Harris Shaikh-Lab 2-Streak, Negative Stain, Collection.docx

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Nova Southeastern University *

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3400

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Feb 20, 2024

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Dr. Crump – BIOL3400 Lab Lab 2 NAME: Harris Shaikh Lab 2 Demo Streaking, Microbe Ubiquity, and Negative Staining 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 2: Collecting microbes, streaking them out on a plate, and performing a key staining technique for imaging under the microscope. Also, you will receive your unknown bacteria and begin to work with it. 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 2) ● Practice streaking a plate virtually (this link does not work with all browsers) http://learn.chm.msu.edu/vibl/content/streakplate.html ● Fill out the pre-lab questions in this worksheet 2. During Lab: ● I will demonstrate aseptic technique again, how to collect a sample, how to streak a plate, and how perform a negative stain. Also, I will introduce you to the unknown bacteria project/lab report. ● You will practice streaking a plate ( E. coli / S. aureus ), collecting 4 different specimens in the classroom in order to streak a plate, negative staining, and work with your unknown (using NSA plates). ● 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. Group/unknown plate number: Pre-Lab Questions (from videos/online): 1. Why is it important to smear the nigrosin on the slide for negative staining? It is crucial to smear the nigrosin onto the slide for negative staining because it will dry and serve as the background. Later, you will add the bacteria sample to the nigrosin stain and smear the entire slide, making it easier to see the bacteria against the dark stain background. 1

Dr. Crump – BIOL3400 Lab Lab 2 2. What makes extremophiles ‘extremophiles’? Provide two places on earth where they are found and why they live there. The ability to survive in harsh conditions when most other creatures would really perish is what distinguishes "extremophiles" from other types of organisms. The Gulf of California is home to an archaeal extremophile, Methanopyrus kandleri strain 116, which thrives at an extremely high temperature of 122 °C (252 °F, the highest temperature ever recorded). Methanopyrus kandleri strain 116 is able to survive there because it is able to obtain energy from hydrogen gas, which is present in volcanic vents such as the one located deep below the surface of the Gulf of California. Their second home is Antarctica, where they can withstand temperatures ranging from -272 °C and as high as 151 °C. These microscopic invertebrates, known as tardigrades, are eukaryotic polyextremophiles that exist wherever on Earth as long as there is fresh water available for them to do gas exchange and prevent uncontrolled desiccation. 3. Why is NASA interested in extremophiles? NASA is interested in extremophiles because they can help us comprehend the limits of Earthly life and help robotic probes and human explorers find evidence of life elsewhere in the universe. 4. Why does hand washing work in preventing the spread of viruses and bacteria? Why is generally preferred over low alcohol content based sanitizers? Hand washing is effective in stopping the transmission of germs and viruses because soap helps to mechanically remove microorganisms. Hand cleaning will therefore stop the infection from spreading to other individuals. It is typically chosen over sanitizers with low alcohol concentration because washing and water eliminates all kinds of bacteria from hands, whereas sanitizers work by eradicating specific bacteria from the skin. In Lab Questions 2.1 5. Today you are doing a Quadrant Streak Plate . You will use E coli or S aureus and may do several plates if you wish. What is the purpose of this plating technique? This plating method is used to demonstrate how simple it is to produce microorganisms. Additionally, performing the scientific method of streaking a sample of a bacterium and placing it on an agar plate so the bacteria can subsequently proliferate, divide, and flourish serves the purpose of allowing the bacteria to grow in a controlled environment. The ability to do research and examine the culture that has been produced comes last. 6. Briefly describe the procedure for this technique This approach involves heating the inoculating loop to a red temperature in the Bunsen burner in order to sanitize it. Let it cool. Using near parallel streaks, distribute an isolated colony from the agar plate culture throughout the first quadrant. Immediately, use a back-and-forth motion to gently streak the inoculating loop over a quarter of the plate. After flaming the loop once more, 2

Dr. Crump – BIOL3400 Lab Lab 2 let it cool. Extend the streaks into the second quarter of the plate (area 2) by going back to the edge of area 1 that you previously streaked. After flaming the loop once more, let it cool. Extend the streaks onto the third quarter of the plate (area 3) by going back to the region you recently streaked (area 2). After flaming the loop once more, let it cool. Extend the streaks into the central fourth of the plate (area 4) after going back to the place you recently streaked (area 3). Again, flame your loop. 7. What are the other streaking techniques and why would they be used instead of a quadrant streak? The lab handbook also mentions T-streak and a straightforward zigzag (continuous streak) pattern as other streaking methods. Since the T-streak method is a version of the quadrant streak and only involves three streakings, there is no specific benefit to utilizing it instead of the quadrant streak. Unlike quadrant streak, which is used to isolate a microorganism sample, basic zigzag (continuous streak) patterning is used when the sample has a low cell density and with pure cultures when isolation is not required. 8. Why is it important to not pierce the agar during this process? It is crucial to avoid piercing the agar during this procedure because doing so will cause damage to the other streak lines, a non-uniform inoculum to be applied at the damaged areas of the pierced agar plate, and clustered growth of microbes that could infiltrate the adjacent streak lines. 9. Do you estimate overall bacterial growth to change if these plates were left at 37°C for 2 days? What about 4°C for 2 days? Leaving these plates at 37°C for two days will affect the total estimate of bacterial growth. The ideal temperature is considered to be 37ºC, with the exception of several bacterial species. Because the plate is below the temperature of the human body, the development of microorganisms will be encouraged and fostered while the chance of the germs being harmful to people is reduced. Because it is so cold, bacterial growth will actually stall, stop, or slow down for the 4 degrees. The ideal temperature range for most bacteria to grow in is between 5 and 60 degrees Celsius. 3.6 10. What type of dye is used in the Negative stain? Is it acidic or basic (negative or positive)? The type of dye that was used in the Negative stain was nigrosin which is an acidic negative stain. 3

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Dr. Crump – BIOL3400 Lab Lab 2 11. What part of the cell is stained? Why? All parts of the cell are actually unstained. The bacterial surface's negative charge serves as a repellent to the negatively charged chromogen, preventing the cell from staining against the dye solution's colorful backdrop. 12. What are the advantages/disadvantages of doing a negative stain? Negative stain has the benefits of enabling morphology and cellular bacteria's size, shape, and arrangement in those that are too fragile to resist heat-fixing. Additionally, in cases when precisely identifying the size is essential, a negative stain might be used as there is less cell shrinking. A drawback of using a negative stain particle, develops as the negative stain gets twisted during the staining process. The particle sheds its hydration shell during the drying process. This shell frequently holds the soluble particle in place in a certain configuration, and it can alter form as it deposits on the carbon. Additionally, there are frequently a lot of air bubbles on the slide, which might be problematic when attempting to identify a new bacteria or even one with a circular form. Lab Manual/Post-Lab Questions 1.4 13. Which bacteria did you use for your quadrant streak plate(s)? Look up this organism and provide a few characteristics. Check with another person in lab to see if another group chose a different microbe. We’ll observe the differences next week. E. Coli , M. Luteus, and an unknown microbe were the two types of bacteria employed in the quadrant streak plates. E. Coli is a gram-negative, rod-shaped, non spore-forming bacteria that can be either nonmotile or motile with peritrichous flagella. M. Luteus is a Gram-positive to Gram-variable, nonmotile, tetrad-arranging, bacteria. We now don't know any traits or what to expect from the streak plate in regards to the unknown. 2.1 14. What three items did you swab in the classroom? Are you expecting the growth of these three swabs to look the same? Why or why not? The three different items that were swabbed during the lab were: the bathroom door, the garbage can lid, and the hallway air vent. Since all of the items were swabbed in various locations, and are exposed to different types of microbes, we are expecting different results for each of the swabs. This is primarily due to the different locations in which each swab was taken. Since the microbes near the air vent may travel more, we believe they may exhibit more growth, as compared to the swab of the garbage lid. 4

Dr. Crump – BIOL3400 Lab Lab 2 3.6 15. What organism did you choose for your negative staining? Check with another person in lab to see if another group chose a different microbe. If so, how does the appearance of yours differ from theirs? Is this confirmed by searching the appearance of each bacteria online? The organisms that I have selected for my negative staining are E. coli, M. luteus, and an unknown bacteria. Since I didn't consult with another group, I can't compare the variations in appearance or look up the unknown bacterium online to make sure it's the right one. 16. Think about your observations from last week- specifically using the microscope to examine the pre-prepared microbial slides. Does negative staining give a vastly different appearance than what you observed last week? Can you make out any cell structures? (in either this week or last week) It is true that the look of the negative staining is very different from what I saw last week. The mixed protozoa have cilia all around the membrane, giving them a circular form. The colorful, round, long rods or strings of mixed green algae are both membrane-enclosed. The spirogyra vegetative has a string-like form, is brown, and is less colored. These are significantly different from the negative stain slide of my unidentified bacterium since the latter is extremely tiny and lacks color. 5

Dr. Crump – BIOL3400 Lab Lab 2 Unknown plate 17. Describe the appearance and color of your unknown plate The color of my unknown plate is a yellowish, or gold color, depending on the lighting. The appearance is thick with a vast spread of colonies across the plate. There was a lot of bubbling as well that spread across the unknown plate. 6

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