Controlling Microbial Growth - PDF Free Download

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

Terminology 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

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

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

Physical Methods of Microbial Control 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

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

Physical Methods of Microbial Control Dry Heat Sterilization kills by oxidation Flaming Incineration Hot-air sterilization Hot-air Autoclave Equivalent treatments 170 C, 2 hr 121 C, 15 min

Physical Methods of Microbial Control 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)

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

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

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

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

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

Types of Disinfectants: Surface Active Agents Surface-Active Agents or Surfactants (Soaps and Detergents) Soap Acid-anionic detergents Degerming Sanitizing

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

Aldehydes Types of Disinfectants:Aldehydes Inactivate proteins by cross-linking with functional groups ( NH 2, OH, COOH, SH) Glutaraldehyde, formaldehyde 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

Gas Sterilants and Oxidizing Agents 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

Microbial Characteristics and Microbial Control Figure 7.11

Microbial Characteristics and Microbial Control Chemical agent Effectiveness against Endospores Mycobacteria Phenolics Poor Good Quats None None Chlorines Fair Fair Alcohols Poor Good Glutaraldehyde Fair Good