EXAM 3 ANSWERED STUDY GUIDE LAB PDF

LM-18 – Control of growth with disinfectants Compare / Contrast disinfectant, antiseptic, and emulsifying agent (like soap): Disinfectant - a chemical substance used to kill or inactivate pathogens on non-living surfaces. Disinfectants are typically stronger than antiseptics and m...

LM-18 – Control of growth with disinfectants Compare / Contrast disinfectant, antiseptic, and emulsifying agent (like soap): Disinfectant - a chemical substance used to kill or inactivate pathogens on non-living surfaces. Disinfectants are typically stronger than antiseptics and may be toxic to human tissues. They are commonly used on surfaces like countertops, floors, and medical equipment (e.g., bleach, phenol). Antiseptic – a chemical agent that is safe for use on living tissues to kill or inhibit pathogens, preventing infection. Antiseptics are milder than disinfectants and are applied directly to the skin or mucous membranes (e.g., hydrogen peroxide, iodine). Emulsifying agent (soap) – a substance that helps lift and suspends oil, dirt, and microbes from the skin or surface so they can be rinsed away with water. Soap doesn’t kill microbes but removes them mechanically, reducing microbial load on the skin. Key difference: disinfectants are for non-living surfaces and are typically stronger while antiseptics are used on living tissues. Emulsifying agents (like soap) don't kill microbes but remove them through physical means. What is decimal reduction time (DRT). Decimal reduction in time (DRT) is the time required to kill 90% of a microbial population at a specific temperature or with a specific chemical treatment. It is also called D-value. How is DRT used to evaluate the effectiveness of a given chemical treatment? DRT provides a quantitative measure of a disinfectant or antiseptic’s effectiveness. A lower DRT indicates a more effective treatments as it takes less time to kill a majority of a microbial population. By comparing the DRTs of different treatments, we can assess which chemical is more efficient at reducing bacterial counts under the same conditions. Would you expect to see more bacterial growth after washing your hands with soap or hand sterilizer/why? You would typically expect less bacterial growth after using a hand sanitizer then after washing with soap. Hand sanitizers usually alcohol-based kill bacteria and viruses on contact, providing an immediate reduction in microbial load. Soap on the other hand removes microbes physically through emulsification and rinsing but doesn't necessarily kill them. However, hand washing with soap is effective at reducing dirt, oils, and some microbes from the skin surface and is recommended when hands are visibly soiled. What are a few structures that certain bacteria have to help resist the effects of a disinfectant? Certain bacterial structures help bacteria resist the effects of disinfectants including: Endospores – thick-walled structures that allows bacteria like bacillus and Clostridium to survive extreme conditions including disinfectants. Biofilms - communities of bacteria encased in a protective matrix that adheres to surfaces, making it difficult for disinfectants to penetrate and reach individual cells. Outer membrane in gram negative bacteria – gram-negative bacteria have an additional outer membrane that acts as a barrier to many disinfectants, making them harder to kill than gram positive-bacteria. Efflux pumps - these protein channels actively pump out toxic substances including disinfectants from inside bacterial cells reducing the disinfectant’s effectiveness. LM-19 – Control of growth with antibiotics What’s an antibiotic? How is it that we can sometimes take them to fight a bacterial or fungal infection? An antibiotic is a chemical substance often produced by microorganisms that can inhibit the growth of or kill bacteria and, in some cases, fungi. Antibiotics target specific bacterial structures are functions such as cell wall synthesis or protein production that are essential for bacterial survival but not present in human cells. This sensitivity allows antibiotics to treat bacterial infections without significantly harming the host. However, antibiotics are generally ineffective against viruses. What is the Kirby-Bauer disk diffusion test and how does it work? How is it used to determine a bacteria species sensitivity to the antibiotic? The Kirby-Bauer disk diffusion test is a standardized method to evaluate the effectiveness of antibiotics on a bacterial species 1. A standardized bacterial suspension is spread evenly across an agar plate. 2. Small paper disks impregnated with specific antibiotics are placed on the surface of the agar. 3. The plate is incubated allowing the bacteria to grow and antibiotic to diffuse outward from the disk. Results: After incubation zones of inhibition (clear areas where bacteria did not grow) form around the antibiotic disks. The size of these zones indicates the bacteria sensitivity to each antibiotic. By measuring the diameter of each zone of inhibition the sensitivity or resistance of the bacterial species with antibiotics can be determined. Larger zones indicate sensitivity while smaller or no zones indicate resistance. What is a zone of influence? Minimum Inhibitory Concentration (MIC)? Where is the MIC located on a KB test? Zone of inhibition – the clear area surrounding an antibiotic disc where bacterial growth is inhibited. The diameter of this zone is measured to determine bacterial sensitivity. Minimum inhibitory concentration (MIC) - the lowest concentration of an antibiotic that inhibits visible bacterial growth period although the Kirby-Bauer test doesn't directly measure MIC, the zone of inhibition size can be correlated with MIC values to infer the effectiveness of the antibiotic. Is the MIC in the KB test what is used as a medication? why/why not? Is MIC used as medication? - Not directly. In clinical settings that those prescribed to patients is usually higher than the MIC to ensure sufficient antibiotic levels in the body, accounting for absorption, distribution, and elimination processes that affect drug concentration. Be able to correlate in general terms the results of a Kirby-Bauer test and if a bacteria is sensitive or resistant to the antibiotic. Sensitive (S) - a large zone of inhibition indicates the bacteria are susceptible to the antibiotic and the antibiotic is likely effective at standard doses. Intermediate (I) - a medium sized zone of inhibition suggests a moderate level of sensitivity where higher antibiotic doses may be required. Resistant R - a small or no zone of inhibition means the bacteria are resistant and antibiotic is unlikely to be effective. How can you tell if the antibiotic is bactericidal or bacteriostatic using the KB test? Bactericidal antibiotic - kills bacteria, resulting in a clear and distinct zone of inhibition. Additional tests beyond the Kirby- Bauer test are typically required to confirm bactericidal action (e.g., subculturing within the zone). Bacteriostatic antibiotic - inhibits bacterial growth without killing the bacteria. Bacteriostatic antibiotics may show a zone of inhibition, but it may be less distinct or show regrowth if subcultured. Lab 20 – Disease Key definitions in disease and epidemiology: 1. Epidemiology - the study of distribution, patterns, and determinants of health and disease in populations. Epidemiologists investigate outbreaks, risk factors, and methods of controlling diseases. 2. Fomite - n an inanimate object or surface that can harbor and transmit pathogens (e.g., doorknobs, towels). 3. Disease - a condition that disrupts normal body function, leading to symptoms and possibly signs. Diseases may be caused by infections, genetic factors, or environmental influences. 4. Pathology - the study of disease focusing on its causes, development, and effects on the body. 5. Infection - the invasion and multiplication of pathogens in the body. An infection may lead to disease if it disrupts normal body function. 6. Etiology - the study of the cause or origin of a disease. 7. Symptoms - subjective experiences of illness reported by the patient, such as pain or fatigue that are not directly observable. 8. Signs - objective evidence of disease that can be observed or measured by others like fever or rash. 9. Syndrome - a group of symptoms and signs that when combined characterize a particular disease or condition. 10. Communicable disease - an infectious disease that can be transmitted from one person to another. 11. Non-communicable disease - a disease that cannot be transmitted between people often caused by genetic, lifestyle, or environmental factors (e.g., diabetes, heart disease). 12. Contagious - a term used for communicable diseases that are easily spread from person to person. 13. Sporadic disease - a disease that occurs infrequently and irregularly in a population. 14. Endemic - a disease consistently presents in a population or geographic area (e.g., malaria in certain regions). 15. Pandemic - a widespread endemic that affects multiple countries or continents often involving a new pathogen (e.g., COVID- 19). 16. Epidemic - an increase in the occurrence of a disease above what is normally expected in a population or region. 17. ID-50 (infectious dose 50) - the number of pathogens required to infect 50% of a population. It measures a pathogens infectivity. 18. LD-50 (lethal dose 50) - the dose of a pathogen or toxin required to kill 50% of a population. It measures a pathogens lethality. Define and differentiate acute, chronic, and latent disease. Acute disease - develops quickly and lasts a short time often with intense symptoms (e.g., influenza). Chronic disease - develops slowly and persists over a long period often with less intense symptoms (e.g., tuberculosis). Latent disease - a disease in which the pathogen remains inactive in the body for a time but can reactivate and cause symptoms (e.g., herpes simplex virus). Define, Compare/Contrast direct vs. indirect contact transmission Direct contact transmission - involves direct physical contact between an infected person and a susceptible host allowing pathogens to spread (e.g., through touching, casing, or sexual contact). Indirect contact transmission - involves a pathogen transferred through an intermediary object or surface (fomite) or medium rather than direct person to person contact. This includes transmission via contaminated objects, surfaces, or droplets in the air. Comparison: Direct contact transmission requires close or physical interaction between individuals whereas indirect contact transmission involves a third party or intermediary for pathogen transfer. Indirect transmission can allow for broader spread as fomites or airborne particles can infect individuals who may not have been in close contact with the infected person. Lab 21 - Pathogenic Bacteria Compare and contrast primary vs. opportunistic infection: Primary infection - an infection caused by a pathogen that can cause disease in healthy individuals with normal immune systems. Primary pathogens can initiate infection on their own. Examples include streptococcus pneumoniae causing pneumonia or back mycobacterium tuberculosis causing tuberculosis. Opportunistic infection - an infection caused by a microorganism that typically does not harm a healthy host but can cause disease in individuals with compromised immune systems, disrupted microbiota, or when they enter parts of the body where they don't normally reside. Examples include Candida albicans infection in immunocompromised individuals or Pseudomonas aeruginosa in burned patients. Comparison: primary infections occur in the healthy host and are caused by pathogens capable of causing disease independently. Opportunistic infections occur when host defenses are weakened or compromised, and they are caused by organisms that normally do not cause disease. What is the Snyder test and how does it work? The Snyder test is used to assess susceptibility to dental carries (cavities) by measuring acid production by lactobacilli and other acid producing bacteria in saliva. How/what does it select for? The tests select for acid producing bacteria particularly Lactobacillus species which are associated with cavity formation. How/what does it differentiate for? It differentiates based on the rate of acid production. The Snyder test medium contains glucose and a pH indicator (bromocresol green) that changes color in response to acid production. How do you determine a positive vs. negative result? Positive versus negative result: o Positive result - the medium turns from green to yellow as bacteria produce acid, lowering the pH indicating a higher susceptibility to dental caries. o Negative result - if the medium remains green it indicates lower acid production and less susceptibility to dental caries. What is a blood agar plate? A blood Agar plate is a type of growth medium containing nutrients and sheep's blood, used to grow a variety of bacteria and observe their hemolytic properties. Is it selective or differential? Why? Blood sugar is differential, not selective. It differentiates bacteria based on their ability to lyse red blood cells (hemolysis) which helps in identifying bacterial species based on hemolytic pattern What is hemolysis? The breakdown of red blood cell (RBC) by bacterial enzyme. What are the three “degrees” of hemolysis? How are the three degrees determined using a blood agar plate?  1. Alpha hemolysis - partial hemolysis resulting in greenish or brownish discoloration around the colonies due to partial breakdown of hemoglobin. An example of alpha hemolytic bacterium is streptococcus pneumoniae. 2. Beta hemolysis - complete hemolysis producing a clear transparent zone around the colonies streptococcus pyogenes is an example of beta hemolytic bacterium. 3. Gamma hemolysis - no hemolysis; no change in the color of the auger around the colonies. Enterococcus faecalis is an example of gamma-hemolytic bacterium. What is MSA agar and how does it work? MSA (mannitol salt agar) is both selective and differential growth medium. How/what does it select for? MSA is selective for Halo tolerant salt tolerant bacteria, such as Staphylococcus species due to its high salt concentration (7.5% NaCl) which inhibits the growth of most other bacteria. MSA differentiates based on mannitol fermentation. The medium contains mannitol a (sugar alcohol) and a pH indicator (phenol red). How/what does it differentiate for? How do you determine a positive vs. negative result? Positive result - if organism ferments mannitol acid is produced, lowering the pH and changing the angular color from red to yellow. This indicates mannitol fermentation. Negative result - if the Organism does not ferment mannitol the agar remains red. What pathogenic species is this test primarily used to identify? MSA is primarily used to identify Staphylococcus aureus which ferments mannitol and turns the Agar yellow differentiating it from other non-mannitol fermenting Staphylococcus species like Staphylococcus epidermidis which does not change the color on the agar. Lab 22 – Transformation Differentiate vertical and horizontal gene transfer in bacteria. Vertical gene transfer - this is the transmission of genetic material from a parent cell to offspring during reproduction. In bacteria, it occurs through binary fission where the genetic material is copied and passed down to daughter cells. Horizontal gene transfer - this is the transfer of genetic material between unrelated bacterial cells allowing them to acquire new traits independently of reproduction. Horizontal gene transfer contributes to genetic diversity and adaptation within bacterial population. Compare and contrast the three types of horizontal gene transfer – conjugation, transformation and transduction. 1. Conjugation: o Involves the transfer of genetic material between bacteria cells through direct cell to cell contact. o A donor cell with plasmid (often F- plasmid) extends a pilus to a recipient cell, transferring the plasmid and potentially conferring nutrients such as antibiotic resistance. 2. Transformation: o Bacteria take up free DNA from the environment often released by lysed cells. o If the DNA is compatible it can integrate into the bacterial genome or exist as a plasmid allowing the bacteria to acquire new traits such as antibiotic resistance or new metabolic capabilities. 3. Transduction: o a bacteriophage (virus) transfers genetic material between bacteria.  o During infection, may accidentally package bacterial DNA instead of viral DNA. When it infects a new host, it transfers this DNA introducing new genes into the recipient cell. Comparison: conjugation requires direct cell contact transformation involves DNA uptake from the environment and transduction uses a viral intermediary. Conjugation transfers plasmids while transformation and transduction may transfer chromosomal or plasmid DNA. Define transformation. Transformation is the process by which the bacteria take up foreign DNA from their environment. In laboratory settings, transformation often involves inserting plasmids into bacteria to introduce new genes. Explain the process of inserting a plasmid into bacteria using the “heat shock” method for transformation. 1. Bacteria are placed in a transformation solution typically containing calcium chloride to make the cell membranes more permeable. 2. The plasmid DNA is added to the bacterial cells. 3. The bacterial culture is briefly subjected to a rapid increase in temperature heat shock, usually 0°C to 42°C and then quickly cooled. These sudden temperature change helps the plasmid enter the bacterial cells. Describe the function of the Transformation solution. The calcium ions in the transformation solution neutralize negative charges on both the bacteria cells membrane and the DNA, allowing the DNA to pass more easily into the cell. Explain how transformed bacterial cells are selected for (how you know a cell received a plasmid)? Transformed cells can be identified as selective media that contains an antibiotic. The plasmid used in transformation usually carries an antibiotic resistance gene, which allows only the transformed bacteria (those that received the plasmid) to grow in the presence of the antibiotic. Non transformed bacteria will be unable to grow in this selective medium. Describe the role(s) of the antibiotic resistance and arabinose genes contained in the plasmid, and the arabinose sugar contained in the agar, with the transformation and expression of GFP (green fluorescent protein) in the pGLO experiment. In the pGLO experiment, the plasmid contains: o Antibiotic resistance gene – provides resistance to ampicillin, allowing only bacteria that have taken up the plasmid to survive on media containing ampicillin. o Arabinose operon with GFP gene – the pGLO plasmid includes the gene for green fluorescent protein (GFP) under the control of the arabinose promoter. The GFP gene is only expressed in the presence of arabinose, a sugar that activates the promoter. Role of arabinose in Agar - arabinose and the growth of medium induces the expression of GFP in transformed cells causing them to glow under UV light Explain the expected results (will they grow / will they glow / why or why not) for pGLO and +pGLO on LB, LB +amp, and LB +amp +arab. 1. LB (Luria broth) only -pGLO: growth expected; no plasmid, so bacteria grow normally. +pGLO: growth expected; transformed cells grow without selective pressure.  Result: no glow in either condition, as there is no arabinose to induce GFP expression. 2. LB + Ampicillin -pGLO: no growth expected; without the plasmid, cells are not resistant to ampicillin and will not survive. +pGLO: growth expected; cells that received the plasmid can survive due to ampicillin resistance. Result: no glow, as there is no arabinose to induce GFP expression. 3. LB + Ampicillin + arabinose -pGLO: no growth expected; cells without the plasmid lack ampicillin resistance and will not survive. +pGLO: Growth expected; transform cells with ampicillin resistance survive and arabinose in the medium induces GFP expression. Result: colonies will glow under UV light due to GFP expression activated by arabinose. The presence of ampicillin selects for transformed bacteria (+pGLO), while arabinose enables GFP expression causing fluorescence. Only +pGLO cells on LB + Amp + Arab plates will grow and glow indicating successful transformation and expression of the GFP gene. Lab 23- Mutations Define a mutation. Are mutations good, bad or neutral? explain. Mutation is a change in the DNA sequence that can alter the genetic information in the cell. Mutations can occur spontaneously or be induced by environmental factors and they can be good, bad or neutral. Good - some mutations can be beneficial providing new traits that improve an organism's survival or adaptation to its environment (e.g., genetic disorders in humans). Bad - mutations can also be harmful leading to malfunctions, diseases, or reduced fitness (e.g., genetic disorders in humans). Neutral - many mutations are neutral and have no noticeable effect on the Organism. This is common when mutations occur in non-coding DNA regions or result in synonymous codons. Discuss how physical mutagens (high-energy light) can cause DNA mutations. Physical mutagens such as high energy light (e.g., UV light, X-rays) can damage DNA leading to mutations: High-energy radiation can break DNA strand or cause cross linking leading to structural damage. What specific mutation does UV light cause? UV light specifically UV light causes pyrimidine dimers primarily between adjacent thymine bases. This disrupts the DNA structure and interferes with DNA replication and transcription. Classify the 2 mutation types by the changes they incur on the DNA sequence. (point / frameshift) 1. Point mutation - a single nucleotide exchange which can alter one amino acid in the protein. Example: a base substitution where A replaces G. 2. Frameshift mutation - an insertion or deletion of a nucleotide which shifts the reading frame changing all subsequent codons and potentially altering the entire protein sequence. Example: deletion of one nucleotide causes a shift in how the sequence is read. Identify 4 mutation types by how the DNA or protein is affected. (silent, missense, nonsense, insertion/deletion)  1. Silent mutation - a change in the DNA sequence that does not alter the amino acid sequence due to the redundancy of the genetic code. 2. Missense mutation - a base substitution that changes one amino acid in the protein sequence potentially affecting protein function. 3. Insertion/deletion - addition or removal of nucleotides that can cause frame shifts changing the reading frame and potentially altering the entire downstream amino acid sequence. Describe how we selected for UV induced Rifampin resistance mutations in E. coli bacteria. To select for UV-induced rifampin-resistant mutation in E. coli: 1. Exposure to UV light - E coli cultures are exposed to UV light which can induce mutations including those conferring resistance to antibiotics. 2. Growth or rifampin-containing medium - the treated E. coli are then plated an agar containing rifampin, an antibiotic that inhibits RNA polymerase in non-resistant bacteria. 3. Selection of mutants - only E.coli cells with mutations in the RNA polymerase gene confer resistance to rifampin karma will survive and form colonies on the rifampin plates.