Sterilization & disinfectionSterilization & disinfection(control of microbial growth)(control of microbial growth)

prepared by

Dr.Ihsan Edan Alsaimary

Dis

infe

ctio

nDisinfection is the elimination of pathogens,

except spores, from inanimate objects

Disinfectants are chemical solutions used to clean inanimate objects

(physical processes, e.g., UV radiation, may also be employed to effect disinfection)

Germicides are chemicals that can be applied to both animate (living) and inanimate objects for the purpose of eliminating pathogens

Antiseptics are formulated for application to living tissue

Ste

riliz

atio

nSterilization is the total elimination of all

microorganisms including spores

Typically the last things to die are the highly heat- and chemical-resistant bacterial endospores

Instruments used for invasive procedures must be sterilized prior to use

Moist heat or steam, radiation, chemicals (e.g., glutaraldehyde), and ethylene oxide (a gas) are employed for sterilization

Sterilization by autoclaving, which uses moist heat, is used in most hospital and microbiology laboratory settings

DISINFECTION & STERILIZATION

STERILIZATION

Process of removing microbes inclusive of bacterial endospores

DISINFECTION

process of removing microbes except bacterial endospores

Sanitization: Lowering of microbial counts to prevent transmission in public setting (e.g., restaurants & public rest rooms)Degerming: Mechanical removal of microbes, e.g., from hands with washingSepsis: Bacterial contaminationAntisepsis: Reduction of or Inhibition of microbes found on living tissueGermincides, Fungicides, VirucidesPhysical versus Chemical disinfectantsStatic (stasis) versus Cidal (e.g., bacteriostatic versus bacteriocidal)

Pasteurization

• Disinfection - not sterilization (removes unwanted organisms)

• Mycobacterium tuberculosis

• 63 C for 30 minutes

• 72 C for 15 seconds (HTST)

• Thermodurics– able to survive high temps.

PasteurizationPasteurization

• Involves heating a liquid to temps. sufficient to destroy vegetative organsims

• Equipment is immersed in hot water bath at 63˚C for 30 min.

• Is disinfection, not sterilization. The moist heat coagulates the cellular protein of microbes. Spores are not destroyed d/t being more resistant

Pasteurization (cont’d)Pasteurization (cont’d)

• Prepare equip. by disassembling, washing in detergent, and then rinsing

• After pasteurization remove equip. and place in drying cabinet

• When dry, assemble, package and date equipment• Advantages of pasteur. include: safe for plastics,

less expensive than chemicals, no employee exposure to chemicals

The Ideal Disinfectant Resistant to inactviation

Broadly active (killing pathogens)

Not poisonous (or otherwise harmful)

Penetrating (to pathogens)

Not damaging to non-living materials

Stable

Easy to work with

Otherwise not unpleasant

Disinfectant Performance… Is dependent on Disinfectant concentrations Is dependent on length (time) of administration Is dependent on temperature during administration

(usual chemical reaction 2x increase in rate with each 10°C increase in temperature)

Microbe type (e.g., mycobacteria, spores, and certain viruses can be very resistant to disinfection—in general vegetative cells in log phase are easiest to kill)

Substrate effects (e.g., high organic content interferes with disinfection—stainless steel bench easier to disinfect than turd)

It is easier (and faster) to kill fewer microbes than many microbes

Cle

ansi

ngCleansing is the removal of soil or

organic material from instruments and equipment & may be done, clinically, in four steps:

Rinsing the object under cold water

Applying detergent and scrubbing object

Rinsing the object under warm water

Drying the object prior to sterilization or disinfection

Different Kinds of Bacteria “Death”

1. Bacteriostatic

2. Bacteriocidal

3. Bacteriolytic

Log

Cel

l #

Time

Total cell count

Viable cell count

Res

ista

nce

to K

illin

g Gram-negative bacteria (with their outer membrane) are

generally more resistant than gram-positive bacteria to disinfectants and antiseptics

Stationary-phase (I.e., non-growing) bacteria generally are more resistant than log-phase (I.e., growing) bacteria

Mycobacteria, endospores, and protozoan cysts and oocysts are very resistant to disinfectants and antiseptics

Nonenveloped viruses are generally more resistant than enveloped viruses to disinfectants and antiseptics

Organic matter (such as vomit and feces) frequently affects the actions of chemical control agent

Disinfectant activity is inhibited by cold temperatures Longer application times are preferable to shorter Higher concentrations, though, are not always preferable

to lower concentration (e.g., alcohols)

cidal vs. static

• Bactericidal - kills bacteria

• Bacteristatic - inhibits bacterial growth

• Fungicidal

• Fungistatic

• Algacidal

• Algastatic

Factors that effect Antimicrobial Activity

• 1. Temp

• 2. Time

• 3. Concentration of Antimicrobial agent

• 4. Type of Microbe

• 5. Activity of Microbe

• 6. Presence of organic matter

Targets of Antimicrobial Agents

• 1. Cell membrane

• 2. Enzymes & Proteins

• 3. DNA & RNA

Application of Heat Heat is frequently used to kill microorganisms

Thermal death point (TDP) is the lowest temperature at which all bacteria in a liquid culture will be killed in 10 minutes

Thermal death time (TDT) is the length of time required to kill all bacteria in a liquid culture at a given temperature

Decimal reduction time (DRT) is the length of time in which 90% of a bacterial population will be killed at a given temperature (especially useful in canning industry)

Dry heat kills by oxidation (slow, uneven penetration)

Moist heat kills by protein coagulation (denaturation) so requires lower temperatures or shorter times, but the moisture must penetrate to pathogens to be effective (grease & oil can block)

Moist Heat Moist heat kills microbes by denaturing enzymes

(coagulation of proteins) Boiling (at 100°C, I.e., at sea level) kills many vegetative

cells and viruses within 10 minutes Autoclaving: steam applied under pressure (121°C for 15

min) is the most effective method of moist heat sterilization—the steam must directly contact the material to be sterilized

Pasteurization: destroys pathogens (Mycobacterium tuberculosis, Salmonella typhi, etc.) without altering the flavor of the food—does not sterilize (63°C for 30 seconds)

Higher temperature short time pasteurization applies higher heat for a much shorter time (72°C for 15 seconds)

An ultra-high-temperature, very short duration treatment (140°C for 3 sec.) is used to sterilize dairy products

Ste

riliz

atio

n T

imes 171o C, 60 minutes, dry heat

160o C, 120 minutes, dry heat

149o C, 150 minutes, dry heat

141o C, 180 minutes, dry heat

121o C, 12 hours, dry heat

121o C, 15 minutes, moist heat (but don’t start the clock until entire item is up to temp—e.g., large volumes fluid)

Radiation

• 1. Ionizing Radiation– gamma rays & x-rays

• penetrates most substances

• Used on substances that could be damaged by heat– plastic petri dishes– plastic syringes– catheters– surgical gloves

Radiation• 2. Non-Ionizing Radiation

– UV Light• does not penetrate plastic, glass or proteinaceous

matter

• Used to reduce microbial populations– hospital rooms– nurseries– operating rooms

Thymine Dimers

Filtration• Removes microorganisms from solutions

that might be damaged by heat

– culture media– enzymes– vaccines– antibiotics

Filt

ratio

n: A

ir &

Flu

ids

Phy

sica

l A

ntim

icro

bial

s

Agent Mechanisms of Action Comments

Moist Heat, boiling Denatures proteins Kills vegetative bacterial cells and viruses Endospores survive

Moist Heat, Autoclaving

Denatures proteins 121°C at 15 p.s.i. for 30 min kills everything

Moist Heat, Pasteurization

Denatures proteins Kills pathogens in food products

Dry Heat, Flaming Incineration of contaminants

Used for inoculating loop

Dry Heat, Hot air oven

Oxidation & Denatures proteins

170°C for 2 hours; Used for glassware & instrument sterilization

Filtration Separation of bacteria from liquid (HEPA: from air)

Used for heat sensitive liquids

Cold, Lyophilization (also desiccation)

Desiccation and low temperature

Used for food & drug preservation; Does not necessarily kill so used for Long-term storage of bacterial cultures

Cold, Refrigeration Decreased chemical reaction rate

Bacteriostatic

Osmotic Pressure, Addition of salt or sugar

Plasmolysis of contaminants

Used in food preservation (less effective against fungi)

Radiation, UV DNA damage (thymine dimers)

Limited penetration

Radiation, X-rays DNA damage Used for sterilizing medical supplies

Strong vis. Light Line-drying laundry

Sur

fact

ants

Soa

p &

Det

erge

nts Soaps are sodium or potassium salts of

fatty acids, a natural product

Detergents, instead, are artificial surfactants

While soaps are always negatively charged, some detergents are negatively charged while others are positively charged

One example of a positively charged detergent are quaternary ammonium compounds (a.k.a., quats)

Qua

ts Quats are cationic detergents that act by

disrupting lipid bilayers

Quats are bactericidal, fungicidal, viricidal (enveloped), and amoebicidal

Quats are most effective against Gram-positive bacteria

Quats do not kill endospores, Mycobacteria spp., nor non-enveloped viruses

Quats are rapidly inactivated by organics including cotton and soap

Zephiran Benzalkonium chloride

Cepacol Cetylpyridinium chloride

Hea

vy M

etal

s Ag, Cu, Hg, Ni, Zn, Ag(NO3)2, CuSO4,

ZnCl2, HgCl2 These metals (and metal ions) react with

sulfhydral (–SH) groups of proteins, denaturing proteins

Silver nitrate is used to treat Ophthalmia neonatorum in newborns as caused by Neisseria gonorrhoeae

Oligodynamic action: the ability of very small amounts of heavy metals (especially silver and copper) to exert antimicrobial activity

Hal

ogen

s Halogens are the seventh (VII) column of the

periodic table of elements

Two halogens are regularly employed as antimicrobials: Iodine and Chloride

Iodine: commonly used as an antiseptic against all microbes, fungi, and viruses

Iodine: It inhibits protein synthesis and oxidizes –SH groups of amino acids

Chlorine: Used as a disinfectant (10% bleach)

Chlorine: Hypochlorous acid (HOCl) is a product, formed in water, that is the active form of the disinfectant

Chlorine: Applied in treatment of drinking water, swimming pool, and sewage

Chlorination 1744 discovered in Sweden 1810 identified as an element 1835 first used to control odors 1890’s started to be used as a disinfectant 1896 earliest recorded use in experiments on water

supplies 1897 used in England to sterilize water mains following

typhoid outbreak 1902 first continuous use in water supplies in Belgium 1909 liquid chlorine (compressed gas) became

commercially available Subsequent rapid spread in use of chlorine throughout the

world WWI: Chlorine gas used as chemical warfare agent

Chlorination Hypochlorite may either be added directly (i.e., in the

form of bleach) or created within water by bubbling chlorine gas through the water

Chlorine gas - preferred for medium to large disinfection systems

Sodium Hypochlorite (liquid) - typically used for small disinfection systems and large swimming pools

Calcium Hypochlorite (powder, tablet) - typically used for private swimming pools

For water purification, do not use scented bleach

Bromine sometimes used as a less-smelly alternative

Hypochlorite is less effective in the presence of significant organic compounds

“What is known as modern chemical warfare began during World War I. The first chemical agent to be used was large amounts of chlorine gas, about one hundred sixty tons, which was released from 6,000 pressurized cylinders into the wind by the Germans against the Allies. The chlorine floated in a huge clouds toward the Allies until it reached the Allied lines causing men to die from the effects of the chlorine gas. Because of the large amounts of gas released the chlorine caused large amounts of yellowish fluid to form in the lungs of its victim, also causing eye, nose, and throat burning before causing death by choking.”

http://www.geocities.com/CapeCanaveral/Lab/4239/chemweapons/history.html

Alc

ohol

s Aqueous ethanol (60-95%) and isopropanol

are used as disinfectants

Effectively kill bacteria and fungi but not endospores nor nonenveloped viruses

Fast acting, no residue (evaporate away), no staining

But not very penetrating and no residual activity (once gone gone)

Exert their action by denaturing proteins and dissolving lipids

In tinctures, they enhance the effectiveness of other antimicrobial chemicals

Flammable; also may damage rubber, plastic, etc.

Tin

ctur

e “Formulae: Fresh juice of

Organic Habanero peppers, New Mexico Jalapeno, African Bird peppers and Hatch Chili peppers.

Dosage: Five to thirty drops, three times daily.  CAUTION ~ EXTREMELY HOT!!

Therapeutic Action: Cayenne is the greatest herbal aid to circulation and should be used on a regular basis.  The extract is very concentrated and gets into the bloodstream quickly and makes it a perfect first aid remedy for heart attacks, stroke, fainting, shock, dizziness, hemorrhage, internal and external bleeding.  Use a few drops to 10 droppers full.  It has saved many lives.

Tin

ctur

e II

A tincture is a nonvolatile substance (medicine) presented as an alcohol solution,

e.g., (for fun with numerous [sic])…

“Formulae: Fresh Garlic Juice, Goldenseal root, Usnea lichen, Myrrh gum, Pine resin, Echinacea root juice, Tea Tree oil, Kelp, Black Walnut inner hulls, Oak galls and Cayenne pepper in 80% grain alcohol.

Dosage: Generally for external use but can be used in the oral cavity.  Soak a cotton swab in the tincture and scrub into the infected area, let air dry.  It has a burning sensation. If the wound is tender, just flush it with multiple droppers full of the tincture but no need to scrub it in.

Therapeutic Action: There has never been an infected occur when this formula has been used.  It's excellent for treating any cut or wound and it is anti-bacterial, anti-viral and anti-fungal.  The tree resins in this formula leave an invisible protective, anti-bacterial coating over the wound.  This formula was use on a man in England who had the top of his knee torn off in an automobile accident.  In 24 hours it literally glued his knee back together.  A nurse from Ireland on the scene said in all the years in the hospital, she had never seen such a severe wound close right up and heal, and with no infections.”

Iodi

ne &

Iodo

phor

es

Phe

nol,

Car

bolic

Aci

d, &

Phe

nolic

s Phenol (carbolic acid) and derivatives

Affect plasma membrane, inactivates enzymes, and denature proteins

Stable, persistant, and especially effective when dealing with disinfecting materials contaminated with organics…

… but leave residual films, can irritate skin, don’t kill endospores, and are corrosive to rubber and plastics

Some phenolics are mild enough for use as antiseptics while others are too harsh or otherwise dangerous to be employed on living tissue

Hexachlorophene, Triclosan, Lysol, soap

Oxi

dizi

ng A

gent

s HOOH, hydrogen peroxide, is most common

HOOH is not a terribly effective disinfectant or anticeptic

This is because bacteria and body tissues contain enzymes (catalase) that inactivate hydrogen peroxide

However, the oxygen released upon inactivation can help oxygenate deep wounds and thus kill strict-anaerobe contaminants, e.g., Clostridium tetani

Ozone and peracetic acid are also oxidizing antimicrobial agents

They exert their effect by oxidizing cell macromolecules (e.g., proteins, DNA, etc.)

Gas

eous

Che

mos

teril

izer

s Propylene oxide (C3H6O)

Chlorine gas (Cl2)

Chlorine dioxide (ClO2)

Ozone (O3)

Ethylene oxide (C2H4O)…

…is used to sterilize heat- or moisture-sensitive items

…is used for items damaged by heat or moisture

…is not corrosive, not damaging to delicate instruments, microscopes, disposable plastic instruments and materials

…permeates porous materials …dissipates rapidly from material …but is costly, toxic, carcinogenic,

explosive, and relatively lengthy process

Ethylene OxideEthylene Oxide

• Is a toxic gas that is combined with moisture and heat to sterilize equip.

• Effectiveness depends on equip. prep., gas [ ], humidity, and temp.

• Works by affecting the enzymes, reproduction, and metabolism of microbes.

• Prepare equip. by washing in detergent, rinsing, and allow to dry(ethylene glycol)

Et. O. (cont’d)Et. O. (cont’d)

• After drying, place in porous package

• Expose package to gas [ ] of 800-1000 mg/L that is mixed with CO2 or freon (decr. explosion hazard), 49-57˚C, 30-60% humidity, for 3-4 hours

• After EtO, aerate equip. in special cabinet for 12 hr. - several days, depending on material

Glu

tara

ldeh

yde

Glutaraldehyde is capable of effectiving sterilization—at room

temperature, even against endospores,

and even in the presence of organics,

but achieving sterilization requries

many hours of exposure… and it is nasty stuff to work

with!

GlutaraldehydeGlutaraldehyde

• Chemical used to cold sterilize or disinfect equip. by immersion

• “Cidex” is most common example

• Can be alkaline or acid glutaraldehyde

• Disassemble equip., wash in detergent, rinse, shake dry, and immerse in

glut.

• Alkaline glut. - disinfection after 10 min., sterilization after 10 hours

• Acid glut. - disinf. after 20 min.

Glutaraldehyde (cont’d)Glutaraldehyde (cont’d)

• After immersion, rinse with sterile water or bleach solution and allow to dry in a drying cabinet

• Dermal sensitivity or allergy to fumes with some people

• Good for rubber and plastic, not electronic equipment

• Life of product varies from 14 - 30 days

2% GLUTARLDEHYDE

Sterilization

Disinfection 8-10 Hrs used for :-

Pneumatic circuits. i.e.- ventilator tubing O2 masks ventury devices nebulizer chamber

15-30 Mts used for dis infecting endoscopes Respiratory tubing's

Che

mic

al

Ant

imic

robi

als

Agent Mechanisms of Action Comments

Surfactants Membrane Disruption; increased penetration

Soaps; detergents

Quats (cationic detergent)

Denature proteins; Disrupts lipids

Antiseptic - benzalconium chloride, Cepacol; Disinfectant

Organic acids and bases

High/low pH Mold and Fungi inhibitors; e.g., benzoate of soda

Heavy Metals Denature protein Antiseptic & Disinfectant; Silver Nitrate

Halogens Oxidizing agent Disrupts cell membrane

Antiseptic - Iodine (Betadine) Disinfectant - Chlorine (Chlorox)

Alcohols Denatures proteins; Disrupts lipids

Antiseptic & Disinfectant Ethanol and isopropyl

Phenolics Disrupts cell membrane

Disinfectant Irritating odor

Aldehydes Denature proteins  Gluteraldehyde - disinfectant (Cidex); Formaldehyde - disinfectant

Ethylene Oxide Denaturing proteins Used in a closed chamber to sterilize

Oxidizing agents Denature proteins Hydrogen peroxide – antiseptic; Hydrogen peroxide – disinfectan; Benzoyl peroxide – antiseptic

Eva

luat

ing

Dis

infe

ctan

ts Phenol Coefficient…

…compares efficacy to that of phenol, with greater efficacy indicated with coefficient >1

…Salmonella typhi and Staphylococcus aureus commonly used to determine coefficients

Filter-Paper/Disk Diffusion method…

…placement of disinfectant impregnated filter paper on well-inocated agar

Use-Dilution test…

…drying bacteria to surface followed by exposure to disinfectant and subsequent washing and inoculation of sterile broth

Dis

k D

iffus

ion

Met

hod

Hypochlorous acid

Phenol

Lysol

NisinEscherichia coli

Chemical Antimicrobials

* Type of Disinfectant: H = High level; I = Intermediate level; L = Low level

Asepsis

Sterilization Disinfection

Heat RadiationChemical Heat Chemical

Steam

Dry heat

Gas

Liquid

Gamma Boiling water Phenol

Heat or chloramines solution

Hexachlorophene

70% AlcoholEthylene oxide

Formalin

Glutaric Aldehyde

Asepsis – Summary