Harris Shaikh-Lab 1-Aseptic-Microscopy-Plating.docx

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Feb 20, 2024

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Dr. Crump – BIOL3400 Lab Lab 1 NAME: Harris Shaikh Lab 01 Worksheet Microscopy + Aseptic Techniques/Plating Throughout the semester, you will consult your lab book and Powerpoints to run tests on bacteria. Each week we will be learning new tests. The goal for Lab 1: To learn how to operate the microscope, how to ensure aseptic techniques to prevent contamination, and how to plate bacteria. While you will become familiar with the microscope in Lab 1, we will only discuss the information/theory/steps for aseptic technique and plating in preparation for Lab 2. Content for Lab 1: 1. Prior to Coming to Lab, you will need to: ● Read from lab book: See Lab Syllabus the exact readings ● Watch Videos (linked in Modules under Lab 1) ● Fill out the pre-lab questions in this worksheet 2. During Lab: ● We will discuss safety first ● I will demonstrate how to use the microscope and plate bacteria ● You will look at various specimens under the microscope ● Complete the worksheet 3. After Lab: ● Complete any questions not finished in the lab. You may type or hand-write the answers on the worksheet. ● Turn in the worksheet (via Canvas) by 1 PM on your respective lab day (M or W). Late lab worksheets will incur a penalty. Pre-Lab Questions (from videos/online): 1. What do clinical microbiologists do? In order to help in illness detection, clinical microbiologists conduct a variety of laboratory tests on samples taken from humans, plants, and animals. 2. What two things did the video not articulate that the lab book suggests (hint: 1.17 and 1.22). Why are these two things important? Using the vortex mixer and rotating the tube rather than the loop are the two suggestions made in the lab book that the video did not address. In addition, moving the tube has more control than moving the loop to ensure that the loop 1

Dr. Crump – BIOL3400 Lab Lab 1 never touches the tube's lip, which is to ensure that there is no contamination or the production of any dangerous aerosols. These two factors are important because bacteria can be suspended in a broth, and it's good to make sure the bacteria is fully throughout the broth and not at the very bottom so that bacteria would be easier to catch and the transfer will be more successful. 3. Why is it important to not pierce the agar when streak plating? What is the purpose of incubation? The other streak lines will be damaged, there will be an uneven application of inoculum at the damaged areas on the pierced agar plate, and there will be clustered growth of microbes that could get into the nearby streak lines. This is why it is crucial to avoid piercing the agar when streak plating. The goal of incubation is to encourage the growth of microorganisms while lowering the possibility that, for example, the bacteria may be harmful to people by keeping the plate at a temperature below body temperature. 4. Provide a few step process of proper microscope handling The following is the procedure for handling a microscope properly: use both hands to move the microscope to your workbench. (Not Just One!) -Put the microscope exactly in front of you on the bench, plug it in, and turn it on. Make sure the cord is not hanging off the side of the bench. (If the cord or plug is not secure, it might be extremely dangerous.) -To remove any grease from the stage, use a Kleenex or paper towel. Then, take a bacterium slide and insert it into the clips on the mechanical stage. (To ensure the slide is secure and to prevent it from falling off the microscope.) -Look through the eyepieces of a binocular microscope with both eyes, adjusting the light intensity to your comfort level. Lab Manual Questions 1.3 Aseptic 5. Provide a couple of examples of aseptic procedures and why they are important in the lab. Transfers from plate culture to sterile broth, agar slant culture to sterile agar slant, and broth culture to sterile broth are a few instances of aseptic operations. They are crucial to the lab because they may aseptically transfer germs from the source culture to a sterile medium and encourage development following such transfers, allowing microbiologists to examine the culture that was generated, conduct research, and benefit society. 2

Dr. Crump – BIOL3400 Lab Lab 1 6. What is the importance of a BSL-2 classification? The importance for a BSL-2 classification is that these organisms are extremely dangerous to work with in the lab because they produce aerosols, which can lead to contamination or infection through inhalation. We are usually unaware of the production of the aerosols and they remain suspended in the air long after the procedure has been completed. 7. Briefly walk through the procedure to transfer a culture to a slant. To transfer a culture to a slant, hold the loop like a pencil and flame it from the base to the tip, just like you would with a broth transfer. Like a broth transfer, loosen and remove the culture tube's cap. With the surface facing up, hold the culture tube at an angle. Burn the lip of the tube like a broth transfer. Pick up a tiny bit of growth by touching the sterile loop to the growth on the agar surface. Take care not to snag the loop on the tube's lip when you remove the tube from over the loop. Replace the cap and the lip of the flame tube. Place the vaccinated tube back onto the rack for test tubes. Make sure the loop and wire are evenly orange-hot by flame-testing them from base to tip. For the designated amount of time, incubate the inoculation culture at the designated temperature. 1.4 Streak 8. What is the difference between a mixed culture and pure culture? What is the purpose of a streak plate? The microbiological culture in a mixed culture consists of two or more species, whereas the microbial culture in a pure culture consists of just one species. 9. Name three types of plating techniques There are three different types of plating techniques: T-streak, quadrant streak, and plain zigzag (continuous streak). 10.What are CFUs and what role does streaking have in our understanding of CFUs? Colony-forming units, or CFUs, are made up of individual cells as well as pairs, chains, or clusters of cells. We may examine and identify the organisms that are in the CFUs by using streaking to help separate them. This contributes to our understanding of CFUs. 3.1 Microscopy 11. How do you determine the total magnification of a microscope objective? a. Ocular lens 15X and 10X objective lens 3

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Dr. Crump – BIOL3400 Lab Lab 1 Multiplying the magnification of the eyepiece by the magnification of the objective lens yields the total magnification of a microscope objective. 15X x 10X = 150X b. Ocular lens 15X and 45X objective lens 15X x 45X = 675X. 12.What is the function of the condenser? The condenser's job is to focus light and create more consistent lighting for the specimen. 13.What is resolution? Resolution is the clarity of an image. 14.What is numerical aperture? Numerical aperture is the amount of light that a lens can "capture" from the specimen and construct it to create an image. 15.What is the highest total magnification your lab microscope can achieve? The highest total magnification the lab microscope can achieve is around 1300 X. 16.Which objective is the immersion oil used with? What is the purpose of the oil? The objective the immersion oil is used with is 100 X, which is a 1000 X total magnification. The purpose of the oil is to increase its numerical aperture and, in turn, makes its limit of resolution smaller. In Lab questions 3-3/3-4 Eukaryotic cells 17.What are the major differences between Eukaryotic and Prokaryotic cells? One of the main distinctions between Eukaryotic and Prokaryotic cells, is that Prokaryotic cells lack mitochondria, but nearly all Eukaryotic cells don’t. Every eukaryotic cell has a nucleus and organelles that are all encased in plasma membranes; prokaryotic cells lack these organelles. Eukaryotes have more complex DNA and are greater in size. The cheek cells are larger and pigmented than the bacterial cells from the 4

Dr. Crump – BIOL3400 Lab Lab 1 pond water I saw, and the microorganisms in the pond water are tiny, transparent, and hardly distinguishable from one another. 18.Were you able to successfully mount your cheek cells on the slide with a stain? Describe what they look like. My cheek cells looked very small, and were not as abundant as other microorganisms were in other slides. They looked like they had a nucleus within each of the cells, with a outer layer protecting the cell. 19.What are Protists (describe them with examples)? Did you ID any microorganisms from pond water? Because they have other membrane-bound organelles and a nucleus, protozoa are classified as eukaryotes. Frequently unicellular, these creatures have the ability to form colonies. Protists include things like slime molds, euglena, amoebas, algae, and plasmodium. I could distinguish a rod shaped microorganism in the pond water; however, I was not able to further distinguish any other microorganisms. 5

Dr. Crump – BIOL3400 Lab Lab 1 20.What other organisms did you observe under the microscope? Select three and provide one specific feature of each. (you may also include drawings) Under a microscope, I also saw mixed protozoa eukaryotes, and vegetative spirogyra. The mixed protozoa have cilia all around the membrane, giving them a circular form. The multicolored, round, long rods or strings of mixed green algae are both membrane-enclosed. The spirogyra vegetative has a string-like shape, is brown, and is less colored. 6

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