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1 Name, Student Email Department of Biology, Florida State College at Jacksonville MICROBIOLOGY - MCB2010C Fall 2023/A15-2366 | 8/28/23 – 12/12/23 Dr Lahn Bloodworth

2 Table of Contents Lab 1: Light Microscope …………………………..……………………………………….…….…………………..3 Lab 2: Basic Principles of Aseptic Technique: Bacterial Transferring and Inoculating ………………………………………………………..…………………………………….……………………………………...5 Lab 3: Preparing a Bacterial Smear and Preparing a Simple Stain, Preparing a Gram Stain, and Preparing a Negative Stain………………...……………………………………………………………..…….6 Lab 4: Basic Bacterial Culturing Methods and Isolation of Pure Bacterial Cultures and Preparing a Streak Plate and Preparing a Spread Plate……….…………………………….…………...8 Lab 5: Preparing a Standard Plate Count of Bacteria…………………...…………………………...……9 Lab 6: Utilization/Fermentation of Carbohydrate, Protein, Catabolism, and Respiration Test in Bacteria……………….……………………………………………………………………….………………..…10 Lab 7: Differential and Selective Media and the Kirby-Bauer Procedure for Testing Antibiotic Sensitivity ..... ………………………………………………………………………………………….…...12 Lab 8: Epidemiology: COVID-19 PandemicReport………………………………….……………………..15 Lab 9:Biofilms: Antibiofilm Agents………………………………………………………………….…………….16 Lab 10 Conclusion: Identification of an Unknown Bacterium…………………………….………...17

3 LAB 1: Lab Microscopy PURPOSE : To learn the different parts and components of a light compound microscope and its functions. OBJECTIVES:  Learn how to identify the different parts of a light microscope  Learn how to properly clean clean a microscope  Learn how to properly observe microorganisms using the light microscope PROTOCOL : 1. Wash Hands 2. Don proper PPE 3. Disinfect work bench thoroughly 5. Get compound light microscope 6. Observe slides 7. Put light compound microscope back and cover 8. Disinfect work bench thoroughly 9. Doff PPE 10. Wash hands OBSERVATIONS: Escherichia Coli Gram -/+ Organisms Bacillus anthracis Typical cocci 400X 100X 40X 1000X LM LM LM LM

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5 Lab 2: Aseptic Technique and Inoculation PURPOSE: To learn how to ensure a sterile environment in a lab and prevent contamination to ensure the reliability of the experimental results. OBJECTIVE:  Learn to properly apply aseptic technique  Learn how to properly inoculate PROCEDURE: 1. Wash Hands 2. Don proper PPE 3. Disinfect work bench thoroughly 4. Obtain 1 of each of the following media: a. TSA plate b. TSA deep tube c. TSA slant tube d. TSB tube 5. Label a+b with S. marcescens (G-) and c+d with S. aureus (G+) 6. Inoculate the media respectively 7. Incubate the material at 37 o for 16-24 hours 8. Disinfect work area 9. Doff PPE 10. Wash hands CONCLUSION: TSA Slant TSA Plate TSA Deep TSB Tube

6 Lab 3: Preparing a Bacterial Smear, Preparing a Simple Stain, Preparing a Gram Stain, and Preparing a Negative Stain PURPOSE: To learn how to properly prepare a bacterial smear so that it will adhere the specimen to a slide to enhance the visibility of the bacteria under a microscope. To learn how to properly prepare a simple stain to increase the contrast between the organism and the background so that the size, shape, and arrangement of bacterial cells can be identified. OBJECTIVE:  Learn how to properly prepare a bacterial slide  Learn how to effectively apply a simple stain PART 1 PROCEDURE: SIMPLE STAIN 1. Wash Hands 2. Don proper PPE 3. Disinfect work bench thoroughly 4. Obtain 4 clean microscopic slides 5. Label two slides S. aureus and two slides E. coli a. Two gram stain b. Two simple stain 6. Add 1-2 drops of methylene blue to the smear and wait 120 seconds 7. Rinse with dH20 until it runs clear 8. Dab dry the slide and view under microscope PART 2 PROCEDURE: GRAM STAIN 1. Crystal Violet ➔ 90 seconds, rinse with dH20 2. Iodine ➔ 60 seconds, rinse with dH20 3. Decolorizer ➔ run through, rinse with dH20 4. Safranin ➔ 45 seconds, rinse with dH20 5. Dab the slide dry and then view under microscope 6. Disinfect work area 7. Doff PPE 8. Wash hands CONCLUSION:

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7 S. aureus simple stain E. coli gram stain Lab 4: Basic Bacterial Culturing Methods and Isolation of Pure Bacterial Cultures and Preparing a Streak Plate and Preparing a Spread Plate PURPOSE: To learn how to isolate a pure colony and to be able to distinguish the difference between a streak plate and a spread plate. OBJECTIVE:  Learn to accurately differentiate a streak plate and spread plate  Learn how to properly prepare a streak plate  Learn how to properly prepare a spread plate PROCEDURE: 1. Wash Hands 2. Don proper PPE 3. Disinfect work bench thoroughly

8 4. Obtain 2 TSA plates 5. Label both plates S. marcescens 6. Inoculate the first plate using the streak plate technique 7. SInoculate the first plate using the pread plate technique 8. Incubate both plates at 37 degree for 16-24 hours 9. Disinfect work area 10. Doff PPE 11. Wash hands CONCLUSION: No growth in the third quandrant and minimal growth in the second. Insufficient transfer form first quadrant. Lab 5: Preparing a Standard Plate Count of Bacteria PURPOSE: To be able to properly count colonies on plates. OBJECTIVE:  Learn how to calculate a bacterial sample  Learn hot to properly prepare a plate count PROCEDURE: 1. Wash Hands 2. Don proper PPE 3. Disinfect work bench thoroughly 4. Obtain 8 TSB tubes and 4 TSA plates 5. Label the tubes 10^-1 ➔ 10^-8 and label the plates 10^-6 ➔ 10^-9, S. marcescens 6. Prep a serial dilution using 0.1 ml S. marcescens 7. Plate the plates as below using 0.1 ml diluted S. marcescens

9 Tube Plate 10 -5 10 -6 10 -6 10 -7 10 -7 10 -8 10 -8 10 -9 8. Incubate the plates at 37 o for 16-26 hours 9. Disinfect work bench thoroughly 10. Doff PPE 11. Wash hands CONCLUSION: Lab 6: Utilization/ Fermentation of Carbohydrate, Protein Catabolism, and Respiration Test in Bacteria PURPOSE: To learn how to properly test for respiration, protein catabolism and fermentation in bacteria. OBJECTIVE:  To learn how to correctly identify different biochemical tests PROCEDURE: PART 1 1. Wash Hands 2. Don proper PPE 3. Disinfect work bench thoroughly 4. Obtain the following media: a. 4 Simmons Citrate Slant Tubes b. 4 SIM Deep Tubes c. 4 TSA Slant Tubes

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10 5. Divide all tubes into 2 sets ➔ Label first set E. coli I and the second set S. aureus 6. Inoculate the times accordingly 7. Incubate: a. Simmons Citrate Slants at 37 o for 7 days b. SIM Deep Tubes at 37 o for 4 days TSA Slant Tubes for 37 o for 24 hours 8. Disinfect work bench thoroughly 9. Doff PPE 10. Wash hands CONCLUSION: Simmons Slant E. coli Simmons Slant S. aureus Test performed S. aureus E. coli Simmons slant Simmons slant tube remains green: Negative test One Simmons slant test tube tested positive and the other Simmons slant test tube tested negative. H 2 S test H 2 S test tube did not change H 2 S had a positive test in which the medium had a black ferric sulfide precipitate. H 2 O 2 test H 2 O 2 test had a minimal reaction with some bubbles: Negative test H 2 O 2 test produced a lot more bubbles quickly: Positive test

11 H 2 O 2 Test H 2 S Test Lab 7: Differential and/or a Selective Media and the Kirby-Bauer Procedure for esting Antibiotic Sensitivity PURPOSE: To learn about the differential and selective media and the Kirby-Bauer (disk diffusion) test. OBJECTIVE:  Learn the differential/selective media  Learn the Kirby-Bauer (disk diffusion) method to test the effectiveness of antibiotics against bacteria PROCEDURE: PART 1 1. Wash Hands 2. Don proper PPE 3. Disinfect work bench thoroughly 4. Obtain 2 plates from: a. Blood agar b. McConkey agar c. Mannitol Salt Agar (MSA) d. EMB agar 5. Divide the plates into 2 equal halves 6. Inoculate the plates accordingly by using short streak technique

12 7. Incubate the plates at 37 o for 16-24 hours 8. Disinfect work bench thoroughly 9. Doff PPE 10. Wash hands CONCLUSION:

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13 PROCEDURE: PART 2 1. Wash Hands 2. Don proper PPE 3. Disinfect work bench thoroughly 4. Obtain 2 MHA plates 5. Label the 1 st plate E. coli and the 2 nd plate S. aureus 6. Inoculate the plates accordingly by using the lawning technique 7. Insert the antibiotics disks 7.Incubate the plates at 37 o for 16-24 hours 8. Disinfect work bench thoroughly 9. Doff PPE 10. Wash hands CONCLUSION: Lab 8 Epidemiology: COVID-19 Pandemic Report Antibiotics used and conc. S. aureus E. coli Ampicillin S / IZ(mm)= 35mm R / IZ(mm)= 15mm Clyndamycin S / IZ(mm)= 25mm S / IZ(mm)= 0mm Gentamycin R / IZ(mm)= 23mm R / IZ(mm)= 25mm Penicillin S / IZ(mm)= 38mm R / IZ(mm)= 0mm

14 During the initial outbreak of COVID-19 there was a disappointing lack of communication on all political levels between countries which could have potentially prevented enormous global outbreaks. I believe that the US, along with other countries, have not done a good job at preventing the transmission of the pandemic. China made the first mistake by silencing doctors who tried to warn the government of a “SARS like illness” outbreak (Forman). The US and many European countries failed by reopening their respective countries before shutting down again. Now that more people are more aware, of COVID, people have taken extra precautions to prevent the spread of COVID. Globally, we’ve started to slow the transmission of COVID, but there are still precautions we could take to slow the spread even more. According to the World Health Organization, the number of new cases for COVID have decreased by 55%, and the number of deaths have decreased by 34% as of September 29, 2023(covid). The 3 crucial strategies for intervention for the pandemic are making sure everyone is up to date on their vaccinations, testing for covid if needed, improving personal hygiene and living a healthier lifestyle. Having common sense is the biggest intervention for the pandemic. If you know you’re sick or if you know someone else is sick with COVID, stay away. Making sure you wash your hands and distance yourself from sick family members and friends. Forman R, Atun R, McKee M, Mossialos E. 12 Lessons learned from the management of the coronavirus pandemic. Health Policy. 2020 Jun;124(6):577-580. doi: 10.1016/j.healthpol.2020.05.008. Epub 2020 May 15. PMID: 32425281; PMCID: PMC7227502. "COVID-19 Epidemiological Update - 29 September 2023." World Health Organization,. Lab 9: Antibiofilm Agents

15 Epigallocatechin Gallate (EGCG) is a type of catechin, which is a class of flavonoids and is one of the major polyphenols found in green tea leaves. Green tea, scientifically known as Camellia sinensis, is known for its many properties, which vary from antiangiogenic and anticarcinogenic to antioxidant, antimicrobial, and anti- inflammatory. Green tea has also shown inhibitory effects on the spread of cancer cells to other parts of the body (metastasis). This may be attributed to the interference with certain cellular pathways involved in metastatic processes. Lantibiotics are a class of antibiotics that are characterized by the presence of unusual amino acids known as lanthionines. Lantibiotics exhibit antibacterial properties, often targeting specific types of bacteria . Lantibiotics work by altering the permeability of the membrane, which is accomplished by rupturing the membrane of bacteria in animals, preventing them from generating enzymes. Nisin is the most known and studied lantibiotic, and it is widely used as a food preservative and there has been some research exploring its potential against Streptococcus mutans, the bacteria associated with the formation of dental biofilms and main cause of tooth decay. Work Cited: Calway, Tyler et al. Epigallocatechin Gallate (EGCG) is the most effective cancer chemopreventive polyphenol in green tea, 2012 Nov 8;4(11):1679-91. doi: 10.3390/nu4111679. PMID: 23201840; PMCID: PMC3509513. Chaiyasut, Chaiyavat et al. A Review of the Role of Green Tea (Camellia sinensis) in Antiphotoaging, Stress Resistance, Neuroprotection, and Autophagy. Nutrients. 2019 Feb 23;11(2):474. doi:10.3390/nu11020474. Accessed 15 Nov 2023. Cheng, Yong et al. A review on anti-cancer effect of green tea catechins, Journal of Functional Foods, Volume 74, 2020, 104172, ISSN 1756-4646, https://doi.org/10.1016/j.jff.2020.104172. Accessed 18 Nov 2023. Deng, Dongmei et al. An In Vitro Study on the Effect of Free Amino Acids Alone or in Combination with Nisin on Biofilms as well as on Planktonic Bacteria of Streptococcus mutans . PLoS ONE 9(6): e99513. https://doi.org/10.1371/journal.pone.0099513. Accessed 16 Nov 2023. Mathur, H., Field, D., Rea, M.C. et al. Fighting biofilms with lantibiotics and other groups of bacteriocins. npj Biofilms Microbiomes 4, 9 (2018). https://doi.org/10.1038/s41522-018-0053-6. Accessed 12 Nov 2023.

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16 Lab 10: Unknown Bacteria By following the prokaryotic identification low chart and using the information given, our group concluded that our unknown sample is Staphylococcus aureus . The given information told us that the unknown bacteria culture was purple, indicating that it was gram-positive. Staphylococci and Streptococci are grouped as Gram-positive cocci, however, the presence of clusters and coccus shape led us to identify the bacteria as belonging to the Staphylococci genu s. Since the dichotomous key indicated that Staphylococci are catalase-positive, we chose not to conduct the catalase test. The next step was to perform a mannitol salt agar (MSA) test which is used to identify Staphylococcus aureus . Mannitol salt agar is a medium that contains 7.5% sodium chloride and supports the growth of Staphylococci. To perform the mannitol salt agar test, a small amount of the unknown bacteria is streaked onto a MSA plate. If the bacteria is Staphylococcus aureus , it will ferment the mannitol in the medium, which will cause the pH of the medium to decrease. This will result in a yellow color change in the bacterial growth on the medium. If there are no notable changes on the MSA plate, Staphylococcus epidermidis would be the likely answer. Using aseptic technique, a mannitol salt agar was inoculated with the unknown sample on 11/15/23 and incubated for 24 hours at 37°C. Upon observation on 11/27/23, colonies on the mannitol salt agar containing the unknown sample exhibited a yellow hue, indicating mannitol fermentation. This lead us to identify our unknown sample as Staphylococcus aureus according to the prokaryotic identification flow chart.

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