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

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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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Dr. Crump – BIOL3400 Lab Lab 04 Our known bacteria seemed to be motile because it grew in a scattered, spreading pattern inside the tube. 4.4 MSA 10. You are going to be doing three spot inoculations on plates today with MSA. One for your unknown and two for other bacteria. What bacteria are you using? S. Aureus and E. Coli are the additional bacteria we're using for the MSA plates. 4.5 MacConkey 11. You are going to be doing three spot inoculations on plates today with MacConkey. One for your unknown and two for other bacteria. What bacteria are you using? The other two bacteria that we are using for spot inoculations are called E. Coli and M. Luteus. 5.13 Starch Hydrolysis 12. You are going to be doing three spot inoculations on plates today with an Amylase test. One for your unknown and two for other bacteria. What bacteria are you using? The other two bacteria we are using on the plates are called P. Vulgaris and M. Luteus. Lab Manual/Post-Lab Questions 4.4 MSA 13. Which type of bacteria will grow on this plate (gram – or +)? Explain why. On this plate, Gram-positive bacteria will grow. This is because Gram-negative bacteria, such as E. coli, are salt intolerant (non-halophilic), and because MSA has a high concentration of salt (NaCl), most bacteria will not form colonies on it. 14. Describe characteristic of the organism growing on this plate? How can you tell if the organism is able to ferment a sugar? Explain. The MSA will seek for microbes that can withstand high salt levels, such as Staphylococcus species. We can tell if an organism can ferment sugar because mannitol fermentation creates an 4

Dr. Crump – BIOL3400 Lab Lab 04 acidic byproduct that causes the phenol red in the agar to become yellow. Staphylococci that cause illness, including Staphylococcus aureus, ferment mannitol. 15. Suppose a mistake is made in preparing the MSA plates and the starting pH is 7.8. How would this affect the medium’s sensitivity and specificity? The indicator in mannitol salt agar (MSA) is phenol. The medium's pH is around 6.8, making it acidic in nature. Other parameters for preventing organisms from developing include pH and amino acid makeup. The medium's sensitivity is insufficient for identifying bacilli species, hence a reliable laboratory diagnosis will be impossible. If the pH is altered, bacteria cannot ferment, and the color shift from red to yellow does not occur. 16. How would the resulting plate look (after incubating) if you inoculated MSA using a species of Bacillus ? If we had infected MSA with a Bacillus species, the plate would not have changed color during incubation. The medium's sensitivity is insufficient for identifying bacilli species, hence a reliable laboratory diagnosis is impossible. 4.5 MacConkey 17. What are the active ingredients for MacConkey Agar? The active components include peptone, proteose peptone, lactose monohydrate, bile salts, sodium chloride, neutral red, crystal violet, agar, and distilled water. 18. What can you say about a bacterium that can grow on MacConkey agar? Because MacConkey agar not only selects for Gram-negative organisms by suppressing Gram-positive organisms and yeast, but also identifies Gram-negative species by lactose fermentation, the bacteria may be classed as Gram-negative. 19. How do you know if an organism can ferment sugar or not on this medium? It can be identified because the fermentation of this sugar results in an acidic pH, causing neutral red, a pH indicator, to turn a brilliant pinky-red. Lactose-fermenting bacteria, such Escherichia coli, form brilliant pinky-red colonies. 5.13 Amylase 20. Starch exists in 2 forms. What are they? The two kinds of polysaccharide forms are known as amylose and amylopectin. 5

Dr. Crump – BIOL3400 Lab Lab 04 21. What are the names of the 2 enzymes that break down starch? Starch and glycogen are broken down into glucose by two enzymes called amylase and maltase. 22. How can you determine if Starch hydrolysis has occurred? Iodine should be added to the agar to help comprehend the results of the starch hydrolysis test. Iodine mixes with starch to generate a dark brown color. As a result, starch hydrolysis creates a clean zone around the bacterial growth. 6.1 23. How would you produce a 10 -2 dilution of a 5mL bacterial sample using the full 5ml volume? I would get 495 milliliters of diluent. The calculation is 5.0 ml times 1 divided by 500 ml equals 1/100, or 0.01. 24. You have a 0.05ml of an undiluted culture at a concentration of 3.6X10 6 CFU/mL. You then add 4.95 mL sterile diluents. What is the dilution, and what is the final concentration of cells? The dilution is 1/100, or 0.01; the final concentration is 36000, or 3.6X104. 25. Plating 1.0ml of a sample diluted by a factor of 10 -4 produced 43 colonies. What was the original concentration in the sample? The initial concentration in the sample was 430000, or 4.3X105. 6

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