Today

1
Today

• Assignments
• Pre-lab 10
• Worksheet 9
• Environmental Isolate Report Due for extra credit
• Exercise 9
• Discuss Data and turn in Antibiotic and
  Antimicrobial work sheet (filled out during lab)
• Exercise 10
• UV light
• Finish any last minute work on Environmental
  Isolate

2
Lab Practical Rules

• Students may not talk during the practical
• If so, the privilege of taking the practical will
  be removed
• Students may not be helped on questions unless
  they do not understand what the question is
  asking
• Students must answer all the questions to have
  the quiz graded

• The test is closed notes and book
• Students may not touch or tamper with the scope
  except for the fine focus
• Students may not reach extra credit that would
  push their grade above 100

3
Exercise 10 UV light
4
UV Light

• Flavors of ultraviolet radiation (UV)
• UV-A, UV-B, UV-C
• Shorter wavelengths cause more damage

5
UV Light

• Of the UV light that reaches the earths surface
• 95 is UV-A
• 5 is UV-B
• 0 is UV-C
• UV-C lamps (or germicidal lamps) are used for
  sterilization

6
UV Light

• Other factors affecting UV light
• Seasons
• Summer more UV
• Elevation/Altitude
• Higher elevations more UV
• In Flagstaff12-15 more UV than at sea level
• Ozone Density
• Depletion of ozone more UV

7
UV Light

• UV can damage
• DNA
• Photosynthetic pigments
• Cell membranes
• Free radical production (reactive oxygen species)

8
UV Light

• UV tolerance
• Determines level of damage
• Temporary or Permanent
• Bacterial UV tolerance is dependent upon
• Avoidance strategies
• Repair mechanisms
• Up-regulation of avoidance strategies and repair
  mechanisms upon UV exposure

9
UV Light

• DNA damage
• Pyrimidine dimers
• Adjacent bases permanently bind
• Cytosine, thymine, uracil
• Particularly T-T

10
UV Light

• Pyrimidine dimers
• Blocks or interferes with DNA polymerase
• DNA replication
• Cell division
• Blocks or interferes with RNA polymerase
• DNA transcription
• Gene expression
• RNA
• Protein or enzyme production

11
UV Light

• Photoreactivation repair (or photolyase)
• Breaks dimer bond
• Nucleotide excision repair
• Degrades DNA around dimer, fills in bases
• Post-replication repair (or SOS repair)
• Polymerase skips dimer during replication, fills
  in bases later

12
UV Light

• Nucleotide excision repair
• Cut and degrade chunk of DNA around dimer
• Use other DNA strand to fill in bases
• Chance of error
• Uses energy and materials
• recA gene in Escherichia coli

13
UV Light Purpose

• Compare sensitivity of different bacterial
  species to UV light
• Escherichia coli
• Escherichia coli recA mutant
• Micrococcus luteus
• Yellow pigment
• Micrococcus roseus
• Red pigment

14
UV Light Procedure

• Prepare NA plate
• Draw grid
• Place one loopful of each species in indicated
  positions
• Allow spots to dry (30-60min)

15
UV Light Procedure

• Expose cells to UV WEAR SAFETY GLOVES AND
  GOGGLES!
• Remove the lid
• Cover plate with cardboard
• Place under UV lamp
• Slide cardboard down to expose 60 for 20
  seconds
• Slide cardboard down to expose 40 for 20 sec
• Repeat for 20
• Cover plate with cardboard and remove from lamp

16
UV Light Procedure

• Incubate
• Next week
• Record results
• Last worksheet!

17
Next Week Last Lab!

• Assignments
• Environmental Isolate Report Due
• Exercise 10
• Discuss Data and turn in UV light worksheet
  (filled out during lab)
• Clean up