Unknown Bacteria lab report PDF

Preview

Summary

Download Unknown Bacteria lab report PDF

Description

Elizabeth Heicher Dr. Jabbour, W315A November 22nd, 2020 Identification of Unknown Bacteria Lab Aims: The main focus of this week’s lab was to determine the unknown bacteria. This was done by preforming multiple tests. Each student performed the Gram staining process and the streak plate method as well as a few more different tests. All of the procedures were done to identify the culture within the lab. This lab was done over a three-week period. Methods and Materials: Materials: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

24-hour unknown bacterial culture broth Bunsen burner Inoculating loop Sharpie pen Gloves NA plate MAC plate UTI filter EMB plate Hydrogen peroxide

Methods: 1. The first step was to obtain the unknown culture, one nutrient agar plate, and one MacConkey agar plate. Then it was important to record the number of the unknown bacterium as well as the temperature the bacteria would be incubated at. 2. After recording the number, by using aseptic techniques, the streak plate method can be done. The plate was then incubated at 25°. 3. The next week the gram staining process was used. By using aseptic techniques 2-3 loopfuls of water was transferred onto the slide, then a colony was spread onto the slide to form a thin film. 4. The bacteria were left to air dry. Once dry, heat fix was done by passing the slide 5 times over the flame. 5. The bacteria was then covered with crystal violet and incubated for 1 minute. After 1 minute the slide can be rinsed with DI water and then Iodine can be added and left to sit for 1 minute. 6. The next step was to decolorize by rinsing the slide with alcohol for 2-3 seconds and immediately rinsed with DI water.

7. Finally, the bacteria was covered with Safranin and incubated for 1 minute. After 1 minute, the slide was rinsed with DI water and blotted dry.

8. Once the whole staining process was done the bacteria would indicate whether it was gram positive or gram negative. In this case the bacteria was gram negative. 9. After deciding the bacteria was gram negative it was decided whether there was lactose fermentation or not. For this specific unknown there was no lactose fermentation. If there was lactose fermentation the colonies would produce a color change. 10. Since there was no lactose fermentation the Oxidase test was done to indicate the presence of the oxidase in the bacteria. Bacteria that react with the strips will turn purple indicating the presence of oxidase. The bacteria had no color change. 11. The EMB streak indicated that the bacteria was a non-fermenting bacteria by streaking the plate. 12. Finally, the UTI test was used to allow the gram (-) bacteria to grow. Then color changes could be observed again. The bacteria remained negative with no color change.

Results Table 1. Data obtained from MacConkey Plate Gram Negative Red

Yes

No

Table 2. Steps for Gram Negative Bacteria

+

Growth was seen on the MAC plate

-

After doing the gram staining process, the staining produced a negative result

-

The bacteria grown on the plate was colorless, indicating there was no lactose fermentation

-

-

The oxidase test was done to see if there was any color change. For this particular unknown there was no color change. Streak EMB was used to isolate the gram (-) bacteria. The bacteria had no color change. The UTI test was used to allow the gram (-) bacteria to grow. Then color changes could be observed again. The bacteria remained negative with no color change.

Discussion Bacterial identification is important when it comes to analyzing a specific bacteria. Since bacteria are ubiquitous and can be found on multiple surfaces it is critical to be able to have an understanding of what different bacteria may be considered. Bacteria found on the human body are considered to be microbiota, which are microorganisms that inhabit the human body. Bacteria

can cause disease within the body so being able to identify and isolate the bacteria is essential. There are a few ways to identify bacteria. Selective media can be used to allow the growth of certain organisms while inhibiting the growth of other organisms. Differential media can be used to allow the growth of multiple organisms, but researchers are still able to differentiate between the organisms. Biomedical tests can also be used to recognize chemicals and enzymes produced by bacteria. The rapid ID membrane allows for quick identification of the specified bacteria. The results of each test helped to bring the identification of the unknown closer. The first method was to streak the agar. After streaking the agar, it was found that the plate contained growth which meant that further tests needed to be done. Once the streaking was done the bacteria needed to be stained. By doing the gram staining process, the bacteria produced a gramnegative result. Any lactose fermentation was identified. The bacteria did not contain any lactose fermentation since the colonies were colorless or transparent and took on the color of the medium. An oxidase test was then done to indicate the presence of cytochrome c oxidase which is used in the electron transport chain. Bacteria that react with the strips will turn purple which indicates that oxidase is present. Unfortunately, oxidase was not present because of the lack of a color change. Since Eosin Methylene Blue (EMB) is selective, only gram-negative bacteria is allowed to grow on it. After doing the EMB streak there was growth on the plate. Finally, the UTI test was used to allow the gram-negative bacteria to grow. The bacteria remained negative with no color change. Each test and method were used to identify unknown bacteria. The identified unknown bacterium was found to be S. marcescens. Possible errors could have happened throughout the lab. Errors could have caused colonies to not to grow on the plate or colonies to grow on the plate if proper aseptic techniques were not used. Incubating the plates for too long or not enough time could have changed the

growth as well. Mistakes could have been made when doing each specific test as well. Cross contamination is possible if not properly labeling the items and not heating the loop before taking a sample. If errors did occur it was important to run another test to ensure that all data and results are correct. Conclusion In this lab we were able to become more familiar with the process of identifying unknown bacteria and determining the genus and species of the bacteria. Different methods and tests can be used to identify unknown bacteria and verify it as well. I can take into consideration all that I have learned from this lab and apply it by understanding how important it is to run many different tests to identify bacteria. By doing this lab I have learned that there is always a chance for misleading results due to error. The best thing to do is to run more tests and verify that the results are correct. This will be very helpful and important for my future when performing experiments and analyzing results....