Lab Exercises:
Results:
#8 Quantification lab
#9 Aerobic/ Anaerobic
#12 UV radiation lab
#22 Normal Skin Biota
New Labs:
#14 Antibiotics
#15 Disinfectants
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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)
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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.
Dilutions: Determining number of organism in original culture
 # colonies x (1/volume plated) x (1/dilution)
• Want 30-300 colonies (countable range)
 Helpful things to remember:
• We plated 1 mL of each dilution
• Dilutions ranged from A(10-4-10-7) or B (10-6-10-9)
• 1 over a negative exponent = a positive exponent
 Example: 256 colonies, 1 mL plated, at 10-7
256 x (1/1) x (1/10-7)=
256 x 1x 107=
256 x 107= 2.56x109
Remember to move the decimal for proper scientific
notation (then add that number to the exponent)
Oxygen Requirements
 Boil nutrient agar to drive off O2; cool to just above
solidifying temperature; innoculate; gently swirl
• Growth demonstrates organism’s O2 requirements
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
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Thymine dimer
Thymine
Thymine
Covalent
bonds
Ultraviolet
light
Sugar-phosphate
backbone
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
5.5. Using Chemicals to Destroy Microorganisms
and Viruses
 Potency of Germicidal Chemical Formulations
• Sterilants destroy all microorganisms
• Heat-sensitive critical instruments
• High-level disinfectants destroy viruses, vegetative cells
• Do not reliably kill endospores
• Semi-critical instruments
• Intermediate-level disinfectants destroy vegetative
bacteria, mycobacteria, fungi, and most viruses
• Disinfect non-critical instruments
• Low-level disinfectants destroy fungi, vegetative bacteria
except mycobacteria, and enveloped viruses
• Do not kill endospores, naked viruses
• Disinfect furniture, floors, walls
5.5. Using Chemicals to Destroy Microorganisms
and Viruses
 Selecting the Appropriate Germicidal Chemical
• Toxicity: benefits must be weighed against risk of use
• Activity in presence of organic material
• Many germicides inactivated
• Compatibility with material being treated
• Liquids cannot be used on electrical equipment
• Residues: can be toxic or corrosive
• Cost and availability
• Storage and stability
• Concentrated stock decreases storage space
• Environmental risk
• Agent may need to be neutralized before disposal
Classes of Germicidal Chemicals
20.3. Mechanisms of Action of Antibacterial Drugs
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Cell wall
 Antibacterial
(peptidoglycan)
synthesis
drugs target
β-lactam drugs
Vancomycin
specific bacterial Bacitracin
processes and
structures
• Cell wall
synthesis
• Protein synthesis
• Nucleic acid
synthesis
• Metabolic
pathways
• Cell membranes
Nucleic acid synthesis
Fluoroquinolones
Rifamycins
A
Cell membrane
integrity
Polymyxin B
Daptomycin
B
Metabolic pathways
(folate biosynthesis)
Sulfonamides
Trimethoprim
Protein synthesis
Aminoglycosides
Tetracyclines
Macrolides
Chloramphenicol
Lincosamides
Oxazolidinones
Streptogramins
20.4. Determining Susceptibility of Bacterial Strain
 Conventional Disc Diffusion Method
• Kirby-Bauer disc diffusion test routinely used to
determine susceptibility of bacterial strain to drugs
• Standard concentration of strain uniformly spread on agar
plate; discs containing different drugs placed on surface
• Drugs diffuse outward, establish gradient
• Resulting zone of inhibition compared with specially
prepared charts to determine
whether strain is susceptible,
intermediate, or resistant
• Drug characteristics must
be taken into account (e.g.,
molecular weight, stability,
amount)