MCB2010C_LabNotebook

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Page 1 Lab Notebook Nicole Linebaugh S3814860@students.FSCJ.edu Department of Biology, Florida State College at Jacksonville MCB2010C - MICROBIOLOGY Fall 2023/ 2360Lec/Lab 09/18/2023- 12/12/2023. Dr Lahn Bloodworth December 9, 2023

Page 2 Table of Contents Week 1 Wednesday 20 September 2023………………..……………………………………….…….…3 Greeting, syllabus discussion, course introduction Canvas: Tour & Brief Introduction to the Class Operation Pearson’s MyLab&MasteringMicrobiology Orientation Study Guide for Success in Microbiology Current Issue in Microbiology: Film: Seven Wonders of the Microbe World Lab Notebook/Lab Report GuideLine Film: Introduction to Microbiology Laboratory Safety Lab Operation Discussion Bio-lab Safety Level 1 & 2 (BSL-1 & BSL-2) Guide (Canvas) Lab α: The control of microbial growth practice: Preparation of work area Week 2 Wednesday 27 September 2023………………..…………………………………….…….…5-8 Lab 1: Observing microorganisms through a microscope Effective use and responsible care of the light microscope Obtaining digital images of microscopic views/slides Lab 2: Basic principles of aseptic technique: Bacterial transferring and Inoculating Week 3 Wednesday 4 October 2023………………..……………………………………….…….…8-13 Lab 3: Preparing a bacterial smear Preparing a simple stain, preparing a gram stain, and preparing a negative stain Lab 4: Basic bacterial culturing methods & Isolation of pure bacterial cultures & Preparing a streak plate & Preparing a spread plate Week 4 Wednesday 18 October 2023………………..……………………………………….…….…13-15 Lab 5: Preparing a standard plate count of viable bacteria Lab 6: Utilization of carbohydrate/Fermentation of carbohydrate Degradation of amino acid/Protein catabolism Respiration test

Page 3 Week 5 Wednesday 1 November 2023………………..………………………………….…….…16-18 Lab 7: Differential and selective media & The Kirby-Bauer procedure for testing antibiotic sensitivity Lab 8: Epidemiology: COVID-19 Pandemic Report Week 6 Wednesday 15 November 2023………………..………………………………….…….…18-20 Lab 9: Biofilms: Antibiofilm Agents Lab 10: Identification of an unknown bacterium

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Page 4 Week 1 Wednesday 20 September 2023 Greeting, syllabus discussion, course introduction Canvas: Tour & Brief Introduction to the Class Operation Pearson’s MyLab&MasteringMicrobiology Orientation Study Guide for Success in Microbiology Current Issue in Microbiology: Film: Seven Wonders of the Microbe World Lab Notebook/Lab Report GuideLine Film: Introduction to Microbiology Laboratory Safety Lab Operation Discussion Bio-lab Safety Level 1 & 2 (BSL-1 & BSL-2) Guide (Canvas) Lab α: The control of microbial growth practice: Preparation of work area

Page 5 Week 2 Wednesday 27 September 2023 Lab 1: Observing microorganisms through a microscope Effective use and responsible care of the light microscope Obtaining digital images of microscopic views/slides Objectives: Proficient Utilization and Ethical Handling of the Light Microscope Acquiring Digital Images of Microscopic Views/SlidesAims: Appropriate and Responsible. Purpose: Proper & effective use & functions of light microscope (LM). View and take photo of at least 4 slides with camera in (4x, 10x, 40x, 100x lenses) Proper microscope storage Aseptic wipe before and after Page 6 Provide at least 4 labeled digital images of 4 different slides taken under 4X, 10X, 40X and 100X objective lenses. Figure 1: Escherichia coli (Gram- stain 4X).

Figure 2: Escherichia coli found in humans (Gram-stain 10X) Figure 3: typical cocci bubbles (Gram- stain 40X) Figure 4: bacillus anthracis (Gram-stain 100X)

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Page 7 Lab 2: Basic principles of aseptic technique: Bacterial transferring and Inoculating Objectives: The objectives of this experiment are to apply knowledge of aseptic techniques in order to transfer and inoculate bacterial cultures without introducing contaminating microbes, as well as to study the migration of bacterial cells by observing. Protocol: 1. Obtain the following materials: - 1x TSA plate - 1x TSA slant tube - 1x TSA deep tube - 1x TSB tube 2. Label each media with "S. marcescens" to indicate the specific bacteria being used. 3. Inoculate each media accordingly, using aseptic techniques to prevent contamination. 4. Incubate the inoculated materials at a temperature of 37 for a period of 16-24 hours. ℃ 5. Observe the samples in the next lab session to study the migration of bacterial cells. Observations (Wednesday 4 October 2023): 1. Verify if there is any presence of contamination. 2. Examine the appearance and growth patterns of the colonies. 3. Distinguish between the microbial growth observed in liquid media versus solid media. 4. Capture photographs of the outcomes for documentation purposes 5. Seek clarification or inquire about any uncertainties or concerns that arise. a. The TSA plate appears as a pink or red medium with abundant growth in each colony. Each colony is round in shape, but slightly irregular. There is condensation present on the lid, and the culture adheres well to the agar surface with colonies being slightly raised. b. In the TSA slant tube, the medium has a clear to tan color and exhibits a texture similar to that of the plate. The colonies are less dispersed and tend to be more clumped together. The medium itself appears milky, cloudy, and opaque. c. Within the TSA deep tube, there is cellular growth primarily concentrated in the middle portion of the tube. This growth extends downwards with shades of red and pink toward the top. d. To examine the TSB tube, gently lock it with fingers and tap lightly to mix its contents. Look for cloudiness within the medium, which may appear slightly milky, cloudy, clear, or tan. Upon mixing, the culture becomes cloudy and eventually settles

as sediment at the bottom of the tube. A faintly pink ring of growth can be observed near the meniscus of the tube. Page 8 Week 3 Wednesday 4 October 2023 Lab 3: Preparing a bacterial smear. Preparing a simple stain, preparing a gram stain. Preparing a negative stain Lab 3: Part 1 Purpose/Objective: Photograph 4 slides (2x S. aureus and 2x E. coli) using a simple stain to observe the morphology (size, shape, cellular arrangement) of the bacteria. Protocol: materials: a. 2x beakers of dH2O b. Methylene blue c. Microscopic slides Lab 3: Part 2: Preparing a Simple Stain. Purpose/Objective: The purpose of photographing the 4 slides containing S. aureus and E. coli is to visually capture the morphology, including size, shape, and cellular arrangement, of these bacteria using both gram stain and simple stain techniques. By documenting the appearance of these bacteria under different staining methods, we can gain a better understanding of their characteristics and distinguish between them. 2. Add 1-2 drops of methylene blue to each smear on the slides, enough to cover them. 3. Wait at room temperature (RT°) for 120 seconds for the dye to stain the bacteria. 4. Rinse the slides with dH2O until the water runs clear. 5. Dab dry the slides using a paper towel or bibulous paper. 6. View the stained slides under a light microscope (LM).

S. aureus should have a blue stain”on t’e smear of the prepared slide, indicating that it has taken up the methylene blue dye. Page 9 Lab 3: Part 3. Preparing a negative stain The purpose/objective of the protocol is to differentiate Gram-positive (Gram+) bacteria from Gram-negative (Gram-) bacteria. This differentiation is based on the differences in their cell wall composition and staining properties. The protocol must be followed in a specific sequence to ensure accurate results: 1. Crystal violet: Apply crystal violet stain to the slide and let it sit for 90 seconds. Then, rinse the slide with distilled water to remove excess stain. 2. Iodine: Apply iodine solution (also known as Gram's iodine) to the slide and let it sit for 60 seconds. This step helps to form a complex between the crystal violet and iodine, which enhances the retention of the stain within Gram+ bacteria. Rinse the slide with distilled water after this step. 3. Decolorizer: Gently run a decolorizing agent, such as acetone or ethanol, over the slide until no more purple color is being washed off. This step removes the crystal violet-iodine complex from Gram- bacteria, making them appear colorless. 4. Safranin: Apply safranin stain to the slide and let it sit for 45 seconds. Safranin is a counterstain that stains any remaining colorless cells pink or red. 5. Dab dry the slide: Carefully blot the excess moisture from the slide using blotting paper or a clean cloth. 6. View under LM: Place the prepared slide under a light microscope (LM) and observe the stained bacterial cells. Gram+ bacteria will appear purple/violet due to retaining the crystal violet stain, while Gram- bacteria will appear pink/red due to taking up the safranin counterstain. It is important to note that this protocol should be performed with caution and following proper safety guidelines, including wearing appropriate personal protective equipment (PPE).

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Page 10 Lab 4: Basic bacterial culturing methods & Isolation of pure bacterial cultures & Preparing a streak plate & Preparing a spread plate Objective: The purpose of this protocol is to demonstrate the procedure for isolating a pure colony and to distinguish between the streak plate and spread plate methods.

Page 11 Page 12 Protocol: Each individual should follow these steps: 1. Obtain two TSA plates. 2. Label both plates as S. marcescens. 3. Inoculate the first plate using the streak plate technique. 4. Inoculate the second plate using the spread plate technique. 5. Incubate both plates at 37 for 16-24 hours. ℃ 6. Observe the results in the next lab session on 10/18/23. Streak Plate Method: 1. The specimen can be in liquid or solid form (if it's solid, mix it with a small amount of liquid such as urine, feces, blood, saliva, or skin). 2. Each streak performed dilutes the specimen. 3. This method does not require a large amount of culture. 4. It is a quick and convenient method. 5. The streaks appear isolated; inspect purity by observing only once in the test tube; use a new sterile loop each time and start streaking from the distal edge of the plate towards the center. 6. This method is practical when testing patients with swabs and slant tubes because each streak mechanically separates colonies for identification. Lab 4 Observations on October 18 th , 2023: 1. Consult the lab manual for guidance. 2. Are there any signs of contamination? Consider the differences in bacterial growth patterns between streak plates and spread plates, and determine which method to use and why. 3. Carefully examine the colonies for characteristics such as color, uniformity, edge, margin, etc. 4. Capture images of the plates using a camera or other suitable device.

Page 13 Week 4 Wednesday 18 October 2023 Lab 5: Preparing a standard plate count of viable bacteria. Objective/Purpose: The objective of this experiment is to count the number of colonies on plates containing 30-300 colony forming units (CFUs). Protocol: Each group will perform the following steps: 1. Obtain 8x tubes of TSB (tryptic soy broth) and 4x TSA (tryptic soy agar) plates. 2. Label the tubes as 10^-1 through 10^-8 and label the plates as follows: - Tube labels: 10^-6 through 10^-9 - Plate labels: 10^-5, 10^-6, 10^-7, 10^-8, 10^-9 3. Perform a serial dilution by adding 0.5mL of S. marcescens to each tube. 4. Plate the plates using a .1mL dilution from each tube as follows: Tubes: Plate labeled 10^-5: Use .1mL from tube labeled 10^-1

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Plate labeled 10^-6: Use .1mL from tube labeled 10^-2 Plate labeled 10^-7: Use .1mL from tube labeled 10^-3 Plate labeled 10^-8: Use .1mL from tube labeled 10^-4 Plate labeled 10^-9: Use .1mL from tube labeled 10^-5 5. Incubate the plates at a temperature of 37 for a duration of 16-24 hours. ℃ 6. Observations will be made during the next lab session scheduled for November 1 st , 2023. Page 14 Observations on November 1 st , 2023: Divide the plates into Quadrants then count each quadrant of the colonies If it's uniform, then count the one quadrant and multiply by four. Clump distribution of the colonies. Count everything on the 30-300 colony plates. Dilution must always be negative. The dilution Factor must always be positive. the equations of CFU/ML= # of colonies/ dilution= For example: 280/10^-7= 280*10^7= 2.8*10^9CFU/ML. Lab 6: Utilization of carbohydrate/Fermentation of carbohydrate Degradation of amino acid/Protein catabolism Respiration test Objective/Procedure: The objective of this experiment is to test for respiration, protein catabolism, and fermentation in bacteria. Each group will perform the following protocol: obtain four test tubes - Simmons Citrate Slant, SIM Deep, TSA Slant; divide the tubes into two equal sets; label the first set as E.

coli and the second set as S. aureus; inoculate the tubes accordingly; incubate the tubes at specific temperatures and durations @37C. A. Simmons Citrate Slant: 7 days B. SIM Deep: 4 days C. TSA Slant: 24hrs Page 15 Carbohydrate utilization involves glycolysis, where glucose is broken down into two pyruvic acid molecules along with ADP and ATP. These pyruvic acid molecules can be used in either cellular respiration (with oxygen) or anaerobic cellular respiration (without oxygen) or fermentation. Fermentation occurs in the absence of oxygen and does not involve the Krebs cycle or electron transport chain. Two types of fermentation include alcohol fermentation, which produces ethanol and CO2, and lactic acid fermentation, which produces lactic acids along with small amounts of byproducts. Fermentation results in the production of 2 ATPs, while cellular respiration can produce 36 or 38 ATPs. Lab 6 Observations: Check for Contamination. Check for Growth. Interpret: + test/ - test Bubbles formed vigorously and immediately are considered a positive test. Bubbles formed small and slowly are considered a negative test..

Week 5 Lab 7: Differential and selective media & The Kirby-Bauer procedure for testing antibiotic sensitivity Objective: The aim of this study is to gain knowledge and understanding about the use of specific types of media that can differentiate and select different bacterial species, as well as the Kirby-Bauer test for assessing antibiotic sensitivity in bacteria. Page 16 Protocol: Each group perform the following: 1) Obtain 2 plates of the below: a. MacConkey agar b. Mannitol salt agar c. EMB agar 2) Divide the plates -> 2 equal sets. 3) Label 1st set: E. coli, 2nd set: S. aureus.

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4) Inoculate the plates accordingly by the short streak technique. 5) Incubate the plates at 37F/ 24hrs. 6) Observe Next Lab Lab #7: Disk diffusion Purpose/ Objective: Protocol: Each group perform the following: 1) Obtain 2 MHA plates. 2) Label 1st plate: E. coli. 2nd plate: S. aureus. 3) Inoculate the plates by LAWNING Technique. 4) Incubate the plates at 37F/ 24hrs. 5) Insert antibiotic disks. 6) Observation next lab Lab 7: Observations: 1) Part 1: Biochemical Test a) Is there contamination? b) How should we interpret the results of the tests? 2) Part 2: Kirby-Bauer Technique a) Measure the size of the zones of Inhibition In millimeters. b) Determine if the microorganism is sensitive, resistant, or intermediate based on the conclusion. Lab 8: Epidemiology: COVID-19 Pandemic Report (Canvas) Page 17 Questions What was the purpose of the ceftriaxone? The tetanus toxoid? The primary aim of administering ceftriaxone is to prevent or treat any potential infections that may have arisen from the puncture caused by the catfish fin. This medication helps in inhibiting the growth and spread of bacteria, reducing the risk of infection. The purpose of tetanus toxoid vaccination is to protect against the toxin produced by the bacteria

responsible for causing tetanus. In simpler terms, it functions as a preventive measure against developing the disease. Tetanus is caused by a bacterium called Tetanus which enters the body through cuts and wounds. In this case, if dirt, soil, or manure contaminated the wound resulting from the incident involving the catfish, there was a possibility of contracting Tetanus. Ceftriaxone aims to address any potential infections resulting from the catfish fin puncture, while tetanus toxoid serves to safeguard against the toxins produced by Tetanus bacteria and prevent its occurrence if contaminants enter the wound. What is granulocytosis? Granulocytosis refers to an increase in the levels of granulocytes, including neutrophils, eosinophils, and basophils. This condition occurs as a response by the body to combat infections. What is the most likely cause of the man’s illness and death? The probable reason for the man's sickness and death is likely to be attributed to rabies. On June 4, the person's supervisor informed hospital authorities that he had been bitten by a bat on his right index finger. Rabies testing was conducted on samples of cerebrospinal fluid (CSF), serum, and a skin biopsy, all of which yielded negative results. However, postmortem examination of brain tissue confirmed the presence of rabies through the direct immunofluorescent antibody test. Page 18 What other information do you need to be sure? Determining if the patient has experienced any prior injuries or cuts and, if so, obtaining information regarding the timing of these incidents. Inquire about the patient's vaccination status for Tetanus and provide the date of immunization, if applicable. Confirmation of a rabies diagnosis can be achieved through a fluorescent-antibody test. How could he have been treated? If he had promptly sought medical treatment by administering antibodies targeting rabies prior to the onset of symptoms, following the bat bite, he could have received rabies immune globulin, which would have boosted his immune system's ability to combat the disease

and increased his likelihood of survival. How should the recipient of the platelets be treated? The person receiving the platelets should receive treatment with rabies immune globulin and be informed about the symptoms of rabies. They should also be instructed to promptly seek medical assistance if they experience any signs of illness. Reference(s): Include at least 1 valid reference for your research in the correct format (either APA or MLA) Centers for Disease Control and Prevention. (n.d.). Human rabies -- Texas, 1990. Centers for Disease Control and Prevention. https://www.cdc.gov/mmwr/preview/mmwrhtml/00001920.htm Week 6 Wednesday 15 November 2023 Lab 9: Biofilms: Antibiofilm Agents (canvas) According to research conducted by the Centers for Disease Control and Prevention (CDC), biofilm bacteria exhibit increased resistance to various antimicrobial treatments, rendering them difficult to eliminate. Moreover, biofilm bacteria can withstand harsh conditions and evade the host's immune system, leading to the development of chronic infections. Consequently, it is imperative to develop new treatment options specifically targeting biofilm-associated infections. Scientists have been focusing on the creation of antibiofilm agents that can effectively disrupt biofilms without causing harm to human or domestic animal cells. Page 19 One promising approach involves the use of enzymatic disruptors, such as dispersin B. These enzymes have demonstrated efficacy in breaking down the extracellular matrix that holds biofilms together. By degrading the essential components of biofilms, these enzymes significantly weaken their structure and promote the dispersion of bacterial colonies. This mechanism of disruption offers a hopeful strategy for combating biofilm formation and treating infections associated with them in both humans and animals. Another potential solution lies in quorum sensing inhibitors or QSIs. Quorum sensing is a communication process through which bacteria coordinate behaviors like forming a biofilm. QSIs interfere with this network of communication, disrupting the bacteria's ability to form cohesive communities and robust biofilms. Compounds such as furanone and halogenated furanone have shown promise in inhibiting quorum-sensing pathways. The application of these QSIs could enhance the efficacy of conventional antibiotics in treating chronic infections caused by biofilms. The development of antibiofilm agents that can disrupt biofilms without harming human or domestic animal cells is crucial in addressing the challenges posed by biofilm-associated

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infections. Enzymatic disruptors and quorum-sensing inhibitors offer promising strategies for combatting biofilm formation and treating associated infections effectively. As stated by CDC researchers, "These approaches show great potential in overcoming the resilience of biofilms and improving treatment outcomes." Sources: (CDC.gov - "Biofilms in Healthcare Settings: New Perspectives on an Old Problem," NIH.gov - "Antibiotic Resistance Threats in the United States, 2019," WHO.int - "Water, sanitation and hygiene in health care facilities") Lab 10: Identification of an unknown bacterium (Canvas) In the laboratory experiment by performed a gram stain on Wednesday, November 15th, which allowed us to identify whether our microbe was gram-stain. Upon completion of the gram stain procedure, the microbe exhibited gram-positive characteristics. Next, we needed to ascertain the shape of the microbe and determine whether it was rod-shaped or cocci. Through careful examination under a light microscope, the microbe displayed a cocci shape and formed clusters. This information enabled us to proceed with either utilizing the Gram-Positive Flow Chart or conducting biochemical testing. We opted to perform the Catalase test based on these findings. Subsequently, the Catalase test was conducted, and the microbe produced bubbles, Page 20 indicating a positive result. This confirmed that our bacterium belonged to the Staphylococci genus. To further narrow down the specific strain of Staphylococci possessed, the Mannitol Salt Agar test to observe if fermentation occurred. Following incubation and careful observation, the conclusion was that the bacterium in question was indeed Staphylococcus Aureus Results from Elise LaCombe in Group #1

Sources: Leboffe, M.J., and Pierce, B.E. (2011). A photographic atlas for the microbiology laboratory (4th Ed.). Morton Publishing.

MCB2010C_LabNotebook

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