Lab Exercises: Results: #8 Quantification lab #9 Aerobic/ Anaerobic #12 UV radiation lab #22 Normal Skin Biota New Labs: #14 Antibiotics #15 Disinfectants

 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 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 1:10,000 dilution to 9 ml diluent 1:1,000 dilution to 9 ml diluent 1:10 dilution to 9 ml diluent 1:100 dilution 5 cells/ml 50,000 cells/ml 5,000 cells/ml 500 cells/ml 50 cells/ml Too many cells produce too many colonies to count. Too many cells produce too many colonies to count. Too many cells produce too many colonies to count. Between 30–300 cells produces a countable plate. Does not produce enough colonies for a valid count. 1 ml

Plate counts determine colony-forming units (CFUs) 4.8. Methods to Detect and Measure Microbial Growth Culture, diluted as needed 0.1–1.0 ml 0.1–0.2 ml Spread cells onto surface of pre-poured solid agar. Pour-plate method Spread-plate method Incubate Bacterial colonies appear only on surface. Melted cooled agar Incubate Some colonies appear on surface; many are below surface. Add melted cooled agar and swirl gently to mix. Solid agar Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Dilutions: Determining number of organism in original culture  # colonies x (1/volume plated) x (1/dilution) Want colonies (countable range)  Helpful things to remember: We plated 1 mL of each dilution Dilutions ranged from A( ) or B ( ) 1 over a negative exponent = a positive exponent  Example: 256 colonies, 1 mL plated, at x (1/1) x (1/10 -7 )= 256 x 1x 10 7= 256 x 10 7= 2.56x10 9 Remember to move the decimal for proper scientific notation (then add that number to the exponent)

 Boil nutrient agar to drive off O 2 ; cool to just above solidifying temperature; innoculate; gently swirl Growth demonstrates organism’s O 2 requirements Oxygen Requirements

 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 8.3. Induced Mutations Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Sugar-phosphate backbone Ultraviolet light Thymine Thymine dimer Covalent bonds

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

 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 5.5. Using Chemicals to Destroy Microorganisms and Viruses

Classes of Germicidal Chemicals

20.3. Mechanisms of Action of Antibacterial Drugs  Antibacterial drugs target specific bacterial processes and structures Cell wall synthesis Protein synthesis Nucleic acid synthesis Metabolic pathways Cell membranes AB Nucleic acid synthesis Fluoroquinolones Rifamycins Cell wall (peptidoglycan) synthesis β-lactam drugs Vancomycin Bacitracin Cell membrane integrity Polymyxin B Daptomycin Metabolic pathways (folate biosynthesis) Sulfonamides Trimethoprim Protein synthesis Aminoglycosides Tetracyclines Macrolides Chloramphenicol Lincosamides Oxazolidinones Streptogramins Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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)