Controlling Microbial Growth What factors limit microbial growth? In what situations are large microbial numbers undesirable? Concept of Microbial Control Factors Which Affect Control Temp., species type and status, environment Physical Control Methods Heat: Moist vs. Dry Autoclaving, pasteurization Filtration Cold Desiccation & high osmotic pressure Radiation (UV, gamma rays) Chemical Control Methods Factors which influence effectiveness Dilution, time, pH, organic matter Types of Disinfectants/Antiseptics Phenol and phenolics (e.g.. amphyl) Halogens (Chlorine, iodine, bromine) Alcohols (e.g.. isopropyl alcohol) Heavy metals (Ag, Hg, Cu, Zn) Surface active agents (soaps & detergents) Quaternary Ammonium Cmpds (quats) Aldehydes (e.g.. glutaraldehyde) Microbial growth can be inhibited by temperature, moisture control or by antimicrobial chemicals

Sterilization: Removal of all microbial life Commercial Sterilization: Killing C. botulinum endospores Disinfection: Removal of pathogens Antisepsis: Removal of pathogens from living tissue Degerming: Removal of microbes from a limited area Sanitization: Lower microbial counts on eating utensils Biocide/Germicide: Kills microbes Bacteriostasis: Inhibiting, not killing, microbes Terminology

Bacterial populations die at a constant logarithmic rate. Figure 7.1a

Number of microbes Environment (organic matter, temperature, concentration, biofilms) Time of exposure Microbial characteristics (e.g. glycocalyx,cell wall, resistance) Effectiveness of antimicrobial treatment depends on: Figure 7.1b

Controlling Microbial Growth What factors limit microbial growth? In what situations are large microbial numbers undesirable? Concept of Microbial Control Factors Which Affect Control Temp., species type and status, environment Physical Control Methods Heat: Moist vs. Dry Autoclaving, pasteurization Filtration Cold Desiccation & high osmotic pressure Radiation (UV, gamma rays) Chemical Control Methods Factors which influence effectiveness Dilution, time, pH, organic matter Types of Disinfectants/Antiseptics Phenol and phenolics (e.g.. amphyl) Halogens (Chlorine, iodine, bromine) Alcohols (e.g.. isopropyl alcohol) Heavy metals (Ag, Hg, Cu, Zn) Surface active agents (soaps & detergents) Quaternary Ammonium Cmpds (quats) Aldehydes (e.g.. glutaraldehyde) Microbial growth can be inhibited by temperature, moisture control or by antimicrobial chemicals

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

Moist heat denatures proteins Autoclave: Steam under pressure 15 min at 121 o C at 15 psi Autoclaving Figure 7.2

Pasteurization 63 o C for 30 minutes 72 o C for 15 seconds 140 o C for 1 second Pasteurization reduces spoilage organisms and pathogens

Dry Heat Sterilization kills by oxidation Flaming Incineration Hot-air sterilization Physical Methods of Microbial Control Hot-airAutoclave Equivalent treatments170˚C, 2 hr121˚C, 15 min

Controlling Microbial Growth What factors limit microbial growth? In what situations are large microbial numbers undesirable? Concept of Microbial Control Factors Which Affect Control Temp., species type and status, environment Physical Control Methods Heat: Moist vs. Dry Autoclaving, pasteurization Filtration Cold Desiccation & high osmotic pressure Radiation (UV, gamma rays) Chemical Control Methods Factors which influence effectiveness Dilution, time, pH, organic matter Types of Disinfectants/Antiseptics Phenol and phenolics (e.g.. amphyl) Halogens (Chlorine, iodine, bromine) Alcohols (e.g.. isopropyl alcohol) Heavy metals (Ag, Hg, Cu, Zn) Surface active agents (soaps & detergents) Quaternary Ammonium Cmpds (quats) Aldehydes (e.g.. glutaraldehyde) Microbial growth can be inhibited by temperature, moisture control or by antimicrobial chemicals

Filtration removes microbes by trapping them in filter Low temperature inhibits microbial growth Refrigeration Deep freezing (-20 o C or -80 o C) Lyophilization High pressure denatures proteins Desiccation prevents metabolism Osmotic pressure causes plasmolysis (shrinkage of cytoplasm) Physical Methods of Microbial Control

Radiation damages DNA Ionizing radiation (X rays, gamma rays, electron beams) Nonionizing radiation (UV)- surface sterilization only (Microwaves kill by heat; not especially antimicrobial) Physical Methods of Microbial Control

Controlling Microbial Growth What factors limit microbial growth? In what situations are large microbial numbers undesirable? Concept of Microbial Control Factors Which Affect Control Temp., species type and status, environment Physical Control Methods Heat: Moist vs. Dry Autoclaving, pasteurization Filtration Cold Desiccation & high osmotic pressure Radiation (UV, gamma rays) Chemical Control Methods Factors which influence effectiveness Dilution, time, pH, organic matter Types of Disinfectants/Antiseptics Phenol and phenolics (e.g.. amphyl) Halogens (Chlorine, iodine, bromine) Alcohols (e.g.. isopropyl alcohol) Heavy metals (Ag, Hg, Cu, Zn) Surface active agents (soaps & detergents) Quaternary Ammonium Cmpds (quats) Aldehydes (e.g.. glutaraldehyde) Microbial growth can be inhibited by temperature, moisture control or by antimicrobial chemicals

Principles of effective disinfection Concentration of disinfectant Organic matter pH Time Chemical Methods of Microbial Control

Evaluating a disinfectant Use-dilution test 1. Metal rings dipped in test bacteria are dried 2. Dried cultures placed in diluted disinfectant (according to manufacturer’s instructions) for min at 20°C 3. Rings transferred to culture media to determine whether bacteria survived treatment Chemical Methods of Microbial Control

Figure 7.6 Evaluating a disinfectant Disk-diffusion method Particular species are evaluated in each test Zone of inhibition must be at or beyond a certain diameter

Controlling Microbial Growth What factors limit microbial growth? In what situations are large microbial numbers undesirable? Concept of Microbial Control Factors Which Affect Control Temp., species type and status, environment Physical Control Methods Heat: Moist vs. Dry Autoclaving, pasteurization Filtration Cold Desiccation & high osmotic pressure Radiation (UV, gamma rays) Chemical Control Methods Factors which influence effectiveness Dilution, time, pH, organic matter Types of Disinfectants/Antiseptics Phenol and phenolics (e.g.. amphyl) Halogens (Chlorine, iodine, bromine) Alcohols (e.g.. isopropyl alcohol) Heavy metals (Ag, Hg, Cu, Zn) Surface active agents (soaps & detergents) Quaternary Ammonium Cmpds (quats) Aldehydes (e.g.. glutaraldehyde) Microbial growth can be inhibited by temperature, moisture control or by antimicrobial chemicals

Phenolics: Disruption of the plasma membrane Phenol Hexachlorophene Thymol Pine- Sol/pine oil (turpineol) O-phenylphenol (Lysol, Amphyl)

Halogens Tincture of Iodine (I 2 ); iodine denatures proteins by breaking disulfide bonds Sodium hypochlorite (NaOCl) in bleach is a strong oxidizing agent. Povidone iodines (I linked to organic molecule) as in Betadine; denatures proteins by breaking disulfide bonds Bromine and chlorine produces are strong oxidizers Iodine denatures proteins by breaking disulfide bonds Bromine tablets (BCDMH - bromo-chloro- dimethyl- hydantoin)used in hot tubs, pools, kills by oxidation Chlorine gas used in swim pools - oxidizing agent.

Types of Disinfectants: Alcohols Table 7.6 Alcohols. Ethanol, isopropanol Denatures proteins, dissolve lipids

Controlling Microbial Growth What factors limit microbial growth? In what situations are large microbial numbers undesirable? Concept of Microbial Control Factors Which Affect Control Temp., species type and status, environment Physical Control Methods Heat: Moist vs. Dry Autoclaving, pasteurization Filtration Cold Desiccation & high osmotic pressure Radiation (UV, gamma rays) Chemical Control Methods Factors which influence effectiveness Dilution, time, pH, organic matter Types of Disinfectants/Antiseptics Phenol and phenolics (e.g.. amphyl) Halogens (Chlorine, iodine, bromine) Alcohols (e.g.. isopropyl alcohol) Heavy metals (Ag, Hg, Cu, Zn) Surface active agents (soaps & detergents) Quaternary Ammonium Cmpds (quats) Aldehydes (e.g.. glutaraldehyde) Oxidizing Agents Microbial growth can be inhibited by temperature, moisture control or by antimicrobial chemicals

Heavy metals Mercurachrome antiseptic for wounds Copper sulfate (CuSO 4 ) algicide Zinc chloride (ZnCl 2 )mouthwashes Silver nitrate (AgNO 3) antiseptic for eyes, wounds Denature proteins by breaking disulfide bonds

Surface-Active Agents or Surfactants (Soaps and Detergents) Types of Disinfectants: Surface Active Agents SoapDegerming Acid-anionic detergentsSanitizing

Quaternary Ammonium Compounds (Quats) Benzalkonium chloride (aka Zephiran) Cetylpyridinium chloride (Cepacol) Roccal: lab disinfectant Disruption of plasma membrane, denaturation of proteins Cationic detergents

Aldehydes Inactivate proteins by cross-linking with functional groups (–NH 2, –OH, –COOH, —SH) Glutaraldehyde, formaldehyde Types of Disinfectants:Aldehydes glutaraldehyde Disinfection of hospital equipment, especially in respiratory therapy, for kidney dialysis machines, endoscopes

Acids and Bases: Denaturation of Proteins, Dissolving Membranes NaOH (sodium hydroxide) in some floor cleaners NH 4 OH in ammonia, esp.good at breaking down lipids Benzoic acid in mouthwashes; benzoic acid, sorbic acid, citric acid, and ascorbic acid, in food products

Gaseous Sterilants Denature proteins Ethylene oxide to sterilize hospital equipment, disposable lab plasticware Other oxidizing Agents O 3 (swim pools), H 2 O 2 (skin), Cl 2 (swim pools), peracetic acid (dialysis equipment), bleach (sodium hypochlorite), bromine Steals electrons from biomolecules, causing breakdown Gas Sterilants and Oxidizing Agents

Microbial Characteristics and Microbial Control Figure 7.11

Microbial Characteristics and Microbial Control Chemical agentEffectiveness against EndosporesMycobacteria PhenolicsPoorGood QuatsNone ChlorinesFair AlcoholsPoorGood GlutaraldehydeFairGood

Controlling Microbial Growth What factors limit microbial growth? In what situations are large microbial numbers undesirable? Concept of Microbial Control Factors Which Affect Control Temp., species type and status, environment Physical Control Methods Heat: Moist vs. Dry Autoclaving, pasteurization Filtration Cold Desiccation & high osmotic pressure Radiation (UV, gamma rays) Chemical Control Methods Factors which influence effectiveness Dilution, time, pH, organic matter Types of Disinfectants/Antiseptics Phenol and phenolics (e.g.. amphyl) Halogens (Chlorine, iodine, bromine) Alcohols (e.g.. isopropyl alcohol) Heavy metals (Ag, Hg, Cu, Zn) Surface active agents (soaps & detergents) Quaternary Ammonium Cmpds (quats) Aldehydes (e.g.. glutaraldehyde) Microbial growth can be inhibited by temperature, moisture control or by antimicrobial chemicals