2054, Chap. 7, page 1

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1 2054, Chap. 7, page 1 I. Control of Microorganisms by Physical and Chemical Methods A. Terminology 1. sterilization = destruction of living cells, viable spores, viruses, viroids 2. disinfection = killing, inhibition, or removal of organisms 3. sanitization = reduction of microorganisms to safe health levels 4. antiseptic = chemical agents applied to tissues to kill or inhibit pathogens 5. -cide = kills organisms 6. -lytic = lyses organisms 7. -static = inhibits growth of organisms B. Pattern of microbial death 1. population death is exponential or logarithmic a. straight line on log plot b. less cells killed per unit time as death progresses 2. death rate may slow after significant reduction due to survival of more resistant strains 3. a dead cell cannot reproduce under permissive conditions C. Effectiveness of antimicrobials 1. population death is generally exponential or logarithmic 2. efficiency of antimicrobials influenced by at least 6 factors a. population size - larger population requires more time to die b. population composition - different degrees of resistance between different organisms or structures (1) spores more resistant than vegetative cells (2) Mycobacterium tuberculosis (acid-fast) more resistant than most bacteria (3) young cells more readily destroyed than older cells c. concentration of antimicrobial (1) usually greater concentration = greater effectiveness (2) 70% ethanol more effective than 95% ethanol d. exposure duration (1) longer exposure = greater death (2) sterilization = reduction of survival probability to #10-6 e. temperature; increase usually increases effectiveness of chemical

2 2054, Chap. 7, page 2 f. local environment (1) heat kills better at acid ph (2) efficiency higher with lower organic matter D. Physical methods 1. moist heat a. boiling kills viruses, bacteria, fungi (Table 7.2) b. 10 minutes boiling kills vegetative cells, not endospores c. thermal death time (TDT) = shortest period of time to kill all organisms at a specific temperature under defined conditions d. decimal reduction time (D) or D value = time to kill 90% (1) important to food industry (2) usually assumed population of cells, reduced to 10 0 (3) if D for C. botulinum spores is minutes at 121 o C, 12D = 2.5 minutes (4) z value = increase in temperature required to reduce D to 1/10 its value (a) for C. botulinum, z = 10 o C (b) at 111 o C, D = 2.04 minutes, 12D = 24.5 minutes e. F value = time to kill a population of cells or spores at a specific temperature (usually 121 o C) f. pressurized steam (autoclave) (1) autoclave = fancy pressure cooker (2) combines wet heat with pressure; allows temperatures above 100 o C (3) temperatures above 100 o C required to destroy endospores (4) chamber filled with saturated steam for 121 o C, 15 psi (5) materials exposed for $15 minutes g. pasteurization (1) reduces microorganism numbers but retains flavor of foods, especially dairy, beer, and other beverages (2) brief heating, followed by rapid cooling (3) 63 o - 66 o C for 30 minutes (batch or older method) (4) flash pasteurization (a) 72 o C for 15 seconds

3 2054, Chap. 7, page 3 (b) also called High Temperature Short-Time (HTST) (5) ultrahigh-temperature sterilization (UHT) = 140 o o C for 1-3 seconds h. tyndallization = discontinuous boiling or fractional steam sterilization (1) heat material to 90 o -100 o C for 30 minutes on 3 consecutive days, incubated at 37 o C (2) 1st heating destroys cells but leaves endospores (3) 2nd and 3rd heating destroys germinating endospores 2. Low temperature a. -20 o C stops growth due to a lack of liquid water (1) ice crystals may burst cells (2) low temperature can be used to preserve cells b. refrigeration slows growth but may select for psychrophiles 3. Dry heat sterilization a. 160 o -170 o C for 2-3 hours b. cell constituents oxidize c. less effective than moist heat 4. Filtration a. physical removal of microorganisms (1) excellent for heat sensitive materials (2) can be used with gases b. depth filters = thick layers of fibrous or granular material (1) twisting channels of small diameter (2) microbes removed by physical entrapment and adsorption to filter material c. membrane filters = thin (0.1 mm) membranes (1) made of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene chloride, or other synthetic materials (2) vegetative cells removed with 0.2:m pore size 5. Radiation a. alters DNA, causing lethal mutations b. UV= ultraviolet light (260 nm) (1) doesn't penetrate glass, dirt films, water, many plastics

4 2054, Chap. 7, page 4 (2) often used to sterilize cabinets or entire rooms c. ionizing (e.g., gamma) (1) penetrates objects (2) also called cold sterilization; excellent with heat-sensitive materials (3) used with foods (a) approved and declared safe by FDA and WHO (b) not used extensively yet E. Chemical methods 1. phenolics a. early use by Lister b. Lysol contains a mixture of phenolics c. denature proteins and disrupt cell membranes d. excellent for surfaces, but can cause skin irritation 2. alcohols a. bactericidal and fungicidal, but not sporicidal b. denature proteins and dissolve membrane lipids 3. halogens (e.g., F, I, Cl, Br) a. iodine most common, followed by chlorine b. tincture of iodine = $2% iodine in water/ethanol solution of potassium iodide (1) effective antiseptic (2) stains and may damage skin (3) iodophor = complex of iodine + organic carrier (a) water soluble, stable, nonstaining (b) slow release to prevent skin burns and irritation c. chlorine disinfectant of choice for municipal water supplies and swimming pools (1) added in many forms; forms hypochlorous acid (2) oxidizes several materials, destroying cells but not endospores (3) Halzone tablets used for personal drinking water (4) excellent household disinfectant (a) 1:100 dilution of household bleach (1.3 oz/gal) + 0.7% nonionic detergent (1 oz/gal)

5 2054, Chap. 7, page 5 (b) cleans and kills bacteria 4. heavy metals a. Hg, Ag, As, Zn, Cu used to be common germicides (1) most heavy metals are bacteriostatic, not bactericidal (2) currently using less toxic, more effective germicides (3) 1% silver nitrate added to eyes of infants to prevent ophthalmic gonorrhea; being replaced by erythromycin, which is also effective against Chlamydia and Neisseria (4) silver sulfadiazine used on burns (5) copper sulfate used as algicide in lakes and swimming pools b. combine with proteins (ofen sulfhydryl groups), inactivating or sometimes precipitating them 5. quaternary ammonium compounds a. detergents = organic molecules (non-soaps) that serve as wetting agents and emulsifiers (1) amphipathic molecules (2) effective cleansing agents b. cationic detergents more antimicrobial than anionic detergents (1) quaternary ammonium compounds most popular (a) benzalkonium chloride (b) cetylpyridinum chloride (2) positively charged quaternary nitrogen with long hydrophobic aliphatic chain c. disrupt membranes and may denature proteins d. kill most cells, but not endospores or M. tuberculosis e. often used as disinfectants for food utensils, small instruments, and skin antiseptics 6. aldehydes a. formaldehyde and gluteraldyde most common b. combine with and deactivate proteins c. 2% glutaraldehyde commonly used to disinfect hospital equipment d. sporacidal 7. gases

6 2054, Chap. 7, page 6 a. ethylene oxide is both microbicidal and sporicidal (1) combines with cell proteins (2) penetrates packing materials, even plastic wraps b. explosive, usually done in special sterilizer F. Evaluation of agents (EPA regulates disinfectants, FDA controls antiseptics) 1. phenol coefficient a. potency compared with phenol (1) dilutions inoculated with Salmonella typhi and Staphylococcus aureus and incubated (2) tubes subcultured at 5 min intervals (3) phenol coefficient obtained from highest dilution that kills bacteria after 10 min, but not after 5 min (4) reciprocal of concentration divided by that for phenol for phenol coefficient value b. can be misleading because conditions of test are not realistic (1) clean medium (2) pure cultures 2. use dilution test more realistic than phenol coefficient a. stainless steel cylinders are contaminated with bacterial mixtures, dried briefly, immersed in test disinfectants for 10 min, transferred to culture media, and incubated 2 days b. disinfectant concentration that kills all organisms with 95% confidence level is determined 3. disinfectants can also be tested under "in use" conditions