Principle of sterilization and disinfections

Dr Habsah Hasan Department of Medical Microbiology and Parasitology

Introduction

Infectious disease agents are controlled by:

Public sanitation measures Sterilization and disinfection procedures Chemotherapeutic agents Bodys defense mechanisms

Terminology

Sterilization is the complete elimination or

destruction of all forms of microbial life.

Disinfection : process that eliminates many

or all pathogenic microorganisms on inanimate objects, with the exception of bacterial spores.

accomplished in health care facilities by either physical or chemical processes.

Disinfection is usually accomplished by the use of liquid chemicals or wet pasteurization in health care settings.

Terminology

When chemicals are used for the purposes of destroying all forms of microbiologic life, including fungal and bacterial spores, they may be called chemical sterilants. These same germicides used for shorter exposure periods may also be part of the disinfection process (i.e., high-level disinfection).

Terminology

Antiseptics- disinfectant agents that can be used on body surfaces, such as skin or vaginal tract, to reduce the number of normal flora and pathogenic contaminants

Asepsis

Processes designed to prevent microorganisms from reaching a protected environment. E.g: for procedures used in

the operating room the preparation of therapeutic agents, and technical manipulation in the microbiology lab

Factors influencing efficacy

Prior cleaning of the object; Presence of organic and inorganic load Type and level of microbial contamination Concentration of and exposure time to the germicide The nature of the object (e.g., crevices, hinges, and lumens) Presence of biofilms Temperature and pH of the disinfection process Relative humidity of the sterilization process (e.g., with ETO).

Organic materials

Organic material (pus, blood, saliva, urine and feces) protects organisms from disinfection

Hepatitis B virus in organic matter such as dry blood - resistance to disinfectant

Resistance organisms

Bacterial endospores

Most resistance forms Able to stand 16 hours of boiling

Mycobacteria

Cell surface is rich in wax-like lipids causing resistance to disinfectant but sensitive to heat
Non culturable organisms unable to check post disinfectants viability Proliferate in distilled and deionized water Produce slime that is a barrier to disinfectants.

Hepatitis virus and HIV

Pseudomonas spp

Methods
Physical Chemical methods
Phenols and phenolics Alcohols Halogens Surfactants Alkylating agents

Heat

Filtration
Radiation

Heat
Moist heat
boiling
Steam under atmospheric pressure Steam under atmospheric pressure pasteurization

Dry Heat
Dry heat sterilization

incineration

Steam at atmospheric pressure

Steam contains latent heat generated during vaporization The latent heat is released when steam condenses on a colder surface The condensation release heat and moisture which is necessary for protein coagulation to occur The shrinking of volume of the steam during condensation creates a negative pressure drawing in more steam

Steam under pressureAutoclaving

To obtain a moist heat temperature of 121 C, saturated steam is placed under pressure of 15 lbs per square inch and sterilization achieved after 15 minutes The required time of exposure diminished as the temperature increased 126C 10 mins Sterilization indicator

chemical changes

Chemical imprinted on tapes and wrappers

culture testing for spores

Bacillus stearothermophilus impregnated in ampoules or

filter paper strips

Pasteurization

Utilizes mild heat to destroy pathogenic vegetative forms in liquids without altering the liquids palatibility Used in cows to prevent infectious disease associated with raw milk tuberculosis, brucellosis, Camylopbacteriosis, dysentry, Q fever Two methods

Holding/Batch method

Milk in vessels is pasturised at 620C for 30 minutes Thin films of milk stream over pipe or plates held at 71.7 C for 15 seconds.

Flash/continuous flow method

Moist Heat
Organisms
Most vegetative form of bacteria, yeast and mould, viruses Most vegetative form of bacteria, yeast and mould, viruses

Temperature
600C 800C

Time
30 minutes 5 to 10 minutes

Mould spores Bacterial spores

800C 121

30 minutes 15 mins

Comparison of various conditions required to kill Microorganisms

Methods

sporocidal disinfection

sterilization

Boiling Steam

x x

X X

(atmospheric pressure)

Steam
(under pressure)

NA

Pateurization

Heat
Moist heat
boiling
Steam under atmospheric pressure Steam under atmospheric pressure pasteurization

Dry Heat
Dry heat sterilization

incineration

Dry heat

Biomolecules exhibit less thermosensitivity to dry heat than to moist heat Dry heat is slower and requires temperatures higher than those used in moist heat

Dry heat sterilization

Used at 1600C for 2 hours or 1700C for 1 hour Dry heat temperature below 1400C do not destroy spores within a reasonable time period. Mechanically convected ovens are used for evenly distributed heat Method of choice for sterilizing powders, oils, and thermostable materials that are adversely affected by moisture

Incineration

Used for discardable,combustible items such as mask, gloves and wipes that are soilde with blood and body fluid Flaming is a convenient way to sterilized inexpensive heat tolerant implements

Methods
Physical Chemical methods
Phenols and phenolics Alcohols Halogens Surfactants Alkylating agents

Heat

Filtration
Radiation

Filtration

Porous material to remove organisms Applications of filters

Sterilization of liquids and air Separation of microorganisms of different sizes Preparation of cell-free solutions of toxins, antigens Determination of virus size

Membrane filters

Paper thin,sieve like elements composed of inert cellulose esters Pore size range differs (0.005 to 1 m) Pore size of 0.2 micron able to filter bacteria Filters can be visualized under microscope or cultured E.g

High efficiency particulate air filters (HEPA) filters to filter particles in the air

Reduce particles as small as 0.3 micron with 99.97%

Sterilized serum

Methods
Physical Chemical methods
Phenols and phenolics Alcohols Halogens Surfactants Alkylating agents

Heat

Filtration
Radiation

Radiation

Ultraviolet radiation Ionizing radiation

Ultraviolet radiation

Causes damage to DNA of the microorganisms UV lamps- generate UV rays with wavelenth 240nm to 280nm Application- to reduce microorganisms in air and surfaces of hospital rooms, operating rooms, entryways, nurseries, gymnasium and cafaterias. Problems

Low penetrability. Organisms must be directly exposed to UV Overexposure to UV in human, causes skin erythema and conjunctivitis

Ionizing radiation

Cobalt 60 produces cathode rays and gamma rays Advantage

Avoid heat and moist Allows prepackaging and sealing of items to be sterilized Sterilizing pharmaceuticals and disposable medical supplies such as syringes and catheters.

Application

Disadvantage fabrics tensile strength will be reduced after irradiation

Methods
Physical Chemical methods
Phenols and phenolics Alcohols Halogens Surfactants Alkylating agents

Heat

Filtration
Radiation

Chemical methods

All chemicals used in sterilization and disinfection are grouped as disinfectants. Chemicals used for sterilization (under prescribed condition) are called sterilants or chemosterilizers Chemical sterilants include

Ethylene oxide (ETO) Formaldehyde and glutaryldehyde

Ideal properties of disinfectant

Able to destroy all forms of microorganisms within practical period Soil will not interfere with its reaction Nonirritating, non allergenic and non toxic Non corrosive and non discolouring Demonstrate wettability and penetrability Soluble in water Chemically stable Dilution will not adversely affect its activity Do not have disagreeable odour or expensive

Non of disinfectants meets all the qualification Disinfection must be chosen on the basis of their intrinsic properties and job at hand.

Classes of disinfectants

Phenol and phenolics Alcohols Halogens Surfactants Alkylating agents

Phenols and phenolics

Properties:

have active compounds in the presence of organic matter Stable and persist on surface for a long period. Inactivated by rubber, wood and plastics

Use: disinfecting saliva, feces and similar matter Disadvantage: irritant to skin, Diphenyl compounds used as skin disinfectant: hexachlorophene and chlorhexidine

Chlorhexidine

Greater bactericidal activity than hexacholorophene 4% chlorhexidine used for surgical scrub, hand washing and wound cleansing Persist on the skin, non irritating, not absorbed Action is neutralized by soaps and anionic detergents

Alcohols

Contact time is important for action Properties:

High antimicrobial properties on environmental surfaces

Use: skin and surface disinfectants Disadvantage: Dilution reduce the effectiveness, volatile, flammable and irritates skin Examples: Ethyl and isopropylalcohol 70%

Halogens

Examples: Chlorine and iodine

Chlorine

Use: sanitation, purification and disinfection.

Elemental chlorine water purification Hypochlorite sanitizing agents

Concentration:

Less than 1 ppm- lethal to most vegetative bacteria, inactivates most viruses 5% sodium hypochlorite surface and glassware decontamination

Disadvantage:organic matters interfere with its activities

Iodine

Active compound: tincture of 2% in 50% alcohol Use:

Disadvantage:

wound and skin antisepsis Preoperative skin antisepsis General disinfectants for thermometers and surgical appliances cause tissue necrosis in strong concentration Stain skin

Povidone iodine has less tissue necrosis because the iodine is solubilized in povidone which provide a sustained released of iodine

Surfactants

Examples

Anionic detergents (Soaps)

effective cleansers, little direct antibacterial effect

Cationic detergents (quartenary ammonium compounds)

highly bactericidal, inactive against spores and virus Adsorbed into cotton, cork, dust becomes inactive Little toxicity to skin and mucous membrane E.g. benzalkonium chloride

Alkylating agents

Formaldehyde Glutaryldehyde Ethylene oxide

Formaldehyde

Use:

gas state as fumigants to decontaminate room or laboratory that have been accidentally and extensively contaminated with pathogenic bacteria Liquid as formalin

Disadvantage: tissue irritant

Glutaryldehyde

Activity: wide spectrum. 2% solution is bactericidal, tuberculocidal and virucidal in 10 to 90 minutes, sporocidals in 10 hours Effective sterilizing agent for apparatus that cannot be heat treated such as lens instrument, equipment for respiratory therapy Disadvantage: not use as skin antisepsis because irritation to skin and soft tissue. Commercially available: cidex

Ethylene oxide

Cyclic ether Advantage: It can sterilized without high level of heat and moisture Usage: catheters, disposable medical items, dental handpieces, sutures Disadvantage: toxic if inhaled, vesicant on contact, slow acting, require 4 hours at 50 to 56 C or 6 to 12 hours at room temperature to produce sterility

Vegetative bacteria

Fungi

enveloped virus

Non-enveloped virus

Mycobacteria

Bacterial spores

Phenol and phenolics Alcohols Iodine Chlorine

X +/

X X X

Chlorhexidi ne 4%

Vegetative bacteria

Fungi

enveloped virus

Non-enveloped virus

Mycobacteria

Bacterial spores

Quartenary ammonium compound Formaldehy de

+/-

(10-90 mins)

(10-90 mins)

(10-90 mins)

(10-90 mins)

(10 hours)

Glutaryldeh (10yde (2%) 90 mins) Ethylene oxide

Disinfectant

High-level disinfectants - At similar concentrations

with chemical sterilants but with shorter exposure periods (e.g., 20 minutes for 2% glutaraldehyde), these same disinfectants will kill all microorganisms with the exception of large numbers of bacterial spores Low-level disinfectants may kill most vegetative bacteria, some fungi, and some viruses in a practical period of time (<10 minutes) Intermediate-level disinfectants may be cidal for mycobacteria, vegetative bacteria, most viruses, and most fungi but do not necessarily kill bacterial spores.

Mode of action
Moist Heat Coagulation of protein of the microorganism, death- inactivation of enzymes, changes in nucleic acids and cytoplasm Alteration of protein by oxidation, desiccation and changes of osmotic pressure Porous material to remove organisms damage to DNA of the microorganisms

Dry heat

Filtration Radiation

STERILIZATION AND DISINFECTION TECHNIQUES COMMONLY USED IN HOSPITAL PRACTICE

Objective

Method Steam autoclaving Ethylene oxide

Principle of microbial inactivation Thermal denaturation Thermochemical denaturation

Example of use Metallic surgical instruments Heat-labile surgical instruments Implantable medical devices (catheters, protheses) Heat-labile instruments Water disinfection Skin antisepsis Skin and mucosae antisepsis Flexible endoscopes Flexible endoscopes

Gamma-irradiation Sterilization Gas plasma Chlorine Alcohols Iodophors Aldehydes Disinfection Peracetic acid

Ionizing denaturation Generated free radical induced denaturation Chemical denaturation Chemical denaturation Chemical denaturation Chemical denaturation Chemical denaturation

Common sterilization technique

A: Heat sterilization, including steam or hot air (steam sterilization processing time from 3 to 30 minutes). B: Ethylene oxide gas ( generally 1 to 6 hours processing time plus aeration time of 8 to 12 hours at 50 to 60 C).

C: Hydrogen peroxide gas plasma (Processing time between 45 and 72 minutes; for endoscope or medical device restrictions based on lumen internal diameter and length)
D: Glutaraldehyde-based formulations (> 2% glutaraldehyde; caution should be exercised with all glutaraldehyde formulations when further in-use dilution is anticipated); glutaraldehyde (0.95%) and 1.64% phenol/phenate. One glutaraldehyde-based product has a 5 minute exposure time at 35 C. E: Ortho-phthalaldehyde 0.55% (FDA cleared as high-level disinfectant; passes the Sporicidal Activity Test in 32 hours at 20 C but not cleared as a chemical sterilant). F: Hydrogen peroxide 7.5% (will corrode copper, zinc, and brass). G: Peracetic acid; concentration variable but 1% or less is sporicidal. Peracetic acid immersion system operates at 50 to 56 C. H: Hydrogen peroxide (7.35%) and 0.23% peracetic acid; hydrogen peroxide 1% and peracetic acid 0.08% (will corrode metal instruments).