Controlling Microbial GrowthChapter 7

ObjectivesLearn some terminologiesKnow the factors that affect the effectiveness of anti-microbialsKnow some physical and chemical methods of controlling microbes

Microbial Control MethodsFigure 7.1

TerminologiesSterilization: Destruction/removal of all forms of microbial life from an objectPhysical processesFiltrationHigh temperature IncinerationIrradiation by ultraviolet light or gamma (g) raysChemical processes Ethylene oxide (used in hospitals)Chlorine dioxide (decontamination of buildings)Steam sterilization (microbiology)Mechanical processes Filtration

TerminologiesDisinfection: Use of chemicals (disinfectants) to kill or inhibit microorganisms that cause diseaseDoes not kill all microorganismsUsually toxic and injurious to human tissues

Widely use Chemicals disinfectants:Chlorine (water supplies)Phenols,Biphenols (chlorhexidine), AlcoholsIodineAldehydesQuats (quartenary ammonium salts)Ozone and UV light (newer means)

TerminologiesSanitization: Related to disinfection, however sanitization only reduces the microbial population to acceptable standards.Restaurants/cafeterias sanitize eating utensils to acceptable public health standards.

Antisepsis - Related to disinfection: Chemicals used to treat or prevent infection of living tissues (still toxic)Common antiseptics AlcoholIodineChlorhexidineHeavy metals (silver, mercury)

TerminologiesAntimicrobial agentsAgents that kill or inhibit the growth of microorganisms (cidal or static)cide: suffix indicating agent kills organismsBiocide or germicide: kills microorganismsVirocide: inactivates virusese.g. microbiocides for HIV used in spermacidesFungicide: kills fungi static: agent that inhibit growth Bacteriostatic agent: (inhibits) growth of bacteria

TerminologiesSepsis: bacterial contaminationAsepsis: absence of significant contaminationAseptic technique minimizes contamination

Factors Affecting Antimicrobial EffectivenessPopulation size (larger populations longer to kill)Population composition: different sensitivity to antimicrobial agentsConcentration or intensity of an antimicrobial agentTime of exposure longer exposure more organisms killed Environment conditionsHigh temperatures (more killing)Organic material (decreases killing)Sewage, biofilmsMicrobial characteristics Spores more resistant than vegetative cells

Microbial Characteristics Gram (-) vs Gram (+) bacteria. Pseudomonas aeruginosa - most resistant gram (-)Acid-fast vs non-acid fast Mycobacterium tuberculosis- resistant to aqueous bactericides (why?).Vegetative vs Endospores- most resistant bacteria formsViruses Naked viruses more resistant than enveloped. Protozoan cysts vs trophozoite stageCryptosporidium cysts are resistant to chlorine

Microbial Sensitivity to Chemical Biocides (Fig 7.11)

*Figure 7.1Concentration and Duration of exposure

Physical Control MethodsHeat Common method to destroy microorganismsMoist Vs Dry Steam at 100oC more dangerous that an oven at 325oC (Why?)Moist heat Kills by coagulation of proteinBoiling water not as effective as steam Boiling water not as effective as steam (endospores resistant to boiling)Dry heat kills by oxidation (higher temps and longer time required)

Moist HeatAutoclaves (steam sterilizers)- Routinely used in Microbiology labOperates like a pressure cookerSterilize by moist heat under pressure 15 psi for 15 minutes at 121CMost effective when organisms in small volume or in direct contact with steam

Autoclave or Steam SterilizerOperates in a cycleSaturated steam enters chamber Air evacuated and temperature raised to 121oCPressure applied (15 psi) 15 minSteam condenses on objectsPressure lowered to normal atmospheric pressure before opening the chamber Why is this important for liquids? Lowering pressure so liquids will change to steamChamber opened before pressure falls for solid objects (surgical instruments)- Why?

Commercial sterilization

Used in the canning IndustrySteam sterilization designed to kill heat resistant spores of Clostridium botulinumThe heat treatment reduces the number of spore by 1012

PasteurizationReduces numbers of pathogensUsed when we dont want to change tasteMilk pasteurizationDesigned to kill Mycobacterium tuberculosis (heat resistant pathogen in milk). Also kills pathogenic and non-pathogenic Streptococcus, Brucella and coliforms Low-temperature hold (LTH) 62.8C for 30 minutesHigh-temperature short-term (HTST): 72C for 15 secondsUltra-high temperature (UHT): Process (5 sec cycle 74oC 140C 74oC)

Dry HeatDry heat kills microbes by oxidation. Higher temperatures and longer periods required. IncinerationFlaming Oven (170C, 2 hours)

Mechanical MethodsFiltrationCan reduce microbial population in liquids Sterilize heat-sensitive materials (heat labile)Can also reduce microbial populations in air Two typesDepth filtering Membrane filters 0.1 mm thickPore sizes from 0.2 mm

Low TemperatureRefrigeration is bacteriostatic Most pathogens do not growListeria monocytogenes grows in refrigerator Freezing: slow freezing creates ice crystals

RadiationNon-ionizing (UV) and Ionizing radiation

UV- (Non ionizing radiation)Not penetratingCan damage eyesDamages DNAThymine dimers in DNAGermicidal lampsvaccine disinfection

RadiationIonizing radiation: Gamma (g) rays, X-rays, or high energy electron beams (most energetic)Cause mutations and deathLow level ionizing rad. used on spices, certain meats and vegetablesHigh energy rad. used to sterilize medical supplies

Limiting WaterDesiccation : bacteriostaticlyophilization used to preserve culturesOsmotic pressure high concentrations of salt or sugarMolds and yeasts most resistant to desiccation and high salt conc.

Chemical Methods of Microbial ControlChemicals can be used as: Disinfectants, Sterilants,AntisepticsSurface-active agents (surfactants)Chemical food preservatives[Antibiotics]

Evaluating DisinfectantsSusceptibility testMake serial dilution of disinfectant madeKnown amounts of Staphylococcus aureus and Salmonella enterica added to each dilutionsIncubate at 20 or 37oC for 5 or 10 minutes.Remove aliquots and inoculate growth mediaDetermine the lowest dilution of disinfectant that kills bacteria at 10 min but not at 5 min exposure (D).Compare to phenol dilution (P) that does the same Phenol Coefficient value = D/PValue greater than 1 Disinfectant more effective

MIC: Minimal inhibition concentrationMBC: Minimal bactericidal concentrationConc. of disinfectant

Evaluating DisinfectantsUse-dilution testMetal rings dipped in test bacteria and dried,Dried cultures placed in disinfectant for 10 min at 20C.Rings then transferred to culture media to determine whether bacteria survived the treatment.Use information obtained to determine lowest dilution to be used for disinfection.

Evaluating DisinfectantsDisc Dilution Methods:Inoculate bacteria on nutrient agar platesPlace filter discs impregnated with disinfectant on plates and incubateMeasure zones of inhibition

Phenols and Phenolics:DisinfectantsDisrupt cell membranesPhenol used as standard Measure effectiveness of disinfectants (Staphylococcus aureus or Salmonella typhi)Active in presence of organic matterStablePersist for long periods after applicationActive against M. tuberculosis

Phenols

Originally used by Joseph ListerLysol mixture of phenols used as general disinfectantHexachlorophene- excellent skin disinfectantEffective against S. aureusBut is neurotoxic in ratsTriclosan used in antibacterial soaps and toothpaste Broad spectrum of activity

Chlorhexidine Disinfectant (member of biguanides)Widely used by dentistsAlso used as skin antisepticLow toxicityUsed on skin and mucous membranes

HalogensDisinfectantsIodine (I2) used in solutionTincture (iodine+alcohol)Iodophors (iodine+organic carrier)Betadine and Isodine, WescodyneStain skin and garmentsEffectiveness- all bacteria, spores, fungus and virusesChlorine (Cl2) used to disinfect water suppliesEffective against wide range of microbesDisadvantage:CorrosiveInactivated by organic matterForms chlorine disinfection by-products (DBPs) linked to bladder cancerChlorine gas forms bleach (hypochlorite) in waterChloramines are chlorine and ammonia

AlcoholsDisinfectantsEthanol or Isopropanol widely usedDenature proteins and damage cell membraneUsed to clean skin prior to surgeryEvaporate quicklyMore active as 70% aqueous solutions (instead of 100%)

Heavy MetalsDisinfectantsDenature proteinsSilver nitrate (topical cream)Mercury (phenylmercury) in eyedrops and contact lenses preservativesCopper sulfate (algicide)Zinc (mouthwash, paints)

SurfactantsDecrease surface tension or can damage lipid membranesSoaps and detergentsCleaning value (little value as disinfectants)Acidianionic sanitizersUsed to clean dairy utensils and equipmentQuaternary ammonium compoundsMicrobicidalFungicides, amoebicidal, and virucidal (enveloped viruses)

Quaternary Ammonium Compounds (Quats)Common quat (Zephiram, Cepacol)Used in mouthwash Pseudomonas auriginosa not affected, can grow in quats.More effective against G(+)Not effective against endospores or mycobacteria

AldehydesFormaldehyde (formalin) and glutaraldehydeCross-link protein moleculesDisinfect instrumentsUsed to preserve tissues for pathology

Chemical SterilizationGas SterilizationEthylene oxide and Chlorine dioxidePlasmasGas excited by an electromagnetic field to generate a mixture of nuclei and free electrons Form free radicles that can damage microbial structures. Use: surgical devises with long hollow tubes, catheters, laparoscopic instruments.Supercritical fluidsCompressed CO2 with both gas and liquid properties (use: bone, tendons, ligaments etc)PeroxygensHydrogen peroxide, peracetic acid, etc.

Gas SterilizationEthylene oxideDenatures proteinsHigh penetration powerKills all microbes and spores But require lengthy exposure timeUsed in hospitals as a chemical sterilant (beddings, mattresses) (chambers resemble autoclaves) Chlorine dioxideShort-livedUsed to sterilize buildings and contents (anthrax endospores)Initial treatment of water supplies before chlorination

Oxidizing AgentsInterfere with metabolism (especially of anaerobes)PeroxygensHydrogen peroxideCommon antiseptic Benzoyl peroxidePeracetic acid (washing fruits and vegetables)OzoneUsed by the GVRD as primary water disinfectant (allowing chlorine to be used in lower conc.)

Microbial Characteristics & ControlGram (-) vs Gram (+) bacteria Pseudomonas & Burkholderia spp. - most resistant Gram ve (porins)Acid-fast vs non-acid fast Mycobacterium tuberculosis- resistant to aqueous bactericides (why?)Vegetative vs endospores most resistant bacteria formsViruses Naked viruses more resistant than enveloped (protein vs lipid coat)Protozoan cysts vs vegetative (trophozoite) protozoa.Cryptosporidium cysts are resistant to chlorine

Other Antimicrobials AntibioticsExamples discussed here are not useful for ingestion or injection to treat diseaseE.g. Nisin to inhibit endospore-forming spoilage bacteria in cheesePlasmasGas is excited by electromagnetic field to form plasma state of matterFree radicals in plasmas destroy microbes and sporesUsed to sterilize long, narrow, hollow surgical instruments

Other Antimicrobials Supercritical fluidsWhen CO2 is compressed into supercritical state it has both gas and liquid propertiesMicrobes are exposed to supercritical fluids inactivatedEndospores inactivated at lower temperatures (45oC)Used to disinfect medical implants (bone, tendons, ligaments from donors)

Chemical Food PreservativesSulfur dioxideSodium benzoateSorbic acidCalcium propionateSodium nitrate and sodium nitrite

Heat PreservationMust know temperature and time needed to kill critical bacteriaThermal death time (TDT)Minimum time required to kill all bacteria in a liquid culture at a particular temperature

Antiseptic EffectivenessThe steeper the downward slope of the killing curve, the more affective the antiseptic is. Which antiseptic is most effective?

Commercial Sterilization

Used in the canning IndustrySteam sterilization designed to kill heat resistant spores of Clostridium botulinumMore-resistant endospores of thermophilic bacteria may survive

Limiting WaterDesiccation : bacteriostaticlyophilization used to preserve culturesOsmotic pressure high concentrations of salt or sugarMolds and yeasts most resistant to desiccation and high salt conc

************Gram + is more resistive than gram negativeCysts- similar to endospore in bacteriaTrophozoite- vegetative cell in protozoan****Moist heat kills by denaturing proteins, whereas dry heat kills by oxidation (charring)Water molecules also wrap around object much more easily********Vaccines, contact lens solutions are sterilized in this manner. Some bio safety cabinets.*******Catheters and bandages can be sterilized using radiation****MIC:lowest concentration that exhibts no growthMBC:lowest concentration that exhibits no growth after culturing*********Phenol has an aromatic ring and is a hydrophobic structure- it is active in the presence of organic matterRecall the ide of biofilms producing carbohydrates (they are hiding) to protect from the organic matterThats why phenol is good******You need for it have both hydrophobic and hydrophilic properties. 100% enters the cell and only damage the outer layer of proteins on the surface and a protective layer is formed.70% on the other hand enters much slowly and then damages more of the inside of the cell******These are water based disenfectant and so is not affective against Gram (-) as it has two different layers********Sodium nitrite is popular for meat preservatives. Nitrites preserve the nice red color on meats.******