Microbiology Methods PDF

D-value The amount of time it takes for a specific protocol to cause the death of 90% of the population, is called the decimal reduction time (DRT) or D-value. Action of Antimicrobial Agents Alteration of cell membranes Cells burst due to osmotic effects when damaged Damage to...

D-value The amount of time it takes for a specific protocol to cause the death of 90% of the population, is called the decimal reduction time (DRT) or D-value. Action of Antimicrobial Agents Alteration of cell membranes Cells burst due to osmotic effects when damaged Damage to cell proteins or DNA  Need to know Most resistant this table Factors Affecting the Efficacy of Antimicrobial Methods Prions Relative Susceptibility of Microorganisms Bacterial endospores Germicide classification Cysts of Cryptosporidium (protozoan) High-level germicides Mycobacteria Cysts of other protozoa Kill all pathogens, including endospores Small nonenveloped viruses Intermediate-level germicides Active-stage protozoa (trophozoites) Kill fungal spores, protozoan Fungal spores cysts, viruses, and pathogenic Most Gram-negative bacteria bacteria Vegetative fungi Low-level germicides Large nonenveloped viruses Kill vegetative bacteria, fungi, Most Gram-positive bacteria protozoa, and some viruses Enveloped viruses Most susceptible  Biosafety Levels Four levels of safety in labs dealing with pathogens: Biosafety Level 1 (BSL-1) Handling pathogens that do not cause disease in healthy humans Biosafety Level 2 (BSL-2) Handling moderately hazardous agents Biosafety Level 3 (BSL-3) Handling microbes in safety cabinets Biosafety Level 4 (BSL-4) Handling microbes that cause severe or fatal disease Heat-Related Methods Moist Heat Boiling Kills vegetative bacteria cells Fungi Protozoan trophozoites Most viruses Boiling time is critical Different elevations require different boiling times Endospores, protozoan cysts, and some viruses can survive boiling  Heat-Related Methods Moist Heat Autoclaving Pressure applied to boiling water prevents steam from escaping Boiling temperature increases as pressure 30 increases Pressure above normal (psi) 25 Autoclave conditions: 121ºC, 15 psi, 15 minutes 20 15 10 5 0 100 110 120 130 140 Temperature (ºC)  Heat-Related Methods Moist Heat Pasteurization Used for milk, ice cream, yogurt, and fruit juices Not sterilization Heat-tolerant microbes survive Pasteurization of milk Ultrahigh-temperature pasteurization 140ºC for one to three seconds, then rapid cooling Treated liquids can be stored at room temperature  Heat-Related Methods Dry Heat Used for materials that cannot be sterilized with moist heat Denatures proteins Requires higher temperatures for longer time than moist heat Liquids transfer heat more efficiently Incineration is the ultimate means of sterilization  Refrigeration and Freezing Decrease microbial metabolism, growth, and reproduction Refrigeration halts growth of most pathogens Some microbes can multiply in refrigerated foods Listeria Slow freezing more effective than quick freezing Organisms vary in susceptibility to freezing  Desiccation and Lyophilization Desiccation (drying) inhibits growth due to removal of water Lyophilization (freeze-drying) used for long-term preservation of microbial cultures Prevents formation of damaging ice crystals Sublimation  Filtration equipment used for microbial control. Filtration Depending on type of filter can remove Nonsterile medium Protozoan cysts Membrane filter Bacteria Viruses To vacuum pump Sterile medium  Radiation Ionizing Radiation Wavelengths shorter than 1 nm Electron beams, gamma ray, some X rays Ions disrupt hydrogen bonding Oxidize double covalent bonds Create hydroxyl radicals  Radiation Nonionizing Radiation Wavelengths greater than 1 nm Excites electrons New covalent bonds Affects 3-D structure of proteins and nucleic acids UV light pyrimidine dimers in DNA does not penetrate well Suitable for disinfecting Air transparent fluids surfaces of objects Chemical Methods of Microbial Control Affect microbes' cell walls, cytoplasmic membranes, proteins, or DNA The effect varies with differing environmental conditions Often more effective against Enveloped viruses Vegetative bacteria cells Fungi Protozoa Chemical Methods of Microbial Control Phenol and Phenolics Effective in presence of organic matter Remain active for prolonged time Was commonly used in health care settings, labs, and homes Have disagreeable odor and possible side effects Not shown to be effective Unknown health risks In 2013 FDA ruled triclosan and some other phenols will no longer be marketed Hand washing just as effective Chemical Methods of Microbial Control Alcohols Intermediate-level disinfectants More effective than soap in removing bacteria from hands Swabbing of skin with alcohol prior to injection removes most microbes  Chemical Methods of Microbial Control Halogens Intermediate-level antimicrobial chemicals Denaturation proteins Widely used Iodine Iodine tablets Chlorine Bleach Disrupts disulfide bonds Bromine Fluorine Chemical Methods of Microbial Control Oxidizing Agents Peroxides, Ozone, and Peracetic acid Kills by oxidation of enzymes High-level disinfectants and antiseptics Hydrogen peroxide can disinfect and sterilize surfaces Ozone Treatment of drinking water Industrial use Peracetic acid is an effective sporicide used to sterilize equipment  Chemical Methods of Microbial Control Surfactants "Surface active" chemicals Reduce surface tension of solvents Soaps and detergents Soaps have hydrophilic and hydrophobic ends Good degerming agents Not antimicrobial Detergents are positively charged organic surfactants Quatenary ammonium compounds (quats) Low-level disinfectants Disrupt membranes Chemical Methods of Microbial Control Heavy Metals Heavy-metal ions Combine with sulfur in cysteine Denature proteins Low-level bacteriostatic and fungistatic agents Thimerosal Used to preserve vaccines Copper Interferes with chlorophyll controls algal growth