Methods of Sterilization

Sterilization:
 Differentiate between sterile (inability to reproduce) and sterilization (free from
microbes).
 Example of sterilization: (Use of knife)
 Definition: The process by which an article or surface or medium is freed/devoid
of all the micro-organisms (completely)either in the vegetative or spore state.
 It is an absolute process, i.e either it is sterile/asterile (0% or 100%) but not
intermediate/inbetween.
 Differentiate sterilization from disinfection (spores are not necessarily killed),
anti-septics (used to prevent the growth of microbes on living tissues),
Importance of sterilization in industries
 To prevent decomposition and spoilage of foods.
 To prevent contamination of sterile products such as ophthalmics,
injectables/parenterals, surgical dressings, sterile containers used for
diagnostic tests (urine sample containers), surgical instruments and
equipments (scalpels, sutures etc).
 To prevent transmission of pathogenic micro-organisms from causing
diseases in plants, animals and in humans.
 To prevent contamination of unwanted microbes in pure cultures and in
other microbial experiments performed for research.
 To prevent contamination in aseptic areas – preparation, testing and
packing areas of injectables, antibiotics, enzymes etc.,.
 To prevent cross-contamination in industries that employ micro-
organisms for the production of fermented products such as breweries.
 The various methods of sterilization
 are: methods of sterilization:
Physical
- Dry heat sterilization – By denaturing their respective enzymes.
- Moist heat/Steam sterilization – By coagulation of proteins.
- Radiation/Cold sterilization – By destroying the genetic
material.
- Mechanical methods/Filtration – By using a barrier.
 Chemical methods of sterilization:
- Gaseous sterilization
- Use of disinfectants
 The method of sterilization to be used is chosen based on
material that need to be sterilized, type of microbes (that need to
be killed) involved, length of time of sterilization.
Methods of sterilization by Dry Heat
 Sun-light : Oldest method, that uses light from the sun (consisting
of heat and U.V) to remove moisture from the substance so that
micro-organisms cannot grow (as they require moisture for
growth & reproduction). However spores are unaffected.
 Red-heat : The materials are sterilized by holding them on a flame
of Bunsen burner till red hot. Used in cases of: inoculating
wires/loops, forcep points, spatulas.
 Flaming: The materials are sterilized by passing them over the
flame of Bunsen burner for few times (3-4) without allowing them
to stand for too long. Used in cases of: glass slides, mouth of
culture tubes, cover slips, scalpels, etc.,
 Incineration: It is an efficient method for
destroying the contaminated or ineffective
materials of hospital/laboratory waste such
as : soiled dressings, patient bedding etc.,. by
reducing them to ashes in an incinerator.
Methods of sterilization by Dry Heat
 Hot-air oven: Most widely used method of sterilization by dry heat.
Substances that are not thermo-labile and can withstand temperatures
upto 250 degree C can be sterilized by hot-air oven.
 The sterilization temperature and holding time for hot-air oven operation
are:
- At 140 degree C for 3 hours.
- At 160 degree C for 1 hour.
- At 180 degree C for 30 minutes.
 Used for : glass-ware, forceps, scissors, sealing materials (liquid paraffin,
greases), dry powders, swab sticks.
 Controls: Browne’s tube, thermocouples, biological control (discussed
later).
 Precaution: After sterilization, do not open the oven immediately, let the
temperature reach around 50 degree C and then remove the sterilized
materials to use or store them.
Methods of sterilization by Moist Heat
 Temperature below 100 degree C:
- Pasteurization: Moderate temperature for a definitive period of time is used – Milk.
# Holder method : 62.8 degree C for 30 minutes.
# Flash method : 71.7 degree C for 15 seconds. (HTST-High temperature short time)
140 degree C for 3 seconds 74 degree C (UHT-Ultra High
Temp.)
* UHT is gaining importance with success around the globe as the organoleptic
characters of the sterilized material are not altered – high importance in food industries.
 Temperature at 100 degree C:
- Boiling water: At atmospheric pressure – drinking water for consumption, baby feed
bottles are sterilized by this method.
- Tyndallization: Steam at 100 degree C for 3 successive days in Koch or Arnold
steamer – where spores get converted to vegetative cells in the meanwhile – used for
egg, serum, sugar based media.
Methods of sterilization by Moist Heat
 Temperature above 100 degree C:
- Autoclave: Autoclaving means sterilizing by saturated steam under high pressure.
Steam is generated and circulated inside a closed/air-tight vessel, the temperature rises
to 121 degree C and pressure increases to 15psi, maintained for 15-20 mins for
satisfactory sterilization. Steam circulates inside the jacket and is supplied under high
pressure to the inner chamber. Saturated steam is more efficient in sterilizing than hot
air as it produces greater lethal action by penetrating easily into the porous materials
like cotton wool stoppers, cloth wrappers, culture media etc.,.
Methods of sterilization by Radiation
(Costly)
It is also called as cold sterilization.
 It is used to sterilize heat sensitive materials.
 Based on their wavelength and penetrating power, radiation can be divided into:
- ionizing radiation: They have more energy and will ionize the target molecules –
highly lethal to DNA – X-rays, Gamma rays, Cathode rays. Used to sterilize antibiotics
such as benzyl pencillin, streptomycin; vitamins such as ascorbic acid; lactose;
sulphonamides; talc, etc.,.
- non-ionizing radiation: IR;Ultra-violet radiation from a UV lamp -
Used to reduce air-borne contamination in the
maintenance of aseptic areas and rooms
within the processing environment; biological
fluids such as plasma and vaccines are also
sterilized by UV rays; now-a-days being used
in water filters too.
Methods of sterilization by Filtration
 It is also called as mechanical method of sterilization.
 The microbes are entrapped inside the filtering material because the pore size of the
filter is less than that of the microbial cell size.
 It is used to sterilize heat sensitive materials – liquid substances such as solutions of
sugars, urea, enzymes and antibiotics.
 The filter efficiency is affected by its pore size, positive or negative pressure (vacuum
pump can be used), nature of the liquid to be filtered.
 The filtering unit/assembly must be sterilized before use.
 The filters used are :
-Asbestos/Seitz filter - disposable made up of magnesium trisilicate – pore size of 0.01 to
5µ.
-Sintered glass/Fritted glass/Morton glass filters - Borosilicate glass packed in disc
moulds and adhered by heat – can be cleaned easily but are EXPENSIVE.
-Membrane/Cellulose filters - Made up of Cellulose and cellulose esters, 150µ thick, with
pore size of 0.45±0.2µm
Methods of sterilization by Filtration
 HEPA FILTERS:
 High Efficiency Particulate Air Filters.
 Microbes larger than 0.3µm are entrapped in HEPA filters.
 Used in ICU’s, IV preparation rooms, antibiotic preparation and packing zones.
 Also used in laminar air flow chambers.
Chemical methods of Sterilization
 In this type of sterilization technique the dependency is on the concentration of the
chemical and its exposure time.
 These are mainly of 2 types:
- Gaseous: Formaldehyde, Ethylene oxide – MOST WIDELY USED – Mixed with carbon
dioxide to mask its flammability, respective conc. and exposure times are mentioned below:

Concentration Exposure time

(mg/L) (hrs)
44 24
88 10
442 4
884 2
- Liquids: They are actually disinfectants and antiseptics such as alcohols (tincture),
phenols (Dettol), cresols (Lysol), halogens, dyes, aldehydes etc.,.