Copyright © 2011 Pearson Education Inc. Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville M I C R O B I O L O G Y WITH DISEASES BY TAXONOMY, THIRD EDITION Chapter 9 Controlling Microbial Growth in the Environment

Copyright © 2011 Pearson Education Inc. Terminology of Microbial Control Sterilization - eliminate all microbial life Disinfection - eliminate pathogens Antisepsis- eliminate pathogens from living tissue Sanitization - reduce microbial population to meet acceptable health standards Biocide/Germicide - kills microbes Bacteriostasis - inhibits microbial growth

Copyright © 2011 Pearson Education Inc. Basic Principles of Microbial Control

Copyright © 2011 Pearson Education Inc. Action of Disinfectants 1.Alterations in cell walls and membrane – Cell wall maintains integrity of cell – Cells burst due to osmotic effects when damaged – Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell – Cellular contents leak out when damaged – Nonenveloped viruses have greater tolerance of harsh conditions

Copyright © 2011 Pearson Education Inc. Action of Disinfectants 2. Damage to proteins and nucleic acids – Protein function depends on 3-D shape – Extreme heat or certain chemicals denature proteins – Chemicals, radiation, and heat can alter or destroy nucleic acids – Can produce fatal mutants – Can halt protein synthesis through action on RNA

Copyright © 2011 Pearson Education Inc. Relative susceptibilities of microbes to control methods Figure 9.2

Copyright © 2011 Pearson Education Inc. Physical Methods of Microbial Control 1.Heat 2.Desiccation (drying) 3.Filtration 4.Osmotic pressure 5.Radiation

Copyright © 2011 Pearson Education Inc. Physical Methods of Microbial Control HEAT Fast, inexpensive & most common Alters proteins, enzymes & dehydrates Two Types: – Moist Heat – Boiling – Steam under pressure (autoclaving) – Pasteurization – Ultrahigh-temperature sterilization – Dry Heat – Incineration – Drying ovens

Copyright © 2011 Pearson Education Inc. Boiling – not recommended for sterilization – Boiling time is critical – Different elevations require different boiling times – Endospores, protozoan cysts, and some viruses can survive boiling – 10 minutes to kill most vegetative bacteria, fungi, many viruses – 30 minutes to kill hepatitis A and protozoan cysts – But does not kill endospores (can survive 20 hrs) – Not effective if > 1 million microbes/ml. Moist Heat

Copyright © 2011 Pearson Education Inc. Moist Heat Autoclave: used to sterilize Steam under pressure kills spores 121 o C,15 psi, 15 mins Figure 7.2

Copyright © 2011 Pearson Education Inc. Spore strip autoclaved along with materials Strip placed in broth; if no growth occurs, assumed sterilization successful Autoclave Controls

Copyright © 2011 Pearson Education Inc. Autoclave packaging Steam must penetrate wrapped material and touch organism

Copyright © 2011 Pearson Education Inc. Pasteurization Decrease number of heat-sensitive pathogens without altering food quality Today, also used for milk, ice cream, yogurt, and fruit juices Not sterilization ; Heat-tolerant and heat-loving microbes survive Ultrahigh-temperature sterilized liquids can be stored at room temp.

Copyright © 2011 Pearson Education Inc. Dry Heat Incineration Hot-air oven – Used for reusable materials that cannot withstand moist heat – Glassware, metals Hot-air ovenAutoclave Equivalent treatments170˚C, 2 hr121˚C, 15 min Material must be clean – organic matter insulates Microwaves will not sterilize; should not be used for home canning

Copyright © 2011 Pearson Education Inc. Physical Methods of Microbial Control Refrigeration and Freezing – Decrease microbial metabolism, growth, and reproduction – Chemical reactions occur slower at low temperatures – Liquid water not available – Psychrophilic microbes can multiply in refrigerated foods – Refrigeration halts growth of most pathogens – Slow freezing more effective than quick freezing – Organisms vary in susceptibility to freezing

Copyright © 2011 Pearson Education Inc. Physical Methods of Microbial Control Dessication and Lyophilization – Drying inhibits growth due to removal of water – Lyophilization used for long-term preservation of microbial cultures; prevents formation of damaging ice crystals – Microbes are more resistant to drying if in organic debris (mucus, pus, feces, blood, etc.) – Susceptibility to desiccation (drying) varies with species : Spores and viruses are often resistant to drying Neisseria gonorrhoeae survives drying up to 1 hour. Mycobacterium tuberculosis survives drying for months

Copyright © 2011 Pearson Education Inc. The use of dessication as a means of preserving apricots Figure 9.8

Copyright © 2011 Pearson Education Inc. Filtration Removes microbes from liquids/air Used for heat-sensitive liquids – Antibiotics, vaccines HEPA filter – High-Efficiency Particulate Air filter

Copyright © 2011 Pearson Education Inc. The role of HEPA filters in biological safety cabinets Figure 9.10 High Efficiency Particulate Air filters remove 99% of air particles use in surgery, burn units, CDC Level IV areas

Copyright © 2011 Pearson Education Inc. Physical Methods of Microbial Control Biosafety Levels – Four levels of safety in labs dealing with pathogens – Biosafety Level 1 (BSL-1) – Handling pathogens that do not cause disease in healthy humans – Biosafety Level 2 (BSL-2) – Handling of moderately hazardous agents – Biosafety Level 3 (BSL-3) – Handling of microbes in safety cabinets – Biosafety Level 4 (BSL-4) – Handling of microbes that cause severe or fatal disease

Copyright © 2011 Pearson Education Inc. Osmotic Pressure High concentrations of salt or sugar in foods to inhibit growth 10-15% salt or 50-70% sugar Cells in a hypertonic solution of salt or sugar lose water; cell desiccates Fungi have greater ability than bacteria to survive hypertonic environments

Copyright © 2011 Pearson Education Inc.

Radiation

Copyright © 2011 Pearson Education Inc. Radiation IONIZING RADIATION damages DNA – X-rays, Gamma rays used to sterilize – Electron beams – effective at killing but do not penetrate well – Gamma rays – penetrate well but require hours to kill microbes – Use on heat sensitive equipment – Plastic syringes, surgical gloves and supplies, catheters and sutures – Spices, fruits, vegetables, poultry, red meat ResourcesForYou/Consu mers/ucm htm

Copyright © 2011 Pearson Education Inc. Radiation NONIONIZING RADIATION – UV light – Can be used on food and water – Suitable for disinfecting air, transparent fluids, and surfaces of objects; i.e. Nurseries, burn units, surgery, wastewater and sewage treatment – UV light must touch organism to kill; cannot penetrate glass, oil, plastic, or paper

Copyright © 2011 Pearson Education Inc. Chemical Methods to Control Microbial Growth Only a few chemicals are used for sterilization Many can be used as antiseptics Affect microbes’cell walls, cytoplasmic membranes, proteins, or DNA Often more effective against enveloped viruses and vegetative cells of bacteria, fungi, and protozoa

Copyright © 2011 Pearson Education Inc. The Selection of Microbial Control Methods Germicides classified as high, intermediate, or low effectiveness – High-level kill all pathogens, including endospores – Intermediate-level kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria – Low-level kill vegetative bacteria, fungi, protozoa, and some viruses

Copyright © 2011 Pearson Education Inc. Chemical Methods of Microbial Control – Aldehydes – Gaseous Agents – Oxidizing agents – Halogens – Phenols – Alcohols – Heavy Metals – Surfactants From High level to Low level germicides

Copyright © 2011 Pearson Education Inc. Inactivate proteins and nucleic acids kills bacteria and viruses – Glutaraldehyde – 10 minutes of 2% solution used to disinfect hospital equipment. If exposure time increased to 10 hours – can sterilize. – Formaldehyde – Gas- irritating vapors, possible carcinogen – Liquid – formalin- – Used to kill bacteria and viruses in vaccines – Preserve biological specimens Aldehydes

Copyright © 2011 Pearson Education Inc. Ethylene Oxide Gas – Microbicidal and sporicidal gases to sterilize (1-3 hours) – Denature proteins and DNA – Used for prepackaged supplies, sutures, artificial heart valves, can penetrate fabric – Disadvantages – Can be hazardous to people – Often highly explosive – Chlorine dioxide gas – Water treatment, food processing equipment, Gov’t bldg after anthrax attack, space probes

Copyright © 2011 Pearson Education Inc. Oxidizing Agents – Kill by oxidation (e- loss) of microbial enzymes – High-level disinfectants (when vaporized) and antiseptics Hydrogen peroxide – does not work well on open wounds; blood contains catalase; best used against anaerobic organisms in deep puncture wounds Benzyl peroxide – acne Ozone – treat water, air conditioner cooling towers Peracetic acid – sterilize medical equipment and food processors

Copyright © 2011 Pearson Education Inc. Halogens Chlorine – denatures proteins Intermediate level disinfectant 10% Sodium hypochlorite (bleach) – kills HIV Chloramines – chlorine and ammonia –, Used on eating utensils and glassware Purify water ½ tsp. bleach in 2 gallons water for 30 minutes Chlorine gas used to disinfect large spaces – federal office buildings

Copyright © 2011 Pearson Education Inc. Halogens Tincture of iodine = Iodine plus alcohol Good antiseptic Can purify drinking water with 5 drops in 1 quart water, 30 minutes Iodophors = Iodine plus detergent Less irritating as wound antiseptic e.g. Betadine for skin, Wescodyne for preop,Isoprep for presurgical scrub

Copyright © 2011 Pearson Education Inc. Bromine Often used in hot tubs because it evaporates more slowly than chlorine; sometime used in place of chlorine in swimming pools, cooling towers, etc. Fluorine (fluoride) Disrupts bacteria in biofilms; used in toothpaste and drinking water Halogens

Copyright © 2011 Pearson Education Inc. Phenols and Phenolics Phenol (carbolic acid) Used by Joseph Lister Organic matter interferes with its activity Strong odor, caustic to skin Phenol derivative = Phenolic compound Less toxic, more germicidal activityLess toxic, more germicidal activity – Intermediate- to low-level disinfectants – Denature proteins and disrupt cell membranes – Effective in presence of organic matter – Remain active for prolonged time – Commonly used in health care settings, labs, and homes

Copyright © 2011 Pearson Education Inc. Phenolics Triclosan

Copyright © 2011 Pearson Education Inc. Phenolic- Lyso l

Copyright © 2011 Pearson Education Inc. Triclosan widely used in soaps, hand lotion, toothpaste, sponges, antibacterial soaps, mouthwash, toys, cutting boards, etc. Phenolic compoundsResistance?? Potential carcinogen??

Copyright © 2011 Pearson Education Inc.

Alcohols Denature proteins, dissolve lipids Intermediate level DOES NOT kill bacteria endospores or fungal spores Ethanol, isopropanol Hand sanitizers Evaporates quickly Must use rubbing action until dry Be careful when using glycerin

Copyright © 2011 Pearson Education Inc. Hand Sanitizers

Copyright © 2011 Pearson Education Inc. Alcohols

Copyright © 2011 Pearson Education Inc. Heavy Metals Low-level bacteriostatic and fungistatic agents – 1% silver nitrate in NB eyes – Silver ointment for severe burns – Metallic silver on catheters to prevent UTI’s – Thimerosal used to preserve vaccines – Copper sulfate controls algal growth in reservoirs, fish tanks, swimming pools, and water storage tanks; interferes with chlorophyll – Zinc used in mouthwash

Copyright © 2011 Pearson Education Inc. Quaternary Ammonium Compounds – “Quats” (cationic= + charged) Low level disinfectant Kills fungi, viruses, some bacteria Does not kill endospores OR Pseudomonas aeruginosa Surface-Active Chemicals or Surfactants (anionic= - charged) Soap Decrease molecule surface tension; aids in mechanical removal Surfactants (Detergents)

Copyright © 2011 Pearson Education Inc. Factors Affecting the Efficacy of a Disinfectant : 1.Site to be treated Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects 2.Number of microbes 3.Relative susceptibility of microbes Prions and endospores require high level disinfectants 4.Time of exposure 5.Presence of organic matter 6.Temperature and pH 7.Concentration of chemicals used