EXAM THREE NOTES

	EXAM THREE NOTES
	Bacterial Growth – Oxygen Effects Toxic Oxygen - Forms Singlet oxygen Superoxide free radicals Peroxides Hydroxyl free radicals Toxic Oxygen - Neutralization –Catalase: 2 H₂O₂ à 2 H₂O + O₂ –Superoxide dismutase: 2 O₂ - + 2 H+ à O₂ + H₂O₂ –Peroxidases: H₂O₂ à H+ + H₂O Oxygen Classification aerobic - grows in presence of oxygen obligate aerobe - requires oxygen anaerobic - grows in absence of oxygen obligate anaerobe - will only grow in absence of oxygen facultative anaerobe - will grow both anaerobically or aerobically Oxygen Classification Aerotolerant - only grow anaerobically but will tolerate oxygen Microaerophillic - growth occurs only in presence of low concentrations of oxygen Bacterial Growth – Culture & Media Media Components Carbon Source Nitrogen Source Minor Nutrients Solid Support If not liquid media Carbon Sources Usually sugars Alternatives include: Fats or lipids Proteins or amino acids Fix CO₂ Nitrogen Sources Usually ammonium ion Alternatives include: Nitrate ion Nitrite ion Amino acids Nucleic acids Other compounds containing nitrogen Minor Nutrients Sulfur in cystiene and methionine Phosphate DNA RNA energy intermediates Iron electron transport Magnesium kinases Other metals Some organisms very particular Solid Support Gelatin Melts near room temperature Digested by proteases which are produced by many bacteria Agar derived from algal polysaccharide Bacterial Growth Introduction Population vs. Cellular Growth Exponential vs. Arithmetic Growth Bacterial Growth - Binary Fission Bacterial Growth Curve Lag Phase Log Phase Stationary Phase Death Phase Calculations - Number of cells Bacterial Growth Calculations - Number of Generations Bacterial Growth Calculations - Generation Time Time for single cell to double Bacterial Growth Measurement of Growth Biomass Viable Counts Total Counts Most Probable Number (MPN) Biomass Dry weight (increase in mass) Turbidity (using a spectrophotometer) Metabolic activity (O₂ used or CO₂ evolved) Viable Counts Plate counts Membrane filters Total Counts Direct Microscopic Count Counting Chamber Electronic Counter Most Probable Number 5 tubes at 10 mL, 1 mL and 0.1 mL sized inoculum Use combinations with table to calculate the most probable number Confidence limits set range of likely number of cells per 100 mL
	Control of Microbial Growth Definitions Sterilization - Processes that kill kill living organisms including spores Disinfection - Destruction of vegetative pathogens (not spores) Antisepsis -Destruction of vegetative pathogens on living tissue Degerming - Removal of microbes from a limited area (alcohol swab prior to injection) Sanitization - Lowers microbial counts Physical Methods of Control Temperature Effects Filtration Desiccation Osmotic Pressure Radiation Effects of Temperature Moist Heat Dry Heat Pasteurization Refrigeration Temperature - Moist Heat Autoclave: 120-130°C (15-20 lbs/in²) Tyndalization Effect on instruments Foil or cloth wrap Must use on liquids Temperature - Dry Heat Glassware and instruments 160-170°C for at least 90 min Usually foil wrap Incineration (loops & needles) Temperature - Pasteurization Milk: 63°C for 30 min (old) Milk: 72°C for 15 sec (new) Juices Temperature - Refrigeration Does not usually kill Slows metabolism although psychrophillic organisms can still grow Best between 0° and 7° C Freeze thaw cycles can kill some organisms Filtration Used for heat sensitive liquids like serum or urea containing media 0.45µ or 0.22µ pores in membrane Often used commercially with beer, wine and fruit juices Desiccation Inhibits growth more than kills Dried meats and vegetables Freeze dry processes in foods Osmotic Pressure High concentrations of salts or sugars inhibit growth of bacteria; examples includes jams and jellies, sugar and salt cured meats Radiation Short wave (X-rays, gamma rays) high penetration power; breaks DNA Non-ionizing (UV) longer wave; no penetrating power; forms thymine dimers Organisms contain multiple repair systems Chemical Methods Phenols Halogens Alcohols Surface active agents Aldehydes Heavy metals Acids & bases Gases Peroxygens Phenols & Phenolics Lister & carbolic acid penetrate plasma membrane & precipitate proteins phenol coefficient - chemical agents evaluated relative to phenol Halogens Chlorine & drinking water Ca(OCl)₂ bleach Iodine Betadine (Iodine and organic solvent) Chloramines Alcohols 70% ethanol isopropanol require some water to be effective used in conjunction with other agents Surface Active Agents soaps - emulsification of oils increases bacterial removal - deodorant soaps contain triclocarban or triclosan acid-anionic agents used in dairy industry quaternary ammonium compounds - work best against G+ bacteria, less effective against G-; also kill fungi protozoa and viruses; Psuedomonas resistant Aldehydes react with a variety of organic functional groups: -NH₂, -OH, -SH Formaldehyde Glutaraldehyde used for embalming Heavy Metals usually toxic Silver nitrate used on infants eyes (replaced by antibiotics) Copper sulfate used as algicide Mercury toxicity and resistance Zinc chloride and oxide in toothpaste Acids and Bases pH extremes inhibit growth of bacteria acid (pickles and tomatoes) trisodium phosphate in detergent Propionic acid in bread Benzoic acid in soft drinks Gases ethylene oxide - strong oxidizer and alkylating agent very penetrating possible carcinogen does not damage instruments but expensive to use Peroxygens - Oxidizing Agents Ozone - being used in water treatment Hydrogen peroxide -surface disinfecting Benzoyl peroxide - skin treatment Peracetic acid - used in food processing because residue is non-toxic
	Antibiotics / Chemotherapy History Properties Testing Spectrum of Antimicrobial Action Modes of Action Survey of Drugs History Quinine for malaria Willow bark for treating fever Paul Ehrlich - staining of bacteria led to ideas for chemotherapy Fleming (1928) observed the effect of Penicillium of on Staphylococcus Flory & Chain (1940) developed penicillin and clinically tested it Properties Selective toxicity (e.g. sulfanilamide mimics PABA in folic acid synthesis) Sources Microorganisms Synthetic agents Plants Testing Broth dilution Agar dilution Disc diffusion Broth dilution MIC - minimal inhibitory concentration; smallest concentration that stops growth Successive dilutions inoculated with same number bacteria Turbidity measure when compared to control (could also do dilutions & plate counts) MBC - minimal bactericidal concentration Agar dilution Dilute drug into agar at varying concentrations Can test multiple species of bacteria Not very quantitative Disc-Diffusion Discs with known concentrations of antibiotics seeded onto “lawn” of bacteria Zone of clearing around disk a measure of effectiveness of antibiotic Spectrum of Antimicrobial Activity Selectively toxic drugs; uses differences between prokaryotic and eukaryotic cells Broad spectrum - both G+ and G- Antibiotic effect, e.g. penicillin and Candida albicans Modes of Action Bactericidal vs. Bacteriostatic Cell Wall Protein Synthesis Plasma membrane Nucleic Acid Synthesis Essential Metabolites Bactericidal vs. Bacteriostatic Bactericidal - kills Bacteriostatic - inhibits growth but once remove growth can resume Inhibition of Cell Wall Synthesis Uniqueness of bacterial cell wall Prevent peptidoglycan synthesis or peptide cross-linking from forming Penicillins & cephalosporins Inhibition of Protein Synthesis Bacterial protein synthesis significantly different than eukaryotic e.g. 70S vs. 80S ribosome or elongation & termination factors Amyloglycosides (streptomycin and gentamicin) Injury of Plasma Membrane Alteration in permeability Interference with required consituents, e.g. sterols in fungal lipid membranes Polymixin B (bacteria) Amphotericin B or miconazole (fungal) Essential Metabolites Para-aminobonzoic acid is an essential cofactor used by bacteria to synthesize folic acid (a vitamin that functions as a coenzyme in the synthesis of nucleic acid precursors) animals ingest folic acid Sulfanilamide is an analog of PABA Inhibition of Nucleic Acid Synthesis Nucleic acid synthesis especially mRNA and DNA Rifampin and quinolones Limited utility because of RNA’s and DNA’s essential role in both prokaryotic and eukaryotic cells Survey of Drugs - Cell Wall Synthesis Penicillins (G, V) Semisynthetic penicillins (Ampicillin) Monobactams Vancomycin - Glycopeptide topical Cephalosporins Bacitracin - bacterial origin; topical use Isoniazid - tuberculosis Ethambutol - tuberculosis Survey of Drugs - Protein Synthesis Amyloglycosides (Streptomycin, neomycin Gentamicin) Tetracyclines - Bacteriostatic Chloramphenicol Macrolides - Erythromycin - Bacteriostatic Survey of Drugs - Plasma Membrane Polymyxin B - topical; works against G- Survey of Drugs - Nucleic Acids Rifampin Quinolones Fluorquinolones Survey of Drugs - Essential Metabolites Sulfonamides