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CHAPTER7 MICROBIAL CONTROL Microbial contamination • Sepsis refers to microbial contamination. Also a term for blood infection/septicemia • Asepsis is the absence of significant contamination. • Aseptic surgery techniques prevent microbial contamination of wounds. Terminology • Bacteriostatic: inhibits bacterial growth • Bactericidal: something capable of killing bacteria • Antiseptic: an agent that is used to inhibit/kill bacterial growth on skin and mucus membranes • Disinfectant: an agent that is used to inhibit/kill bacterial growth on inanimate objects 1 Terminology • Sterilization: Removal of all microbial life • Commercial Sterilization: Killing C. botulinum endospores • Degerming: Removal of microbes from a limited area • Sanitization: Lower microbial counts to safe public health standards • Biocide/Germicide: Kills microbes History behind microbial control • Joseph Lister was the first to introduce the use of carbolic acid to reduce bacterial infections in hospitals (1860s) • Ignatz Semmelweis regarded as the “Father of Infection Control”, physicians used chlorinated lime to cleanse hands (1850s) 2 What factors influence the success of microbial control? • Is the organism an endospore former? • What type of organism are you targeting? • What type of environment are you treating? Bacteria die at a constant logarithmic rate Figure 7.1a Effectiveness of treatment • Number of microbes • Environment (organic matter, temperature, biofilms) • Time of exposure • Microbial characteristic Figure 7.1b 3 Are all bacteria equally sensitive? What parts of a bacterial cell are sensitive to physical treatments and chemicals? • Plasma membrane • DNA and proteins Physical Methods of Microbial Control • Heat – Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min. – Thermal death time (TDT): Time to kill all cells in a culture – Decimal reduction time (DRT): Minutes to kill 90% of a population at a given temperature 4 Physical Methods • Heat – Dry: kills by oxidation (incineration, flaming, hot air) – Moist: promotes coagulation of proteins • Boiling (100oC) • Autoclave(121oC, 15lbs/sq inch) • Pasteurization reduces spoilage organisms and pathogens • Equivalent treatments – Classic: 63°C for 30 min – High-temperature short-time/HTST: 72°C for 15 sec – Ultra-high-temperature/UHT: 140°C for <1 sec – Thermoduric organisms survive Autoclave 5 Autoclaves work due to steam under pressure What if the substance is heat labile? • Filtration is the best choice • Pore sizes can be either .45um or .22um Physical Methods • Low temperature: freezing does not kill bacteria, most cultures are stored at -80oC – Refrigeration (0⁰C to 7⁰C)reduces metabolic rate – lyophilization • High Pressure: apply to liquids such as fruit juice; denatures proteins • High Osmotic Pressure: use high concentration of salt or sugar; plasmolysis • Dessication: remove the water and bacteria can remain viable but do not grow 6 Physical Methods of Microbial Control • Radiation damages DNA – Ionizing radiation (X rays, gamma rays, electron beams) – Nonionizing radiation (UV) – (Microwaves kill by heat; not especially antimicrobial) Figure 7.5 Chemical Methods • Most are only able to reduce the numbers of organisms, not achieve sterility • Types of chemicals – – – – – Phenol and phenolics Halogens Alcohols Heavy metals Soaps 7 Chemical Methods of Microbial Control • Evaluating a disinfectant – Use-dilution test 1. 2. 3. Metal rings dipped in test bacteria are dried Dried cultures placed in disinfectant for 10 min at 20°C Rings transferred to culture media to determine whether bacteria survived treatment Evaluation of chemicals: disc diffusion method • Phenol • Phenolics – coal tar (cresols) – O-phenylphenol (Lysol) • Bisphenols. Hexachlorophene (Phisohex), Triclosan – Disrupt plasma membranes Figure 7.7 8 Types of Disinfectants • Biguanides. Chlorhexidine – Disrupt plasma membranes Chemical control • Halogens – Oxidizing agents – Iodine • Tincture • Iodophor • Skin disinfectant and wound treatment; some patients are allergic to iodine -Chlorine Drinking water, swimming pools, etc. Bleach is hypochlorous acid (HOCl) Chloride dioxide-gas used for area disinfectant Chemical Control • Alcohols – Mechanism of action is protein denaturation, can also dissolve lipids – Most common are ethanol and isopropanol – Does not kill endospores or viruses 9 Chemical control • Heavy metals exert oligodynamic action • Denature proteins • Silver, copper and mercury – – – – Silver nitrate: NBs Silvadine: burns Mercury: paints Zinc: mouthwashes Chemical control • Surfactants (soaps) – Little value as an antiseptic • Acid-anionic surface-active sanitizers are important in cleaning of dairy equipment and utensils • Quats: if foams, contains quats – Mouthwashes – Some bacteria (Pseudomonas) can grow DISINFECTANTS • Surface-action agents or Surfactants Soap Degerming Acid-anionic Sanitizing detergents Bactericidal, Quarternary Denature ammonium proteins, compounds disrupt Cationic plasma detergents membrane 10 Types of Disinfectants • Chemical Food Preservatives – Organic Acids • Inhibit metabolism • Sorbic acid, benzoic acid, calcium propionate • Control molds and bacteria in foods and cosmetics – Nitrite prevents endospore germination – Antibiotics. Nisin and natamycin prevent spoilage of cheese Types of Disinfectants • Aldehydes – Inactivate proteins by cross-linking with functional groups (–NH2, –OH, –COOH, — SH) – Glutaraldehyde: sterilizing, used on hospital equipment – Formaldehyde:preserve biological specimens – Both used in embalming Types of Disinfectants • Gaseous Sterilants – Denature proteins – Ethylene oxide: Sterilizes; used to sterilize spacecraft 11 Types of Disinfectants • Peroxygens – Oxidizing agents – O3, H2O2, – peracetic acid (sterilize food processing and medical equipment) Are all bacteria equally sensitive? Which antiseptic works the best? 12 Microbial Characteristics and Microbial Control Chemical agent Phenolics Quats Chlorines Alcohols Glutaraldehyde Effectiveness against Endospores Mycobacteria Poor Good None None Fair Fair Poor Good Fair Good 13 14