Lab 9 Puzzle with Two Sides.

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Lab 9 Puzzle with Two Sides. Purpose: The purpose of this lab is to have comprehension on how bacteria will grow in petri dishes. The purpose of growing bacteria in a petri dish with Agar is to see the types present and in what quantities. The purpose of using the Zigzag method is to reproduce the pure culture and increase the quantity . The purpose of doing gram staining lab is differentiate the two major classes of bacteria gram negative and gram-positive group by coloring cell red or violet. Gram stain is used test bacterial infection the test will portray if the patient has a gram positive or gram negative. A gram stain may be used to treat fungal infection. The result shows the classification like mold or yeast Hypothesis: In this lab I predict that in this lab I will be able to culture bacteria in a petri dish using the Zigzag method. I predict to successfully achieve bacteria colonies for gram staining by diluting the bacteria through 1-2,2-3,3-4,4 -41/2. I expect i will be able to observe, explain and differentiate bacterial arrangment and shape. I expect the bacteria to be gram negative due to the red color appearance, rod shaped and have a single arrangement. Introduction Dichotomous keys are a crucial scientific tool that distinguishes between various creatures based on their observable characteristics. Dichotomous keys guide users to the proper identification by providing a sequence of statements with two alternatives for each step (National Park Service, 2018). Dichotomous key help in identification process. When a gram positive or gram-negative bacterium is detected, the key then sends the user toward one group of questions and away from the other. By directing non-expert users to examine the well-known, significant creatures, dichotomous keys make it possible for them to identify organisms, which is greatly helpful. The ability to ask questions and provide valuable information based on results is crucial because many people may not know how to discern different types of bacteria based on arrangement cells or gram stain results, for example.The Greek words "di" for "two" and "tome" for "cutting instrument" are the source of the name. As the name implies, a dichotomous key uses a sequence of questions with two alternative responses to arrive at the answer for species identification. By eliminating, each response reduces the number of potential candidate species (BD Editors, 2017). Bergey's Manual of Determinative Bacteriology is one specific kind of dichotomous key for identifying bacteria. The Manual of Systematics of Bacteria and Archaea by Bergey For researchers working at the forefront of microbiology, the BMSAB is a vital tool. The most thorough and reliable explanation of bacterial and archaeal variety is found in Bergey's work (Wiley to Publish Bergey’s Manual of Systematics of Archaea and Bacteria, n.d.). Zig-zag growth is the technique used to grow bacteria. The gram stain method separates gram positive and gram negative groups by coloring these cells red or violet, which is how it differs from bacterium pigment. The reason gram positive bacteria stain violet is because their cell walls have

a thick layer of peptidoglycan, which keeps the crystal violet stain on the bacteria's surface. On the other hand, the reason why Gram negative bacteria stain red is because their peptidoglycan wall is thinner and does not hold on to the crystal violet color throughout the decolorization process. (Petersen & McLaughlin, 2021) Materials Required: Growing Bacteria in a Petri Dish 1. Petri Dish with agar (Hughes, 2020, “growing bacteria”). 2. Micro incinerator (Hughes, 2020, “growing bacteria”). 3. Inoculation loop (Hughes, 2020, “growing bacteria”). 4. Bacteria (Hughes, 2020, “growing bacteria”). 5. Marker. 6. Paper towel. 7. Water. 8. Soap. 9. spraying bleach solution. 10. Hibiclens. 11. Compound Microscope number 20 12. Slide (Hughes, 2020, “gram staining”). 13. Timer (Hughes, 2020, “gram staining”). 14. Distilled water (Hughes, 2020, “gram staining”). 15. Cristal violent (Hughes, 2020, “gram staining”). 16. Gram’s iodine (Hughes, 2020, “gram staining”). 17. Safranin (Hughes, 2020, “gram staining”). 18. Immersion oil (Hughes, 2020, “gram staining”). 19. Gloves. 20. Bleach solution in a spray bottle. 21. Paper towel. 22. Alcohol (Hughes, 2020, “gram staining”). 23. Bibulous paper (Hughes, 2020, “gram staining”). 24. Lens paper (Hughes, 2020, “gram staining”). 25. Slide rack (Hughes, 2020, “gram staining”). 26. Tongs (Hughes, 2020, “gram staining”). 27. Bunsen burner (Hughes, 2020, “gram staining”). 28. Striker Procedure : Growing Bacteria in a Petri Dish 1. When entering the lab ensure you have all your hair tied up, closed shoes to cover entire foot and have a protective clothing on top of your home cloths. Sterilize the working table by spraying bleach solution and wipe with a paper towel. 2. Remove jewelry that are on your hands to the wrist, roll up your sleeves to perform hand washing. Turn on the faucet and adjust the water temperature to your comfortable temperature, wet your hand with water and apply anti bacteria hand wash soap and scrub your hands to include 4 inch above the wrist. Scrubbing to remove the dirt for at least 1

minute then rinse the soap with running water. If you touch the sink or anything repeat the whole hand washing process and dry your hands with a paper towel and discard it to the correct bin and pick other clean paper towels and shut the faucet off and dispose the paper towels in the disposal bin. Do not touch your face, eye and mouth while working in microbiology lab. 3. Carefully put on your face mask and wear latex gloves. 4. Gather all the materials and instruments needed for the lab work i.e. petri dish from the refrigerator, Micro incinerator from the storage. 5. Turn on the Micro incinerator by plugging it on to the power using the power cable and switch it on at the on/off button which will turn the light on at the switch to show it is on and let it warm for 10 mins. You know it is ready to use when the inside has an orange glow. 6. Using a marker label the petri dish from the outside to read 1-2, 2-3,3-4 and 4- 41/2 in a square like shape at the far corners of the Petri dish with the numbers following each other at the corners (i.e. label 1 on the upper left corner, 2 at the left lower corner, 3 on the right lower corner and 4 on the upper right corner). 7. Sterilize your inoculating loop by inserting it at the Micro incinerator without touching any sides for 10 second until the wire turn orange then remove the inoculation loop from the micro incinerator and let it cool down for 30 seconds. Remove your petri dish that has the bacteria to culture from the incubator and carefully open it. Using your dominant hand, insert your sterile inoculating loop in the petri dish containing the concentrated colony and scoop a small sample, close it return to the incubator. Transfer the scooped bacteria to the labeled petri dish using the zig zag technique. Apply the scooped bacteria at the labeled petri dish number 1 along the line to number 2 using thin zigzag movements. (Hughes, 2020, “growing bacteria”). 8. When done drawing the zigzags from 1 to 2, sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. (Hughes, 2020, “growing bacteria”). 9. Using the sterile inoculating loop, dilute the bacteria by drawing a zigzag movement from number 2 to 3 by touching where the zigzag ended at the number 2 corner and draw along the line to corner number 3. Sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. 10. Using the sterile inoculating loop, dilute the bacteria further by drawing a zigzag movement from number 3 to 4 by touching where the zigzag ended at the number 3 corner and draw along the line to corner number 4. Sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. Using the sterile inoculating loop, dilute the bacteria by drawing a zigzag movement from number 4 to half way towards number 1 by touching where the zigzag ended at the number 4 corner and draw along the line midway between 4 and 1 to avoid contamination of the diluted bacteria and we will have achieved colonies. sterilize inoculating loop by inserting it to the micro incinerator for 10 seconds until the wire turn orange and remove it to allow it to cool down for 30 seconds. 11. Label on the petri dish and place in the incubator facing upside

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down to allow growth of bacteria downwards. 12. When done, switch off the micro incinerator and remove power cable from the socket putting it away to storage table. Discard appropriately any disposable waste materials from the lab work and put away other materials and equipment to appropriate storage at the end of the lab work. Then disinfect the working table by spraying bleach solution and wipe with a paper towel. 13. Remove the gloves and face mask and discard in the appropriate disposal bin and perform hand washing. Turn on the faucet wet your hand with water and apply anti bacteria hand wash soap and scrub up to 4 inch above the wrist, scrubbing thoroughly for at least 1 minute then rinse the soap with running water and apply a dime size amount of hibiclens, rub hands for 30 seconds then rinse it off. If you touch the sink or anything repeat the whole hand washing process and dry your hands with a paper towel and discard it in to the correct disposal bin. Use another paper towel to shut the faucet and dispose it. Roll down your sleeves and can leave the lab when done. Procedure: Gram staining 1 . When entering the lab ensure you have all your hair tied up, closed shoes to cover entire foot and have a protective clothing on top of your home cloths. Sterilize the working table by spraying bleach solution and wipe with a paper towel. 2. Remove jewelry that are on your hands to the wrist, roll up your sleeves to perform hand washing. Turn on the faucet and adjust the water temperature to your comfortable temperature, wet your hand with water and apply anti bacteria hand wash soap and scrub your hands to include 4 inch above the wrist. Scrubbing to remove the dirt for at least 1 minute then rinse the soap with running water. If you touch the sink or anything repeat the whole hand washing process and dry your hands with a paper towel and discard it to the correct bin and pick other clean paper towels and shut the faucet off and dispose the paper towels in the disposal bin. Do not touch your face, eye and mouth while working in microbiology lab. 3. Carefully put on your face mask and wear latex gloves. 4. Gather all the materials and instruments needed for the lab work i.e. petri dish from the refrigerator, Micro incinerator from the storage. Gather the instruments and materials to use i.e. the microscope from the storage shelf, slides, crystal violet, immersion oil, gram iodine, safranin, alcohol, timer and pencil. Pick microscope #20 from the shelve while moving the microscope to the lab table one hand holding the arm while the other holding the base. 5. Turn on the Micro incinerator by plugging it on to the power using the power cable and switch it on at the on/off button which will turn the light on at the switch to show it is on and let it warm for 10 mins. You know it is ready to use when the inside has an orange glow.

6. Prepare the slide by placing the slide on the slide rack, sterilize the inoculating loop for 10 seconds and let it cool for 30 seconds and run distilled water through it leaving a drop of water on the slide that you will transfer to the slide. Sterilize the inoculating loop. With the inoculating loop just touch on the bacteria colony without scooping the whole of it and mix it up to the drop of water at the slide and give it a good mix and separate the cells to the entire slide. Ensure your petri dish with the colonies is upside down. Slide should be turbid but can see through it. 7. Turn on the power button of the slide warmer. 8. Set up a Bunsen burner check the Striker is working properly by pushing down and over to give a spark turn the gas until you can hear it light the Bunsen burner using a Striker turn the flame down until you get two flames you have five second to light the Bunsen burner. If you take more than 5 second you have to switch the gas off wait for a minute before you try again. 9. With use of tong run the slide through the middle of the Bunsen burners flame 4 times and then slide will be heat fixed and safe to work with. 10. Place the prepared smear # 7 on the slide rack and flood the entire slide with crystal violet for 1 min. Gram positive has up to 40 layers peptidoglycan while gram negative has about 2-3 layers and cells will turn purple (Hughes, 2020, “gram staining”). 11. Slightly slant the slide on the staining tray rinse the slide with distilled water front and the back until the water becomes clear be careful you don’t interchange the back and the front (Hughes, 2020, “gram staining”). 12. Flood the slide with Gram’s iodine if it starts drying up add more iodine. Iodine attaches crystal violet into bacteria cell acting as mordant (Hughes, 2020, “gram staining”). 13. After 1 minute open and turn down and squeeze to have enough alcohol add alcohol wait 2-3 seconds as soon as its added to the slide start counting. Rinse the slide immediately with distilled water the more you wait it will take color out of gram positive. Alcohol takes the color out and gram positive have 40 layers it will take colors from couple of layers but not all so the gram positive will still look purple while gram negative will be colorless (Hughes, 2020, “gram staining”).

14. Apply the safranin to the slide make sure it doesn’t dry up. Leave it for 1 minute. safranin gets attached to fat in gram positive. Gram positive will be purple while gram negative will be red. Lean the slide on the staining tray rinse the slide and the gloves with distilled water front and the back until the stain is removed and water becomes clear (Hughes, 2020, “gram staining”). 15. Blot the distilled water off the slide with Bibulous paper. Be careful not to wipe the cells off the slide (Hughes, 2020, “gram staining”). 16. Put up the microscope the distance from the edge should be greater than 6 inch plug the power cable one end on the microscope and other end to the power source lower the stage make sure the objective is red. If the microscope objective is not red use nose piece to select red objective turn on micro scope (Hughes, 2020, “microscope”). 17. Using the coarse focus lower the stage. Insert the slide on the stage and fasten it with clips. Centre the stain into the light Adjust the stage to the highest level. 18. Begin to view your sample #7 the focus should be on scanning objective red use a fine focusing knob to view and use the iris to regulate the light. Start with a low-magnification objective to find the area of interest on your slide. Use the nose piece to turn to the next objective yellow use the fine adjustment to focus then move to the next objective blue once in focus turn in between blue and white objective add a drop of immersion oil right where the light hit the slide move the white objective in place and use fine knob to focus. Always use the correct lens when doing oil immersion as it will be damaged. 19. If you do not successfully view your slide under the oil immersion. Use the coarse knob lower the stage down move the objective between white and blue dab the oil out of white objective and check all other objectives and make sure they are free from oil. move the slide to a different spot using the nose piece turn from blue to yellow then to red objective start focusing from red objective then yellow, blue move the nose piece between blue and white add immersion oil then move to white and focus Once you have put oil in all spots the slide you cannot use it again you need to prepare another slide let the tutor know to provide another slide, go back to the beginning of the process and make another slide to view. 20. When done with microscope lower the stage remove the slide clean the microscope with

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lens paper use the nose piece to move objective from white to blue then to yellow then red. Dab the oil out from the oil immersion lens objective check all objectives to make sure they are free from oil use a new piece of lens paper each time you clean the objective. Immersion oil will penetrate and damage microscope components and objectives not suited for immersion. Remove excess oil using a lens cleaning tissue with a single sweep across the lens. Keep wiping the objective front lens with a clean piece of tissue for each wipe until no trace of oil. 21. Dispose the used slides to the sharp’s container, put away the other materials back to storage shelves. 22. Lower the stage turn off the microscope plug off the cable from the microscope and power source transport microscope 20 back to the shelf. 23. Then disinfect the working table by spraying bleach solution and wipe with a paper towel. 24. Remove the gloves and face mask and discard in the appropriate disposal bin and perform hand washing. Turn on the faucet wet your hand with water and apply anti bacteria hand wash soap and scrub up to 4 inch above the wrist, scrubbing thoroughly for at least 1 minute then rinse the soap with running water and apply a dime size amount of hibiclens, rub hands for 30 seconds then rinse it off. If you touch the sink or anything repeat the whole hand washing process and dry your hands with a paper towel and discard it in to the correct disposal bin. Use another paper towel to shut the faucet and dispose it. Roll down your sleeves and can leave the lab when done. Results: All possible results Based on the bacterial cell wall's unique staining characteristics, gram stain distinguishes between different types of bacteria. Gram positive bacteria are identified by their blue to purple staining, whereas gram negative bacteria are identified by their red to pink staining. This is due to differences in peptidoglycan layer thickness. With gram stain, there are four possible outcomes. Four types of gram reactions: gram positive, gram negative, gram non-reactive and gram variable. Based on the way the bacteria respond to the Gram stain, the categories are identified. They will either remain purple or turn pink or red. Bacteria are Gram-positive if they continue to be purple. The bacteria are Gram-negative if they turn pink or Red. The most prevalent forms of the bacteria on the slide are spherical (cocci) or rod-shaped (bacilli), with cocci appearing individually, in pairs, in groups of four, in clusters, or in chains (Gram Stain, 2021). In the event that the Gram stain test comes out positive, it indicates the presence of bacteria in your sample. If your test is positive, the results typically provide details regarding the type of organism that was on the sample slide. Such as, both gram-positive and gram-negative bacteria

are recognized. Bacteria can be spherical (cocci) or rod-shaped (bacilli). Additional features of the bacteria: size, proportionate amount (number), and/or organization, if any. Other cells whether red blood or white blood cells are present, as well as whether bacteria are intracellular in other cells. fungus: Gram stains can be used to detect the presence of molds or yeasts, which are examples of fungus (Cleveland Clinic, 2022). Atypical bacteria are either Gram-positive or Gram-negative bacteria that stay dry and do not change when stained with gram stain (Which Bacteria Cannot Be Gram Stained?, n.d.) Organisms that are gram-variable cannot be classified as either negative or positive. The presence of gram-positive or gram-negative staining organisms indicates the presence of living things in the smear. These organisms could either be harmful or not. In order to comprehend the results, more identification is needed (“Gram Stain: Reference Range, Interpretation, Collection and Panels,” 2020). F1gure 1 (Gram Stain: MedlinePlus Medical Encyclopedia Image, n.d.) Figure 2 Gram variable (Sermet et al., 2022). Conclusion: Restate Explain: Results Uncertainty Q1. Q2

References Sermet, K., Kipnis, E., Duployez, C., Wallet, F., Dessein, R., & Le Guern, R. (2022). Photo Quiz: Gram-Variable Staining in Anaerobic Bacteremia. Journal of Clinical Microbiology , 60 (1). Retrieved on November 1, 2023 https://doi.org/10.1128/jcm.00328- 21 BD Editors. (2017, April 28). Dichotomous Key - Definition, Examples, Types & Function | Biology Dictionary. Biology Dictionary. https://biologydictionary.net/dichotomous-key/ Cleveland Clinic. (2022, March 16). Gram Stain: What It Is, Purpose, Procedure & Results. Cleveland Clinic. https://my.clevelandclinic.org/health/diagnostics/22612-gram-stain Gram stain Information | Mount Sinai - New York. (n.d.). Mount Sinai Health System. https://www.mountsinai.org/health-library/tests/gram-stain Gram Stain. (2021, January 27). Testing.com. https://www.testing.com/tests/gram-stain/#:~:text=What%20does%20the%20test %20result Gram stain: MedlinePlus Medical Encyclopedia Image . (n.d.). Medlineplus.gov. Retrieved on, November 1 2023 from https://medlineplus.gov/ency/imagepages/19955.htm Gram Stain: Reference Range, Interpretation, Collection and Panels. (2020). EMedicine. https://emedicine.medscape.com/article/2093371-overview#a2 Hughes,M.(2020) Gram Stain Video . Www.youtube.com. https://www.youtube.com/watch? v=a_T8g8FJWto Hughes,M.(2020) Growing Bacteria in a Petri Dish . Www.youtube.com. https://www.youtube.com/watch?v=f5NnDsl0qs0

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National Park Service. (2018, January 9). Using Dichotomous Keys - Teachers (U.S. National Park Service). Www.nps.gov. https://www.nps.gov/teachers/classrooms/dichotomous- key.htm Petersen, J., & McLaughlin, S. (2021, April 29). Introduction to Staining. Biology LibreTexts. https://bio.libretexts.org/Courses/North_Carolina_State_University/ MB352_General_Microbiology_Laboratory_2021_(Lee)/04%3A_Staining_Techniques/ 4.01%3A_Introduction_to_Staining Which Bacteria Cannot Be Gram Stained? (n.d.). Byjus.com. https://byjus.com/question- answer/which-bacteria-cannot-be-gram-stained/ Wiley to publish Bergey’s Manual of Systematics of Archaea and Bacteria. (n.d.). EurekAlert! https://www.eurekalert.org/news-releases/487320

Lab 9 Puzzle with Two Sides.

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