BIOL 3340

1. BIOL 3340 Chapter 8

2. Chapter 8 Control of Microorganisms by Physical and Chemical Agents

3. Figure 8.1-Microbial Control Methods

4. Definition of Frequently Used Terms in Microbial control methods Sterilization • destruction or removal of all viable organisms from an object or habitat. Disinfection • killing, inhibition, or removal of pathogenic organisms that may cause disease:substantial reduction of total population. • Disinfectants • agents, usually chemical, used for disinfection; not necessary kills viable spores

5. More Definitions… Sanitization • reduction of microbial population to levels deemed safe (based on public health standards) Antisepsis • prevention of infection or sepsis. of living tissue by microorganisms using antiseptics. • antiseptics • chemical agents that kill or inhibit growth of microorganisms when applied to tissue-should not be toxic as disinfectants to kill host tissues.

6. Antimicrobial Agents • agents that kill microorganisms or inhibit their growth e;g Chemotherapy- chemical agent to kill or inhibit growth of microorganisms within host tissues. • -cidal agents to kill • -static agents to inhibit growth

7. -cidal Agents -cide • suffix indicating that agent kills • germicide • kills pathogens and many nonpathogens but not necessarily endospores • include bactericides, fungicides, algicides, and viricides

8. -static Agents -static • suffix indicating that agent inhibits growth • include bacteriostatic and fungistatic

9. The Pattern of Microbial Death A microbial population is not killed instantly population death usually occurs exponentially as growth rate (Fig 8.2) 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 nonculturable (VBNC) condition once they recover they may regain the ability to reproduce and cause infection

10. …Microbial death Decimal Reduction Time (D): D is the time required to kill 90% of the microorganisms or spores in a sample under specified conditions. Microorganisms is defined as dead when they don’t grow or reproduce when inoculated in culture medim.

11. Fig 8.2-Pattern of Microbial death-an exponential plot of survivors against mins of exposure to heating at 121 0C. Note D121is decimal reduction time which is 1 min to kill 90%

12. Conditions Influencing the Effectiveness of Antimicrobial Agent Activity Population size: • larger populations take longer to kill than smaller populations Population composition: • microorganisms differ markedly in their sensitivity to antimicrobial agents. Bacterial spores are much more resistant to Microbial agents e.g Mycobacterium tuberculosis.

13. More Conditions… Concentration or intensity of an antimicrobial agent: • usually higher concentrations or intensities kill more rapidly • relationship is not linear Duration of exposure: longer exposure to antimicrobial agents more organisms killed

14. More Conditions… Temperature: • higher temperatures usually increase amount of killing Local environment: • many factors (e.g., pH, viscosity and concentration of organic matter) can profoundly impact effectiveness • organisms in biofilms are physiologically altered and less susceptible to many antimicrobial agents. Organic matter in biofilms protects biofilm microorganisms.

15. The Use of Physical Methods in Control • Heat • Low temperatures • Filtration • Radiation

16. Moist Heat Sterilization • must be carried out above 100oC which requires saturated steam under pressure • Moist heat destroys viruses, bacteria and fungi • carried out using an autoclave (Fig8.3) also known as Steam Sterilizer • effective against all types of microorganisms including spores • degrades nucleic acids, denatures proteins, and disrupts membranes

17. The Autoclave or Steam Sterilizer Figure 8.3-Autoclave Steam Sterilizer

18. Table 8.2

19. MeasuringHeat-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 (def previously)

20. Pasteurization • controlled heating at temperatures well below boiling- Louis Pasteur • used for milk, beer and other beverages • process does not sterilize but does kill pathogens present and slow spoilage by reducing the total load of organisms present

21. Dry Heat Sterilization • less effective than moist heat sterilization, requiring higher temperatures and longer exposure times • items subjected to 160-170oC for 2 to 3 hours • oxidizes cell constituents and denatures proteins

22. Filtration • reduces microbial population or sterilizes solutions of heat-sensitive materials by removing microorganisms • also used to reduce microbial populations in air

23. Filtering Liquids Depth filters • thick fibrous or granular materials bonded in thick layers that remove microorganisms by physical screening(size), entrapment, and/or adsorption to the surface of the filter materials. • Solution contain microorganisms are sucked under vacuum • membrane filters • porous membranes with defined pore sizes that remove microorganisms primarily by physical screening. This has replaced Depth Filters.

24. Figure 8.5-Membrane Filter Sterilisation

25. Figure 8.6-Membrane Filter

26. Filtering Air • surgical masks used in hospitals and Labs • cotton plugs on culture vessels • high-efficiency particulate air (HEPA) filters used inlaminar flow biological safety cabinets (remove 99.97% of particles) Figure 8.7 (a)-Laminar Floor

27. Ultraviolet (UV) Radiation • UV (260nm) quite lethal is limited to surface sterilization because it does not penetrate glass, dirt films, water, and other substances. • UV prevent replication and transcription of Microbial DNA. • has been used for water treatment Figure 7.9

28. Ionizing Radiation • Excellent sterilization agent e.g Gamma radiation • penetrates deep into objects • destroys bacterial endospores; not always effective against viruses • used for sterilization and pasteurization of antibiotics, hormones, sutures, plastic disposable supplies, and food

29. Figure 8.8-Sterilization with Ionization; Radiation machine which uses Cobalt 60 as a Gamma radiation to sterilize fruits, veg, fish, meat, etc..

30. Chemical Control Agents -Disinfectants and Antiseptics Phenolics • commonly used as laboratory and hospital disinfectants • act by denaturing proteins and disrupting cell membranes • tuberculocidal, effective in presence of organic material, and long lasting • disagreeable odor and can cause skin irritation

31. Alcohols • bactericidal, fungicidal, but not sporicidal • inactivate some viruses • denature proteins and possibly dissolve membrane lipids

32. Halogens • any of five elements: fluorine, chlorine, bromine, iodine, and astatine • iodine and chlorine are important antimicrobial agents

33. Halogens - Iodine • skin antiseptic • oxidizes cell constituents and iodinates proteins • at high concentrations may kill spores • skin damage, staining, and allergies can be a problem • iodophore • iodine complexed with organic carrier

34. Halogens - 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

35. 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

36. Ammonium Compounds These are detergents that have antimicrobial activity and are effective disinfectants • organic molecules (cleansing agents) with hydrophilic and hydrophobic ends for food utensils, small instruments and skin antiseptics. • act as wetting agents and emulsifiers • Because of its positively charged nitrogen, cationic detergents/ammonium compound, are effective disinfectants. • Hhey disrupt microbial membrane , may denature protein. • kill most bacteria, but not Mycobacterium tuberculosis or endospores • safe and easy to use, but inactivated by hard water and soap • E.g Benzalkonium chloride and Cetylpyridinium

37. Aldehydes • highly reactive molecules • sporicidal and can be used as chemical sterilants • combine with and inactivate nucleic acids and proteins • E.g Formal dehyde and glutaraldehyde

38. Sterilizing Gases • used to sterilize heat-sensitive materials such as disposable petri dishes, syringes, heart lung machine components, sutures, catheters • microbicidal and sporicidal • combine with and inactivate proteins • E.g Ethylene oxide gas (EtO)

39. Figure 8.11-Ethylene Oxide Sterilizer

40. Chemotherapeutic Agents • chemicals that can be used internally to kill or inhibit the growth of microbes within host cells (covered later in book) • their selective toxicity allows them to target the microbe without harming the host • most are antibiotics, chemicals synthesized by microbes that are effective in controlling the growth of bacteria

41. Evaluation of Antimicrobial Agent Effectiveness Testing of antimicrobial agents is a complex process regulated by US federal agencies such as: • Environmental Protection Agency (EPA)_ regulates disinfectants • Food and Drug Administration (FDA)- agents used against humans and animals

42. Biological Control of Microorganisms Biological Control: • control of pathogenic microorganisms by predation on one-another, • Physical barrier • Viral, fungal, bacterial mediated lysis, • Toxin-mediated

43. Bibliography • Lecture PowerPoints Prescott’s Principles of Microbiology-Mc Graw Hill Co. • http://en.wikipedia.org/wiki/Scientific_method • https://files.kennesaw.edu/faculty/jhendrix/bio3340/home.html