chapter 9: control of microbial growth · sterilization > commercial sterilization ... •high...
TRANSCRIPT
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Chapter 9:Control of Microbial Growth
1. Physical Methods
2. Chemical methods
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Important Terminology (pg. 263-264)
sterilization > commercial sterilization > disinfection = antisepsis > degerming > sanitization
Also, a microbicidal agent kills microbes whereasa microbistatic agent inhibits growth without killing
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Rates of Microbial Death (pg. 264)
Constant percentageof the extant population
is killed each minute
1 min
90% die
1 min
90% die
Num
ber o
f liv
ing
mic
robe
s
1 2 3 4 5 6 7 8 9 10Time (min)
109
108
107
106
105
104
103
102
101
100
rate of death is constant, but the time required to
kill ALL organisms depends on
population density
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1. Physical Methods ofMicrobial Control
Chapter Reading – pp. 268-275
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Physical Methods to Control Growth1) Temperature
2) Filtration
3) Dessication
4) Osmotic Pressure
• physical removal of microorganisms
5) Radiation
• removal of water
• high concentrations of solutes (salts, sugars)
• high energy emissions that cause molecular damage
• high or low temperatures that limit microbial growth
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Treatment with Heat
1) temperature
2) amount of moisture
3) length of exposure
• water is much more effective at transferring heat than dry air, causing proteins to denature & coagulate
• larger microbial populations and larger materialsrequire longer exposure times
Heat denatures proteins & other macromoleculesat a rate that depends on 3 factors.
Thermal Death Point (TDP)• lowest temperature at whichALL organisms killed in 10’
Thermal Death Time (TDT)• time required to kill ALLorganisms at a given temp.
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Sterilization by Autoclavingmanual
exhaust toatmosphere
pressuregauge
safetyvalve
valve for steamto chamber
exhaustvalve
steamjacket
material tobe sterilized
steamair
door
thermometer steam supplytrap
Pres
sure
abo
ve n
orm
al (p
si)
100Temperature (°C)
30
110 120 130 140
25
20
15
10
5
0
Autoclaves are chambers of high pressure steam used for sterilization
• method of choice for heat-tolerant, small-size material
• inexpensive to use, non-toxic
higher pressures = higher temperatures
(w/o loss of moisture)
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cap that allowssteam to penetrate
flexible plastic vialcrushable
glass ampule
nutrient mediumcontaining pHcolor indicator
endospore strip
Incubation
After autoclaving, flexiblevial is squeezed to break
ampule and releasemedium onto spore strip.
Yellow mediummeans spores areviable; autoclavedobjects not sterile
Red mediummeans spores
were killed; autoclavedobjects are
sterile
Verification of Target Temperature“Indicators” are important to verify the necessarytemperature was reached for the required time:
• test vials of endospores • “autoclave tape” with indicator
chemicals that change color
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Pasteurization
• thermophilic organisms survive, however they don’tcause spoilage or disease (why not?)
A process of heating for specific periods of time to eliminate spoilage, pathogens without damaging the food product (e.g., food, wine, beer).
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Low Temperatures
DessicationThe elimination of moisture by dessication is amicrobistatic treatment.
• microbes cannot metabolize & grow but are typically NOT killed and thus can grow if moisture is restored
Low temperatures can be microbicidal and/ormicrobistatic:
• refrigeration is microbistatic by simply slowing down or eliminating microbial growth, it does NOT kill
• freezing can be microbicidal due to the formation of icecrystals, though some organisms can survive freezing
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FiltrationFilters with pore sizes smaller than microbial cells (0.2 µm) can effectively sterilize liquids
• receptacle to capture filtrate must be sterile
• more costly than heatsterilization
• best method for thesterilization of liquidsthat cannot tolerate high temperatures
• vacuum pressure pullsliquid through filter
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Treatment with Radiation
High energy electromagnetic radiation• short wavelength UV, x-rays, gamma rays
High energy particle radiation• e.g., electron beams
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Ionizing vs Nonionizing RadiationIonizing radiation• has high enough energy to cause the removal ofelectrons from atoms
• results in free radicals (usu. .OH from water)
Nonionizing radiation• energy is too low to remove electrons but can cause other types of damage:
• x-rays, gamma rays, electron beams
• e.g., UV radiation which causes specific DNA damage
**Both types of radiation can be used to sterilize**
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safety glassviewscreen
outside
exhaust HEPAfilter
blower
supply HEPAfilter
light
high-velocityair barrier
Physical Barriers
Microbial contamination can be minimized with the use of:
• safety cabinets
• HEPA filters
• lab coats, gloves, surgical masksSAFETY
CABINET
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2. Chemical Methods ofMicrobial Control
Chapter Reading – pp. 265-267, 275-281
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Effectiveness of ChemicalsChemicals rarely achieve sterility (usually disinfection, antisepsis) & their effects can be quite variable:
• effectiveness varies depending on the organism
• may not make contact with all organisms present• e.g., dense microbial populations or biofilms
• can be inhibited by various organic molecules• e.g., lipids and proteins that may bind to it
The choice of chemical agent depends on:• target organism(s)• degree of microbial control needed • material to be treated (e.g., countertop, human skin)
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Types of Chemical Disinfectants• phenol-based compounds (aka “phenolics”)
• alcohols (ethanol, isopropanol…)
• halogens (chlorine, iodine…)
• surfactants (quarternary ammonium ions or “quats”)
• peroxygens (hydrogen peroxide, ozone…)
• aldehydes (formaldehyde…)
• gaseous chemosterilizers (ethylene oxide…)
• “preservatives” (benzoic acid, sulfur dioxide…)
• heavy metals (silver, mercury, copper…)
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Phenol-based CompoundsPhenol was one of the first chemical disinfectants • damages microbial plasma membranes • can be irritating to human tissues
**especially effective against themycobacteria and their lipid-rich cell walls**
Many derivatives of phenolhave been developed that are less irritating but as effective:• O-phenylphenol or cresol
(used in “Lysol”) • bisphenols (used in antibacterial
soaps, kitchenware)
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AlcoholsEthanol (CH3-CH2OH) and isopropanol (CH3-CHOH-CH3)are most commonly used.• denature proteins, disruptmembrane lipids
• effective against most fungi & bacteria, NOT endosporesand viruses w/o envelopes
• NOT very effective on openwounds (poor contact)
• MOST effective when mixedwith water (necessary fordenaturation to occur)
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HalogensHalogens are the “salt-forming” elements (F, Cl, Br, I)w/7 valence electrons (group VIIA of the periodic table).
Many compounds that contain chlorine or iodine are effective disinfectants:
• “bleach” (sodium hypochlorite: NaOCl)
• “iodine” (I2 mixed as a tincture with an aqueous alcohol)
• halogens are thought to be oxidizing agents (remove e-) that damage and denature proteins
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PeroxygensPeroxygens such as hydrogen peroxide (H2O2) and ozone (O3) damage macromolecules via –OH radicals• overwhelm the protective enzymes of aerobic organisms
• peroxyacetic acid can even kill endospores
Aldehydes (-HC=O)Formaldehyde & glutaraldehyde crosslink and inactivate proteins (to sterilize) however they are irritants and thus not used as antiseptics (good for embalming!).
• effective for treating open wounds
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Gaseous ChemosterilizersGaseous chemicals used to sterilize in a closedchamber (usually ethylene oxide or chlorine dioxide):• denatures proteins, requires >4 hrs to sterilize
• can be toxic to humans (carcinogenic)
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Hydrophobic tail
Heavy MetalsCompounds that contain metals such as silver (Ag), mercury (Hg) & copper (Cu):
SurfactantsDetergents that disrupt membranes
• interact with & denature proteins to inhibit microbial growth
• detergents containing quaternary ammonium (NH4
+) ions or “quats” are the most effectiveand most widely used
*NOT effective against endospores & mycobacteria Quaternary ammonium ions (quats)
Ammonium ion
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Resistance Rankings
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Key Terms for Chapter 9
• sanitization, microbicidal, microbistatic
• autoclave, pasteurization
• thermal death point, thermal death time
• ionizing vs nonionizing radiation
• sterilization, disinfection, antisepsis, degerming
• phenolics, aldehydes, peroxygens, halogenssurfactants, “quats”
Relevant Chapter Questions MC: 1-6, 8-12, 14, 16-9 SA: 1-8, 10-13