Sterilization & Disinfection
Lecture-9
Dr Sadia Ikram
• Microorganisms are the agents of contamination,
infection and decay.
• Hence it becomes necessary to remove them from
materials & areas.
• Early civilization practiced salting, smoking, pickling
and exposure to sunlight .
• In mid 1800s Lister developed Aseptic techniques to
prevent contamination of surgical wounds.
• Prior to this development:
• Nosocomial infections (hospital acquired infections)
caused death in 10% of surgeries.
• Up to 25% mothers delivering in hospitals died due
to infection.
Bacteriostatic Agent:
An agent that inhibits growth of bacteria,
but does not necessarily kill them.
Bactericidal:
Agent that kills bacteria. Most do not kill spores.
Sporicidal: An agent that kills spores.
Sterilization & Disinfection
• Process used to achieve sterility, an absolute term meaning the
absence of all viable microorganisms (vegetative forms & spores).
Or
• Killing or removal of all microorganisms, including bacterial & viral
spores, which are highly resistant.
• Disinfection: Process which reduces the number of
contaminating micro-organisms.
or
• Reducing the number of pathogenic microorganisms to the point
where they no longer cause diseases, leaving spores.
• Uses:
1. Required for instruments & materials used in
procedures involving penetration into sterile
body parts, e.g. Surgical operations, I.V.
infusions, injections etc.
2. Required for media, reagents & equipment
used in laboratory practices.
Flaming
Microwave ovens
1. STERILIZATION BY HEAT
•More effective
•Coagulates and denatures enzymes & structural
Moist proteins/ kills Micro organisms
Heat
•Less effective
•Destructive oxidation of essential cell constituents/
Dry kills Micro organisms
a. STERILIZATION BY MOIST HEAT
• Moist heat coagulates & denatures enzymes &
structural proteins & kills micro organisms.
i. Pasteurisation: Process of killing of pathogens in
milk but does not sterilize it .
• Milk is heated at 63oC for 30min (HOLDER METHOD)
• At 72oC for 15-20 Sec (FLASH PROCESS)
ii. Tyndallisation /Intermittent Sterilization
• For media containing sugar and gelatin exposure of
100oC for 20 min for 3 successive days is used.
iii. Boiling: At 100oC
• Kills vegetative forms of bacterial pathogens.
• Hepatitis virus: Can survive up to 30 minutes of
boiling.
• Endospores: Can survive up to 20 hours or more of
boiling.
iv. STEAM UNDER PRESSURE - AUTOCLAVE
Invented by Charles Chamberland in 1879.
Principle: Pressure used to produce high temperature steam to
achieve sterilization.
Normally water boils at 100oC.
When water heated in closed container, pressure inside rises,
correspondingly increasing the boiling temperature. (121oC)
Steam formed is thus superheated.
Superheated steam condenses cooler objects
Release thermal energy (Leading to denaturation of microbial
proteins).
•Autoclave consists of a vertical or
a horizontal cylinder.
•One end has an opening which is
meant for keeping materials to be
sterilized.
•Lid is provided with a Pressure
gauge, to measure the pressure.
•A safety valve is present to permit
the escape of steam from the
chamber.
•Articles to be sterilized are placed AUTOCLAVE
in the basket provided. Temperature: 121oC.
Pressure: 15lb/inch2
Holding time: 15-20 minutes.
Uses of Autoclave
• Sterilization of:
• Culture medias
• Surgical instruments
• Hospital linen etc.
STERILIZATION CYCLE
• Heating time
• Hold time
• Cooling time
Physical Methods of Sterilization
• Kills by effecting oxidation of bacteria.
b. Sterilization By Dry Heat:
•Oven utilizes dry heat to sterilize articles.
1. Hot air oven
•A holding period of 160oC- 180oC for 1-2 hr
is desirable.
• It has thermostat controlling the
temperature.
•Double walled insulation keeps heat in &
conserves energy.
•Used for materials: that withstand high
temp for longer time. e.g. glassware when
needed dry.
• Materials likely to be affected by steam or
into which steam can’t penetrate e.g. oils,
powders.
2.FLAMING
• Used for:
• Inoculation loop or
Wireloop, tip of
forceps and
spatulas.
• Held in a bunsen
flame till they are
red hot.
3.Incineration
• At high temperature, efficient method for
sterilization and disposal of contaminated material.
• Used for disposal of pathological waste, materials,
surgical dressings, sharp needles, other clinical &
hospital waste.
4. Microwave Ovens
• Heating effect not uniform, hence not very reliable.
2. STERILIZATION BY RADIATION
Two types of radiations : NON –IONIZING
IONIZING
• Ionizing radiations:
• ɣ rays, X-rays, accelerated electrons.
• High penetrative power
• No appreciable increase in temperature – COLD
STERILISATION
• Large scale industrial process.
• Used to sterilize large amounts of
pre-packed single-use items
e.g. disposable syringes, catheters,
gloves etc.
Non- Ionizing radiation:
• Electromagnetic rays with longer wavelength.
• Absorbed as heat.
• Can be considered as hot air sterilization.
• Used in rapid mass sterilization of pre-packed
Syringes and catheters.
Eg: UV rays
Chemical Agents
•Sterilizing
Gases •Ethylene oxide
•Sterilizing agents: Glutaraldehyde
Liquids & formaldehyde
•Disinfectant………
STERILIZATION BY GASEOUS PROCESS
• Ethylene oxide: Alkylates bacterial proteins & nucleic acids.
• Highly penetrative, non corrosive. Colorless with a sweet ethereal
smell.
• Effective against all types of microorganisms including viruses and
spores
• USES: Used in industry for single-use, heat sensitive medical
devices. e.g. prosthetic heart valves.
• Especially used for sterilizing: Heart-lung machines, respirators,
sutures, dental equipments, books and clothing, Glass, metal and
paper surfaces ,plastics, oil, some foods and tobacco.
Sterilization by Liquids
• Glutaraldehyde & formaldehyde: Denatures protein & nucleic acids
of bacteria.
• For sterilization of fiberoptic endoscopes, corrugated rubber
anesthetic tubes, Face masks, Plastic endo-tracheal tubes, Metal
instruments,
DISINFECTION BY CHEMICAL AGENTS
Chemical agents act on the following sites of
bacteria:
1. Disruption of the cell membrane
2. Modification of proteins/Protein coagulation
3. Modification of nucleic acids
Disruption of Cell Membranes
a. Alcohol: 70% Ethanol widely used to clean skin before
immunization or venipuncture. Acts by disorganizing lipid
structure in membranes & denatures.
b. Detergents: Surface-active agents composed of long-chain,
lipid-soluble, hydrophobic portion & polar hydrophilic group
(cation or anion, or nonionic group). Interact with lipid in cell
membrane through their hydrophobic chain and with
surrounding water through their polar group & disrupt
membrane. Quaternary ammonium compounds
(benzalkonium chloride used for skin antisepsis).
c. Phenols: First disinfectant used in operating room (Lister in
1860s), rarely used today as it is too caustic. Damage
membranes & denature proteins.
Hexachlorophene used in germicidal soaps, cresol (active
ingredient in Lysol).
Modification of Proteins
a. Chlorine: Powerful oxidizing agent, Disinfectant to purify water supply & to
treat swimming pools. E.g. Hypochlorite (bleach, Clorox), disinfectant used in
home & hospitals.
b. Iodine: Inactivates sulfhydryl-containing enzyme: Most effective skin
antiseptic, used before obtaining blood culture & installing I/V catheters (To
avoid contamination with skin flora Staphylococcus epidermidis.e.g. Tincture
of iodine & Iodophors
c. Heavy Metals: Mercury & silver have greatest antibacterial activity. Act by
binding to sulfhydryl groups & blocking enzymatic activity. E.g. Silver nitrate
drops useful in preventing gonococcal ophthalmia neonatorum. Silver
sulfadiazine used to prevent burn wound infections.
d. Hydrogen Peroxide: Oxidizing agent, attacks sulfhydryl groups & inhibits
enzymatic activity. Used as antiseptic to clean wounds & disinfect contact
lenses. (Effectiveness limited by catalase produced by organisms, that
degrades H2O2).
e. Acids & Alkalis: Denature proteins. Mycobacterium tuberculosis relatively
resistant to 2% NaOH. Weak acids (benzoic, propionic, citric acids used in food
preservatives as they are bacteriostatic).
Modification of Nucleic Acids
• Dyes stain microorganisms & inhibit their growth.
• Crystal violet (gentian violet),used as skin
antiseptic. Binding of positively charged dye
molecule to negatively charged phosphate
groups of nucleic acids).
• Malachite green: (component of Löwenstein-
Jensen's medium, used to grow M. tuberculosis).
Inhibits growth of unwanted organisms in
sputum during 6-8week incubation period.
Sterilization by Filtration
Filtration helps to remove bacteria from heat
labile liquids such as serum & solutions of
sugar, antibiotics and fluids.
• Types of filters:
• Membrane filters
• Candle filters: Widely used for purification of
water.
• Asbestos filters
• Sintered glass filter
Membrane filters:
work by physically trapping particals larger than pore size
• Made of cellulose esters or other polymers,
Pore size < 0.45 μm.
• Uses: For parenteral fluid like antibiotic &
blood products.
• Separation of toxins from bacterial cultures,
preparation of bacterial cultures.
• Water purification & analysis
