Lab Exercises:
#9 Aerobic/ Anaerobic
#12 UV radiation lab
#8 Quantification lab
#22 Normal Skin Biota
Pre lab due: 1/24/15
Post lab due: 2/0715
Oxygen Requirements
 Boil nutrient agar to drive off O2; cool to just above
solidifying temperature; innoculate; gently swirl
• Growth demonstrates organism’s O2 requirements
Providing Appropriate Atmospheric Conditions
 Aerobic
• Most obligate aerobes and facultative anaerobes can be
incubated in air (~20% O2)
• Broth cultures shaken to provide maximum aeration
• Many medically important bacteria (e.g., Neisseria,
Haemophilus) grow best with increased CO2
• Some are capnophiles, meaning require increased CO2
• One method is to incubate in candle jar
 Microaerophilic
• Require lower O2 concentrations than achieved by
candle jar
• Can incubate in gas-tight container with chemical packet
• Chemical reaction reduces O2 to 5–15%
Providing Appropriate Atmospheric Conditions
 Anaerobic: obligate anaerobes sensitive to O2
• Anaerobic containers useful if microbe can tolerate brief
O2 exposures; can also use semisolid culture medium
containing reducing agent (e.g., sodium thioglycolate)
• Reduce O2 to water
• Anaerobic chamber provides more stringent approach
8.3. Induced Mutations
 Radiation: two types
• Ultraviolet irradiation forms thymine dimers
• Covalent bonds between adjacent thymines
– Cannot fit into double helix; distorts molecule
– Replication and transcription stall at distortion
– Cell will die if damage not repaired
– Mutations result from cell’s SOS repair mechanism
• X rays cause single- and
double-strand breaks in DNA
– Double-strand breaks
often produce lethal
deletions
• X rays can alter nucleobases
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Thymine dimer
Thymine
Thymine
Covalent
bonds
Ultraviolet
light
Sugar-phosphate
backbone
Repair of Thymine Dimers
 Several methods to repair damage from UV light
• Photoreactivation: light repair
• Enzyme uses energy from light
• Breaks covalent bonds of
thymine dimer
• Only found in bacteria
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Photoreactivation
Covalent bonds
5'
3'
G C G A T T G A C G
C G C T AA C T G C
3'
3'
G C G A T T G A C G
C G C T A A C T G C
• Excision repair: dark repair
• Enzyme removes damage
• DNA polymerase, DNA ligase
repair
5'
3'
5'
Thymine dimer distorts the
DNA molecule.
5'
An enzyme uses visible light to
break the covalent bond of the
thymine dimer, restoring the DNA
to its original state.
Excision repair
Covalent bonds
5'
3'
3'
G C G A TT G A C G
C G C T AA C T G C
Cut
5'
3'
5'
Cut
A T T GA
G
G C
C G
C G C T A A C T G C
5'
3'
Thymine dimer distorts the
DNA molecule.
3'
5'
3'
G C G A T T G A C G
C G C T A A C T G C
5'
An enzyme removes the
damaged section by cutting
the DNA backbone on either
side of the thymine dimer.
The combined actions of DNA
polymerase and DNA ligase fill in
and seal the gap.
Repair of Thymine Dimers
 Several methods to repair damage from UV light
(continued…)
• SOS repair: last-ditch repair mechanism
•
•
•
•
Induced following extensive DNA damage
Photoreactivation, excision repair unable to correct
DNA and RNA polymerases stall at unrepaired sites
Several dozen genes in SOS system activated
– Includes a DNA polymerase that synthesizes even in
extensively damaged regions
– Has no proofreading ability, so errors made
– Result is SOS mutagenesis
4.8. Methods to Detect and Measure
Microbial Growth
 Viable cell counts: cells capable of multiplying
• Can use selective, differential media for particular species
• Plate counts: single cell gives rise to colony
• Plate out dilution series: 30–300 colonies ideal
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Adding 1 ml of culture to 9 ml of diluent results in a 1:10 dilution.
Original bacterial
culture
to 9 ml diluent
1:10 dilution
to 9 ml diluent
1:100 dilution
50,000
cells/ml
5,000
cells/ml
500
cells/ml
1 ml
Too many cells
produce too
many colonies
to count.
1 ml
Too many cells
produce too
many colonies
to count.
1 ml
Too many cells
produce too
many colonies
to count.
to 9 ml diluent
1:1,000 dilution
50
cells/ml
1 ml
Between 30–300
cells produces a
countable plate.
to 9 ml diluent
1:10,000 dilution
5
cells/ml
1 ml
Does not produce
enough colonies
for a valid count.
4.8. Methods to Detect and Measure
Microbial Growth
• Plate counts determine colony-forming units (CFUs)
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Spread-plate method
Solid agar
Incubate
Culture, diluted
as needed
0.1–0.2 ml
Bacterial colonies
appear only on surface.
Spread cells onto surface
of pre-poured solid agar.
Pour-plate method
0.1–1.0 ml
Melted cooled agar
Incubate
Add melted cooled agar
and swirl gently to mix.
Some colonies appear on
surface; many are below surface.
22.1. Anatomy, Physiology, and Ecology
 Skin prevents entry, regulates body temperature,
restricts fluid loss, senses environment
• Epidermis: surface layer made from layers of flat cells
• Outermost are dead and filled with water-resistant keratin
• Constantly flake off and replaced
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• Dermis: nerves, glands, blood
and lymphatic vessels
• Subcutaneous tissue:
fat, other cells that
support skin
Hairs
Epidermis
Sebaceous
gland
Arrector pili
(smooth muscle)
Skin
Dermis
Hair follicle
Nerve
Vein
Artery
Sweat gland
Fat
Subcutaneous
tissue (hypodermis)
22.1. Anatomy, Physiology, and Ecology
• Outermost layers bathed in secretions
• Sweat delivered via fine tubules from sweat glands;
evaporates, leaves salty residue that inhibits microbes
• Sebaceous glands open into hair follicles, secrete oily
sebum that keeps hair and skin soft, water-repellant
• Normal microbiota adapted to dry, salty, cool habitat
• Use substances in sweat, sebum as nutrients; byproducts inhibit other microbes (e.g., breakdown of
sebum yields fatty acids that are toxic to many bacteria)
• Too dry, salty, acidic, and toxic for most pathogens
– Those that tolerate often shed with dead skin cells
• Normal microbiota can be troublesome
• Body odor from bacterial breakdown of odorless sweat
• Some are opportunistic pathogens
22.1. Anatomy, Physiology, and Ecology
• Different regions of skin have different inhabitants
• Drier back may have only 1,000 bacteria per cm2
compared with more than 10 million in groin, armpit
• Most microbial inhabitants in three groups
• Diphtheroids: oily regions (forehead, upper chest, back)
– Propionibacterium most common: obligate anaerobes,
grow within hair follicles
• Staphylococci: salt-tolerant, use nutrients and produce
antimicrobial substances
active against other
Gram-positive bacteria
• Malassezia: tiny lipiddependent yeasts
22.2. Bacterial Skin Diseases
 Few bacteria invade intact skin directly
• Strands of hair provide route of invasion
 Acne Vulgaris: commonly begins at puberty
• Signs and Symptoms
• Enlarged sebaceous glands, increased sebum secretion
• Hair follicle epithelium thickens, sloughs off in clumps
• Blockage yields large accumulations of sebum, produces
blackheads and whiteheads
• Causative Agent
• Propionibacterium acnes, which multiplies in sebum
22.2. Bacterial Skin Diseases
 Acne Vulgaris (continued…)
• Pathogenesis
• Metabolic products cause inflammatory response
• Neutrophils recruited, release enzymes that damage
follicle wall; follicle may burst to yield abscess
– Collection of pus (living and dead neutrophils, bacteria, and
tissue debris)
• Epidemiology
• Most people have P. acnes on skin throughout lives
• Increased incidence of acne during puberty likely due to
excess sebum production due to increased hormones
• Treatment and Prevention
• Usually mild; medications available
• Squeezing lesions ill-advised, can rupture follicles
22.2. Bacterial Skin Diseases
 Hair Follicle Infections: generally mild
• Signs and Symptoms
• Folliculitis is inflammation, causes red bumps (pimples)
• If infection extends to adjacent tissues, yields furuncle
– Localized redness, swelling, tenderness, pain
– Pus may drain from the boil
• May worsen to form carbuncle
– Large area of redness, swelling, pain, draining pus
– Fever often present
• Causative Agent
• Commonly caused by Staphylococcus aureus
– Produces identifying coagulase and clumping
factor, which are virulence factors
22.2. Bacterial Skin Diseases
 Hair Follicle Infections (continued…)
• Pathogenesis
• S. aureus attaches to cells of hair follicle, multiplies,
spreads to sebaceous glands
• Infection produces inflammatory response
– Neutrophils recruited
– Plug forms from inflammatory cells, dead tissue
• Deeper spread yields furuncle
• Can increase to form carbuncle
• If infection enters bloodstream,
can spread, reach heart,
bones, brain
Staphylocci
 S. aureus- pathogen, many virulence factors
 S. epidermidis- commensal, not typically
pathogenic
 Differentiating Staphylococci species:
• Mannitol Salt Agar (MSA) plates
• S. aureus can ferment mannitol
– Results in yellow color change
• S. epidermidis can NOT ferment mannitol
– Results in no color change (agar remains pink/red)
• Coagulase test
• S. aureus coagulates rabbit plasma
• S. epidermidis can NOT coagulate rabbit plasma