48 hours of incubation, resulting in a negative test result. Bacteria, like E. Coli, are considered to have no flagella. Motility experiment on Staphylococcus epidermidis - Gather some S. epidermidis from the plate containing S. epidermidis. Once the bacteria are on the inoculation loop, place the loop directly in a straight stabbing fashion, into the motility test tube, labeled S. epidermidis. No reagents were used in this experiment. Wait 48 hours for incubation to see if bacteria have grown exactly where you placed it, or if it has moved. The S. epidermidis still looked the same after 48 hours of incubation, resulting in a negative test result. Bacteria, like S. epidermidis, are considered to have no flagella.

In carbohydrate fermentation we are testing whether the bacteria is capable of fermenting the sugars glucose, fructose, and mannitol. For this phenol red broth is used because they contain a pH indicator called phenol red and in the presence of alkaline conditions, phenol red turns red. So, they're more bases or neutral or basic pH, but in the presence of acids, it turns yellow. So, if it will turn yellow, we know that acids are produced as a byproduct of fermentation. Due to fermentation organisms can also produce gas. To see if the organism produces gas due to fermentation of the sugars we use little Durham tubes to collect the gas that is produced due to fermentation. So if the tubes color to yellow or we see a gas bubble in the Durham tube after incubation, then it would mean that the organism ferments that sugar. Carbohydrate Fermentation experiment on Staphylococcus epidermidis – When each of the three reagents, glucose, fructose, and mannitol is added to three phenol red test tubes containing S. epidermidis and small, empty Durham test tubes placed in each of the three phenol red test tubes. We incubate these for 24 hours to see if the solution had turned yellow or if any gas bubbles had formed. We can see that the solution has turned slight red, so the test is negative, and we know that acids and gas are not produced as a byproduct of fermentation of the sugars. Carbohydrate Fermentation experiment on Saccharomyces cerevisiae – When each of the three reagents, glucose, fructose, and mannitol is added to three phenol red test tubes containing S. cerevisiae and small, empty Durham test tubes placed in each of the three phenol red test tubes. We incubate these for 24 hours to see if the solution had turned yellow or if any gas bubbles had formed. You can see that the solution with reagent fructose has turned slight yellow and there are gas bubbles, so the test is positive, and we know that acids and gas are produced as a byproduct of fermentation of the fructose. Whereas the rest two solutions with reagents glucose and mannitol have turned slight red, so the test is negative, and we know that acids and gas are not produced as a byproduct of fermentation of the glucose and mannitol.

Section V: Sources

(1) Clinical Microbiology C453: Unit 7: Performance Assessment/Microbiological Techniques HTTPS://WGU-NX.ACROBATIQ.COM/ENUS/COURSEWARE/PAGE/WGU_C453_01MAY21_CLINICAL_MICROBIO_1/WBP_LESSON_1__PERFORMAN CE_ASSESSMENT_TASK_2...