STERILIZATION
Mrs. Anjila Pal
Lecturer
Department of Microbiology
CMCTH, Bharatpur
TERMINOLOGIES
• Cleaning plays an important preparatory role before sterilization
or disinfection, by removing soil and other dirt and reducing the
microbial burden, making sterilization more effective
• Sterilization: The process by which an article, surface or medium
is freed of all living microorganisms either in vegetative or spore
state
• Results in reduction of ≥106 log CFU of microorganisms and their
spores
• Sterilant: chemical agents that are capable of destroying spores
• Asepsis: state of complete absence of viable pathogenic
microorganisms in any environment
Sterilization methods
A. Physical methods B. Chemical methods
1. Sunlight 1. Gas
2. Heat 2. Liquid
3. Filtration
4. Radiation
5. Sound (sonic) waves
SUNLIGHT
• Is a natural method of sterilization of water in tanks, rivers and lakes
• Germicidal effect due to combined effect of UV and heat rays in
tropical countryside
• Bactericidal activity
• Role in spontaneous sterilization
HEAT
• Most reliable and universally applicable method of sterilization
• Heat can be used for sterilization in two forms:
1. Dry heat
2. Moist heat
• Dry heat kills by protein denaturation, oxidative damage, and toxic
effect of elevated level of electrolytes
• Moist heat kills by coagulation and denaturation of proteins
Dry heat sterilization
1. Flaming
• Inoculating wire or loop, tips of forceps,
searing spatulas are sterilized by holding them
almost vertically in a Bunsen burner flame till
they become red hot
Uses: Glass slides, scalpels and mouth of culture
tubes are sterilized by passing them through the
Bunsen flame without allowing them to become
red hot
2. Incineration
• An excellent method for safely
destroying biomedical waste by burning
them to ashes
• Incinerators regularly employed in the
hospitals
Uses: destruction of syringes, needles,
dressings, bandages, animal carcasses and
pathology samples
• Not for metals and heat-resistant glass
materials
3. Hot air oven
• Most widely used method of sterilization by dry heat
• Sterilization is achieved by conduction
• The hot-air oven is electrically heated and fitted with fan for even
distribution of hot air in the chamber
• It is also fitted with thermostat that ensures circulation of hot air of
desired temperature in the chamber
• Temperature and time of sterilization:
160ºC for 2 hours
170ºC for 1 hour
180ºC for 30 minutes
• Once electrically heated to desired temperature, heat is absorbed
by the surface of the item to be sterilized
• Penetrates to the center until entire item reaches the desired
temperature
Uses:
Glassware such as test tubes, measuring cylinder, flasks, petri
dishes, pipettes
Metal instruments such as forceps, scissors, scalpels
Non-aqueous materials and powders
Oils and glycerol
Precautions:
• Chamber should not be overloaded
• Materials arranged in a manner to allow free circulation of air
• Materials should be perfectly dry
• Test tubes, pipettes and flasks should be wrapped with
newspaper
• Rubber and inflammable materials should not be placed
• Allowed to cool below 80ºC (about 2 hours) before opening
Sterilization controls:
1. Physical method: Thermocouples
2. Chemical method: Browne’s tubes No. 3 (red to green)
3. Biological method: Spores of Clostridium tetani or Bacillus
subtilis subsp. niger
Moist heat sterilization
Sterilization by moist heat is classified as follows:
1. Sterilization at a temperature below 100ºC
2. Sterilization at a temperature of 100ºC
3. Sterilization at a temperature above 100ºC
1. Sterilization at a temperature below 100ºC
Pasteurization: milk is pasteurized in two ways:
Holder method: 63ºC for 30 minutes
Flash method (HTST): 72ºC for 15-20 seconds
• Followed by rapid cooling to 13ºC or lower
Vaccine bath:
• Bacterial vaccines are heat-inactivated in water bath at 60ºC
for 1 hour
• Serum or body fluids sterilized by heating at 56ºC for 1 hour
in a water bath on several successive days.
Inspissation:
• Heating at 80 to 85ºC for 30 minutes on 3 successive days
• Instrument used is inspissator
• Uses: Lowenstein-Jensen and Loeffler’s serum media are
sterilized
2. Sterilization at a temperature of 100ºC
i. Boiling
• Boiling of water for 10 to 30 minutes
• Addition of 2% sod. bicarbonate promote sterilization
Uses: Disinfection of surgical instruments prior to processing
ii. Steam sterilizer
• Free steam for 90 minutes
• Uses: disinfect selective heat-labile culture media
Koch and Arnold’s steam sterilizer
o Articles placed on perforated shelf
oMost of vegetative forms killed
oThermophiles persist
iii. Tyndallization:
• Exposure to steam at 100ºC for 20 minutes on 3 successive days
• Vegetative cells killed during first heating
• More resistant spores germinate
• Killed on successive heating on 2 nd or 3rd day
Uses: Heat sensitive culture media containing egg or serum or
carbohydrates
3. Sterilization at temperature above 100ºC
Autoclave
Principle of autoclave:
Water boils when its vapor pressure equals that of the
surrounding atmosphere. When pressure inside a closed vessel
increases, the boiling temperature of water also increases. The
saturated steam having high penetrative power, on contact with
cooler surface condenses to water and releases its latent heat to
that surface
For example, nearly 1600ml steam at 100ºC and at atmospheric
pressure condenses to 1ml water at 100ºC and releases 518
calories of heat
Sterilization conditions:
• 121ºC at 15 psi pressure for 15 minutes
• 126ºC for 10 minutes at 20 psi
• 133ºC for 3 minutes at 30 psi
Uses:
• Sterilizing culture media, rubber material, dressing materials,
gowns, dressing, linen, gloves, etc.
• Water containing materials
• Materials which cannot withstand higher temperature
Sterilization controls
1. Physical control (Thermocouples)
2. Chemical control (Browne’s tube)
3. Biological control
• Spores of Geobacillus stearothermophilus
• Paper impregnated with 106 spores in envelopes
• Spores require exposure of 12 minutes to be destroyed
Ultra High Temperature (UHT) Sterilization:
Uses: dairy industry
• 134ºC for 1-2 seconds
• All non-sporing pathogens like mycobacteria, brucellae and
salmonellae destroyed
FILTRATION
Excellent way to reduce the microbial population in solutions of
heat-labile materials such as sera and sugar solutions or antibiotics
Types of filters:
i. Membrane filters
• Made up of cellulose acetate or esters or other polymers
• Come in wide range of average pore diameters (APD)
• Pore size range of 0.1-5µm available, 0.22µm most commonly used
• Used for bacteriological examination of water in hospital settings
• Also to remove bacteria from heat labile liquids
ii. High-efficiency particulate air (HEPA) filter
• Removes 99.97% of 0.3µm particles from the filtered air
• Used in laminar air flow systems, biological safety cabinets,
operation theatre
• Also in surgical mask and N95 respirators
RADIATION
1. Ionizing radiation:
• An excellent sterilizing agent with high penetrating power
• Includes high energy radiations like X-rays, gamma rays and
cosmic rays
• Gamma radiation emitted from cobalt-60 source is used for
sterilization of antibiotics, hormones, sutures and vaccines and
prevent food spoilage
• Since there is no detectable increase in temperature; cold
sterilization
2. Non-ionizing radiation:
Are electromagnetic rays with wavelengths longer than those of
visible light
i. Infrared radiations: rapid mass-sterilization of disposable
syringes and catheters
ii. Ultraviolet radiation: wavelength of 240-280 nm has
marked bactericidal activity
• Denatures bacterial protein and interferes with DNA replication
Uses: Disinfection of enclosed areas such as biosafety cabinets,
operation theatres and entryways
SOUND (SONIC) WAVES
• High frequency sound (sonic) waves beyond the range of
human hearing disrupt cells
• Sonication transmits vibrations through a water-filled chamber
to induce pressure changes and create intense points of
turbulence that can stress and burst cells in the vicinity
• Above 15000 cycles per second
• Gram-negative rods are most sensitive
Uses: dental and some medical offices to clear debris and saliva
from instruments before sterilization
CHEMICAL METHODS
Ethylene oxide sterilizer
• ETO is a colorless liquid used for gaseous sterilization
• It is highly penetrating gas with sweet ethereal smell
• Sterilization is carried out in ethylene oxide sterilizer
• Low temperature 55˚C
• Duration of cycle: 12-14 hours
• It is highly inflammable and explosive at concentrations greater
than 3%
• Mixed with inert gases, 10% CO2 or N2 makes it less
explosive
• Acts by alkylating amino, carboxyl, hydroxyl and sulfhydryl
groups of protein molecules within the microbes and spores
• Also reacts with DNA and RNA
Uses: used by CSSD for sterilization of moisture or heat labile
articles such as heart-lung machines, suture materials, dental
equipment, catheters, respirators
Sterilization control: spores of Bacillus atrophaeus
Plasma sterilization: Plasma, the fourth state of matter
• Hydrogen peroxide used as chemical sterilant
• Radio frequency applied to create an electromagnetic field
• Generate a state of plasma containing free radicals of
hydrogen and oxygen
• Gaseous state consists of ions, free electrons or neutral
particles
• Cycle run for 24-75 min
• Low temperature 37-44˚C
• Sterilize by denaturing DNA of all microorganisms
• Used by CSSD for sterilization of materials and devices that
cannot tolerate high temperature and humidity
• Such as some plastics, electrical devices, and corrosion-
susceptible metals such as arthroscopes, urethroscopes,
laparoscope, micro and vascular instruments
Sterilization control: Geobacillus stearothermophilus
Central Sterile Supply Department (CSSD)
• An integrated place in hospitals and other health care facilities
where physical cleaning, washing, packing and sterilization of all
hospital linen, medical devices, surgical instruments and
equipment are carried out
• It plays an important role in the infection control process of all
hospitals and health care settings
• There is a unidirectional work flow
Thank you
