Physical and Chemical Control of Microbes

Chapter 11

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Learning Objectives

• Distinguish between sterilization, disinfection, antisepsis and decontamination

• Define “-static” and “-cidal”

• Identify factors affecting microbial death rate

• Name four categories of cellular targets for physical and chemical agents

• Name and describe six methods of physical control of microorganisms

• Give examples, describe modes of action and application, and discuss advantages and disadvantages of the following groups of chemical agents: halogens, phenolics, alcohols, hydrogen peroxide, detergents, heavy metals, and aldehydes

Some Important Terms

• Sterilization - kill all vegetative cells and spores

• Disinfection - reduces number of pathogens on an inanimate surface (sanitation)

• Decontamination - makes contaminated surfaces safe to handle by reducing the number of microbes present

• Antisepsis - killing microbes on living tissue

• BacteriostaticInhibits growth

• BactericidalKills cells

• BateriolyticKills and lyses cells

Antimicrobial Effects

Microbial Death

• Begins when a certain threshold of concentration and time is reached

• Proceeds in a logarithmic manner

• Young cells die faster than old cells

Microbial Death Rate

• Affected by many factors:

• Number of microorganisms

• Type of microorganism

• Temperature and pH

• Concentration of the agent

• physiological state

• other substances (solvents, organic matter, inhibitors)

Cellular Targets

• The cell wall

• The cell membrane

• Cellular synthetic processes (DNA, RNA)

• Proteins

Physical (Dry Heat)

• Incineration - flaming your loops

• Baking - requires long periods

• 150° - 180C for 2-4 hours

• Advantages: cheap and easy

• Disadvantages: materials must withstand high temperatures and be dry (not aqueous)

Physical (Moist Heat)

• Boiling - will not kill endospores, used for disinfecting drinking water, food, to sanitize materials for babies

• Tyndallization – non-pressurized steam, intermittent sterilization, used for heat-sensitive materials.

• Pasteurization - High heat, short time

• Autoclaving – Steam under pressure

Pasteurization

• Commonly used with juice, beer, milk, and other dairy products to prevent spoilage

Batch - 63°C for 30 min

High Temperature Short Time - 72°C for 15-20 sec

Ultra-High Temperature - 134°C for 1-2 sec

• Does not kill spores and thermodurant bacteria

• Target: Salmonella, Brucella, Campylobacter jejuni, Listeria monocytogenes, Coxiella burnetii, Mycobacterium bovis.

(a) (b)

Recorder

Pressure regulator

Safety valve

Exhaust to atmosphereSteam fromjacket to chamberor exhaust from chamber

Steam jacket

Condensateto wasteTemperature-

sensingbulb

Controlhandle

Steam fromJacket to chamber

Strainer

Steamto jacket

Steamsupplyvalve

DoorgasketDischarge

Steamtrap

Autoclaving• Commonly use in the

laboratory

• Temperatures higher than boiling

• Use steam pressure - 15 p.s.i. above normal (2 atm)

• 121°C for 20 min

• Kills all endospores

• Home pressure cookers do the same thing

Thermal Death Measurements

Both time and temperature have to be considered

• Thermal death point -the lowest possible temperature that will achieve complete killing within ten minutes

• Thermal death time -the minimum time to achieve complete killing in a liquid solution at a given temperature

Physical (Cold)

• Freezing - kills some cells due to ice crystal formation

• Refrigeration – does not kill bacteria, only slows down growth. Ignorance of this fact results in food poisoning

• Lyophilization preservation of microbes by freezing and drying.

• Advantages: many products tolerate cold better

• Disadvantages: very little killing and is expensive

Physical (Filtration)

• Pass liquid or gas through a filter with sufficiently small pore size

• HEPA - filter out > 0.3 µm particles

• Advantages: No thermal damage

• Disadvantages: viruses not eliminated and must be either liquid or gas

Filter

Sterilizedfluid

(a)

VacuumPump suction

Filter

Liquid

Pore

(b)

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b: © Fred Hossler/Visuals Unlimited

Ionizing vs Non-ionizing radiation)

• Ionizing Radiation: ejects electrons from an atom, causes ions to form

• Gamma ray - very good penetration

• X-ray - less penetration

• Non-ionizing radiation raises electrons in an atom to a higher energy state

• Ultraviolet - damages DNA, with poor penetration

Ultraviolet Damage

• Wavelenth: 200-300 nm

• Poor penetrating power

• Pyrimidine dimers

• Mutations induced

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O

OODetailsof bonding

Thyminedimer

Normalsegment ofDNA C

G

UV

TT

O

CH3CH3

G

C

AACA

T TTG

TGT

A C A A

T

Radiation

• Advantages: very effective with little product damage (“cold” sterilization)

• Disadvantages: dangerous materials need shielding, and lack of public trust.

Chemical Treatments

• Chemotherapeutics - disease treatment

• Disinfectants - cleaning surfaces

• Many are available today (hundreds)

• Choice is based upon:

Nature of the object

Kinds of microbes targeted

Desired effect

Chemical (Oxidizers)

• Damage proteins and membranes

• Halogens

• Chlorine - disinfectant (added to water)

• Iodine - antiseptic (tincture of iodine, betadine)

• Hydrogen peroxide (H2O2)

• 3% is a weak antiseptic

• Your body and many bacteria can break this down enzymatically

Chemical (Phenolics)

• Denature proteins

• Disrupt membranes

• Joseph ListerExamples: phenol,lysol, chlorhexidine

• Effective on surfaces

• Many of these aretoo toxic to apply to tissue

OHOH OH CH3

CH3

Phenol(basic aromaticring structure)

p-cresolo-cresol

OHCl

OHOH

Cl

Hexachlorophene(a bisphenol)

Chlorophene(a chlorinated phenol)

Cl

Cl

ClCl

CH2

Cl

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Chemical (Alcohols)

• Denature proteins

• Disrupt membranes

• Examples: ethanol, and isopropanol

• Most effective at 50-70%

• Increased plasmolysis after damage

• These are commonly used for antisepsis

Chemical (Surfactants)

• Amphiphilic compounds

• Disrupt membranes

• Quaternary ammonium compounds (quads)

• Charged nitrogen

• Four hydrophobic groups

• Example - cepacol,and roccal

N

+

Cl–

(b)

(a)

Hydro carbon chain (C number from 8 to18)

R1

Benzalkonium chloride

R3

R2 R4

+

CH3

CH2

CH2N+CNH2N+

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Gold foilSilver amalgam

Oligodynamic Effect

• Inhibition byheavy metals

• Silver

• Copper

• Mercury

• Gold

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© Kathy Park Talaro/Visuals Unlimited

Chemical (Alkylators)

• Damage proteins or DNA by addingcarbon adducts

• Examples: formalin, glutaraldehyde, and ethylene oxide

• Highly noxious

• Ethylene oxide isused to sterilize products via gas

OO

O O

O O

Amino groups inpeptidoglycan

Cross-linking withmicrobial protein

Polyglutaraldehyde

Glutaraldehyde

Polymerization

G+G–

N

NN

N

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