Micro Test 3

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Micro Test 3
2015-04-25 10:39:25
Micro Test

Micro Test 3
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  1. Why classify organisms?
    • Establish similarities
    • Can influence therapy
    • Predict common characteristics/features (growth, habits)
    • To place in appropriate group which allows you to predict characteristics of organisms
    • Common language
    • facilitates the retrieval f information and appropriate study
    • DNA
  2. Viability Stains
    • Living Organisms
    • Asses viability
    • Includes:
    • Methalin Blue Yeast Viability
    • Trypan blue exclusion
  3. Methalin Blue Yeast Viability
    • Specimen w/ Cata Albacins and stain w/ methalin blue and look for yeast cells.
    • Dead cells = blue
    • Live Cells = clear
    • Hep. Vaccine is made in yeast
  4. Trypan Blue Exclusion
    • Used to test viability of bone marrow donations.
    • Get membrane of bone marrow, add trypan blue.
    • Viable Cell = Will not accumulate trypan blue
    • Dead Cell = Stains blue
  5. Cell Viability and Viruses
    • Relevant in virus detentions because the virus is associated with characteristics cytopathogenic effects such as cell death and rounding.
    • If cells are dead = cannot tell if virus or not so you have to show viability.
    • Any change seen after is due to the virus cuz the virus killed the cell and that is how you can infer it.
  6. Basic principles of Microscopy
    • ID organisms and diagnose infections
    • Resolution
    • Magnification
    • Contrast
    • Formula: R=lamda/2NA
    • Most useful unit: micrometer = 10^6
  7. Microscopy Forumula
    • Deals with resolution
    • R=lamda/2NA
    • - Lamda = wavelength
    • - NA = numeric aperture
    • - Smaller R is a better value
    • Smaller wavelength = better resolution
  8. Types of Microscopes Used
    • Unaided Human Eye
    • Compound Light Microscope
    • Scanning electron microscope
    • Transmission electron microscope
    • Scanning tunneling microscope
    • Atomic Force Microscope
  9. Unaided human eye
    200 µm
  10. Compound Light Microscope
    200 nm-10 mm
  11. Scanning Electron Microscope
    0.4 nm - 1mm
  12. Transmission Electron Microscope
    0.078 nm - 100 µm
  13. Scanning tunneling Microscope
    0.01 nm - 10 nm
  14. Atomic Force Microscope
    1 nm - 10 nm
  15. Benefits of Immersion Oil
    • Cannot see in focus unless you have immersion oil because it has the same refractive index as glass and it actually extends the objective lens (focus light) and it has to have contact with the oil.
    • Basically you can see better because it acts as another lens
  16. Probe Microscopy
    Used for specific antibodies not useful clinically
  17. Why Classify?
    • Indicates specific characteristics of organisms
    • More on notes, listen to voice
  18. Taxonomic Hierarchy
    • Domain - bacteria, archaea, eukarya
    • Kingdom - Animalia, Plantae, fungi
    • Phylum
    • Class
    • Order
    • Family
    • Genus
    • Species
  19. Taxonomic Kingdom Categories
    • Animalia
    • Plantea
    • Fungi
    • Protista
    • Prokaryotae
  20. Linnaus and Taxonomic Categories
    • Linnaus's goal was to classify organisms to catalog them.
    • Modern goal is to understand relationships among groups of organisms
    • - Reflect phylogenetic hierarchy
    • - Emphasis on camparison of organisms genetic material
    •    - Led to proposal to add domain
  21. Taxonomic ID Classifications
    • Physical characterisitcs
    • Biochemical tests
    • Serological tests
    • Phage typing
    • Analysis of nucleic acids
  22. Serology
    • Antibody - antigen reactions
    • Antibodies found in blood of animals that are raised with particular anitbody
  23. Serological Classifications
    • Inc of antibody content is typically indicative of infection.
    • Can look at relative concentrations of IGG vs IGM
  24. IGG vs IGM in Serological Classifications
    • IGG = the longest half life and has the highest concentrations.
    • Think of below as a point on a graph:
    • During an infectious process the first antibody produced is IGM and the second AB produced is IGG. So if a patients blood has more IGG than IGM it means that it's an old infection. If there is more IGM than IGG it means that is a recent infection.
  25. Antibody Levels
    • Antibody levels document response to to vaccination and to confirm protection.
    • To do this you measure the Titer.
    • All about concentration ratios.
  26. Titer
    • A way of expressing concentrations. To get titer you need to use serial dilution.
    • Measures antibody levels to determine if you have contracted an infection
  27. Phage
    • Bacterial virus
    • Also have plaques
    • Specific to each strain
    • Can tell because it kills bacteria and you get lyses.
  28. Plaques in Phages
    Absence of bacteria and where ever the virus infects bacteria you get the holes which are plaques
  29. Basis of Phages
    • Phage typing enables the the indicative/definitive ID of different strains of bacteria that establish the origin or the source of infection.
    • Identical strains are usually infected by the same phage pattern
  30. Uses of Phages
    • You have 3 patients: S. Aureus for example.
    • Patient A has a phage pattern of 1, 8, 16, and 32.
    • Patient B has a phage pattern of 1, 12, 24, and 68. 
    • Patient C has a phage pattern of 2, 4, 10, and 12
    • Doctor has a phage pattern of 2, 4, 10, and 12.
    • Q. Where did the S. Aureus come from.source?
    • A. The source is the doctor because the phage patterns match
  31. Taxonomic Keys
    • Consist of dichotomous keys
    • In the biochemical unknown you have either gram + or gram - and in morphology it could be a rod or coccus
  32. Dichotomous Keys
    • Shows characteristics of organisms so if you look at the biochemical profile you will end up with the definitive ID for the species.
    • Think of this as a flow chart we do in lab to ID the organisms, it's either this or that and you follow steps
  33. What is the difference between citrobacter and enterobacter
    Citrobacter doesn't produce acetoin
  34. T/F. Humans are capable of anerobic respiration?
  35. VP test
    Detects acetoin and butanediol
  36. MR test
    Detects mixed acid fermentation
  37. Metabolism Definition
    • Total of all biochemical reactions in an organism.
    • Basic feature of Life
    • Consists of anabolic and catabolic reactions
  38. Anabolic reaction
    • Synthetic reaction.
    • Makes new macro molecules for cells.
    • Linked with ATP production/use
  39. Catabolic Reactions
    • Degradative. 
    • Breaks down macro molecules to release energy as ATP
    • Link by ATP production/use
  40. Why is Metabolism Clinically Relavent. 2 Examples
    • 1. Infection associated with distinctive odors produced by volatile end products of microbial metabolism.
    • - Whiff test for G. Vaginalis
    • 2. Lactate relevance in sepsis and septic shock
  41. Whiff Test for G. Vaginalis procedure
    • Add potassium hydroxide to cervical scraping slide, then you will get a distinctive fishy amine odor which confirms diagnosis of g. Vaginalis
    • #1 of why Metabolism is relevant cuz of odor
  42. DNA Gyrase
    • Unwind DNA in order to replicate.
    • Can be in bacterial form or in eukaryotic form that is found in humans
  43. Antibody Level Applications With Titer: Single Static Titer USE IGG VS IGM FOR SEROLOGY RELAVENCE
    Single Static Titer: You can determine if a patient is infected by taking the ratio of titer of the patient vs the gen. population titer. If the gen. population has a ratio of 1:16 and the patient has a ratio of 1:32 that means that the patient is infected because the antibody levels are 1 fold higher.
  44. High Titer
    • Means that you have a high concentration with the most antibodies. So you have to dilute that more.
    • Think of it as 10 spoons of sugar and 2 spoons of sugar in different cups. 10 spoons will be sweeter so you have to put more water to make it normal.
  45. Definitive ID: Biochemical Profile
    • Part of metabolism
    • Bacteria ID by biochemical profile (end products).
    • Enzymes Present
    • Substrates Utilized
  46. E. Coli Vs. Enterobacter part of Biochemical Profile
    • They are differentiated by IMVIC tests
    • E. Coli: Positive for Indole and Methyl Red. Negative for Vp and Citrate
    • Enterobacter: Positive for VP and citrate, negative for indole and methul red
  47. Volatile End Products
    • Part of Metabolism
    • C. Defficile ID is based on production of isocaproic acid in feces.
    • Anaerobes ID by gas chromatography detection of end products
  48. Methods of Control
    • Physical/chemical methods use of heat/others
    • Depend on inhibiting key enzymes, protein denaturation, inhibition of metabolic pathways by antibiotics
  49. Patient Presentation
    • Infectious diseases associated with disturbances in homeostasis and metabolic activity.
    • Sepsis and septic shock. Arterial blood gases, vital signs, blood ph, other
  50. Stages of Metabolism
    • Glycolysis
    • Krebs Cycle
    • Oxidative Phosphorylation
    • Follows the order above one happens after the other
  51. Krebs Cycle
    • Produces NADH and FADH, ATP, CO2
    • Occurs with aerobic and anaerobic respiration not with fermintation
  52. Oxydative Phosphorylation
    • Involves and Electron Transport Chain
    • Involved in Aerobic and Anaerobic Respiration
    • Read about electron transport chain to understand
  53. What are enzymes?
    Substance produced by a living organism that acts as a catalyst to bring about specific biochemical reactions
  54. Clinical Applications of Enzyme Activity
    • Fluodroquinolones and bacterial DNA gyrase
    • Sulfur Drugs as antibiotics, will deal with specificity of action
  55. Action of Fluodroquinolones (FQ) on Bacterial Gyrase
    • FQ's (antibiotic)
    • Will have more affinity on the bacterial gyrase and very little in eukaryote gyrase.
    • - It will inhibit bacteria in the bacterial gyrase which is based on affinity (more binding capabilities)
    • FQ prefers to bind to bacterial gyrase because of the affinity. Specifically inhibit bacteria replication which is the goal
  56. Sulfur Drugs
    • Specificity relies on the difference in the pathways of enzymes used to make folic acid.
    • When treating with sulfur drugs it will inhibit bacterial pathways cuz of the different enzymes and the sulfur drugs actually PABA which mimics folic acid synthesis.
    • So if you unhibit it that means the folic acid stops and tha prevents DNA synthesis and as a result the bacteria are inhibited
  57. Six Categories of Enzymes
    • Hydrolases
    • Isomerases
    • Ligases or polymerases
    • Lyases
    • Oxidoreductases
    • Transferases
  58. Enzyme Activity
    • Lower activation energy
    • - makes reactions easier to achieve so reaction is faster
    • Measured in how fast reactions occur
    • Provides a physical site for the collision of reactions
  59. Things that influence Enzymes
    • Temperature
    • pH
    • Enzyme substrate concentrations
    • Presence of inhibitors
  60. Things that inhibit Enzymes
    • Substances that block enzyme active site
    • Do not denature enzymes
    • Three types
  61. How Enzymes are Inhibited
    The reaction rate slows down or terminates activity
  62. Three types of Enzyme Inhibitors
    • Competitive inhibitor
    • Allosteric inhibition
    • Feedback Inhibition
  63. Competative Inhibitor
    Inhibit by competing with substrate and you can overcome it by inc. substrate concentration
  64. Allosteric Inhibition
    • Could be activator or inhibitor. 
    • Inhibitions occurs when the allosteric inhibitor binds to an active site of an enzyme which changes the active site to an inactive site because it changes shape of the binding site and makes it inactive
  65. Feedback Inhibition
    • Org. needs an end product.
    • Shuts down when there is enough and when decreases it starts again.
  66. Glycolysis Stages
    • Initial reactions Energy-
    • Energy-Investment Stage
    • - Spend money to make money
    • Lysis Stage
    • - Destruction on voice 
    • Energy-conserving stage
    • - Recover ATP
  67. Why is lactate relevant in sepsis and septic shock?
    • #2 Of why metabolism is relevant.
    • Critical in diagnosis of cryptic septic shock
    • Relevant because during sepsis and septic shock you have ischemia, so you diminish normal metabolic activity since you lack oxygen. Because of that you get lactate produced at much higher concentrations than normal as a by product of fermentation
  68. Rybozymes
    Are autocatalytic they are the exception and are not proteins like other enzymes
  69. Michalis Menten Plot
    • Km = 1/2 of Vmax and is a measure
    • Vmax = Substrate concentrations where reactions are fastest/highest possible. (where the graph curves)
    • Vmax is achieved with all active sites are saturated with substrate
    • Km = indicative of the affinity of substrate for the active site of the enzyme
    • Read in notes and listen to lecture
  70. Glycolysis
    • Breaks down glucose to form pyruvates
    • Produces 4 ATP and 2 NADH, but uses 2 ATP for the proccess and nets 2 ATP and 2NADH
    • Converts exogonic reaction to endogonic reaction
  71. Lysis Stage
    • Step 2 of Glycolysis
    • Breaks down glucose to form pyruvates
  72. Energy Conserving Stage
    • Stage 3 of Glycolysis
    • Energy Recovery Stage
    • Converts pyruvate to make ATP and NADH
  73. Energy Investment Stage
    • Stage 1 of Glycolysis
    • Spend money to make money into pyruvate
  74. Pyruvate Conversion Possibilities
    Can be fermented, can go through oxidative phosphorylation when oxygen is present in aerobic respiration, or can be broken down anerobically using alternate acceptors
  75. Hyperbolic Oxygen
    Useful as an adjunct to treating flesh eating disease, dicubibus ulcers, gangrene, and wounds that do not heal.
  76. Why Anaerobes and Anaerobes
    They cannot survive with oxygen because they don't have the oxygen protective enzymes
  77. Alternate Anaerobic Acceptors
    Sulfate, nitrate, carbonate, and thiosulfate
  78. Glycolysis and Krebs Cycle
    • Are major amphibolic pathways. Function in both anabolic and catabolic activity
    • All major macro molecules in any org. are cycled, synthesized, and broken down through Krebs and Glycolysis
  79. Largest # of bacteria per gram of fecal matter is?
    Anaerobic bacteria
  80. Krebs Cycle Produces
    • 2 molecules of ATP
    • 2 molecules of FADH
    • 6 molecules of NADH
    • 4 molecules of CO2
    • FADH and NADH do not produce ATP until reaching the electron transport chain
  81. Electron Transport Chain
    Most significant ATP production occurs here
  82. What is involved in having a successful Electron Transport Chain
    Need to have an intact membrane, electron carriers and proteins that flow out
  83. ATP Synthase
    • Only way that the protons can get back into the membrane of the electron transport chain.
    • Uses chemiosmosis to work which has 3 parts loose, tight, and open
  84. Chemiosmosis
    • Protons pumped out of electron transport chain.
    • Then Protons build up outside of membrane
    • Then protons enter membrane through ATP Synthase which then follows below steps
    • Loose = accepts things
    • Tight = Groups together
    • Open = Releases back into electron transport chain
  85. T/F. Life and death can be defined as metabolism
  86. T/F. Life is a tiny electron current that is directed by the sun.
  87. Proton Motive Force (PMF)
    Force implies potential energy due to electron and proton flow
  88. Aerobic Respiration
    Oxygen serves as final electron acceptor
  89. Anaerobic Respiration
    Molecule other than oxygen serves as final electron acceptor
  90. Question? An aerobic org. was fed radioactive labeled oxygen where does this oxygen end up?
  91. Question? A fuel tank was painted with cyanide paint to prevent microbial growth, 2 days after the bacteria were recovered from the tank. What process was being used to produce ATP?
  92. Alternatives to Glycolysis
    • Pentose phosphate pathway
    • Entner-Doudoroff pathway
  93. Fermentation
    • Uses organic molecule within cell as final electron acceptor
    • Sometimes cells cannot completely oxidize glucose by cellular respiration
    • This provides the cells with source of NAD
  94. Similarities between Aerobic, Anaerobic Resp. and Fermentation
    • All 3 process are common to glycolysis
    • All produce ATP substrate level phosphorylation
  95. Differences between Aerobic, Anaerobic Resp. and Fermentation
    • Different end products are produced
    • Aerobic = most efficient
    • Aerobic & Anaerobic = Role of CO and cyanide
    • Aerobic & Anaerobic = Include Oxidative phosphorylation
    • Aerobic & Anaerobic = Has electron transport chain