Control of Microbial Growth

1. LECTURES IN MICROBIOLOGY Control of Microbial Growth Sofronio Agustin Professor LESSON 7

2. Lesson 7 Topics Controlling Microorganisms: • Physical Means • Chemical Means

3. Overview Various Microbial Control Methods

4. Antimicrobial Action • -static agents- inhibit growth of microbes Ex:bacteriostatic, fungistatic • -cidal agents - destroy or kill microbes Ex: bactericidal, fungicidal

5. Antimicrobial Resistance • Highest resistance - bacterial spores and prions. • Moderate resistance - some bacteria, protozoan cysts, fungal (sexual) spores, naked viruses. • Least resistance - most bacteria, fungal (asexual) spores and hyphae, enveloped viruses, yeast, protozoan trophozoites.

6. Comparative Resistance Relative resistance of bacterial endospores and vegetative cells

7. Terms • Sterilization • Disinfection • Antisepsis • Sanitation • Degerming

8. Sterilization • Process of complete removal or destruction of all viable microbes including endospores. • Example: Use of physical or chemical agents on surgical instruments, and commercially packaged foods.

9. Disinfection • Use of physical or chemical agent (disinfectant) to destroy vegetative bacteria and other microbes except endospores. • Example: Use of bleach solution on bench tops.

10. Antisepsis • Use of physical or chemical agent (disinfectant) to destroy vegetative pathogens on living organisms. • Example: Scrubbing surgical patients with chemical agents.

11. Sanitization • Reduction of the number of microbes to acceptable levels established by public health standards. • Sanitization of utensils in restaurants.

12. Degerming • Physical removal or reduction of microbes from human skin. • Ex: Washing of hands with germicidal soap.

13. Efficacy • Number of microorganisms • Target microbe - bacteria, fungi, spores, viruses. • Temperature and pH • Concentration of agent • Its mode of action • Interfering substances - solvents, debris, saliva, blood, feces.

14. Factors Factors that influence the effectiveness of antimicrobial agents

15. Mode of Action Effects on: • Cell wall • Cell membrane • Nucleic acid synthesis • Protein synthesis • Protein function

16. Cell Wall • Effects on bacteria and fungi: • Block cell wall synthesis • Degrade cellular components • Destroy or reduce its stability • Ex: Penicillin, detergents, alcohols

17. Cell Membrane • Effects on all microbes including enveloped viruses: • Bind and penetrate membrane lipids • Loss of selective permeability resulting in leakage of cytoplasmic contents. • Ex: Surfactants - surface active agents.

18. Surfactant Action The effect of surfactants on the cell membrane.

19. Nucleic Acid Synthesis • Irreversible binding to microbial DNA results in: • Ceasation of transcription and translation • Mutations • Ex: Formaldehyde and ionizing radiation

20. Protein Synthesis Ribosome binding stops translation and prevents peptide bond formation. Ex: The antibiotic chloramphenicol

21. Protein Function • Blockage of protein (enzyme) active sites prevents binding of substrate. • Protein denaturation. • Ex: Heat, acids, alcohols, phenolics, metallic ions.

22. Protein Function Effects of heat, pH, and blocking agents on protein function.

23. Physical Methods • Heat • Radiation • Filtration

24. Heat • Moist heat: • Coagulation of proteins • Denaturation of proteins • Dry heat: • Dehydration • Denaturation • Oxidation (burning to ashes)

25. Moist Heat • Boiling water • Tyndallization • Pasteurization • Steam and pressure

26. Boiling Water Boiling at 100 ˚C for 10-30 minutes kills most non-spore forming pathogens. Ex: Boiling of baby bottles and unsafe drinking water Note: Hepatitis B virus needs 1 hour boiling to be killed.

27. Tyndallization • Intermittent sterilization using free-flowing steam for 30 to 60 minutes. • Ex: Used on heat-sensitive media, canned foods. • Note: This will not destroy spores.

28. Pasteurization • Use of moist heat at : - 63 ˚C for 30 minutes (Classical) - 72 ˚Cfor 15 seconds (HTST) - 140 ˚C for < second (UHT) • Used in milk industry, wineries, breweries. • Prevents transmission of milk-borne diseases caused by: Salmonella, Campylobacter, Listeria, Mycobacteria.

29. Steam and Pressure • Used in media preparation and glassware sterilization. • Ex: Autoclave and pressure cooker • Autoclave setting at 121 ˚C,15 p.s.i. pressure for at least 15 minutes effectively destroys spores.

30. Autoclave

31. Dry Heat • Hot air • Incineration • Requires greater temperature and exposure time than moist heat

32. Hot Air • Ex: Hot air oven • Effective at 150˚C to 180˚C for 2-4 hrs • Useful for sterilization of glasswares and oils

33. Incineration Destroys microbes to ashes or gas • Bunsen flame - up to 1870˚C Ex: Sterilization of loops and needles. • Furnace - 800˚C to 6500˚C Ex: Incineration of animal carcasses.

34. Incinerator An infrared incinerator using flame to burn or oxidize materials into ashes.

35. Cold and Desiccation • Cold temperatures - reduce microbial activity except psychrophiles. • Desiccation or dehydration - removal of water stops metabolic activity of microbes. • Lyophilization –a freeze drying method used to preserve microbes and vaccines.

36. Radiation • Ionizing: - Gamma rays (High energy) - X-rays (Intermediate energy) - Cathode rays (Lowest energy) • Nonionizing: • Ultraviolet light

37. Radiation Effects • Ionizing radiation: • Ejects orbital electrons from an atom • High energy - penetrates liquids and solids effectively. • Nonionizing radiation: • Raises atoms to a higher energy level • Low energy - less penetrating • UV - formation of pyrimidine dimers

38. Cellular Effects of Radiation

39. Ultraviolet Radiation UV radiation cause the formation of pyrimidine dimers on DNA.

40. Applications of Radiation Ionizing radiation: • Alternative sterilization method • Materials sensitive to heat or chemicals • Some foods (fruits, vegetables, meats) Nonionizing radiation: • Alternative disinfectant • Germicidal lamp in hospitals, schools, food preparation areas (inanimate objects, air, water)

41. Gamma Irradiation (a) Gamma radiation machine used to sterilize fruits, vegetables, meats, fish, and spices (b) Radora symbol

42. UV Irradiation UV treatment system used to disinfect water.

43. Filtration • Removes microbes and spores from liquids and air • Perforated membrane of varying pore sizes v (0.22 to 0.45 um) • Applications: • Liquids that are sensitive to heat (serum, vaccines, media) • HEPA filtration of operating rooms etc.

44. Membrane Filter Membrane filtration system. (b) Membrane filter close-up.

45. Chemical Methods Chemical agents commonly used in healthcare

46. Classes of Chemical Agents • Halogens • Phenolics • Surfactants • Hydrogen peroxide • Detergents and soaps • Heavy metals • Aldehydes • Gases • Dyes, acids, and alkali

47. Halogens Chlorine -disinfectant and antiseptic • Disrupt sulfhydryl groups in amino acids Iodine - topical antiseptic • Disruption is similar to chlorines

48. Phenol and Phenolics • Differ by functional groups attached to their aromatic rings. • Disrupts cell walls and membranes. • Ingredient in soaps up to kitty litter • Examples: Hexachlorophene, Triclosan

49. Phenolics

50. Alcohols • Dissolve membrane lipids, disrupt cell surface tension, denatures proteins. • Used for skin degerming. • Examples: Ethyl alcohol, Isopropyl alcohol • 70% concentration the best and most effective concentration to use.