Lab Exercises: #9 Aerobic/ Anaerobic #12 UV radiation lab #8 Quantification lab #22 Normal Skin...

Lab Exercises:

#9 Aerobic/ Anaerobic#12 UV radiation lab#8 Quantification lab#22 Normal Skin Biota

Pre lab due: 1/24/15Post lab due: 2/0715

Boil nutrient agar to drive off O2; cool to just above solidifying temperature; innoculate; gently swirl• Growth demonstrates organism’s O2 requirements

Oxygen Requirements

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

Providing Appropriate Atmospheric Conditions

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- anddouble-strand breaks in DNA

– Double-strand breaksoften produce lethaldeletions

• X rays can alter nucleobases

8.3. Induced Mutations

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Sugar-phosphatebackbone

Ultravioletlight

Thymine

Thymine

Thymine dimer

Covalentbonds

Several methods to repair damage from UV light

Repair of Thymine Dimers

• Photoreactivation: light repair• Enzyme uses energy from light

• Breaks covalent bonds of thymine dimer

• Only found in bacteria

• Excision repair: dark repair• Enzyme removes damage

• DNA polymerase, DNA ligase repair

AT

GC

CG

GC

TA

TA

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TCG

GC

An enzyme uses visible light tobreak the covalent bond of thethymine dimer, restoring the DNAto its original state.

Thymine dimer distorts theDNA molecule.

Excision repair

Photoreactivation

Thymine dimer distorts theDNA molecule.

An enzyme removes the damaged section by cutting the DNA backbone on either side of the thymine dimer.

The combined actions of DNApolymerase and DNA ligase fill inand seal the gap.

Covalent bonds

Covalent bonds

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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

Repair of Thymine Dimers

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

4.8. Methods to Detect and Measure Microbial Growth


Adding 1 ml of culture to 9 ml of diluent results in a 1:10 dilution.

Original bacterialculture

to 9 ml diluent1:10,000 dilution

to 9 ml diluent1:1,000 dilution

to 9 ml diluent1:10 dilution

to 9 ml diluent1:100 dilution

5cells/ml

50,000cells/ml

5,000cells/ml

500cells/ml

50cells/ml

Too many cellsproduce toomany coloniesto count.



Between 30–300cells produces acountable plate.

Does not produceenough coloniesfor a valid count.

1 ml1 ml1 ml1 ml1 ml

• Plate counts determine colony-forming units (CFUs)

4.8. Methods to Detect and Measure Microbial Growth

Culture, dilutedas needed

0.1–1.0 ml

0.1–0.2 ml Spread cells onto surfaceof pre-poured solid agar.

Pour-plate method

Spread-plate method

Incubate

Bacterial coloniesappear only on surface.

Melted cooled agar

Incubate

Some colonies appear onsurface; many are below surface.Add melted cooled agar

and swirl gently to mix.

Solid agar


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

• Dermis: nerves, glands, bloodand lymphatic vessels

• Subcutaneous tissue: fat, other cells thatsupport skin

22.1. Anatomy, Physiology, and Ecology

Subcutaneoustissue (hypodermis)

Skin

Dermis

Epidermis

FatSweat glandArtery

Vein

Nerve

Hair follicle

Arrector pili(smooth muscle)

Sebaceousgland


Hairs


• 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; by-

products 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


• 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 lipid-dependent 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


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


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


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

Lab Exercises: #9 Aerobic/ Anaerobic #12 UV radiation lab #8 Quantification lab #22 Normal Skin...

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