Chapter 7: Control of microorganisms

1. Chapter 7: Control of microorganisms Many bacteria cause disease and food spoilage: the need exists to kill or inhibit the growth of these bacteria Sterilization - removal or destruction of all living cells, viable spores, viruses and viriods Disinfection - removal or destruction of pathogens (spores and some other microorganisms remain) Sanitization - reduction of microbial population to safe levels Antisepsis - prevention of infection (accomplished by antiseptics) Bactericide - substance that kills bacteria Bacteriostatic - substance that prevents growth of bacteria

3. The Pattern of Microbial Death • Microorganisms are not killed instantly • Population death usually occurs exponentially, slows down at later stages due to the survival of more resistant forms • When do you consider a population to be dead? • microorganisms were previously considered to be dead when they did not reproduce in conditions that normally supported their reproduction • however we now know that organisms can be in a viable but non-culturable (VBNC) condition • Once they recover they may regain the ability to reproduce and cause infection

4. Measuring Heat-Killing Efficiency • Thermal death time (TDT) • shortest time needed to kill all microorganisms in a suspension at a specific temperature and under defined conditions • Decimal reduction time (D or D value) • time required to kill 90% of microorganisms or spores in a sample at a specific temperature

5. Effect of exposing bacteria to 121 degrees Celcius Pattern of microbial death Microorganisms often die logarithmically (i.e. the population will be reduced by the same fraction at regular intervals), not instantaneously D value = 1 min

6. Conditions influencing effectiveness of antimicrobial agents • Population size • Population composition (e.g. spores Vs fast growing cells, Mycobacterium Vs E. coli) • Concentration or intensity of agent • Duration of exposure to agent • Temperature • Local environment (e.g. pH, presence of organic material)

7. Physical methods of control • Heat • Low temperature • Filtration • Radiation

8. Z value determination. * Note exponential temperature dependence Measuring heat-killing efficiency * Z value - the increase in temperature required to reduce D to 1/10 its value * F value - time in minutes at a specific temperature required to kill a population of spores or cells

9. Examples of D and z values Note: canned food is usually exposed to high temperatures. The heating process during canning destroys ~ half of vitamins A and C, riboflavin, and thiamin.

10. Heat • Moist heat: steam sterilization • # Effective against all types of microorganisms; degrades nucleic acids, denatures proteins, and disrupts membranes • # Autoclaves are used to kill endospores; uses steam under pressure to achieve temperatures above boiling • Pasteurization: controlled heating at temperatures below boiling • # Does not sterilize; kills pathogens and reduces levels of spoilage microorganisms, used for milk, beer, juice, etc. • # Traditional method: 63 ºC for 30 minutes; flash pasteurization: 72 ºC for 15 seconds • # Ultrahigh temperature (UHT) sterilization: milk heated at 140 to 150 ºC for 1 to 3 seconds. Products can be stored at room temperature for 1 to 2 months • Dry heat sterilization • # Less effective, requiring higher temperatures and longer exposure times (160-170oC for 2 to 3 hours)

11. Table 7.2

12. Dry Heat Incineration • bench top incinerators are used to sterilize inoculating loops used in microbiology laboratories Figure 7.4

13. Low temperature • Refrigeration: storage at 4 ºC slows microbial growth (only used for short-term storage) • Freezing: storage at - 20 ºC stops microbial growth (does not kill microorganisms) • Freezing at -30 to -70 ºC used to preserve microbial samples

14. Can be used to sterilized or reduce the microbial population of heat-sensitive liquids • Removes microorganisms rather than destroying them • Solutions often forced through filters by pressure or a vacuum Filtration

15. Membrane filters: Porous membrane about 0.1 mm thick; pore size of 0.2 um diameter removes most cells but not viruses Air filtration * Laminar flow biological safety cabinets: employ high efficiency particulate air (HEPA) filters that remove 99.97 % of particles larger than 0.3 um. Filtration

16. Ultraviolet (UV) radiation Near 260 nm; lethal but does not penetrate glass; used to sterilize air or exposed surfaces Radiation • Ionizing radiation • Penetrates deep into objects, but not always effective against viruses • Gamma radiation from Cobalt 60 often used • Used to treat meat, fruits, vegetables and spices, antibiotics, hormones, plastic disposable supplies.

17. Most commonly used agents for disinfection and antisepsis Chemical agents

18. Phenolics • Phenol first used by Lister • Phenol and derivatives used as disinfectants in hospitals and labs • Effective in the presence of organic material • Can cause skin irritation • Denature proteins and disrupt cell membranes

19. Alcohols • Not effective against spores or lipid-containing viruses • Ethanol and isopropanol most commonly used (at 70-80 %) • Act by denaturing proteins and possibly dissolving membrane lipids

20. Halogens • Include fluorine, chlorine, bromine, iodine and astatine • Iodine used as a skin disinfectant • Chlorine used to disinfect water • Both act by oxidizing cell material and iodinating or chlorinating molecules • -Iodophore • iodine complexed with organic carrier

21. Halogens… • e.g., chlorine • oxidizes cell constituents • important in disinfection of water supplies and swimming pools, used in dairy and food industries, effective household disinfectant • destroys vegetative bacteria and fungi, but not spores • can react with organic matter to form carcinogenic compounds

22. Heavy Metals • e.g., ions of mercury, silver, arsenic, zinc, and copper • effective but usually toxic • combine with and inactivate proteins; may also precipitate proteins

23. Quaternary Ammonium Compounds

24. Quaternary Ammonium Compounds • Detergents • organic molecules with hydrophilic and hydrophobic ends • act as wetting agents and emulsifiers • Cationic detergents are effective disinfectants • kill most bacteria, but not Mycobacterium tuberculosis or endospores • safe and easy to use, but inactivated by hard water and soap

25. Aldehydes • Formaldehyde and gutaraldehyde are the most commonly used • Are highly reactive molecules • Inactivate proteins and DNA by cross-linking alkylating molecules

26. Sterilizing Gases Figure 7.7 • Used to sterilize heat-sensitive materials • Microbicidal and sporicidal • Combine with and inactivate proteins

27. Evaluation of Antimicrobial Agent Effectiveness • Complex process regulated by US federal agencies • Environmental Protection Agency • Food and Drug Administration

28. Potency of disinfectant compared to phenol Coefficient greater than 1 indicates agent is more potent than phenol Not always indicative of potency during normal use Evaluating antimicrobial agent effectiveness Phenol coefficient