Week08-01 (lec) Micro 20 (Microbial control I).pptx

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Chapter 11

Microbial Control I

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11-2

Controlling Microorganisms (1 of 2)

• Controlling exposure to potentially harmful

microbes is essential to our lives, and it has

been a concern also in the past

• Early protection attempts laid the foundation

for microbial control methods today:

–

Burning wood releases formaldehyde

–

Herbs, perfume, and vinegar contain mild

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11-3

General Considerations in Microbial

Control

• Physical, chemical, and mechanical methods

to destroy or reduce undesirable microbes in

a given area (decontamination)

• Primary targets are microorganisms capable

of causing infection or spoilage:

–

Vegetative bacterial cells and endospores

–

Fungal hyphae and spores, yeast

–

_{Protozoan trophozoites and cysts}

–

_Worms

–

Viruses

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11-4

Relative Resistance of Microbes

(1 of 2)

• Highest resistance

–

Prions, bacterial endospores (

Bacillus,

Clostridium

)

• Moderate resistance

–

Protozoan cysts; naked viruses

–

Bacteria with no endospores but resistant

walls:

–

Mycobacterium

,

Pseudomonas

,

Acinetobacter

,

other gram(-)

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11-5

Relative Resistance of Microbes

(2 of 2)

• Least resistance

–

Most bacterial vegetative cells

–

Fungal spores and hyphae

–

Yeasts

–

Enveloped viruses

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11-6

The Resistance of Endospores

(1 of 2)

TABLE 11.1

Relative Resistance of Bacterial Endospores

and Vegetative Cells to Control Agents

Method

Endospores

*

Vegetative

Forms

*

Relative

Resistance

**

Heat (moist)

120°C

80°C

1.5x

Radiation (X-ray) dosage

4,000 grays

1,000 grays

4x

Sterilizing gas (ethylene oxide)

1,200 mg/l

700 mg/l

1.7x

Sporicidal liquid (2% glutaraldehyde)

3 hours

10 min

18x

*

Values are based on methods (concentration, exposure time,

intensity) that are required to destroy the most resistant

pathogens in each group.

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11-7

The Resistance of Endospores

(2 of 2)

S.J. Jones, C.J. Paredes, B.Tracy, N. Cheng, R.Sillers, R.S. Senger, E.T. Papoutsakis, “The

transcriptional program underlying the physiology of clostridial sporulation,”

Genome Biol

.,

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11-8

Terminology and Methods of Control

(1 of 2)

• Sterilization

–process to destroy all viable

microbes

• _{Microbicidal agents}

_{(germicides) –}

antimicrobial agent aimed at destroying a certain

group of microorganisms (bactericide, fungicide,

virucide, sporicidal)

–

Disinfection

– use of a physical process or a

chemical agent (disinfectant) to destroy vegetative

pathogens but not bacterial endospores

–

Antisepsis

– application of chemical agents

(antiseptics) directly to exposed body surfaces,

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11-9

Terminology and Methods of Control

(2 of 2)

• Agents that cause microbistasis

–

antimicrobial agent aimed at temporarily prevent

microbes from multiplying

• _{Methods that reduce the numbers of}

microorganisms

–

Sanitation

– any cleansing technique that

removes microorganisms from inanimate surfaces

to reduce the potential for infection and spoilage

–

Degermation

– reduction of microbial load from

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TABLE 11.2 Microbial Control

Terminology

Term Definition Example

Decontamination The destruction, removal, or reduction in _{number of undesirable microbes} Asepsis, disinfection, sanitization, _{degermation, sterilization}

Sepsis The growth of microorganisms in the _tissues The presence of infected wounds, blood _infection

Asepsis Techniques that prevent the entry of _{microorganisms into sterile tissues} Cleansing the skin with iodine prior to _{surgery, using sterile needles}

Antiseptic Chemicals applied to body surfaces to _{destroy or inhibit vegetative pathogens} lodophors, antibacterial soap, _{chlorhexidine}

Disinfection Destruction of vegetative pathogens on _{inanimate objects} 5% bleach, boiling water

Sanitization Cleansing technique that removes microorganisms and debris from

inanimate surfaces Dishwashing, laundering clothes

Degermation Cleansing technique that removes microorganisms and debris from living

tissue Surgical handscrub, alcohol wipes

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11-11

Microbial Death

• Hard to detect, microbes often reveal no

conspicuous vital signs to begin with

• Permanent loss of reproductive capability,

even under optimum growth conditions

• Factors

that affect microbial death:

–

Number of microbes

–

Nature of microbes in the population

–

_{Temperature and pH of environment}

–

_{Concentration or dosage of agent}

–

Mode of action of the agent

–

Presence of solvents, organic matter, or

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Practical Concerns in Microbial

Control

Selection of method of control depends on

circumstances:

• _{Does 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 and is it}

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Antimicrobial Agents’ Modes of

Action (1 of 4)

Cellular targets of physical and chemical agents:

• Cell wall

–becomes fragile and cell lyses

(some antimicrobial drugs, detergents, and

alcohol)

• Cell membrane

– loses integrity

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11-14

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11-15

Antimicrobial Agents’ Modes of

Action (3 of 4)

Cellular targets of physical and chemical agents:

• Protein and nucleic acid synthesis

–

prevention of replication, transcription,

translation, peptide bond formation, protein

synthesis (

chloramphenicol, ultraviolet

radiation, formaldehyde

)

• Protein function

– disrupt or denature

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11-16

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Physical Methods of Control

• The vast majority of microbes are readily

controlled by abrupt changes in their

environment

• Some physical methods are:

–

Heat (moist and dry)

–

_{Cold temperatures}

–

_Desiccation

–

Radiation

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11-18

Mode of Action and Relative

Effectiveness of Heat (1 of 2)

• Moist heat

– lower temperatures and

shorter exposure time; coagulation and

denaturation of proteins, which halts cellular

metabolism

• Dry heat

– moderate to high temperatures;

dehydration, alters protein structure;

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11-19

Mode of Action and Relative

Effectiveness of Heat (2 of 2)

TABLE 11.3

Comparison of Times and

Temperatures to Achieve Sterilization with Moist

and Dry Heat

Temperature

Time to Sterilize

Moist Heat

121°C

15 min

125°C

10 min

134°C

3 min

Dry Heat

121°C

600 min

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11-20

Methods of Moist Heat Control

1. Sterilization with Steam Under Pressure

• Autoclave

(15 psi/121

o

C/10-40min) Pressure

increases steam temperature

• _{Steam must reach surface of item being sterilized}

• Item must not be heat or moisture sensitive

• Produces denaturation of proteins, destruction of

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11-21

Methods of Moist Heat Control

4. Pasteurization

• Heat is applied to kill potential agents of infection

and spoilage without destroying the food flavor or

value

–

Flash method

: 71.6

°

C, 15 sec

• _{Not sterilization – kills non-spore-forming}

pathogens and lowers overall microbe count;

does not kill endospores or many nonpathogenic

microbes

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Dry Heat: Hot Air and Incineration

(1 of 2)

Dry heat uses higher temperatures than moist

heat

• Incineration

– flame, electric heating coil,

infrared incinerators

–

Ignites and reduces microbes and other

substances

–

_{Very common practice in the microbiology lab}

• Hot air (Dry) ovens

– heated, circulated air

(150

o

C-180

o

C, 12min-4h)

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11-23

Dry Heat: Hot Air and Incineration

(2 of 2)

Dry heat incineration.

Infrared incinerator with shield to

prevent spattering of microbial samples during flaming.

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11-24

Using Heat for Sterilization and

Disinfection (1 of 3)

TABLE 11.4

Applications Using Heat for Sterilization and

Disinfection

Form of Heat _{Temperature/Time}Equipment Medical and Commercial Uses and _Limitations

Moist heat: Employs steam, boiling, or hot water; mode of action coagulates proteins

Steam under pressure

Autoclave, pressure cooker; 121°C, pressure of 15 psi for 10 to 40 minutes, depending on load size; kills endospores.

Sterilization of heat-resistant materials made of glass, cloth, rubber, metal; media; a few plastics; canned food, spices. Decontamination of

cultures. Not appropriate for moisture-repellent oil or powder.

Unpressurized steam (tyndallization)

Arnold's Sterilizer—100°C for 30 minutes on 3 days; items are incubated between

treatments to allow endospores to germinate.

Limited uses in sterilizing some media and foods that cannot be

autoclaved; can be used to disinfect but not sterilize some medical

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11-25

Using Heat for Sterilization and

Disinfection (2 of 3)

The “TABLE 11.4” continues on this slide.

Form of Heat

Equipment Temperature/

_Time

Medical and Commercial Uses

_{and Limitations}

Pasteurization

Pasteurizer; flash method

works at 71.6°C for 15

seconds; ultrahigh

temperature (UHT) uses

134°C for a few seconds.

Disinfection of milk and dairy

products to destroy milk-borne

pathogens; UHT can sterilize for

longer shelf life. Fruit juices,

beer, and wine may be

pasteurized to destroy

contaminants and preserve the

products.

Boiling water

Water bath or pan heated to

100°C; items are placed

in water for 30 minutes

to kill vegetative

pathogens.

Limited to disinfection and

sanitization of heat-resistant

household objects such as eating

utensils, clothing, sick room

supplies, baby supplies, bedding,

and water in emergencies;

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11-26

Using Heat for Sterilization and

Disinfection (3 of 3)

The “TABLE 11.4” continues on this slide.

Form of Heat _{Temperature/Time}Equipment Medical and Commercial Uses and _Limitations

Dry heat: Employs dried hot air, flame, or heat coil; works by

dehydration or combustion

Dry oven Electric or gas chamber heated to 150°C to 180°C for 2 to 4 hours

Materials that can withstand hot temperatures and dehydration;

glassware, metals, powders, oils. Poor choice for liquids, rubber, plastics; time-consuming.

Incineration Bunsen burner flame up to 1,800°C; small infrared incinerators to 800°C

Furnaces, incinerators up to 6,500°C For sterilizing tips of inoculation

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11-27

Effects of Cold and Desiccation

• Cold

–

Microbiostatic – slows the growth of microbes

–

Refrigeration 0–15

o

C and freezing < 0

o

C

–

Used to preserve food, media, and cultures

• Desiccation

–

Gradual removal of water from cells, leads to

metabolic inhibition

–

_{Not effective microbial control – many cells}

retain ability to grow when water is

reintroduced

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11-28

Physical Methods of Control:

Radiation (1 of 2)

• Radiation:

Energy emitted from atomic

activities and dispersed at high velocity

through matter or space

• Types of radiation suitable for microbial

control:

1. Ionizing radiation



Gamma rays



X rays



Cathode rays

2. Noniodizing radiation

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11-29

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11-30

Cellular Effects of Irradiation (1 of 4)

1. Ionizing radiation

– deep penetrating

power sufficient energy to cause electrons to

leave their orbit

– Gamma rays, X rays, cathode rays

– Breaks DNA

2. Nonionizing radiation

– little penetrating

power

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11-31

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11-33

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11-34

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11-35

Physical Methods of Control:

Filtration (2 of 2)

Filtration

: Physical removal of microbes by passing a gas

or liquid through filter

• Used to sterilize heat sensitive liquids and air in

hospital isolation units and industrial clean rooms

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11-36

Chemical Agents in Microbial Control

• Disinfectants, antiseptics, sterilants,

degermers, and preservatives

• Some desirable qualities of germicides:

–

_{Rapid action in low concentration}

–

_{Solubility in water or alcohol, stable}

–

Broad spectrum, low toxicity

–

Penetrating

–

Noncorrosive and nonstaining

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11-37

Factors that Affect Germicidal

Activity of Chemicals

• Nature of the material being treated

• Degree of contamination

• Time of exposure required

• Concentration of the chemical agent.

Strength and chemical action of the germicide

expressed in various ways (dilution factor

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11-38

Germicidal Categories

• Halogens

• Phenolics

• Chlorhexidine

• Alcohols

• Hydrogen peroxide

• Aldehydes

• Gases

• Detergents & soaps

• Heavy metals

• Dyes

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11-39

Halogens (1 of 2)

The

halogens

used in germicidal preparations

are:

• Chlorine

– Cl

₂

, hypochlorites (chlorine

bleach), chloramines

–

_{Denaturate proteins by disrupting disulfide}

bonds

–

Intermediate level

–

Unstable in sunlight, inactivated by organic

matter

–

Water, sewage, wastewater, inanimate

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11-40

Halogens (2 of 2)

• Iodine

- I

₂

, iodophors (betadine)

–

Interferes with disulfide bonds of proteins

–

_{Intermediate level}

–

Milder medical and dental degerming agents,

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11-41

Applications of Halogens (1 of 4)

TABLE 11.7

Applications of Halogen Compounds

Form of Chlorine Primary Applications How Delivered/Concerns

Chlorine Gas (Cl₂) Large-scale disinfection of drinking water, sewage, and wastewater.

Chlorination to a concentration of 0.6 to 1.0 parts of chlorine per million parts of water destroys most vegetative pathogens.*

Hypochlorites (HClO) Bleach

Used extensively in sanitization and disinfection of food equipment, treatment of swimming pools, spas, drinking water, and fresh foods; for wound antisepsis and routine

medical and household disinfection, deodorizing, and stain removal.

Common household bleach is a 5% solution of sodium hypochlorite; dilutions of 1:10-1:1,000 are highly effective germicides.

Chloramines (Dichloramine, Halazone)

An alternative to pure chlorine in treating water supplies; also as sanitizers and disinfectants; for treating wounds and skin surfaces.

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11-42

Applications of Halogens (2 of 4)

The “TABLE 11.7” continues on this slide.

Form of Iodine

Primary Applications

How Delivered/Concerns

lodophors

Most common iodine for skin and

mucous membranes; antiseptic

prep for surgery and injections;

for surgical handscrubs; to

disinfect equipment and

surfaces; possibly for burns; and

may be an alternative preventive

for eye infections in newborns.

A complex of iodine and a

neutral protein polymer

provides slow release and

reduced toxicity or irritation

of tissues; less prone to

staining. Common products

are povidone-iodine (PVI;

Betadine), which contain 2%

to 10% available iodine.

Elemental Iodine:

Aqueous or

Tinctures

Topical antiseptic prior to surgery;

sometimes for burned or injured

skin. Medium–level disinfection

for plastic instruments,

thermometers; tablet form

available for disinfection of

contaminated water.

Weak solutions of 1% to 3% in

water or in alcohol tinctures.

Aqueous solutions or tinctures

of 5% to 10%; somewhat

limited by their toxicity and

tendency to stain.

*

Some cyst-forming protozoans such as

Giardia

and

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11-43

Phenols and Its Derivatives (1 of 2)

Phenol

(carbolic acid) is an acrid, poisonous

compound

Disrupt cell walls and membranes and

precipitate proteins

• Low to intermediate level

• Bactericidal, fungicidal, virucidal (not

sporicidal)

–

_{Lysol and Triclosan – antibacterial additive to}

soaps

• Toxicity of phenolics makes them a

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11-45

Alcohols (1 of 2)

• Only

ethyl

and

isopropyl

are suitable for

microbial control

• Intermediate level

–

_{Concentrations of 50% and higher dissolve}

membrane lipids and coagulate proteins of

vegetative bacterial cells and fungi

–

Water is needed for proteins to coagulate,

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11-46

Alcohols (2 of 2)

A nurse takes advantage of an alcohol gel hand cleansing

station. These are very common in hospitals and clinics

to encourage hand antisepsis.

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11-47

Hydrogen Peroxide (1 of 2)

• Produce highly reactive hydroxyl-free radicals

that damage protein and DNA while also

decomposing to O

₂

gas – toxic to anaerobes

• Antiseptic at low concentrations; strong

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11-48

Hydrogen Peroxide (2 of 2)

Disinfection of contact lenses with peroxide-based

cleaner. Note the bubbles produced, indicating the

release of oxygen gas.

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11-49

Aldehydes (1 of 2)

• Kill by alkylating protein and DNA

–

Glutaraldehyde



In 2% solution (Cidex)



High level



Used as sterilant for heat sensitive instruments

–

Formaldehyde



Formalin – 37% aqueous solution



Intermediate to high level

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11-50

Aldehydes (2 of 2)

Technician treats surgical instruments with

glutaraldehyde prior to heat sterilization.

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11-51

Detergents and Soaps (1 of 2)

• Detergents

: Polar molecules, surfactant

–

Quaternary ammonia compounds

(

quats

)

act as surfactants that alter membrane

permeability of some bacteria and fungi

– Very low level

• Soaps:

alkaline compounds

– Mechanically remove soil and grease

containing microbes

– Weak microbicides, destroy only highly

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TABLE 11.11 Applications of

Detergents and Soaps (3 of 3)

Soaps

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11-55

TABLE 11.12 Applications of Heavy

Metals

Form of Metal Primary Applications How Delivered/Concerns

Organic Mercury

Thimerosal (Merthiolate) and

nitromersol (Metaphen) are weak antiseptics and infection

preventives; can be preservatives in cosmetics and ophthalmic solutions.

Tinctures (0.001% to 0.2%) are fairly effective but a poor choice for broken skin because they are toxic and can delay healing.

Mercurochrome is now considered one of the poorest of antiseptics.

Silver Sulfadiazine Ointment

Added to dressings, it effectively prevents infection in second- and third-degree burn patients.

A yellow ointment containing silver salts and a sulfa drug.

Sliver Nitrate (AgNO₃) Primarily as a topical germicide for mouth ulcers and occasionally root canals.

1% to 2% solutions have been used as antiseptics, but they discolor skin and other tissues.

Metallic Silver

Incorporated into catheters to prevent urinary tract infections in the

hospital; added to paint, plastics, and steel as a way to control

microbial survival on items such as toilet seats, stethoscopes, and even walls and floors in homes.

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11-56

Dyes as Antimicrobial Agents

• Aniline dyes are very active against

gram-positive species of bacteria and various fungi

• Sometimes used for antisepsis and wound

treatment

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11-57

Acids and Alkalis

• Low level of activity

–

Organic acids prevent spore germination and

bacterial and fungal growth

–

Acetic acid inhibits bacterial growth

–

_{Propionic acid retards molds}

–

_{Lactic acid prevents anaerobic bacterial growth}

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11-58

TABLE 11.13 Active Ingredients of

Various Commercial Antimicrobial

Products

Product Specific Chemical Agent Antimicrobial Category

Lysol and Clorox Sanitizing Wipes Dimethyl benzyl ammonium chloride Detergent (quat) Tilex and Lysol Mildew Remover Sodium hypochlorites Halogen

Ajax and Dial Antibacterial Hand

Soap Triclosan Phenolic

Lysol Disinfecting Spray Alkyl dimethyl benzyl _{ammonium/ethanol} Detergent (quat)/alcohol

ReNu Contact Lens Solution Polyaminopropyl biguanide Chlorhexidine Wet Ones Antibacterial Moist

Towelettes Benzethonium chloride Detergent (quat) Noxzema Triple Clean Triclosan Phenolic

Scope Mouthwash Ethanol Alcohol Purell Instant Hand Sanitizer Ethanol Alcohol

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