Control of Microbial Growth Sofronio Agustin Professor Sofronio Agustin Professor LECTURES IN MICROBIOLOGY LECTURES IN MICROBIOLOGY 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 Terms  Sterilization  Disinfection  Antisepsis  Sanitation  Degerming

6 Sterilization  Complete removal or destruction of all viable microbes including endospores.  Application: Surgical instruments and commercially packaged foods.  Commercial sterilization: Killing C. botulinum endospores.

7 Disinfection  Use of physical or chemical agents (disinfectant) to kill vegetative bacteria and other microbes except endospores.  Example: 10% Bleach solution on bench tops.

8 Antisepsis  Use of physical or chemical agents to kill pathogens on living tissue.  Example: Scrubbing surgical patients with chemical agents.

9 Sanitization  Reduction of microbial counts to acceptable levels of public health standards.  Example: Sanitization of eating utensils in restaurants.

10 Degerming  Physical removal or reduction of microbes from a limited area.  Ex: Washing of hands with germicidal soap.

11 Efficacy Depends On  Number of microbes  Target microbe characteristics  Environmental factors - temperature, pH, biofilms  Concentration and mode of action of agent  Organic matter - sputum, blood, feces

12 Factors Factors that influence the effectiveness of antimicrobial agents

13 Mode of Action  Damage to cell wall  Alteration of membrane function  Damage to proteins  Damage to nucleic acids

14 Damage to Cell Wall Effects on bacteria and fungi:  Blockage of cell wall synthesis  Degradation of cell wall components  Reduction of its stability and integrity Ex: Penicillin, detergents, alcohols

15 Injury to Cell Membrane Effects on microbes including enveloped viruses:  Bind and penetrate membrane lipids  Loss of selective permeability resulting in leakage of cytoplasmic contents. Ex: S urfactants - surface active agents.

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

17 Effects on Nucleic Acids Irreversible binding to microbial DNA results in:  Ceasation of transcription and translation  Mutations Ex: Formaldehyde and ionizing radiation

18 Effects on Proteins  Blockage of enzyme active sites prevents binding of substrate.  Protein denaturation. Example: Heat, acids, alcohols, phenolics, and metallic ions.

19 Effects on Proteins Effects of heat, pH, and blocking agents on protein function.

20 Physical Methods  Heat  Radiation  Filtration

21 Effects of Heat Moist heat:  Coagulation of proteins  Denaturation of proteins Dry heat:  Dehydration  Denaturation  Oxidation (burning to ashes)

22 Effects of Heat  Thermal death point (TDP): Lowest temperature at which all cells in a culture are killed in 10 min.  Thermal death time (TDT): Time needed to kill all cells in a culture.  Decimal reduction time (DRT): Minutes to kill 90% of a population at a given temperature.

23 Moist Heat  Boiling  Tyndallization  Pasteurization  Steam under pressure

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

25 Tyndallization  Intermittent use of free-flowing steam for 30 to 60 minutes. Ex: Used on heat-sensitive media, canned foods.  Note: This will not destroy spores.

26 Pasteurization  Pasteurization reduces spoilage caused by microbes and kills pathogens.  Thermoduric microbes survive Methods :  Classical: 63 ˚C for 30 minutes  High Temperature Short Time: 72 ˚C for 15 seconds  Ultra High Temperature: 140 ˚C for < 1 second  Used in milk industry, wineries, breweries.  Prevents transmission of milk-borne diseases caused by: Salmonella, Campylobacter, Listeria, Mycobacteria.

27 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.

28 Autoclave

29 Dry Heat  Flaming  Hot air  Incineration  Requires greater temperature and exposure time than moist heat

30 Hot Air Hot air oven  Effective at 170˚C for 2 hrs  Useful for sterilization of glasswares and oils

31 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.

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

33 Radiation  Ionizing radiation:  X-rays  Gamma rays  Electron Beams  Nonionizing radiation:  Ultraviolet light  Microwave -kills by heat, not especially microbicidal.

34 Radiation

35 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

36 Cellular Effects of Radiation

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

38 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)

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

40 UV Irradiation UV treatment system used to disinfect water.

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

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

43 Cold and Desiccation  Cold temperatures - reduce microbial activity except psychrophiles.  Refrigeration  Freezing  Desiccation - dehydration stops microbial metabolism.  Lyophilization – freeze drying in a vacuum preserves microbes and vaccines.

44 Classes of Chemical Agents  Phenols and Phenolics  Biguanides  Halogens  Alcohols  Heavy metals  Surfactants - detergents and soaps  Aldehydes  Peroxygens  Gases  Dyes, acids, and alkali

45 Phenol and Phenolics  Phenol - carbolic acid  Phenolics- Lysol  Bisphenols- Hexachlorophene Triclosan  Disrupt plasma membranes  Ingredients in cutting boards, kitty litter

46 Biguanides  Used as surgical hand scrubs and preoperative skin preparation  Strong binding affinity to skin and mucus membranes  Disrupt plasma membranes Ex: Chlorhexidine

47 Halogens  Oxidizing agents  Disrupt sulfhydryl groups in amino acids Iodine - topical antiseptic  Tincture or Iodophor Chlorine -disinfectant and antiseptic  Hypochlorous acid (HOCl) -germicidal  Calcium hypochlorite used in dairy and restaurant industries

48 Alcohols  Dissolve membrane lipids, denatures proteins.  Used for skin degerming.  Ethanol, Isopropanol  70% concentration - most effective

49 Soaps and Detergents Quaternary ammonium (quats):  Cationic  Denature proteins and disrupt cell membrane  Low-level disinfectant in the clinical setting Soaps and detergents:  Fatty acids, oils, sodium or potassium salts  Sanitizing and degerming agents  More effective if mixed with germicides

50 Detergent Action Positively charged region of the detergent binds with bacteria and the uncharged region integrates into the cell membrane

51 Hand Scrubbing Comparison of efficacy between nongermicidal and germicidal soaps used in hand scrubbing.

52 Heavy Metals Ag, Hg, Cu  Denature proteins  Oligodynamic action  Preservatives in cosmetics and ophthalmic solutions.  Silver nitrate - Ophthalmia neonatorum prophylaxis.  Silver sulfadiazine - burn patients and catheter tips.

53 Oligodynamic Action

54 Aldehydes  Crosslink with functional groups of proteins on the cell surface (-NH 2, -OH, -COOH, -SH)  Formaldehyde, Glutaraldehyde  Sterilants for surgical and dialysis instruments

55 Aldehyde Effect

56 Gases  Denature proteins and affect functional groups of DNA.  Sterilizes plastic materials Example: Ethylene oxide

57 Gas Sterilization Ethylene oxide sterilization chamber

58 Peroxygens  Oxidizing agents - form hydroxyl free radicals  Effective against anaerobes Examples:  H 2 O 2 - skin and wound cleaner  Peracetic acid - quick method for sterilizing medical equipment  O 3 - water treatment

59 Food Preservatives  Prevents spore germination  Inhibits metabolism  Control molds and bacteria in food and cosmetics Examples:  Organic acids - sorbic, benzoic, acetic acids  Calcium propionate - bread  Nitrite prevents endospore formation in meat products.

60 Summary of Chemical Methods Chemical agents commonly used in healthcare

61 Efficacy of Disinfectants Principles of effective disinfection  Concentration of disinfectant  Presence of organic matter  Temperature  pH  Time

62 Evaluation of Efficacy Use-Dilution Test:  Metal rings dipped in test bacteria are dried.  Dried rings placed in disinfectant for 10 minutes at 20 o C.  Rings transferred to media to determine bacterial death/survival.

63 Evaluation of Efficacy Disk-Diffusion Method (Kirby-Bauer)

64 Efficacy of Chemical Agents Chemical agentEffectiveness against EndosporesMycobacteria PhenolicsPoorGood QuatsNone ChlorinesFair AlcoholsPoorGood GlutaraldehydeFairGood

65 Antimicrobial Resistance

66 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.

67 Comparative Resistance Relative resistance of bacterial endospores and vegetative cells