Micro Test 4

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Micro Test 4
2015-05-17 00:36:45
Micro Test

Micro Test 4
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  1. How to culture organisms
    You have to give them what they need and what they like. It's that simple
  2. Colony
  3. Bacteria and food
    They need a lot of food because they multiply quickly. No food = death, unless they are spores
  4. Spore Formation
    Are formed during harsh environments. It's a signal to convert the organism into sport in order to survive.
  5. CFU
    Colony forming units. The cells are actually clones of the original cell
  6. Microbial growth and results
    • Growth - Increase in a population of microbes
    • Results - An aggregation of cells arising from single parent cell
  7. Nutrients: Chemical and Energy Reqs.
    Major Nutrients - Carbon, energy, and electrons
  8. Carbon nutrient groups
    • Autotrophs
    • Heterotrophs
    • Usually come from a source like beef extracts.
  9. Autotrophs
    • Self feeding organisms. They make their own food like green plants and photosynthetic org. as for microbes algae and cyanobacteria.
    • Carbon nutrient groups
  10. Heterotrophs
    • Depend on preformed organic material. Need other source of food, can't make their own.
    • Carbon nutrient groups
  11. Energy Nutrient Groups
    • Chemotrophs
    • Phototrophs
  12. Chemotrophs
    They use chemical reactions to produce energy required for growth. Like we do with metabolism
  13. Phototrophs
    Use the sun as their energy source
  14. Undefined/Defined mediums
    • Defined - Shows exactly the amounts for the exactly that is known. 
    • Undefined - Mcconkeys is undefined
  15. Four basic groups of organisms
    Carbon dioxide, Organic compounds, and chemical compounds
  16. Auto.
    All they need is carbon dioxide mixed with light or chemical compounds
  17. Hetero
    Preformed organic compounds. Mixed with light of pchemical compunds
  18. FTM
    Fluid Thogrycollade
  19. T/F: Oxygen is a toxic biological molecule to all life forms.
    True cuz oxygen utilization makes hyperactive free radicals that are extremely toxic. The free radicals break down cell walls, cell membranes and cause mutations in DNA. That is why you need the oxygen protective triad otherwise we would not be able to use oxygen and evolution would not have occured.
  20. Toxic Oxygen
    • Produced by metabolic action.
    • Since oxygen makes hyperactive free radicals you have use hyperbolic oxygen to treat gangrene, wounds that do not heal etc.
  21. Four Toxic Forms of Oxygen
    • Singlet oxygen
    • Superoxide radicals
    • peroxide anion
    • Hydroxyl radical
  22. Bacteria that Oxygen affects
    • Aerobes
    • Anaerobes
    • Facultative anaerobes
    • Aerotolerant anaerobes
    • Microaerophiles
  23. Aerobes
    • Have all 3 of the oxygen protective triad and can survive and grow in oxygen.
    • Need to have 21% oxygen present
    • Organism related: Pseudomonas
  24. Anaerobes
    • Don't have any of the oxygen protective triad so they die in the presence of oxygen and cannot grow.
    • Organism related: Clostridium
  25. Facultative Anaerobes
    • Can grow with our without oxygen, they usually have at least 1 or 2 of the oxygen protective triad. Prefers to grow in oxygen cuz ATP production is better.
    • Organism related: S. Aureus
  26. Aerotolerant Anaerobes
    • Typically have 1 of the oxygen protective triad. Not killed at the presence of oxygen. They can only tolerate/withstand oxygen not used for growth. Because they have inductive superoxide desmotase
    • Oxygen is lethal @ high concentrations
    • Organism Related: Bacteroides
  27. Microaerophiles
    • Can only grow under reduced oxygen 5-10% and increase CO2 8-10%.
    • Organism Related: Campylobacter
  28. http://microbeonline.com/oxygen-requirements-for-pathogenic-bacteria/
    Used for pp9 on 4-19 notes about aerobes to microaerophiles
  29. Nitrogen
    • Gets recycled from amino acids and nucleotides
    • Cyanobacteria can fix nitrogen and convert nitrogen from the atmosphere
  30. Temperature
    • If too low = activity slows and membranes become rigid and fragile
    • It too high = proteins are denatured and deactivated and membranes become too fluid
  31. Four categories of microbes based on temperature ranges for growth
    • Psychrophiles
    • Mesophiles
    • Thermophiles
    • Hyperthermophiles
  32. Psychrophiles and Temperature
    • Love low temp
    • Growth: -5 to 20°C
  33. Mesophiles and Temperature
    • Love medium temp
    • Growth: 15 to 45°C
    • These are most likely to infect humans because of our body temp is the same
  34. Thermophiles and Temperature
    • Love high temp
    • Growth: 45 to 80°C
  35. Hyperthermophiles and Temperature
    • Love super hot temp like boiling water and stuff
    • Growth: 65 to 105°C
  36. Listeria Monocytogenes
    • Is a Psychrotrophic bacteria. They are really mesophiles but can adapt to psychrophiles that is why they grow in cold environment. 
    • Clinical significance of Listeria: Septic abortion, neonatal sepsis, sepsis and septic shock, fetal death
  37. pH requirements
    • Organisms are sensitive to changes in acidity
    • Neutrophiles grow best in a narrow range around neutral pH
    • Acidophiles grow best in acidic habitats
    • Alkalinophiles live in alkaline soils and water
  38. Physical effects of water
    • All organisms require water
    • Used in many metabolic reactions
    • Dehydration is the leading cause of death
    • Endospores and cysts are the exception to dehydration
  39. Dessication
    • Part of dehydration, difference between pickling and curing.
    • In fact we use this when there is a high salt conent.
    • Can also be used to make ham and jelly
  40. Osmotic Pressure
    • Pressure is exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane.
    • Restricts organisms to certain environments
  41. Hypotonic solutions
    Have lower solute concentrations
  42. Hypertonic solutions
    Have greater solute concenrtations
  43. S. Aureus and salt tolerance
    It is salt tolerant and not a halophile
  44. Halobacter salinarum and salt
    • 12-25% salt tolerant = halophile. 
    • Can be found in salt marshes, ocean, and saltered sea/dead sea
  45. Facultative Halophile
    • Vibrio species/ocean.
    • Vibrio cholera
    • Vibrio vulunificus
  46. Vibrio Cholera
    • Transmitted by fecal contamination
    • Can affect human body because theya re facultative and not obligate.
    • Which means they don't need salt to start growing
  47. Vibrio Vulunificus
    Contaminated shell fish
  48. Betadiene
    • High molecular weight polysaccharide
    • This is the adaptive response found in facultative halophiles.
  49. Halophile
    Grow in salt
  50. How to distinguish between obligate and facultative halophoes
    • They are distinguished by the range of salt.
    • The obligate need 15-25% salt to grow while the facultative need much smaller salt concentrations and can start at 0%
  51. Know biofilms
    Look it up online
  52. Synergistic Relationships
    Use environment to grow in the same place and exploit it to their own advantage
  53. Symbiotic Relationships
    Usually are between 2 or more organisms. Grow together in the same environment so they grow much faster and the effect is greater than what is expected
  54. How to get an appropriate specimen. On written
    • 1. Should contain viable organisms.
    •    - Protozoa are the exception cuz they do not      have to be viable in order to be ID. They            are the only preserved media.
    • 2. Free from contamination
    • 3. Specimen must be representative of te infectious process
    • 4. Must be taken from the infection site and must also be free of org from neighboring sites
    • 5. Processed ASAP or stored under conditions to preserve viability
    • 6. Place in container to preserve growth or viability
    • 7. Procedure for sampling must be appropriate
    • 8.Taken in quantities adequate to permit work up
    • 9. Label appropriately, including patients name, source, specimen etc.
    • 10. Send to the lab in timely fashion
  55. Inoculation
    • Take bacteria and put it on something that will make it grow so proper nutrients are needed.
    • Viruses, chlamydia need to be alive because the cannot be grown in artificial medium
  56. When to culture for isolation?
    For viruses and bacteria only, sometimes fungi
  57. How are the five I's relavent?
    Patient comes in to hospital and they are ill and they want it diagnosed and treated. You collect the specimen and process in lab which then becomes the basis of CNS. Have to isolate org, then Id by biochemistry, then do the susceptibilit of it. more on voice?
  58. Standard Plate Count/ Serial Dilution
    Was not designed for ID of org but to count how many bacteria is present
  59. Describe streak for isolation as done clinically with adequate detail to document isolation.
    Involves flaming and cooling of loop every time you streak. You drag 4 times and the 5th time 2 squiggles in the middle. The idea is to perform serial dilutions over the entire plate.
  60. The most accepted and typical method to ??? (on voice 5-6-15)
    Quadrant streak isolation is the answer.
  61. Types of Culture Media
    • Defined media
    • Complex media
    • Selective media
    • Differential media
    • Anaerobic media
    • Transport media
  62. Defined Media
    • Every single known ingredient is established as well as the concentrations, volume, proportions are all established. 
    • Everything is established about the medium
  63. Complex Media
    Vast majority of these are used clinically
  64. Selective Media
    Mcconkeys, manitol salt agar (MSA), EMB are selective because the allow only certain bacteria to grow
  65. Selective and differential for fecal coliforms in mcconkeys and EMB
    Such as E.Coli
  66. Why is MSA selective?
    • Because it has 5% salt and it is inhibitoy for most bacteria other than S. Aureus.
    • S. Aureus will grow while others wont
  67. Why is Mcconkeys inhibitory?
    • It has inhibitory crystal violet and will inhibit gram - bacteria as well as isolate them. Ex. E.Coli cuz it's a fecal coliform and is gram -
    • Ferments lactose and turns it pink
  68. Why is EMB selective and differential?
    • Fecal coliforms have distinct representation on it like iridescent, blue black colonies w/ or w/o metallic sheen.
    • Inhibits growth of gram +, favors growth of gram - and also gives ID because it has media that is utilized for these bacteria. This is why it is selective and differential. 
    • Vigorous fermentation of lactose and glucose will produce the above results with color etc.
  69. Differential Media
    The reactions are specific for the organism that is what makes it differential since it can tell which org is present based on its documented reaction
  70. Transport Media
    Specimen DNA, to transfer the bacteria in order to preserve it so it gets to the lab in a viable form for it to be examined.
  71. Media used for fungal growth?
    Sabourads dextrose agar because it's acidic and fungi prefer acidic conditions. Selective but not differential
  72. Blood Agar
    • Generalize but are not specific
    • Show hemolysis which is the destruction of red blood cells.
    • Shows:
    • Beta hemolysis
    • Gamma hemolysis
    • Alpha hemolysis
  73. Beta hemolysis
    Complete lysis
  74. Alpha Hemolysis
    Partial grading of blood
  75. Gamme Hemolysis
    No hemolysis
  76. CAMP Test
    ID group B streptococci based on their formation of a substance. Like a bow tie
  77. A gram + coccus is shown to be beta hemolytic and gives a + CAMP reaction?
    • Asnwer is: Streptococcus agalactiae because it's a group B.
    • or Streptococcus pyogenes
  78. Streptococcus Pyogenes
    Group B which causes strept throat
  79. A gram + spore forming rod is shown in reverse CAMP
    Clostridium perfringenese the enzymes produced by this literally digest skin cells that allow bacteria to persist and infect
  80. Clinical Relevance of CAMP
    • Related clinically because group b strept. should be screened for anyone contemplating pregnancy, both male and female.
    • Male especially if they are donating sperm because it will transmit.
  81. Pregnant Females Screening and CAMP relavence
    • All pregnant females need to be screened for group B strept. Generally treated in the last trimester. Her partner should also be treated and tested because you can reinfect.
    • This can cause sepsis since 40% of neonatal sepsis and meningitis is cause by ecoli and streptococcus galactia?
  82. Special Culture Techniques
    • Animal and cell culture
    • Low-oxygen culture
    • Enrichment culture
  83. Animal and Cell Culture
    Used for chlamydia and viruses
  84. Low-Oxygen Culture
    • Grow low oxygen microaerophiles.
    • Bacteria that need very tiny oxygen amounts to grow.
  85. A patient has diarrhea for 2 or 3 days, very watery stools and reports drinking raw milk. Campylobacter suspected. What environment would you provide to allow isolation of campylobacter?
    • A. Aerobic
    • B. Anearobic
    • C. Microaerophilic
    • D.None of the above
    • Answer is C. Microaerophilic
  86. Preserving Cultures
    • Refrigeration - Stores for short periods of time
    • Deep-Freezing - Stores for years
    • Lyophilization - Stores for decades
  87. Generation Time
    • Time required for bacterial cell to grow and divide.
    • For humans it's 9 months
  88. Microbial Grow and Clinical Importance
    • They rapidly multiply, since they divide so rapidly even small numbers can lead to full virus infections.
    • # of bacteria determine the virulence.
    • High virulent org. need fewer # of bacteria to cause disease.
  89. Virulence and Ex.
    • Ability to cause disease measured by # required.
    • Ex1. E. Coli OK157 causes hemolytic uremia which produces ~10-100 CFU so they are highly virulent
    • Ex2. Salmonela has a infectious dose of 10^6 through 10^9 CFU to cause infection is it is less virulent than E. Coli
  90. Lag Phase
    • During this phase they are preparing to divide and is a period of intense metabolic activity, not to be interpreted as being inactive or dormant.
    • Time spent in lag phase depends on what environment it came from. Rich = less time in lag phase.
  91. Log (exponential) Phase
    Phase of most active growth and they are dividing in their maximum rate, it's linear and it's the only part of the curve that is associated with linear increasing.
  92. Stationary Phase (Batch culture only)
    • Continuous culture doesn't have this stage. It's synchronized. 
    • The bacteria are dieing as fast as they are produced so they = a steady state. 
    • Should not be confused with absence of growth or them stopping dividing.
  93. Death (decline) Phase
    • Continuous culture doesn't have a death phase. 
    • Linear decrease in #'s (exponential), and is happening because of exhaustion of nutrients and accumulation of waste products which are inhibitory.
  94. Synchronous Culture
    • All bacteria are in the same stage of growth. Simultaneous division.
    • They are multiplying as rapidly as possible because they have abundant nutrients and the inflow matches the outflow.
    • Same rate of inflow and outflow which maintains the same growth.
  95. Wash out of bacteria in culture
    The bacteria numbers decrease significantly and there is no more culture left because of the inflow and the outflow
  96. Continuous Culture
    • There is no accumulation of cell death or waste products because they all go into the outflow.
    • The material from the inflow and the outflow is the same and when that happens all the growth is synchronized and because of that they are in the exponential rate of growth.
  97. Continuous Culture Advantages and Disadvantages
    • Advantage:
    • It allows you to study physiological and adaptive responses in an organism
    • Disadvantage:
    • The body is not a continuous culture so you can't predict how it will react unlike the controlled culture.
  98. What is similar to a continuous culture?
    Biofilms because as along as you have a solid substrate and nutrients you ahvre accumulation of bacteria
  99. Quarum Sensing
    • Form of communication that bacteria use to talk to each other.
    • If you stop this they wouldn't be able to talk to each other and then you can break up the biofilm essentially. Now you can control the biolfilm growth and formation
  100. Serial Dilutions and Viable Plate Counts
    • Part of the direct method.
    • The dilution series is relevant because you can't count them all and that is why you do a series since if you do not know how many # of bacteria are present. That is why you cannot do 1 dilution you need a series.
  101. Why do we use a 1 micro-meter loop and not an ordinary loop that is metal?
    • The loop is calibrated because we have a known value which was 1 micro-meter.
    • Relevant in UTI definition because we are looking for bacteria isolation that is = or greater than 10^5 CFU/ml and that is why you need a clean catch or a mid stream.
  102. Membrane Fultration
    • Part of Direct Methods
    • on voice
  103. Microscopic Counts
    • Part of direct method
    • You look for micro-colonies that means the bacteria is viable and if they are single cell that means they are dead.
  104. There is a plate with 200 bacteria on it and it's a 10^-6 dilution. What is the original concentration since the dilution was made?
    • 200x10^6 not 200x10^-6
  105. What are the acceptable #'s when doing a standard plate count to warrant estimation of growth?
    • 30-300 are the acceptable numbers.
    • If below 30 or above 300 they are no acceptable
  106. Electronic Counters
    • Part of Direct Method
    • You can label cells coming through and then easily count them individually
  107. Direct Methods of Measuring Microbial Reproduction
    • Serial dilution and viable plate counts
    • Membrane filtration
    • Microscopic counts
    • Electronic counters
  108. Indirect Methods of Measuring Microbial Reproduction
    • Metabolic Activity
    • Dry Weight
    • Turbidity
  109. Metabolic Activity
    • Part of the Indirect Method
    • Shows if they are viable but doesn't say (rest on voice)
  110. Dry Weight
    • Part of the Indirect Method
    • You have to dry the material and then you have to dry it to the point where the is no change in dry weight at all (no water)
    • Can't tell if dead or alive
  111. Turbidity (Optical Density)
    • Part of the Indirect Method
    • Principle is if you have a lot of bacteria the bacteria will block the light and you wont have the light going through.
  112. Optical Density
    • How many bacteria are present in the mixture. If high OD then you have more bacteria and if you have low OD then you have less bacteria.
    • Doesn't say if they are dead or alive because they still impede the passage of light that is why it is indirect.
    • OD reading are linear up to 1 OD. Reading that are greater than 1 are not reliable.
  113. % Transmittance can be converted to OD
    • 2-log10%transmission
    • This is the way to get around OD, you can also dilute the sample till it falls into the curve of the 1 OD
  114. The vast majority of bacteria have not been cultured. It is estimated that only 3-5% have actually been cultured.
    True statement
  115. T/F: Gene Replacement Therapy is No Longer a Myth?
  116. Procedure of Human Gene Therapy
    • Take gene and insert it into a specific virus that doesn't infect humans and use it as a shuttle.
    • Cells from the target tissue are removed from the patient.
    • The cells are grown in large numbers in tissue culture plates
    • Then transfered back to patient
    • Use virus as a shuttle to transfer the new gene into the cell to infect the cells and then you put the cells back into the patient
  117. Deoxyribose
    • 5 carbon sugars
    • A & G = purine
    • C, T, & U = pyramidine
  118. How are DNA strands positioned?
    They are anti parallel cuz the leading and lagging strands are in different positions.
  119. Leading DNA Strands
    • This strand is right side up
    • Information is being received by this strand and goes from 5' --> 3'
    • It is also a continuous strand
  120. Lagging DNA Strand
    • This strand is upside down
    • Discontinuous cuz the orientation is not correct and goes from 3' --> 5'
  121. # of hydrogen bonds between purines and pyramadines
    • 2 hydrogen bonds between A and T
    • 3 hydrogen bonds between G and C
  122. Relevance of hydrogen bonds between purines and pyramidines
    • Cuz the thermal stability of the DNA molecules is determined by it's G (guanine) and C (cytozine) ratio/content
    • The higher the content the more heat stable the DNA is.
    • Thermophiles and Hyperthermophiles will have the higest G and C content/ratio cuz they need it in order to survive in those conditions.
  123. Flow of Information in Genetic Coding
    DNA to RNA to Proteins.
  124. Transcription
    • DNA to RNA
    • Copying it word for word otherwise shits gets fucked up
  125. Translating
    RNA to Protein
  126. Exceptions to the Flow of Information in Genetic Coding
    • HIV, retroviruses
    • Retroviruses use RNA to make DNA by using the enzyme called reverse transcriptase.
    • This can be used to control replication of virus like HIV by inhibiting the reverse transcriptase
  127. HAART
    • Highly Aggressive Retrovital Therapy
    • Used to treat HIV/AIDS
  128. DNA Replication
    • DNA copying itself.
    • When copying it makes 4 strands, 2 new and 2 old.
    • Goes through cycles
  129. How DNA Replication Cycles Work
    • With each cycle 50% of DNA represents new synthesized DNA and the other half represents old DNA.
    • Cycle 1: 50%
    • Cycle 2: 25%
    • Total: 50 + 25 = 75%
  130. T/F: Definitive ID of Microbes in a mixed sample requires isolation in pure culture to ID by DNA probes
    False, cuz once DNA probes bind, it only binds to the specific sequence which was obtained or present in the organism from which the probe was made.
  131. Clinical Applications of DNA
    • DNA Probes:
    • Probe binding is based on complementary base pairing (A & T and G & C) and recognizes organism with that same sequence. 
    • Greatest % of binding is associated with the DNA obtained from the same organism
  132. Features of DNA Probe
    • The sequence is known. Must be shown to be present in all strains of the same species.
    • Must not be subject to mutation or variation
    • Must be stable
    • Must be unique to the organism and not present in related strains or species
  133. Why would a DNA probe be clinically useful?
    • Highly specific and reproducible.
    • Rapid
    • Cost Effective
    • Time saving
    • Provides information that facilitates effective patient monitoring.