Harris Shaikh - Lab 4-PlateCount-MSA-Mac-Starch

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Nova Southeastern University *

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3400

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Biology

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Feb 20, 2024

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Dr. Crump – BIOL3400 Lab Lab 04 NAME: Harris Shaikh Lab 04 Standard Plate Count, MSA, MacConkey, Starch Hydrolysis/Amylase 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 goals for Lab 4: ● To identify different bacterial properties and in turn be able to identify bacterial species, including your unknown, from differential and selective media ● To count colonies from last week’s serial dilutions Content for Lab 4: 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 4) ● Fill out the pre-lab questions in this worksheet 2. During Lab: ● I will teach how to plate bacteria on differential and selective media. ● See above under goals of what you will be doing. ● 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 two things do MSA plates have within them? How do each of those things impact the presence of colony growth and the resulting color change? MSA plates consist of two ingredients: sugar mannitol and neutral red, a pH indicator. This medium exclusively supports salt-loving bacteria, whereas bacteria that can ferment mannitol flourish on MSA, which has an impact on colony development. When an organism ferments mannitol, it produces an acidic byproduct, which turns the phenol red in the agar yellow. 2. Why is MacConkey’s agar considered to be both selective and differential? 1

Dr. Crump – BIOL3400 Lab Lab 04 Because it contains bile salts, which inhibit most gram-positive bacteria, MacConkey agar only works against gram-negative enteric bacteria. Lactose is also present, separating gram-negative lactose fermenters from gram-negative nonfermenters. 3. What colors are we looking for in the starch hydrolysis test? What do they represent? We are looking for blue, purple, or black in the starch hydrolysis test since these are the colors that Iodine takes on in the presence of starch, depending on the concentration. A clearing around the bacterial growth also indicates the presence of hydrolyzed starch. 4. What are differential and selective media types? How do they differ? Selective media promotes the development of a wanted organism while inhibiting or eliminating the growth of non-desired organisms. Differential media makes use of target species' biochemical characteristics, which frequently results in a noticeable shift as target organisms develop. In Lab questions 6.1 1. Describe the differences in appearance of your four plates. Do you see a gradient between the five? Yes, there is a difference between the four. The four plates have different appearances, with plate one having the most obvious development and the others being more difficult to identify. Plate four has a lower bacterial concentration. 2. Fill out the following chart related to the total dilution from the initial broth and the number of colonies you spot. Input the number of colonies counted. Feel free to section off the plates with a marker to make counting easier (like in 6.1). If you’re observing more than 300, make sure to write TNTC, or if you have less than 30 enter TFTC. Plates 2 3 4 5 Dilution from source broth 1/100 1/1000 1/10,000 1/100,000 Count TFTC TNTC TNTC TNTC 2

Dr. Crump – BIOL3400 Lab Lab 04 3. What are the average number of colonies estimated across the plates? Unable to calculate due to error in plate colony count. We were told to write TNTC, due to irregular colony growth. 4. Calculate the OCD. OCD=CFU/(DxV) 5. How does your fifth and last plate look compared to the others? The first plate looked to have no colony growth whatsoever. However, the last plate was described as having multiple colonies, which were growing at a sporadic rate. The other plates also had a growth rate consistent with their plate number and dilution. However, due to the errors implemented above, we didn’t have a proper rate of growth. 6. Did you have any issues last week with the dilution process? What mistakes did you make and how would those have influenced your results? (That’s ok if you made mistakes!) Yes, we made dilution errors because we unintentionally transferred too little fluid from one test tube to the next. This might have impacted our findings since there may not have been as much of a difference between the colonies on the first plate and the colonies on the last plate. This is because the bacteria's concentration may not be as low as predicted. 5.28 Motility 7. View the results of your motility test from the previous lab. Were you able to distinguish a difference between the S. aureus and E. coli tubes? Describe them. Yes, there was a difference between the E. coli and S. aureus tubes. The germs in the S. Aureus tube mostly persisted in one area. The motile bacteria had crowded the E. coli tubes, causing a dispersion of growth throughout the tube. 8. Which of the two organisms you used for the motility test appear to be motile? Does that correspond with the actual answer? (you can check online) Of the two species tested for motility, E. Coli appears to be more motile than S. aureus. This relates to the actual answer obtained on the internet. 9. Based on the results above, does your unknown bacteria seem to be motile? Why? 3

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