Sterilization and Disinfection

Microorganisms are ubiquitous

Early days:

Nosocomial infections caused death in 10% of surgeries

Up to 25% mothers delivering in hospitals died due to infection

In mid 1800s “aseptic techniques” Semmelweiss and Lister to prevent contamination of surgical wounds.

Sepsis: decay or putrid. Indicates bacterial contamination.

Asepsis: Absence of significant contamination.

Aseptic techniques are used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery.


Killing or removing all forms of microbial life (including endospores) in a material or an object.


Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases. Usually, involves the removal of vegetative or non-endospore forming pathogens.

Disinfectant: Applied to inanimate objects.

Antiseptic: Applied to living tissue (antisepsis- prevention of infection).


Number of organisms & spores affects the rapidity of S & D

Higher the Bio-burden the more extensive the process of S & D

Minimize Bio-burden by prior cleaning

Cleaning - the removal of soil or organic material from instruments and equipment

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

The method of disinfection or sterilization selected should be based on

1. The type of device,

2. How it is used,

3. The manufacturer’s recommendation.



Enters sterile tissue or Vascular System

Ex: Implants, Scalpels needles, surgical and dental equipments, endoscopic accessories

PROCESS: Sterilization, Steam under pressure, Dry heat, Chemical gas sterilizers

Liquid sterilants


Touches mucous membrane or broken skin

Ex: Endoscopes, endo-tracheal tubes, and anesthesia equipment

Process: High Level Disinfection (exposure time >20 min)

Ex: Gluteraldehyde 2%,

Stabilized Hydrogen peroxide 6%

OR wet pasteurization after detergent cleaning

OR Household bleach (Sod. Hypochlorite 5.25% 1000ppm available chlorine= 1:50 dilution)


Touches intact skin:

Ex: Stethoscopes, tabletops, floors, bedpans, furniture

Intermediate level disinfection (exposure time <10 min

Ex: Ethyle or isoprpopyle alcohol (70% - 90%), phenolic or Iodophore germicidal detergents Quaternary Ammonium germicidal detergents

Household bleach (Sod. Hypochlorite 5.25% 1000ppm available chlorine= 1:50 dilution)

Environmental surfaces, such as bedside tables, are usually not sources of infections.

However, if soiled, they could be a source of contamination to hands or other objects which may have contact with the patient.

Therefore, environmental surfaces should be cleaned regularly with approved hospital disinfectants

When selecting a method of disinfectantion/sterilization, consider how

the item is to be used,

what it is made from, and

What the device manufacturer recommends for disinfection/sterilization.

Methods of sterilization and disinfection

  1. Physical
  2. Chemical


Kills microorganisms by denaturing their enzymes and other proteins.

Heat is frequently used to kill microorganisms

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

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

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

1. Moist Heat:

Kills microorganisms by coagulating proteins.

Moist heat is more effective than dry heat.

v Boiling: Heat to 100oC or more at sea level. Kills vegetative forms of bacterial pathogens, almost all viruses, and fungi and their spores within 10 min or less. Endospores and some viruses are not destroyed quickly. However brief boiling will kill most pathogens.

Hepatitis virus: Can survive up to 30 minutes of boiling.

Endospores: Can survive up to 20 hours or more of boiling.

Reliable sterilization with moist heat requires temperatures above that of boiling water.

Steaming under atmospheric pressure at 100oC for 90 min

Intermittent sterilization for 20 min each time- tynderlization

Steam under high pressure

Autoclave: Chamber which is filled with hot steam under high pressure. Preferred method of sterilization, unless material is damaged by heat, moisture, or high pressure.

Temperature of steam reaches 121oC at twice atmospheric pressure.

Most effective when organisms contact steam directly or are contained in a small volume of liquid.

All organisms and endospores are killed within 15 min.

Require more time to reach center of solid or large volumes of liquid.

v Pasteurization: Developed by Louis Pasteur to prevent the spoilage of beverages. Ex: beer, milk, wine, juices, etc.

Classic Method of Pasteurization: Milk was exposed to 65oC for 30 min.

High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72oC for 15 sec.

Ultra High Temperature Pasteurization (UHT): Milk is treated at 140oC for 3 sec and then cooled very quickly in a vacuum chamber.

Advantage: Milk can be stored at room temperature for several months.

2. Dry Heat: Kills by oxidation effects.

Direct Flaming: Used to sterilize inoculating loops, needles and mouths of culture bottles.

Incineration: Effective way to sterilize disposable items (paper cups, dressings) and biological waste.

Hot Air Sterilization: Place objects in an oven. Require 1-2 h at 160-170oC for sterilization.


Bactericidal activity for spontaneous sterilization

Combined effect of UV and heat


Removal of microbes by passage of a liquid or gas through a filter. Used to sterilize heat sensitive materials like vaccines, enzymes, antibiotics, and some culture media.

High Efficiency Particulate Air Filters (HEPA): Used in operating rooms and burn units to remove bacteria from air.

Membrane Filters: Uniform pore size. Used in industry and research. Different sizes:

ü 0.22 and 0.45um Pores: Used to filter most bacteria. Don’t retain spirochetes, mycoplasmas and viruses.

ü 0.01 um Pores: Retain all viruses and some large proteins.

Low Temperature

Effect depends on microbe and treatment applied.

Refrigeration: Temperatures from 0 to 7oC. Bacteriostatic effect. Reduces metabolic rate of most microbes so they cannot reproduce or produce toxins.

Freezing: Temperatures below 0oC.

ü Flash Freezing: Does not kill most microbes.

ü Slow Freezing: More harmful because ice crystals disrupt cell structure.

ü Over a third of vegetative bacteria may survive 1 year.

ü Most parasites are killed by a few days of freezing.


In the absence of water, microbes cannot grow or reproduce, but some may remain viable for years. After water becomes available, they start growing again.

Susceptibility to dessication varies widely:

  • Neisseria gonnorrhea: Only survives about one hour.
  • Mycobacterium tuberculosis: May survive several months.
  • Viruses are fairly resistant to dessication.
  • Clostridium spp. and Bacillus spp.: May survive decades.

v Spores are unaffected

Osmotic Pressure: high concentrations of salts and sugars increase the osmotic pressure and create a hypertonic environment.

Plasmolysis: As water leaves the cell, plasma membrane shrinks away from cell wall. Cell may not die, but usually stops growing.

Yeasts and molds: More resistant to high osmotic pressures.

Staphylococci spp. that live on skin are fairly resistant to high osmotic pressure.


Three types of radiation kill microbes:

1. Ionizing Radiation: Gamma rays, X rays, electron beams, or higher energy rays. Have short wavelengths (less than 1 nm).

Dislodge electrons from atoms and form ions.

Cause mutations in DNA and produce peroxides.

Used to sterilize pharmaceuticals and disposable medical supplies.

Disadvantages: Penetrates human tissues. May cause genetic mutations in humans.

2. Ultraviolet light (Non-ionizing Radiation) :Wavelength is longer than 1 nm. Damages DNA by producing thymine dimers, which cause mutations.

Used to disinfect operating rooms, Laboratories, nurseries, cafeterias.

Disadvantages: Damages skin, eyes. Doesn’t penetrate paper, glass, and cloth.

3. Microwave Radiation

Wavelength ranges from 1 mm to 1 m.

Heat is absorbed by water molecules.

May kill vegetative cells in moist foods.

Bacterial endospores, which do not contain water, are not damaged by microwave radiation.


Mechanisms of Action


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


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


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

Chemical methods

Ideal disinfectant

Resistant to inactivation

Broadly active (killing pathogens)

Not poisonous (or otherwise harmful)

Penetrating (to pathogens)

Not damaging to non-living materials


Easy to work with

Otherwise not unpleasant

1. Phenols and Phenolics:

Phenol (carbolic acid) was first used by Lister as a disinfectant.

Rarely used today because it is a skin irritant and has strong odor.

Used in some throat sprays and lozenges.

Phenolics are chemical derivatives of phenol Ex: Hexachlorophene, Triclosan, Lysol, soap

Cresols: Derived from coal tar (Lysol).

Biphenols: Effective against gram-positive staphylococci and streptococci. Used in nurseries. Excessive use in infants may cause neurological damage.

Chlorhexidine is a relatively nontoxic skin antiseptic

Destroy plasma membranes and denature proteins. can irritate skin, don’t kill endospores, and are corrosive to rubber and plastics

Advantages: Stable, persist for long times, and remain active in the presence of organic compounds.

2. Halogens: Effective alone or in compounds.

A. Iodine:

Tincture of iodine (alcohol solution) was one of first antiseptics used.

Combines with amino acid tyrosine in proteins and denatures proteins.

Stains skin and clothes, somewhat irritating.

Iodophors: Compounds with iodine that are slow releasing, take several minutes to act. Used as skin antiseptic in surgery. Not effective against bacterial endospores. Betadine , Isodine

B. Chlorine:

When mixed in water forms hypochlorous acid:

Cl2 + H2O ------> H+ + Cl- + HOCl (Hypochlorous acid)

Used to disinfect drinking water, pools, and sewage.

Chlorine is easily inactivated by organic materials.

Sodium hypochlorite (NaOCl): active ingredient of bleach.

Chloramines: Consist of chlorine and ammonia. Less effective as germicides.

3. Alcohols:

Kill bacteria, fungi, but not endospores or naked viruses.

Act by denaturing proteins and disrupting cell membranes.

Evaporate, leaving no residue.

Used to mechanically wipe microbes off skin before injections or blood drawing.

Not good for open wounds, because cause proteins to coagulate.

§ Ethanol: Drinking alcohol. Optimum concentration is 70%.

§ Isopropanol: Rubbing alcohol. Better disinfectant than ethanol. Also cheaper and less volatile

4. Heavy Metals:

Include copper, selenium, mercury, silver, and zinc.

Oligodynamic action: Very tiny amounts are effective.

A. Silver:

1% silver nitrate used to protect infants against gonorrheal eye infections until recently.

B. Mercury

Organic mercury compounds like merthiolate and mercurochrome are used to disinfect skin wounds.

C. Copper

Copper sulfate is used to kill algae in pools and fish tanks.

D. Selenium

Kills fungi and their spores. Used for fungal infections.

Also used in dandruff shampoos.

E. Zinc

Zinc chloride is used in mouthwashes.

Zinc oxide is used as antifungal agent in paints.

5. Quaternary Ammonium Compounds (Quats):

Widely used surface active agents.

Cationic (positively charge) detergents.

Effective against gram positive bacteria, less effective against gram-negative bacteria.

Also destroy fungi, amoebas, and enveloped viruses.

Zephiran, Cepacol, also found in lab spray bottles.

Pseudomonas strains that are resistant and can grow in presence of Quats are a big concern in hospitals.

Advantages: Strong antimicrobial action, colorless, odorless, tasteless, stable, and nontoxic.

Disadvantages: Form foam. Organic matter interferes with effectiveness. Neutralized by soaps and anionic detergents.

6. Aldehydes:

Include some of the most effective antimicrobials.

Inactivate proteins by forming covalent cross-links with several functional groups.

A. Formaldehyde gas:

Excellent disinfectant.

Commonly used as formalin, a 37% aqueous solution.

Formalin was used extensively to preserve biological specimens and inactivate viruses and bacteria in vaccines.

Irritates mucous membranes, strong odor.

Also used in mortuaries for embalming.

B. Glutaraldehyde:

Less irritating and more effective than formaldehyde.

One of the few chemical disinfectants that act as a sterilizing agent.

A 2% solution of glutaraldehyde (Cidex) is:

u Bactericidal, tuberculocidal, and viricidal in 10 min.

u Sporicidal in 3 to 10 h.

Commonly used to disinfect hospital instruments.

Also used in mortuaries for embalming.

7. Gaseous Sterilizers:

Chemicals that sterilize in a chamber similar to an autoclave.

Denature proteins, by replacing functional groups with alkyl groups.

A. Ethylene Oxide:

Kills all microbes and endospores, but requires exposure of 4 to 18 h.

Toxic and explosive in pure form.

Highly penetrating.

Most hospitals have ethylene oxide chambers to sterilize mattresses and large equipment.

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

8. Peroxygens (Oxidizing Agents):

Oxidize cellular components of treated microbes.

Disrupt membranes and proteins.

A. Ozone:

Used along with chlorine to disinfect water.

Helps neutralize unpleasant tastes and odors.

More effective killing agent than chlorine, but less stable and more expensive.

Highly reactive form of oxygen.

B. Hydrogen Peroxide:

Used as an antiseptic.

Not good for open wounds because quickly broken down by catalase present in human cells.

Effective in disinfection of inanimate objects.

Sporicidal at higher temperatures.

Used by food industry and to disinfect contact lenses.

C. Benzoyl Peroxide:

Used in acne medications.

D. Peracetic Acid

One of the most effective liquid Sporicides

Kills bacteria and fungi lessthan 5 min.

Kills endospores and viruses within 30 min.

Used widely in disinfection of food and medical instruments because it does not leave toxic residues.


Mechanisms of Action



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


Oxidizing agent
Disrupts cell membrane

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


Denatures proteins; Disrupts lipids

Antiseptic & Disinfectant
Ethanol and isopropyl


Disrupts cell membrane

Irritating odor


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

Disinfectants that Inactivate
Hepatitis B Virus (HBV)

The following common disinfectants have been shown to inactivate HBV:

glutaraldehyde (2%),

iodophor (80 ppm),

Isopropyl alcohol (70%),

Sodium hypochlorite (500 ppm).

Disinfectants that Inactivate
Human Immunodeficiency Virus (HIV

The following common disinfectants have been shown to inactivate HIV:

glutaraldehyde (2%),

Hydrogen peroxide (.3%),

Ethyl alcohol (50%),

Isopropyl alcohol (70%),


Sodium hypochlorite (50 ppm).

Special Considerations

Creutzfeldt-Jacob Disease (CJD) Agent:

The agent that causes CJD is a prion. It is extremely resistant to normal inactivation methods.

The following sterilization/disinfection techniques are recommended:

Steam sterilization for at least 30 min. at a temperature of 132° C (269° F) (121° C [249° F] is ineffective) Method for treatment of contaminated material. 18 min. at 134° C - 138° C (273° F - 280° F) also is effective.

Alternatively, immersion in 1 N sodium hydroxide (a caustic solution) for 1 hr. at room temperature followed by steam sterilization at 121° C (249° F) for 30 min. may be used for critical and semi-critical items.

Non-critical patient care items or surfaces (e.g., autopsy table, floors) may be disinfected with either

undiluted or up to 1:10 dilution of bleach, or

1 N sodium hydroxide at room temperature for 15 min.

Important facts

Never use low-level disinfectants to disinfect critical devices that will enter sterile tissue or the vascular system or semi-critical devices that touch mucous membranes or broken skin.

Do not use glutaraldehyde or other high-level disinfectants to clean environmental surfaces or other noncritical devices.

Always use the right dilution of disinfectant and proper contact time.

Sometimes stronger concentrations of disinfectants do not inactivate microorganisms but can be toxic to the user or damage the item being cleaned.

Always use safety precautions and protective equipment according to manufacturer’s instruction for sterilization and disinfection

Decreasing order of resistance of microorganisms to disinfectants/sterilantsPrions



Non-enveloped virus



Enveloped virus

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