Exercise 10 Effect of Ultraviolet Light on Microbial Growth

1
Exercise 10Effect of Ultraviolet Light on
Microbial Growth

• Ultraviolet Light Exercise
• Antimicrobials Worksheet

2
Sowhat is UV?

• Energy range just beyond the violet end of the
  visible spectrum.

3
Ultraviolet Light

• Types of ultraviolet radiation (UV)
• UV-A, UV-B, UV-C
• Shorter wavelengths more damaging

4
Ultraviolet 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

5
Ultraviolet Light

• Seasonal changes
• Summer more UV
• Elevational differences
• Higher elevations more UV
• Ozone layer
• Depletion of ozone more UV
• Here in Flagstaff
• 12-15 more UV than at ocean level

6
Ultraviolet Light

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

7
Ultraviolet Light

• Bacterial UV tolerance is dependent upon
• Avoidance strategies
• Repair mechanisms
• Up-regulation of avoidance strategies and repair
  mechanisms upon UV exposure

8
Ultraviolet Light

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

9
Ultraviolet LightThymine Dimers
10
Ultraviolet LightThymine Dimers
11
Thymine Dimers

• Thymine-Thymine dimers greatly increase the
  chance that the wrong base or an extra base will
  be inserted or deleted during replication.

12
Ultraviolet LightPyrimidine 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

13
Ultraviolet LightRepair Mechanisms
14
Ultraviolet LightRepair Mechanisms

• 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

15
Ultraviolet LightRepair Mechanisms
16
Ultraviolet 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
• Essential in the repair of mutated DNA

17
Ultraviolet Light ExercisePurpose

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

18
Procedure

• Draw grid and label plate
• Apply one loopful of culture to each square
• Wait for culture to dry

19
Ultraviolet Light ExerciseProcedure

• Expose cells to UV
• WEAR SAFETY GLASSES AND GLOVES

You must wear safety glasses and gloves when
exposing your plate to the UV lamp!!!! If not you
will lose all technical points!!!
20
Ultraviolet Light ExerciseProcedure

• Expose cells to UV
• 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