Physical and Chemical Control of Microbes Chapter 11 Copyright © The McGraw-Hill Companies, Inc) Permission required for reproduction or display.

Learning Objectives Distinguish between sterilization, disinfection, antisepsis and decontamination Define “-static” and “-cidal” Identify factors affecting microbial death rate Name four categories of cellular targets for physical and chemical agents Name and describe six methods of physical control of microorganisms Give examples, describe modes of action and application, and discuss advantages and disadvantages of the following groups of chemical agents: halogens, phenolics, alcohols, hydrogen peroxide, detergents, heavy metals, and aldehydes

Some Important Terms Sterilization - kill all vegetative cells and spores Disinfection - reduces number of pathogens on an inanimate surface (sanitation) Decontamination - makes contaminated surfaces safe to handle by reducing the number of microbes present Antisepsis - killing microbes on living tissue

Bacteriostatic Inhibits growth Bactericidal Kills cells Bateriolytic Kills and lyses cells Antimicrobial Effects

Microbial Death Begins when a certain threshold of concentration and time is reached Proceeds in a logarithmic manner Young cells die faster than old cells

Microbial Death Rate Affected by many factors: Number of microorganisms Type of microorganism Temperature and pH Concentration of the agent physiological state other substances (solvents, organic matter, inhibitors)

Cellular Targets The cell wall The cell membrane Cellular synthetic processes (DNA, RNA) Proteins

Physical (Dry Heat) Incineration - flaming your loops Baking - requires long periods 150°  C for 2-4 hours Advantages: cheap and easy Disadvantages: materials must withstand high temperatures and be dry (not aqueous)

Physical (Moist Heat) Boiling - will not kill endospores, used for disinfecting drinking water, food, to sanitize materials for babies Tyndallization – non-pressurized steam, intermittent sterilization, used for heat-sensitive materials. Pasteurization - High heat, short time Autoclaving – Steam under pressure

Pasteurization Commonly used with juice, beer, milk, and other dairy products to prevent spoilage Batch - 63°C for 30 min High Temperature Short Time - 72°C for sec Ultra-High Temperature - 134°C for 1-2 sec Does not kill spores and thermodurant bacteria Target: Salmonella, Brucella, Campylobacter jejuni, Listeria monocytogenes, Coxiella burnetii, Mycobacterium bovis.

(a)(b) Recorder Pressure regulator Safety valve Exhaust to atmosphere Steam from jacket to chamber or exhaust from chamber Steam jacket Condensate to waste Temperature- sensing bulb Control handle Steam from Jacket to chamber Strainer Steam to jacket Steam supply valve Door gasket Discharge Steam trap Autoclaving Commonly use in the laboratory Temperatures higher than boiling Use steam pressure - 15 p.s.i. above normal (2 atm) 121°C for 20 min Kills all endospores Home pressure cookers do the same thing

Thermal Death Measurements Both time and temperature have to be considered Thermal death point - the lowest possible temperature that will achieve complete killing within ten minutes Thermal death time - the minimum time to achieve complete killing in a liquid solution at a given temperature

Physical (Cold) Freezing - kills some cells due to ice crystal formation Refrigeration – does not kill bacteria, only slows down growth. Ignorance of this fact results in food poisoning Lyophilization preservation of microbes by freezing and drying. Advantages: many products tolerate cold better Disadvantages: very little killing and is expensive

Physical (Filtration) Pass liquid or gas through a filter with sufficiently small pore size HEPA - filter out > 0.3 µm particles Advantages: No thermal damage Disadvantages: viruses not eliminated and must be either liquid or gas Filter Sterilized fluid (a) Vacuum Pump suction Filter Liquid Pore (b) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. b: © Fred Hossler/Visuals Unlimited

Ionizing vs Non-ionizing radiation) Ionizing Radiation: ejects electrons from an atom, causes ions to form Gamma ray - very good penetration X-ray - less penetration Non-ionizing radiation raises electrons in an atom to a higher energy state Ultraviolet - damages DNA, with poor penetration

Ultraviolet Damage Wavelenth: nm Poor penetrating power Pyrimidine dimers Mutations induced Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. O OO Details of bonding Thymine dimer Normal segment of DNA C G UV T T O CH 3 G C A A CA TTTG TG T A C AA T

Radiation Advantages: very effective with little product damage (“cold” sterilization) Disadvantages: dangerous materials need shielding, and lack of public trust.

Chemical Treatments Chemotherapeutics - disease treatment Disinfectants - cleaning surfaces Many are available today (hundreds) Choice is based upon: Nature of the object Kinds of microbes targeted Desired effect

Chemical (Oxidizers) Damage proteins and membranes Halogens Chlorine - disinfectant (added to water) Iodine - antiseptic (tincture of iodine, betadine) Hydrogen peroxide (H 2 O 2 ) 3% is a weak antiseptic Your body and many bacteria can break this down enzymatically

Chemical (Phenolics) Denature proteins Disrupt membranes Joseph Lister Examples: phenol, lysol, chlorhexidine Effective on surfaces Many of these are too toxic to apply to tissue OH CH 3 Phenol (basic aromatic ring structure) p-cresolo-cresol OH Cl OH Cl Hexachlorophene (a bisphenol) Chlorophene (a chlorinated phenol) Cl CH 2 Cl Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Chemical (Alcohols) Denature proteins Disrupt membranes Examples: ethanol, and isopropanol Most effective at 50-70% Increased plasmolysis after damage These are commonly used for antisepsis

Chemical (Surfactants) Amphiphilic compounds Disrupt membranes Quaternary ammonium compounds (quads) Charged nitrogen Four hydrophobic groups Example - cepacol, and roccal N + Cl – (b) (a) Hydro carbon chain (C number from 8 to18) R1R1 Benzalkonium chloride R3R3 R2R2 R4R4 + CH 3 CH 2 N+N+ C N H 2N + Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Gold foilSilver amalgam Oligodynamic Effect Inhibition by heavy metals Silver Copper Mercury Gold Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. © Kathy Park Talaro/Visuals Unlimited

Chemical (Alkylators) Damage proteins or DNA by adding carbon adducts Examples: formalin, glutaraldehyde, and ethylene oxide Highly noxious Ethylene oxide is used to sterilize products via gas OO OO OO Amino groups in peptidoglycan Cross-linking with microbial protein Polyglutaraldehyde Glutaraldehyde Polymerization G+G+ G–G– N N N N Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.