The Control of Microbial Growth

Control of Microorganisms by Physical and Chemical Agents1. To destroy pathogens and prevent their transmission 2. To reduce or eliminate microorganisms responsible for the contamination of water, food, and other substancesDefine the following key terms related to microbial control : sterilization, sterilant, disinfection, disinfectant, sanitization, antisepsis, antiseptic, germicide, bactericide, bacteriostaticControl of Microorganisms 3Relative resistance of microbesHighest resistanceBacterial endosporesModerate resistancePseudomonas sp.Mycobacterium tuberculosisStaphylococcus aureusProtozoan cystsLeast resistancemost vegetative cellsFungal sporesenveloped virusesYeastProtozoan trophozoitesSepsis refers to microbial contaminationAsepsis is the absence of significant contaminationAseptic surgery techniques prevent microbial contamination of woundsThe Terminology of Microbial ControlSterilization: Removing all microbial life. All living cells, viable spores, and viruses destroyed or removed. When sterilization is achieved by a chemical agent, the chemical is called a sterilant. (Commercial sterilization: Killing C. botulinum endospores).Disinfection: Removing pathogens. Killing, inhibition, or removal of microorganisms that may cause disease. The Terminology of Microbial ControlThe usual definition of sterilization is the removal or destruction of all forms of microbial life; how could there be practical exceptions to this simple definition? Sanitization: reduced of microbial population to levels that are considered safe by public health standard.Antisepsis: Removing pathogens from living tissue with chemical agents.Biocide/germicide: Kills microbes. Germicide kills pathogens but not necessarily endospores.Bacteriostasis: Inhibiting, not killing microbes, but prevent growth.The Terminology of Microbial ControlThe usual definition of sterilization is the removal or destruction of all forms of microbial life; how could there be practical exceptions to this simple definition?

A Microbial Death CurveHow is it possible that a solution containing a million bacteria would take longer to sterilize than one containing a half-million bacteria?

Population Death Rate Is ConstantExample: Microbial Heat-Killing experiment9Factors that influence action of antimicrobial agentsNumber of microbesNature of microbes in the populationTemperature & pH of environmentConcentration or dosage of agentMode of action of the agentPresence of solvents, organic matter, or inhibitors

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1011Cellular targets of controlCell wallCell membraneCellular synthetic processes (DNA, RNA)Proteins

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14Practical concernsDoes the application require sterilization?Is the item to be reused?Can the item withstand heat, pressure, radiation, or chemicals?Is the method suitable?Will the agent penetrate to the necessary extent?Is the method cost- and labor-efficient & is it safe?15Methods of Physical ControlHeatCold temperaturesDesiccationRadiationFiltration

Heat is very effective (fast and cheap). 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 cultureDecimal Reduction Time (DRT): Minutes to kill 90%of a population at agiven temperature

1. Heat16Heat is frequently used to kill microorganisms.2.Moist heat kills microbes by denaturing enzymes.

181. Heat (Cont)Moist heat use of hot water or steamMode of action denaturation of proteins, destruction of membranes & DNASterilization Autoclave 15 psi/121oC/10-40minDisinfectionPasteurization 0.22 mIn what situation is filtration the only practical way to eliminate undesirable microbes?

Filtration Air filtration using high efficiency particulate air (HEPA) filters. Effective to 0.3 mMembrane filters for fluids.Pore size for bacteria: 0.2 0.4 mPore size for viruses: 0.01 m

Fig 7.436Physical Methods of Microbial ControlCompare the effectiveness of moist heat (boiling, autoclaving, pasteurization) and dry heat.Describe how filtration, low temperatures, high pressure, desiccation, and osmotic pressure suppress microbial growth.Explain how radiation kills cells.

Learning ObjectivesChemical Methods of Microbial ControlFew chemical agents achieve sterility.Consider presence of organic matter, degree of contact with microorganisms, and temperature Disinfectants regulated by EPAAntiseptics regulated by FDAUse-dilution testMetal rings dipped in test bacteria are dried.Dried cultures of S. choleraesuis, S. aureus, and P. aeruginosa are placed in disinfectant for 10 min at 20C.Rings are transferred to culture media to determine whether bacteria survived treatment.

Disk-diffusion Method Disk of filter paper is soaked with a chemical and placed on an inoculated agar plate; a zone of inhibition indicates effectiveness.

Fig 7.63940Chemical controlHalogensPhenolicsChlorhexidineAlcoholsHydrogen peroxideDetergents & soapsHeavy metalsAldehydes

Types of DisinfectantsPhenol = carbolic acid (historic importance)Phenolics: Cresols (Lysol) - disinfectantBisphenols Hexachlorophene (pHisoHex, prescription), hospitals, surgeries, nurseriesTriclosan (toothpaste, antibacerial soaps, etc.)

Phenol and derivatives disrupt plasma membranes (lipids!) and lipid rich cell walls (??) Remain active in presence of organic compoundsPFig 7.7Pseudomonas very resistant to triclosan41421. HalogensChlorine Cl2, hypochlorites (chlorine bleach), chloraminesDenaturation of proteins by disrupting disulfide bondsCan be sporicidal

Iodine - I2, iodophors (betadine)Denature proteinsCan be sporicidalMilder medical & dental degerming agents, disinfectants, ointments

ChlorineOxidizing agentWidely used as disinfectantForms bleach (hypochlorous acid) when added to water. Broad spectrum, not sporicidal (pools, drinking water)IodineMore reactive, more germicidal. Alters protein synthesis and membranes.Tincture of iodine (solution with alcohol) wound antisepticIodophors combined with an organic molecule iodine detergent complex (e.g. Betadine). Occasional skin sensitivity, partially inactivated by organic debris, poor sporicidal activity.

Halogens

442. PhenolicsDisrupt cell membranes & precipitating proteins; bactericidal, fungicidal, virucidal, not sporicidalLysol triclosan- antibacterial additive to soaps45

463. ChlorhexidineHibiclens, HibitaneA surfactant & protein denaturant with broad microbicidal propertiesNot sporicidalUsed as skin degerming agents for preoperative scrubs, skin cleaning & burns474. AlcoholsEthyl, isopropyl in solutions of 50-90%Act as surfactants dissolving membrane lipids and coagulating proteins of vegetative bacterial cells and fungiNot sporicidalEthyl (60 80% solutions) and isopropyl alcoholDenature proteins, dissolve lipidsNo activity against spores and poorly effective against viruses and fungiEasily inactivated by organic debrisAlso used in hand sanitizers and cosmetics

Table 7.6

Alcohols

495. Hydrogen peroxideWeak (3%) to strong (25%)Produce highly reactive hydroxyl-free radicals that damage protein & DNA while also decomposing to O2 gas toxic to anaerobesStrong solutions are sporicidal

Hydrogen Peroxide: Oxidizing agent Inactivated by catalase Not good for open woundsGood for inanimate objects; packaging for food industry (containers etc.)3% solution (higher conc. available)Esp. effective against anaerobic bacteria (e.g.:Effervescent action, may be useful for wound cleansing through removal of tissue debris

50516. Detergents & soapsQuaternary ammonia cpds act as surfactants that alter membrane permeability of some bacteria & fungiNot sporicidalSoaps- mechanically remove soil and grease containing microbes52

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Soaps and DetergentsMajor purpose of soap: Mechanical removal and use as wetting agentDefinition of detergentsAcidic-Anionic detergents Anion reacts with plasma membrane. Nontoxic, non-corrosive, and fast acting. Laundry soap, dairy industry.Cationic detergents Quarternary ammonium compounds (Quats). Strongly bactericidal against against wide range, but esp. Gram+ bacteria

Surface Acting Ingredients / SurfactantsSoapDegermingAcid-anionic detergentsSanitizingQuarternary ammonium compounds(cationic detergents)Strongly bactericidal, denature proteins, disrupt plasma membrane54557. Heavy metalsSolutions of silver & mercury kill vegetative cells in low concentrations by inactivating proteinsOligodynamic actionNot sporicidal56

Heavy MetalsOligodynamic action: toxic effect due to metal ions combining with sulfhydryl (SH) and other groups proteins are denatured.Mercury (HgCl2, Greeks & Romans for skin lesions); ThimerosalCopper against chlorophyll containing organisms AlgicidesSilver (AgNO3): Antiseptic for eyes of newbornsZinc (ZnCl2) in mouthwashes, ZnO in antifungal in paint

57The oligodynamic effect (Greek oligos = few, dynamis = force) was discovered in 1893 by the Swiss KW Ngeli as a toxic effect of metal-ions on living cells, algae, molds, spores, fungus, virus, prokaryotic and eukaryotic microorganisms, even in relatively low concentrations. This antimicrobial effect is shown by ions of: mercury, silver, copper, iron, lead, zinc, bismuth, gold, aluminium and other metals.588. AldehydesGlutaraldehyde & formaldehyde kill by alkylating protein & DNAglutaraldehyde in 2% solution (Cidex) used as sterilant for heat sensitive instruments formaldehyde - disinfectant, preservative, toxicity limits use59

Aldehydes (alkylating agents)Inactivate proteins by cross-linking with functional groups (NH2, OH, COOH, SH)Glutaraldehyde: Sterilant for delicate surgical instruments (Kills S. aureus in 5, M. tuberculosis in 10 min)Formaldehyde: Virus inactivation for vaccinesChemical Sterilants for heat sensive materialDenature proteinsEthylene oxide

Aldehydes and Chemical SterilantsSulfur dioxidewineOrganic acids Inhibit metabolismSorbic acid, benzoic acid, and calcium propionateControl molds and bacteria in foods and cosmeticsSodium nitrate and nitrite prevents endospore germination. In meats. Conversion to nitrosamine (carcinogenic)

Chemical Food PreservativesPlasmaLuminous gas with free radicals that destroy microbesUse: Tubular instruments, hands, etc.

Microbial Characteristics and Microbial ControlFig 7.11the end64Gases & aerosolsEthylene oxide, propylene oxide, betapropiolactone & chlorine dioxideStrong alkylating agents, sporicidal65