Lab Exam 1 Study Guide Fall 2024 PDF

Fall 2024 Lab Exam 1 Study Guide for Labs 1-6 and all associated case studies. - **Lab 1** 1. Which groups of microbial agents (Bacteria, Fungi, Protozoa, and Viruses) can be cultured in the microbiology laboratory? Bacteria (majority of samples) prokaryotic, Fungi (yeast and molds)...

Fall 2024 Lab Exam 1 Study Guide for Labs 1-6 and all associated case studies. - **Lab 1** 1. Which groups of microbial agents (Bacteria, Fungi, Protozoa, and Viruses) can be cultured in the microbiology laboratory? Bacteria (majority of samples) prokaryotic, Fungi (yeast and molds) eukaryotic, Viruses (some). Which group(s) is/are not cultured in clinical microbiology? Protozoa -- unicellular eukaryotes. These are a sub kingdom of the kingdom protisa 2. Be able to provide two purposes for bacterial cultivation in clinical microbiology. Culture and Sensitivity testing (routine culture): for bacteria and yeast. Sample evaluation is important for **1. Identification of species: diagnostic testing** -- of the bacteriological media/medium. These are in the form of broth or agar. **2.** **Antibiotic Susceptibility: determines effectiveness of multiple antibiotics.** 3. Be able to define bacteriological media. Bacteriological media (medium, sin): Substrate used to culture bacteria What is the purpose of using bacteriological media? To grow / culture bacteria in laboratory, it is necessary to propagate sufficient numbers of cells for analysis as its impossible to work with one microscopic cell. 4. What is broth media? Liquid media. What is the purpose of using broth media? Used to grow large numbers of cells (\~10\^9 cells/mL) Define the term turbidity. Turbidity: cloudiness/opacity of broth that indicates growth. What are the clinical applications of turbidity? Turbidity of urine and bodily fluids indicates an infection. 5. What is agar media? Solid media that cannot be liquified or consumed by the bacteria. Define the term colony. A discrete cell mass that represents a population of bacteria that is derived from a single cell. 6. What is the purpose of assessing the appearance of colonies on agar? Clues for identifying the organism by knowing the location, agar, and broth results in identifying the correct species. Skin, mouth, and intestines is common NFOP locations. For agar specifically, color and shape provide helpful information. Like if its round, small, flat, clumped, yellow, thick etc. 7. Be able to define the term opportunistic pathogen. Typically associated with human tissues without harm, but can cause disease under certain conditions. Dysbiosis, sepsis, weak immune systems. 8. What is the purpose of routine hand washing? 9. Be able to explain the **[value]** of routine hand washing in reducing transmission of certain microbes. It removes germs from hands which can spread them quickly through inanimate surfaces (fomites) or our bodies like mouth, eyes, nose, ears, etc. NF OP and other pathogens can all turn into diseases if entered a sterile site. But NF OP can be fine on epidermis surfaces as long as its superficial not deep. Which microbes are not removed by routine hand washing? Transient flora -- colonize people for hours or weeks but do not establish themselves permanently. Hard to remove in handwashing. And NF residential flora, even harder to remove in handwashing. 10. What is a fomite? Inanimate surface that harbors potential pathogens, found in high touch surfaces. What types of pathogens can be found on fomites? Bacteria, viruses, yeast, mold 11. Be able to provide multiple examples of clinically relevant fomites: a. Patient environments: healthcare workers, patients, door handles, bed rails, walls, telephones, tv remotes, reusable blood pressure cuffs, stethoscopes etc. respiratory droplets (sneezing) b. A other common fomite is cell phone fomites contact with finger, mouth (saliva), ear. 12. Be able to cite the serious complications of measles infections. What is the occurrence (ratio) of patients who develop a serious complication? 13. Be able to cite the at-risk populations for measles infections. 14. Define the chain of infection. A model that explains the spread of a communicable disease from one individual (host) to another. 15. Be able to cite **the six parts of the chain of infection**. Which parts are most susceptible to intervention strategies? 1. Pathogen (infectious agent) - Viruses - Bacteria - Yeasts/molds - Protozoa - Worms 2. Reservoir (where the pathogen comes from) - Infected human - Infected animal - Environment 3. Portal of exit (how the pathogen leaves the reservoir) - Coughing - Sneezing - Vomiting - Diarrhea - Airborne (dust, soil particles) - Insect (vector) - Surfaces (fomites) 4. Mode of transmission: listed below c. Direct contact: skin-skin, sexual contact, saliva contact, blood contact (injections) that transmits the pathogen. d. Droplet: large droplets of saliva or mucous that travel short distances \2 meters from the origin point that transmits the pathogen. h. **Fomite: contaminated surfaces due to direct contact (skin), droplet/aerosols, and vehicle modes that transmits the pathogen to a susceptible individual.** 5\. Portal of entry: how the pathogen enters the new host (food/water, air, etc) 6\. Susceptible host= newly infected individual. \- Intervention strategies target steps **3, 4, 5, and 6** - **Take-Home Assignment 1** Be able to define clinical microbiology and be able to cite the three purposes of the BIO 39 lab. Clinical microbiology- refers to the prevention diagnosis and treatment of infectious diseases as well as new clinical applications of microbes for the improvement of health. Purpose of BIO 39 LAB: a. Recognitions diagnoses, and treatment of infectious diseases b. Cover standard practices in infection prevention and control (risk mitigation). c. Interpretation of laboratory and radiology tests related to infectious diseases. 5. Be able to list all the standard precautions. 1. Hand hygiene (hand washing) 1infection control practice 2. Use of PPE personal protective equipment a. Gloves b. Mask and respirators c. Gown d. Show covers e. Hair caps 3. Respiratory hygiene and couch etiquette 4. Environmental cleaning to reduce pathogen transmission events 5. Sterilization of medical wastes, soiled linens reusable medical equipment and instruments to destroy all microbial agents 6. Isolation precautions are used to prevent the transmission of infectious agents from patient-to-patient. This may include physical separation of the patient from the general population. 7. Strict observance of [aseptic techniques]. 6. Be able to define aseptic techniques, - performing clinical techniques without causing infection or contamination of patient tissue, medication, or patient environments. provide clinical examples of aseptic techniques,- examples include: hang hygiene, applying antiseptics to exposed body surfaces, maintaining a sterile field and equipment, PPE, sterile gloves, dispose of contaminated wastes properly. 7. Be able to define all of Koch's Postulates, provide three examples of Koch's Postulates, and explain the role of the ICP. Purpose: in order to prove the germ theory of a disease. A procedure to establish the specific cause of disease in all cases of infection. Postulate 1: Find evidence of a particular microbe in every case of disease Postulate 2: isolate that microbe from an infected subject and cultivate it in a culture in the laboratory; characterize it fully. Postulate 3: Inoculate a susceptible healthy subject with laboratory, isolate and observe the same reluctant disease. Postulate 4: Reisolate the same agent from this subject. Three examples of Koch's postulates: beer pasteurization, in 1875 Koch used this experimental system to show that anthrax was caused by a bacterium called bacillus anthracis, PPT - Koch's Postulates PowerPoint Presentation, free download - ID:2507035 examples that the postulates could not be used to determine infectious causation... 1. Obligate intercellular pathogens 2. Diseases (polymicrobial infections) 3. Asymptomatic carriers Also this example Define ICP- the healthcare provider is responsible for educating the patient about the risks and harm they can cause if they do not follow orders of confinement or treatment. Ex. Since the infected individual infected so many people through her profession cooking as a chef. 8. Be able to define and cite the health range of the following vital signs: d. Heart rate (pulse) -- 60-100 beats/min bpm e. Respiration rate -- 12-16 (10) breaths/min bpm f. Body temperature- 98.6 F (37C) fever is temp above 100.4F 38C associated with infection g. Blood pressure- 90/60- 120/80 lower is hypotension (associated w bloodstream infection/sepsis), higher is hypertension. i. Measure of arterial pressure when the heart beats (systolic) and rests (diastolic) (blood pressure moves RBCs through the vasculature) h. Oxygen saturation- 95-100% ii. A measure of the amount oxygen bound to hemoglobin in Red blood cells RBCs 9. Be able to define the ABCs of emergency medicine: airways, breathing, circulation. 10. Be able to define a fever. -- as temporary increased body temp above 100.4f (per the vital signs assessment) the increase in body temp is commensurate with the severity of the infection. 101f =infection 107.5=severe infection 11. Be able to cite the healthy range of white blood cells (WBCs). Healthy range of wbc is 4,500-11000 WBC/ul What is the significance of increased WBCs with regards to infections? \>12000WBC/ul is elevated amount. 16,000 wbc=infection 37,000 wbc-sever infection. WBC= cellular component of the immune system, 12. Be able to define the term burn. An injury to the skin or other tissue primarly cuasaed by heat or due to radiation, radioactivity, electricity, friction or contact with chrmicals. Thermal (heat) burns occur when some or all of the cells in the skin or other tissues are destroyed. 13. Be able to cite how an airway issue, breathing issue and circulation issue would affect vital signs. PG 4 of lab 2.. airway issue causes the breathing issues by lack of 02 and this an in turn cause circulation to not be effectively exchanging o2 to the body. This would cause an increase in heart rate, low blood pressure, and increased breathing in the lungs. 14. Be able to explain how heat inactivation destroys microbes. Heat denaturation of proteins, DNA membranes by [Heat denaturation refers to the process where **heat alters protein conformation by disrupting weak forces**](https://www.bing.com/ck/a?!&&p=dcacbe3fb58b801fJmltdHM9MTcyODUxODQwMCZpZ3VpZD0yOWVhZGM0OC04ZDk4LTYzOTEtMjg0ZC1jZWE1OGNkNDYyZDQmaW5zaWQ9NTkzNg&ptn=3&ver=2&hsh=3&fclid=29eadc48-8d98-6391-284d-cea58cd462d4&psq=heat+denaturation&u=a1aHR0cHM6Ly9iaW9sb2d5cmVhZGVyLmNvbS9wcm90ZWluLWRlbmF0dXJhdGlvbi1ieS1oZWF0Lmh0bWw&ntb=1)[**^1^**](https://www.bing.com/ck/a?!&&p=67e0901f083dfdf2JmltdHM9MTcyODUxODQwMCZpZ3VpZD0yOWVhZGM0OC04ZDk4LTYzOTEtMjg0ZC1jZWE1OGNkNDYyZDQmaW5zaWQ9NTkzNw&ptn=3&ver=2&hsh=3&fclid=29eadc48-8d98-6391-284d-cea58cd462d4&psq=heat+denaturation&u=a1aHR0cHM6Ly9iaW9sb2d5cmVhZGVyLmNvbS9wcm90ZWluLWRlbmF0dXJhdGlvbi1ieS1oZWF0Lmh0bWw&ntb=1). [For proteins, heat exposure above 50°C generally denatures them by increasing kinetic energy and breaking weak hydrogen bonds and dispersion forces](https://www.bing.com/ck/a?!&&p=a873e9926b593c54JmltdHM9MTcyODUxODQwMCZpZ3VpZD0yOWVhZGM0OC04ZDk4LTYzOTEtMjg0ZC1jZWE1OGNkNDYyZDQmaW5zaWQ9NTkzOA&ptn=3&ver=2&hsh=3&fclid=29eadc48-8d98-6391-284d-cea58cd462d4&psq=heat+denaturation&u=a1aHR0cHM6Ly9iaW9sb2d5cmVhZGVyLmNvbS9wcm90ZWluLWRlbmF0dXJhdGlvbi1ieS1oZWF0Lmh0bWw&ntb=1)[**^1^**](https://www.bing.com/ck/a?!&&p=6d3130455a8d669dJmltdHM9MTcyODUxODQwMCZpZ3VpZD0yOWVhZGM0OC04ZDk4LTYzOTEtMjg0ZC1jZWE1OGNkNDYyZDQmaW5zaWQ9NTkzOQ&ptn=3&ver=2&hsh=3&fclid=29eadc48-8d98-6391-284d-cea58cd462d4&psq=heat+denaturation&u=a1aHR0cHM6Ly9iaW9sb2d5cmVhZGVyLmNvbS9wcm90ZWluLWRlbmF0dXJhdGlvbi1ieS1oZWF0Lmh0bWw&ntb=1). [In the case of DNA, heating causes the double-stranded DNA to unwind and the hydrogen bond](https://www.bing.com/ck/a?!&&p=a55a55d23b472ffaJmltdHM9MTcyODUxODQwMCZpZ3VpZD0yOWVhZGM0OC04ZDk4LTYzOTEtMjg0ZC1jZWE1OGNkNDYyZDQmaW5zaWQ9NTk0MA&ptn=3&ver=2&hsh=3&fclid=29eadc48-8d98-6391-284d-cea58cd462d4&psq=heat+denaturation&u=a1aHR0cHM6Ly9lbi53aWtpYm9va3Mub3JnL3dpa2kvU3RydWN0dXJhbF9CaW9jaGVtaXN0cnkvTnVjbGVpY19BY2lkL0ROQS9ETkFfRGVuYXR1cmF0aW9u&ntb=1) 15. Be able to define the function of the autoclave. Be able to cite three uses of the autoclave in medicine. What types of items are sterilized by the autoclave? The use of pressure to increase the boiling point of water to 121 C for 20 minutes i. Destroys all micros, enzymes, and biomolecules j. Autoclaved items are biologically sterile. k. For sterilization of iii. Microbial media iv. Surgical instruments v. Biomedical waste - Lab 2 1. Define aseptic techniques **Aseptic technique** is the collection of procedures and **techniques** designed to prevent the introduction of unwanted organisms into a pure culture or into the laboratory environment. - sterile gloves - sterile gowns - masks for the patient and healthcare provider - sterile drapes 2. Be able to define the **[purpose]** of clinical skin treatment. To remove all organisms from the skin, including staphylococci. Consistent proper clinical hand washing has been statistically demonstrated to decrease the risk and number of HAIs by reducing the overall microbial load on healthcare providers and clinical surfaces and lowers the risk of provider-patient transmission and infection. Failure to follow protocols is a major risk to breach infection control policy an can prolong hospital stays or even cause death PURPOSE- to remove all microbes from skin, including skin microbes, requires the use of antiseptics. 3. What is the mechanism of action of 70% ethanol? Disrupt lipids, denatures protein What are the clinical applications of 70% ethanol? Venipuncture IV line etc., (small areas of skin) 4. What is the mechanism of action of chlorohexidine gluconate (CHG, hibiclens)? Functions to disrupt cell walls and proteinsWhat are the clinical applications of CHG?hibiclens clinical soap, clinical hand sanitizer.. pre-surgical skin and hand treatment 5. What is the mechanism of action of betadine? Iodine compound is an oxidizing agent: destroy all biomolecules What is the clinical application of betadine? Pre-surgery skin treatment in surgical suite left on skin 10-20 minutes prior to surgery 6. Be able to compare and contrast the environments of the skin and the mouth in terms of normal flora bacterial growth with regards to moisture levels, pH, tonicity, nutrient concentration and competition. Which environment would produce environmentally resistant and sensitive species? 7. Be able to explain **[why]** some normal flora from the skin and mouth become opportunistic pathogens. NF can become opportunistic if it gets into sterile tissue sites. Skin NFOP are looking for more nutrients. Oral/GI NFOP are under nutrient constraints because of the high competitive environments. 8. Be able to answer questions 1-5 from the MRSA case study in Lab 2. 1. How does environmental resistance of S. aureus/MRSA contribute to the development of clinical outbreaks? a. Environmental resistance of s. aureus is incredibly susceptible to spreading especially if there is not proper handwashing/PPE/ aseptic techniques. Medical staff, patients, and visitors must use precautions especially if they're infected or in proximity. This bacteria will last 2 weeks on surfaces! 2. Provide three explanations are to how the patient acquired MRSA: b. Short staffed-delay in removind linen from pts room and observing visitors c. Many visitors- can lead to spreading especially if not using antibacterial techniques. d. Nurses- left mid treatment to assist other pts. 3. Provide an explanation as to why the prophylactic antibiotic did not prevent infection with MRSA. Because it was a beta lactam. This infection MRSA is resistant to all Beta lactam. 4. What is your interepetation of vital signs over 48 hrs post surgery?over 48 hrs post surger, the vital signs of pt. 2 has become alarmingly abnormal. The pt is high risk of mortalty and infected with MRSA. Vital signs should be taken every four hours. At 32 hrs he had high heart rate, high respiration rate, less SaO2, fever, and hypotension 5. Using the patient outcome table, provide 2 examples as two why four patients had poor wound healing or died as a result of their MRSA HAI: Age- (over 65 yrs, weak immune system), Cancer patient= decreased immune system. 9. Be able to explain how MRSA is transmitted, signs/symptoms and pathogenesis of MRSA infections, and be able to list the antibiotics that are used to treat MRSA infections from the Lab 2 Required reading on pages 9-10 of Lab 2. a. Sores or boils on the skin b. Swollen, red, painful bumps c. Fluid or pus filled bumps d. Affected area is warm to touch e. Fever f. Chills g. Low blood pressure h. Severe headache i. Shortness of breath 10. What percentage of the general population is colonized with *S. aureus*?.\~30-33% of population is colonized. In nostrils, skin, orooharynx, non-sterile genital tract (transient NF) 11. List the most common infections caused by *S. aureus*- Skin infections -bacteremia, infective endocarditis, skin and soft tissue infections (e.g., impetigo, folliculitis, furuncles, carbuncles, cellulitis, scalded skin syndrome, and others), osteomyelitis, septic arthritis, prosthetic device infections, pulmonary infections (e.g., pneumonia and empyema), gastroenteritis, meningitis, toxic shock syndrome, and urinary tract infections. 12. How likely is a patient to develop a *S. aureus* infection if the patient is already colonized with *S. aureus*?9X more likely to develop infection if colonized with staph aureus 13. Be able to cite the increase in hospital stay and mortality rate for patients with *S. aureus* infections. Increase in hospital stay with S aureus infections is 3x more likely. and 5x increase in risk if in-hospital death compared to uninfected patient's. 14. What percentage of *S. aureus* infections are caused by MSSA strains and MRSA strains? MSSA\~35-50% MRSA\~50-65% 15. Be able to explain why infections caused by MRSA strains are potentially more serious than infection with MSSA strains. Multi-drug-resistant staph aureus is more serious than muilti-drug sensitive staph aureus because it is resistant to many classes of antibiotics. Antibiotic resistance represents treatment failure that allows MRSA to continue destroying patient tissues that lead to poorer patient outcomes. 16. Be able to provide to explain how healthcare providers may incidentally transfer *S. aureus* from themselves to patients or patient to patient. Health care providers can transfer it between patients when proper hand washing is not observed when stethoscopes are not cleaned in between different patients, when gloved hands touch contaminated surfaces such as bedrails prior to working with sterile site tissues (Surgical site infections, IV and ventilator placement, etc) 17. Be able to explain how *S. aureus* environmental persistence may cause HAIs. How long can *S. aureus* survive on dry surfaces? Up to two weeks - Lab 3 18. Define selective media.- promotes or inhibits bacterial growth 19. Define differential media.-colony appearance due to unique biochemical reaction 20. Why are patient screenings for *S. aureus* important prior to surgery? - Patients who are colonized with Staphylococcus aureus are 9x more likely to develop S. aureus post-surgical infection than patients who are not.\ This could cause complications, including infections that could become fatal for that individuals and potential victims of transmission. 21. What is the increased risk for patient colonized with *S. aureus* to develop a surgical site infection? 9x more likely. 22. What is the purpose of using mannitol salt agar (MSA)? MSA is used as a Staphylococcus screen. 23. What is the selective agent in MSA and how does it select for a specific group of bacteria? The selective agent is 7.5% NaCl, which produces a hypertonic environment that kills non-salt tolerant species (Staphylococcal species are selected to survive because it lives on the skin, which is a hypertonic environment.) 24. How does MSA distinguish between different species by colony color? Differential Agent: Mannitol (sugar/alcohol) (MSA) and a pH indicator used by showing a color change that indicates mannitol utilization. j. Cellular respiration (CR)produces alkaline end products. k. Fermentation (F) produces acid end products 25. What is the colony color of *Staphylococcus aureus and Staphylococcus epidermidis* on MSA? Utilization by cellular respiration produces alkaline end products (PINK colonies indicate Staphylococcus epidermidis). Utilization by fermentation produces acid end products (YELLOW on yellow indicates Staphylococcus aureus). 26. What is the purpose of using blood agar? indicates Streptococcus. Specific species are Streptococcus pyogenes and Streptococcus pneumoniae 27. What is the differential agent of blood agar? 5% whole sheep RBCs for hemolysin assessment. 28. What are hemolysins? bacterial toxins that damage or destroy RBCs for acquisition of nutrients (cytoplasm) and iron. 29. Define alpha hemolysis.- Partial damage to RBC's with hemoglobin (Hgh) release. 30. What is the colony color of alpha hemolytic colonies? Hgb stains the colonies green/brown 31. What is the important alpha hemolytic species? Strep. Pneumonia and other NF (oral) 32. Define beta hemolysis. Hemolysis(beta hemolytic strep) loss of RBC's 33. What is the colony color of beta hemolytic colonies? Loss of red color on medium (clear) 34. What is the important beta hemolytic species? Strep Pyogones 35. Define gamma hemolysis. Hemolysis: no hemolysis, no loss of red color of medium l. Characteristics of NF and NF-OP 36. What are the hemolytic patterns of a normal throat culture? 37. Compare and contrast alpha, beta, and gamma hemolysis. 38. What is the function of the optochin antibiotic? m. Optochin is an antibiotic that is only effective against S. pneumoniae so therefore, would create a clear ring indicating no bacteria around the disc. 39. What is the species is indicated by positive optochin test indicate? Pos- No growth around optochin disc S pneumoniae + , growth around and under optichin disc S pnemoniae - 40. What type of bacteria does eosin methylene agar (EMB) select for? G-R What are the selective agents in EMB agar? Eoisin and Methylene blue (MB) - Select for G- cells and against G+ cells. - 1\. PG binding dyes that disrupt PG of G+ cells (lethel) - 2\. Outer membrane 41. What is the differential agent in EMB agar? Lactose fermentation What are the three (3) differential reactions that can be seen on EMB agar? What are the colony colors that are produced by the differential reactions? 1. Strong (vigorous) lactose fermenter: High concentration of acid. Causes eosin and MB to precipitate onto cells. (colonies are metallic green) 2. Moderate lactose fermenter: Moderate acid production causes precipitation of eosin alone. (colonies are pink) 3. Non-lactose fermenter- NO acid production; no dye precipitation (colonies are purple) 42. What is the purpose of using Thayer-Martin agar? - Neisseria isolation 43. What are the specific selective agents in Thayer-Martin agar? Antibiotics n. Vancomycin: kills G+ o. Polymyxin E: kills G- except Neisseria p. Nystatin: kills fungi 44. What two species does Thayer-Martin agar identify? q. Neisseria meningitidis: meningitis and sepsis r. Neisseria Gonorrhea: urinary tract infection 45. Explain why Thayer-Martin media is used at the patient bedside by direct health care providers. Neisseria cells are particularly delicate and do not often survive transport to the laboratory. Due to the number of types of antibiotics found in the Thayer-martin medium. Only the Neisseria species will grow. The anatomical site of sample collection will inform as to which Neisseria species is suspected. 46. What are NAAT tests? Rapid diagnostic tests for identification of pathogens. Short for \"Nucleic Acid Amplification Test\". 47. What do NAAT tests evaluate for diagnosis? detects specific genes from specific pathogens. 48. What are the advantages of using NAAT tests? Results may be available within 4 hours; it allows for more rapid diagnosis and treatment administration of patients. 49. What is the limitation of NAAT? It's available for some but not all pathogens 50. What groups of microbes can be seen by standard microscopy? 1. Bacteria: prokaryotic cells 2. Fungi: eukaryotic cells, yeast, and molds 3. Protozoa: eukaryotic cells 4. Be able to define and know the importance of magnification and resolution. 5. Be able to cite the function of the following parts of the light microscope: a. Condenser -- functions to collect and converge light waves onto the sample. b. Iris diaphragm: controls the amount of light that's leaving the condenser and entering the sample. c. Objective lenes: 4X.- scanning objective, microbes not seen (too small), human cells can be seen! 10X -- defining objective, large eukaryotic microbes can be seen, 40X -- refining objective, small eukaryotic microbes can be seen, and 100X -- oil immersion, bacteria can be seen. d. Ocular lens(10x) second series of lenses for magnification and resolution. 6. Be able to calculate total magnification. Objective x ocular lenses, maximum magnification=1000X. 7. Be able to define refraction and be able to explain why refraction limits magnification and resolution. Bending of light waves with passage through different densities (glass and air). 8. Be able to explain how immersion oil improves magnification and resolution **in detail**. 9. Define the term micrometer. 10. Be able to cite the average bacterial cell ranges (length and width). a. Micrometer- 1/1,000,000m; standard unit of measurement for cells b. Average ranges of bacteria cell: 1-5 micrometers long and 0.5-1 micrometers wide. 11. Be able to compare the size of prokaryotic cells to that of eukaryotic cells and viruses. 12. Be able to define the three major bacterial cell shapes. **Diagnostic** c. Coccus (pl. cocci)- spherical shaped cels: "C". d. Rod or baccilus (pl. bacilli): oblong shaped cells: "R" \*\*\>99% of samples will be C or R e. Spirochete: helical shaped cells. i. Treponema pallidum: Syphilis ii. Borrelia burgdorferi: Lyme diseases -- often occur from tick bites 13. Define the term bacterial arrangements. Clinically significant bacteria may form characteristic cell arrangements (cell groupings) when obtained from a patient sample. Arrangements are an artifact of cell division. As one cell divides to become two, residual cellular materials can hold two or more cells together, thus producing an arrangement. 14. Define the term diplococci. (division in one plane & axis) produces this. Pairs f. Streptococcus pneumoniae: oral NF, OP pneumonia, sepsis, meningitis \>25 years g. Neisseria meningitidis: oral NF, OP meningitis 5-25 years h. Neisseria gonorrhoeae: true pathogen urogenital tract 15. Define the term streptococci. -- cells in chains 5-10+ cells i. Streptococcus pyogenes: true pathogen, pharyngitis (strep throat), skin/soft tissue infections j. Enterococcus faecalis/faecium: NF, intestines; OP UTI, wound, sepsis 16. Define the tetrad and sarcina arrangements. And species. k. Tetrad- group of 4 cells; NF arrangement, Micrococcus luteus l. Sarcina arrangement- group of 8 cells; Pepto streptococcus sp. NF-OP n (deep tissue infections) 17. Define the staphylococci arrangement. Irregular cluster of cells (no patterns) What prominent species forms this arrangement? Staphylococcus aureus: NF skin; OP any sterile site, major HAI agent 18. What is the purpose of air drying a slide. - To prevent cell lysis when exposed to fixing agent. 19. Be able to list the three (3) purposes of slide fixation. s. Adhere cells to the slide t. Kill cells u. Allows dyes to better stain cells. 20. Be able to provide five (5) characteristics of *S. aureus*. m. staphylococci: group of cocci chains n. Clinical species: Staphylococcus aureus: NF skin; OP any sterile site, major HAI agent o. Normal Flora of Skin\ Harsh environment\ Skin bacteria are deposited to sterile tissue with loss of epidermis\ Many species of skin NF and NF-OP species 1. Salt tolerant (growth in hypertonic environments)\ 2. Thermotolerant (to 50oC/122oF)\ 3. Resistance to desiccation: extreme dryness\ 4. Resistance to acidic pH: survival at pH 3.5\ 5. Multiple virulence factors\ 6. Clinical Significance 21. **Define medical nutrition therapy (MNT). -prevention and treatment of diseases and conditions, including severe and chronic infectious diseases, using a patient specific nutrition plan.** 22. Be able to list three (3) benefits of MNT regarding **infectious diseases**. - Functions to support the immune system that helps immune system to efficiently remove pathogens and facilitates regeneration of tissue damaged by pathogen. - MNT can also be used to prevent infection by specific pathogens. Due to certain injuries and conditions, not all patients are capable of eating (by mouth). In such cases, nutrition can be administered to patients by alternate routes in two ways. - Through total enteral nutrition (TEN)-- which refers to a liquid diet that is given through a naso-gastric (NG) tube (feeding tube) that is passed through the nasal passage and rests in the stomach. Given to patients who have a function gastrointestinal (GI) tract and has the benefit of maintaining GI functions. 23. Be able to define total enteral nutrition (TEN) and total parenteral nutrition (TPN). 24. **Be able to compare and contrast the uses of TEN and TPN.** - Lab 4 71. Be able to define the term differential stain. Multiple dyes used to distinguish cells based on chemical or physical differences 72. What is the definition of the Gram stain? Differential stain that distinguishes G+ from G- cells based on PG % 73. What does the Gram stain differentiate? G+ from G- cells based on PG % 74. Will the Gram stain also provide size, shape, and arrangement of bacterial cells? Yes, determines G+ from G- and cell morphology Gram-positive bacteria show blue or purple after gram-staining in a laboratory test. They have thick cell walls. Gram-negative bacteria show pink or red on staining and have thin walls. 75. Know the functions of each of the following reagents for the Gram stain: a. Crystal violet- PG binding dye b. Gram's iodine- stabilizes CV binding to PG c. Decolorizer (methanol)- removes CV from PG; CV will be removed from G- before CV is removed from G+ due to PG% differences. G+ cell will be purple, G- cell will be clear d. Safranin (PG binding dye)- binds the G- PG, cannot bind to G+ due to CV 76. What is the importance of the decolorizer step in distinguishing Gram positive from Gram negative cells? This is what makes it differential G- clear, and G+ purple 77. Why will the safranin dye stain Gram negative cells, but not Gram positive cells? The CV will not allow PG to bind with G+ 78. What is the purpose of the Gram stain? To determine G+, and G- and cell morphology 79. What are the three clinical applications of Gram stain for presumptive assessments? 1. Presumptive identification of species 2. Initial antibiotic selection 3. Presumptive prognosis; severity of disease and apporpriate interventions 80. Be able to explain why the three presumptive assessments of the Gram stain are critically important for patient management. It allows for the physician to properly take care of patient, first by identifying what species, then selecting the correct antibiotic, gauges the severity of the disease to treat accordingly 81. What time frame can Gram stain results be available? \< 1 hour : presumptive identification and antibiotic selection 82. What time frame can Culture and Sensitivity results be available? What information does the Culture and Sensitivity provide for the clinical management of the patient? \~24-48 hours: definitive identification and antibiotic selection 83. Be able to explain the semi-quantitative scale of 0-4+ in the assessment of bacteria, other cellular microbes, white blood cells (WBCs) and epithelial cells. Provides a relative number for bacteria, relative number of WBC (RBC- if vascular damage), and relative number of epithelial cells → 99% of bacteria make a capsule 84. What is the significance of the 0-4+ scale regarding the number of bacterial seen on Gram stain? 0 - none, 1- rare, 2-few, 3-moderate, 4-many; this is the grade for number of bacteria, 4+ tissue bacteria, virulent & destructive 85. What is the significance of white blood cells (WBCs) seen on a Gram stain? To see if there are many WBC trying to fight pathogen- addresses the severity of infection 86. What is the significance of epithelial cells seen on a Gram stain? Provides further information like specimen type and indicators if the patient is infected with pathogen 87\. Know ALL the presumptive Gram stain, shape and arrangement characteristics for bacteria from page 4 of Lab 4: a. Wounds (skin, dermal tissues): - G+C, chains: streptococcus pyogenes - G+C, clusters: staphylococcus aureus ← everywhere except urine - G-R: pseudomonas aeruginosa - G+R: clostridium perfringes, C.histolyticum - G+C, chains, short: Enterococcus faecalis b. Sputum (respiratory sample, bronchioles, lungs)- MUCUS IN R "STERILE TISSUE" - G+C, Pairs: streptococcus pneumoniae - G+C clusters: staphylococcus aureus - G-rods:Klebsiella pneumoniae c. Cerebrospinal fluid (CSF)- brain, meninges (100% certain what pathogen it is) - G+C, pairs: streptococcus pneumoniae - G-C, Pairs: d meningitidis ← 5-25yr/o - G-rods: Haemophilus influenzae d. Urine (renal system) - G-rods: E.coli - most common reason for UTI - G-rods (non-e.coli): enterobacter aerogenes, proteus mirabilis (fecal flora) - G+C, Chains, short: Enterococcus faecalis, eneterococcus faecium - G+C, Clusters : Staphylococcus saprophyticus- only time its is not s.aureus e. Blood - G+C, pairs: streptococcus pneumoniae - G+C, clusters: staphylococcus aureus - G+C, chains, short: Enterococcus faecalis - G-R: E.coli f. Uro-Genital tract - G-C, pairs: Neisseria gonorrhoeae - Spirochete: treponema palladium - Intracellular: chlamydia trachomatis g. Peritoneum/ Peritoneal (perotinitis) - G-R Bacteroides 88. Be able to interpret Gram stain results for bacteria, white blood cells, and epithelial cells. +-----------------+-----------------+-----------------+-----------------+ | | Description | Bacteria | Cells (WBC \^2/ | | | | | | | | | | Epithelial \^3) | +=================+=================+=================+=================+ | | None | 0 | 0 | +-----------------+-----------------+-----------------+-----------------+ | | rare | 1 | 1 | +-----------------+-----------------+-----------------+-----------------+ | | few | 2-10 | 2-5 | +-----------------+-----------------+-----------------+-----------------+ | | Moderate | 11-50 | 6-10 | +-----------------+-----------------+-----------------+-----------------+ | | many | \>50 | \>10 | +-----------------+-----------------+-----------------+-----------------+ 89. Be able to define and compare and contrast the terms from Lab 6 a. Vesicle and Bulla - Epidermal elevations in the skin containing clear fluid, Vesicles are \0.5cm. - Common bacterial species include: s.aureus,and s.pyogenes. b. Impetigo - Infection of the superficial layers of the skin characterized by a blister like rash of gold- brown fluid filled vesicles in primarily children (2-6) No scarring or pain is associated with syndrome. - Primary causes: S. Pyogenes ( group A streptococcus) and S. aureus c. Erysipelas - Infection of the superficial layers of the skin characterized by swollen, bright red patches of skin with defined boarders. - Most cases are caused by Streptococcus pyogenes strains that secrete a specific protein toxin that produces illness. Patient may experience pain,fever, chills, headache, and fatigue d. Abscess - Conical swelling of tissue with redness and pain often with pus. - Pus is a collection of destroyed white blood cells and cellular debris due to the secretion of bacterial toxins (leukocidins). The epidermis remains intact. Severity ranges from minor (superficial tissues) to severe (involvement of subcutaneous tissues). - S. aureus is a primary cause ofabscesses, however there are additional species known to produce abscesses. e\. Pustule - Abscess of the superficial dermis that contains pus. - S. aureus is he most common cause of pustules. Typically less than 0.5 cm. f. Folliculitis - Infection of hair follicles consisting of vesicles and Pustules. - S. aureus is a common cause of folliculitis g. Furuncle - Painful abscess nodule formed in dermal and subcutaneous tissues, enclosing a core containing pus \~1-3 cm. - Typically caused by S.aureus. h. Carbuncle - Very large (3-12 cm), painful abscess with draining pus that involves deep subcutaneous tissues and extensive tissue destruction with fever and sepsis complication. - Typically caused by S. aureus i. Cellulitis - Infection of the dermal and subcutaneous tissues with undefined borders, redness, pain, and high fever(\>104 o F/40 oC). - There are \~14 million cases/year U.S j. Necrosis - Defined as tissue death. Dead tissue must be removed from the body. - Necrosis has both infectious and non-infectious causes k. Necrotizing fasciitis - Rapidly spreading infection of the dermal, subcutaneous, and fascia, tissues with high fever, and severe pain. - Necrotic tissue must be surgical removed and may require amputation ❖Lab5 ===== 90. What is the aerobic and anaerobic percentage of oxygen? Aerobic -20.8% Anaerobic - 0.0% 91. What is the cellular origin of toxic oxygen molecules? O2 passes through PM and collides with H20 in cytoplasm to produce oxygen radicals ( ROS ) that are toxic 92. What is the mechanism of action of toxic oxygen molecules? ROS acts as oxidizing agents - take antioxidants \- There is no way for any cell to prevent the formation of ROS in cytoplasm in O2! 93. Name two specific toxic oxygen molecules. a. Superoxide anion (O2-): O2 + electron; very strong oxidizing agent b. Hydrogen peroxide (H2O2): H2O + O atom: stron oxidizing agent 94. Be able to define the functions of superoxide dismutase, catalase, and peroxidase Detoxifying enzymes: remove O2 radicals from cytoplasm 1. Superoxide dimutase (SOD): converts O2- to H2O2 ( hydrogen peroxide) 2. Catalase: converts H2O2 into H2O & O2 3. Peroxidase: converts H202 into H2O. 95. Be able to define oxygen requirement categories for: CR- CELLULAR RESPIRATION F- FERMENTATION a. obligate aerobe: ALWAYS, CR with O2, no F pathway. - Enzymes: SOD, CATALASE, PEROXIDASE b. facultative anaerobe: SOMETIMES, CR with O2, F with no O2 - Enzymes: SOD catalase c. aerotolerant: tolerant to air, F when O2 present, F when O2 absent - " F all the time" → do not have electron transport chain - Enzymes: SOD and Peroxidase d. obligate anaerobe: F when O2 absent, No tolerance to O2 - " O2 poisonous, they do not have detoxifying enzymes NO ENZYMES! 96. Be able to cite the distribution of SOD, catalase, and peroxidase among the oxygen classifications of bacteria Obligate aerobe- SOD, CATALASE, PEROXIDASE Facultative anaerobe- SOD and CATALASE Aerotolerant- SOD and PEROXIDASE Obligate anaerobe- No Enzymes!!!! 97. Know the growth patterns of obligate aerobe, facultative anaerobe, aerotolerant, and obligate anaerobe species in the presence (20.8%) and absence (0%) of oxygen. a. Obligate aerobe: grow really good in 20.8%, and do not grow in the presence of 0% b. Facultative anaerobe: grow good in 20.8% O2, and fair in 0% O2 → will exhibit a differential growth pattern,more growth on +O2, than -O2 \- Catalase: bubbles (+) c. Aerotolerant: grow poor in 20.8% O2, and poor in 0.0% O2 → equivalent growth, they do not use \- Catalase: no bubbles (-) d. Obligate anaerobe: no growth in 20.8%, and poor growth in 0.0% O2- DIE IN PRESENCE OF 98. What specific species produces coagulase? S. aureus 99. What reaction does coagulase catalyze? Fibrinogen (soluable) → coagulase → Fibrin (insoluble) 100. What are the two purposes of the coagulase enzyme for the bacteria that secretes it? 1. evade the immune system ( immunosubversion) 2. Fibrin barrier during abscess formation 101. Be able to describe positive and negative coagulase reactions. a. Coagulase +: solid clot - s.aureus b. Coagulase -: liquid plasma - s.epidermidis, other staphylococci 102. What is the purpose of using casein (protein) agar for the evaluation of virulence of Staphylococcal isolates? Organisms that secrete a protease exoenzyme will hydrolyze the larger protein casein found in milk to smaller peptide or individual amino acid nutrient sources. 103. What is the purpose of using Rhodamine B (lipid) agar for the evaluation of virulence of Staphylococcal isolates? Lipase-positive organisms inoculated on Rhodamine B agar hydrolze the triglyceride -tributyrin to substrates for glycolysis (glycerol) and citric acid cycle (fatty acids). 104. What is the clinical significance of protease and lipase secretion? Bacteria secrete lipases and protease exoenzymes to hydrolyze large fats and proteins in their surroundings to smaller nutrient sourceds that can be transported across the cellular envelope for utilization by metabolic enzymes in the cytoplasm. 105. What is the purpose of using Sucrose-gelatin agar? Supports excessive capsule production Capsule- thick carbohydrate structures that protect the bacterial cell from the immune system 106. What is the purpose of using Starch agar? What enzymes are evaluated using Starch agar? The agar is used to determine the secretion of a specific enzyme to hydrolyze starch, a large carbohydrate molecule that cannot pass through the cell wall Enzymes: alpha amylase and oligo-1, 6-glucosidedase → contributed to tissue destruction = severity 107. What is the function of carbol fuchshin? Bind to mycolic acids to stain AFB cells red 108. What is the timeframe for return of results of an AFB stain? 24 hours 109\. Be able to cite the differential stain for AFB. Will AFB be seen on Gram stain? Will include a 0-4 + grade for the number of AFB seen on the slide ❖Lab6 ===== 110\. Answer the following regarding urea medium: a. What enzyme is evaluated in urea medium? - G- isolate urease b. What reaction does the enzyme catalyze? Ammonia + CO2 c. How does the product of the enzymatic reaction change the color of the urea Medium? The urea test evaluates decarboxylation of amino acids and requires the enzyme urease. Hydrolysis of urea, a precursor of amino acids, produces ammonia and CO2. The formation of ammonia alkanizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to pink pH 8.1. -production of acidic end products turn the medium yellow- indicating that the urease enzyme is absent. d. What is the color change of the urea medium that indicates a positive reaction? Light orange 111\. Answer the following regarding citrate medium: a. What is the purpose of using citrate medium? Determines if a species can use citrate as a sole carbon and energy source. b. What enzyme is evaluated in citrate medium? Citrate - permease c. What reaction does the enzyme catalyze? Converts citrate to pyruvate that is metabolized via Krebs cycle and cellular respiration d. What is the role of cellular respiration for citrate medium? produces alkaline end products e. What is the color change of citrate medium that indicates a positive reaction? The pH indicator bromothymol blue is green at neutral pH and blue at alkaline pH 112. What is the purpose of the catalase test? Evaluates the presence of an electron transport chain that requires oxygen. 113. What is the purpose of the oxidase test? Determines the presence of cytochrome c in the electron transport chain. 114. What is the group of bacteria that are oxidase positive. pseudomonas and other G- species. 115. What is the purpose of the MRVP test? Evaluates two different glucose fermentation end products that distinguish between Gram negative bacteria 116. What end products are evaluated in the MR test? Evaluates stable acid end products with pH \< 4.5 - methyl red pH indicator 117. What end products are evaluated in a VP test? What color change indicates a VP positive test? Evaluates alcohol end products 118. What is the purpose of the indole test? What color change indicates a positive result? The indole test evaluates the tryptophan broth with your Gram negative unknown species. There should either be a yelllow - or red + 119. What pathway is evaluated in the BCP glucose test? What color changes are associated with cellular respiration, fermentation, and oxidative-fermentation? In BCP glucose the pathway- fermentation (yellow, H2 +) / CR cr- purple or blue top/ purple bottom Fermentation-yellow or purple top/yellow bottom oxidative-fermentation-yellow top purple bottom 120. What enzyme is associated with BCP lactose and sucrose utilization? BCP lactose: beta-galactosidase BCP sucrose-sucrase 121. What enzyme is evaluated using ornithine broth? Why is an anaerobic environment Required? enzyme : orinthine decarboxydase, an anaerobic enviornment is required for the OD enzyme to be active, for this reason a mineral oil is used as a barrier to the diffusion of O2. 122. Be able to diagram the nitrate reduction pathway. What are the two end products that are evaluated in this test? - Species can reduce nitrate (NO3) to Nitrite (NO2)- determined by color change of the broth. - NO3 ---\-\-\-\-\-\-\--\> nitrate reductase ---\-\-\-\-\-\-\-\-\-\-\--\> NO2 ---\-\--\> dentrification ---\-\-\-\-\-\-\--\> N2 123. Be able to answer questions 1-11 on the Necrotizing Fasciitis Case Study. - 1.What is the definition of necrotizing fasciitis? Infection of superficial and deep fascia with extensive tissue destruction -.What are the 4 major characteristics of necrotizing fasciitis 1. Discoloration of skin ( tissues) - caused by necrosis 3.What are the two primary monomicrobial causative agents of a majority of necrotizing fasciitis cases? What is the most common cause of necrotizing fasciitis? - Streptococcus pyogenes {worst in this case} 2. G+, chains - Streptococuus Aureus 3. G+ C, clusters - Vibrio vulnificas ( marine) 4. G-R MOST COMMON CAUSE: polymicrobial {mix of G- & G+}: HAI - most common, associated with diabetes. 4.What is the role of bacterial digestive enzymes in the pathophysiology ( disease process) in necrotizing fasciitis cases? - this rapid, within 24 - 72 Hours - \*secretion of several digestive enzymes ( protases, lipase, glycosidases) - Surgical intervention to remove bacteria - Mortality \~30 % 5.What is the definition of debridement? Why is it necessary in necrotizing fasciitis cases? Debridement- removal of infected tissue, organs Amputation - removal of limb 6.Explain how the wound vac system is used to treat necrotizing fasciitis cases? Negative pressure wound therapy (npwt): wound vac system 1. Vaccuum reduces swelling, promotes blood flow to the wound for improved tissue healing 2. Vacuum decreases bacterial cell division ( growth) 7.What is the presentation in the Emergency Department to the Triage Nurse that suggests necrotizing fasciitis? What do the vital signs suggest? Does the blood work indicate a serious infectious process? Pt presentation: area of discoloration in lower R leg. With intense pain at the site with examination ( disproportional to the area affected) Infection with hypotension : low BP ( SEPSIS!) 8\. What is your interpretation of the surgery Gram stain result? What is the prognosis of the patient? Presumptive: streptococcus pyogenes - very progressive NF, due to high \[enzymes & toxins\] , susceptible to antibiotics 9.Were the initial antibiotics appropriate for the presumptive identified agent? Be able to explain your answer. Antibiotic therapy & I.V. - Clindamycin: good tissue penetration, stron inhibitor of bacterial protein synthesis ---\> decrease ( enzyme & toxins) - Zosyn: cell wall synthesis inhibitor. +BLI 10. Was amputation necessary for this case? Be able to explain your answer. Will physical therapy be required for the patient? Multiple amputations required to remove necrotic tissue, and a year of physical therapy required. 11. What is the purpose of MNT for the patient? PRELABS: