Microbiology Chapter 12 Sterilization and Disinfection

1. Microbiology Chapter 12Sterilization and Disinfection Microbiology 130 R.L. Brashear-Kaulfers

2. Principles of Sterilization & Disinfection • Disinfectants-applied to inanimate objects • Antiseptics- applied to living tissue • Some can be used as both • Sterilization- killing or removal of all microorganisms in a material or on an object • Sterility- no living organisms on/in object • Disinfection- reducing the # of pathogenic organisms on object or in material, so they pose no threat of disease

3. Control of Microbial Growth • 1out of 1 million probability is sterile 1)Total # of microbes present affects the length of time needed to eliminate them 2) Fewer organisms present, shorter time needed for sterility Different microbial agents affect differently Most susceptible phase is logarithmic phase 3) Microorganisms differ in their susceptibility to antimicrobial agents

4. Chemical Antimicrobial Agents • Potency or effectiveness of Chemical Agents: • Bactericidal- killing • Bacteriostatic- growth inhibiting • Ethyl and isopropyl alcohol- 70% effective as it can penetrate more deeply

5. Evaluating Effectiveness • * Phenol coefficient- compare to phenol (carbolic acid) =1 higher is more effective, lower is less effective • Use with Staph typhi and Staph aureas standards • Used to research new disinfectants, but has problems • * Filter Paper Method- uses small filter disks • And look for zone of inhibition-shows some effectiveness but organic matter may interfere with results • * Use-Dilution test: standard prep of bacteria , coated on stainless steel and dipped into dilutions of agents, incubated and observed for no growth* better results than phenol test

6. Disinfectant Selection-Several Qualities • 1) Should be fast acting in presence of organic substances • 2) Be effective against all types of infectious agents without destroying tissue or acting as a poison if ingested • 3) Easily penetrate material to be disinfected, without damaging/discoloring it • 4) Be easy to prepare, stable when exposed to light, heat or other environ-mental factors • 5) Be inexpensive, easy to obtain and use • 6) Not have an unpleasant odor

7. Mechanisms of Action of Chemical Agents • Reactions that affect proteins: denaturation by mild heat, acid, alkali destroys /alters protein • Reactions that affect membranes: protein and lipids- surfactants such as soaps and detergents Do not kill but allow agents into membranes • Reactions that affect cell components: nucleic acids and energy production- lactic acid inhibit fermentation and energy production in bacteria and molds • Reactions that affect viruses: alkylating agents affect DNA or RNA

8. Specific Chemical Antimicrobial Agents • Soaps and detergents: remove microbes, oily deposits and dirt, detergent-cationic (+)(food utensils), or anionic (-) laundry and household cleaners • Quats have amonium disinfectants: BAK, mouthwash • Acids and Alkali: soap is a mild alkali, acid preservatives retard mold growth in breads, margarine, soft drinks • Heavy Metals: selenium, mercury, copper, silver can inhibit bacterial growth such as silver nitrate and mercurochrome and merthiolate -tincture w/alcohol, selenium sulfide kills fungi and spores (dandruff shampoo) Copper sulfate- kills algae in pools

9. Specific Chemical Antimicrobial Agents • Halogens: Hypochlorous acid Cl, I, Br, Cloramine • Alcohols: denature proteins, skin antiseptics • Phenols: disrupt cell membranes • Oxidizing Agents:H2O2 disenfectant • Alkylating Agents: disrupt nucleic acids and protein structures, may cause cancer, formaldehyde, ethylene oxide, glutaraldehyde • Dyes: acridine and methylene blue, crystal violet • Other Agents: plant oils for thyme and clove, nitrates, sulfites, sodium nitrate ( p 338)

10. Physical Antimicrobial Agents- Heat Killing • Heat- thermal death point-temperature • DRT or D value-time needed to kill 90% of organisms • Dry Heat- oxidizes molecules, used for metal objects and glassware, oils and powders. Dry heat penetrates more slowly 171C for 1 hr, 160 C for 2 hrs or 121 C for 16 hrs

11. Physical Antimicrobial Agents- Heat Killing • Moist Heat- widely used • Autoclave at 15 lb/in pressure for 15-20 minutes, temp at 121C to kill spores • Hospital autoclave called prevacuum autoclave, less time to sterilize • Pasteurization - kills pathogens, but not sterile milk = 71.6 C for 15 sec (flash method), or heat at 92.9 C for 30 min (holding method) • UHT-ultrahigh temp 74-140-74C in 5 sec used to make coffee creamers

12. Physical Antimicrobial Agents-Cooling, Refrigeration • Refrigeration- foods 5C , Clostridium spores produce lethal toxins in frig • Freezing- -20C, preserve foods on home and industry, slows rate of microbes so they do not spoil food. Frozen foods should not be thawed and refrozen • Drying- absence of water inhibits enzymes, so it will preserve foods, drying clothes in dryer or in sunshine can destroy pathogens • Freeze-drying- lyophilization is drying from a frozen state to make instant coffee, to preserve cultures of microbes

13. Physical Antimicrobial Agents-Radiation • UV-ultraviolet light-40-390nm 200nm is most effective wavelength for killing by DNA dimers, UV light for sewage treatment in some areas • Ionizing radiation- X rays and gamma rays, .1-40nm very short • Microwave radiation-long wavelengths 1mm-1m • Strong visible light- sunlight, 400-700nm due to UV

14. Physical Antimicrobial Agents-other Methods • Sonic and Ultrasonic waves- • Filtration- passage of material through a filter, use millipore -membrane filters 25um Microbes on filter can be transferred to agar (p346) HEPA filters- clean air and capture microbes • Osmotic Pressure- plasmolysis or loss of water occurs with high concentration of salt, sugar used in jellies, syrup, pickles (p 347)