STERILIZATION

HISTORICAL BACKGROUND

 Ignatz Semmelweis (1816 –1865), a Hungarian

physician, was the pioneer in introducing hand
washing with chlorinated lime water to avoid
infection.
 Joseph Lister (1827–1912), a British physician,
treated his patients’ wounds with phenol to
avoid wound infection.
 STERILIZATION
 It is defined as the process that refers to removal, killing, or
deactivating all forms of life (in particular referring to
microorganisms such as fungi, bacteria, viruses, spores,
unicellular eukaryotic organisms such as Plasmodium, etc.)
 It involves a biocidal agent or microbial removal process from
any product or surface
 Disinfection: Destruction of microorganisms, especially
potential pathogens, on the surfaces of inanimate objects or in
the environment
 STERILIZATION
 Antisepsis: Destruction or inhibition of microorganisms on living
tissues by chemicals (non-toxic and non-irritating)
 Chemical agents- Antiseptic
 Germicide/microbicide: chemical agent that kills pathogenic
microorganisms
 Sepsis: growth of microorganisms in the body or the presence of
microbial toxins in blood and other tissues
 Asepsis: Practice to prevent entry of infectious agents into sterile
tissues and thus prevents infection
 STERILIZATION

 Sanitization: Cleansing technique that mechanically removes

microorganisms to reduce the level of contaminants.
 Sanitizer- compound (e.g., soap or detergent).
 Bacteriostasis- condition where the multiplication of the bacteria is
inhibited without killing them.
 Bactericidal- chemical that can kill or inactivate bacteria. Such
chemicals may be called variously depending on the spectrum of
activity, such as bactericidal, virucidal, fungicidal, microbicidal,
sporicidal, tuberculocidal or germicidal.
 Uses of Sterilization

1. Sterilization for Surgical Procedures and medicines:

Gloves, aprons, surgical instruments, syringes, drugs
and other supplies etc.
2. Sterilization in Microbiological works: Preparation
of culture media, reagents and equipments.
 PHYSICAL METHODS OF STERILIZATION:
Sunlight:
 The microbicidal activity of sunlight is mainly due to the presence of
ultra violet rays in it.
 It is responsible for spontaneous sterilization in natural conditions.
 In tropical countries, the sunlight is more effective in killing germs due
to combination of ultraviolet rays and heat.
 By killing bacteria suspended in water, sunlight provides natural
method of disinfection of water bodies such as tanks and lakes.
Sunlight is not sporicidal, hence it does not sterilize.
 TEMPERATURE (HEAT)

 It is the most common, effective, reliable and productive mean of sterilization

 It is used to sterilize laboratory glassware, laboratory media, surgical instruments
etc.
 ADVANTAGE
 It is economical and easily controllable means of microbial growth
 MECHANISM
 It usually kill microbes by causing denaturation of respective enzymes or
coagulation of
 LIMITATION
 Heat resistance capacity of organisms must be studied carefully and taken
into consideration
Factors affecting sterilization by heat

 Nature of heat: Moist heat is more effective than dry heat

 Temperature and time: temperature and time are inversely proportional. As temperature
increases the time taken decreases.
 Number of microorganisms: More the number of microorganisms, higher the temperature
or longer the duration required.
 Nature of microorganism: Depends on species and strain of microorganism, sensitivity to
heat may vary. Spores are highly resistant to heat.
 Type of material: Articles that are heavily contaminated require higher temperature or
prolonged exposure.
 Certain heat sensitive articles must be sterilized at lower temperature.
 Presence of organic material: Organic materials such as protein, sugars, oils and fats
increase the time required.
 Characterization of Heat Sterilization
 Thermal Death Time (TDT)
 Minimum time required to kill all microbes at a specified temperature.
 Thermal Death Point (TDP)
 Lowest temperature at which all microbes are killed in 10 minutes.
 Heat resistance of each organism is variable hence TDP determines the sensitivity of each organism to
temperature.
 Both TDT and TDP are employed to determine the heat resistance of microbes before sterilization.
 Decimal Reduction Time (DRT)
 It is also termed as D-Value.
 It represents the extent of heat resistance of each microbe.
 It is the time at which almost 90% microbes are killed
 It is usually employed in canning industry for sterilizing fruit concentrates, pulps, vegetables, fish etc.
DRY HEAT

 Red heat
 Articles such as bacteriological loops, straight
wires, tips of forceps and searing spatulas are
sterilized by
holding them in Bunsen flame till they become
red hot. This is a simple method for effective
sterilization of such
articles, but is limited to those articles that can be
heated to redness in flame.
 DRY HEAT

 Flaming
 This is a method of passing the article over a Bunsen flame, but not heating it
to redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides
and cover slips are passed through the flame a few times. Even though most
vegetative cells are killed, there is no guarantee that spores too would die on
such short exposure. This method too is limited to those articles that can be
exposed to flame. Cracking of the glassware may occur.
 DRY HEAT

 Incineration:
 This is a method of destroying contaminated material by burning them in
incinerator.
 Articles such as soild dressings; animal carcasses, pathological material and
bedding etc should be subjected to incineration.
 This technique results in the loss of the article, hence is suitable only for those
articles that have to be disposed.
 Burning of polystyrene materials emits dense smoke, and hence they should
not be incinerated.
 HOT AIR OVEN

 Introduced by Louis Pasteur.

 Articles are exposed to high temperature (160º C) for duration of one hour in an electrically heated
oven.
 Since air is poor conductor of heat, even distribution of heat throughout the chamber is achieved by a
fan.
 The heat is transferred to the article by radiation, conduction and convection.
 Conduction is the process by which heat energy is transmitted through collisions between neighboring
atoms or molecules.
 Convection is a process of displacement of cooler air by hot air.
HOT AIR OVEN (cont..)

 Metallic instruments (like forceps, scalpels, scissors)

 Glasswares (such as petridishes, pipettes, flasks, all-glass syringes)
 Swabs, oils, grease, petroleum jelly and some pharmaceutical
products
 Unsuitable for rubber and plastic
 HOT AIR OVEN (cont..)
 The oven should be fitted with a thermostat control, temperature
indicator, meshed shelves and must have adequate insulation.
 Oven not overloaded
 Materials perfectly dry and arranged to allows free circulation of air
inside the chamber
 Mouths of flasks, test tubes and both ends of pipettes must be plugged
with cotton
 Petri dishes and pipettes wrapped in a paper
 60 minutes at 160º C, 40 minutes at 170ºC and 20 minutes at 180º C.
HOT AIR OVEN (cont..)

 Increasing temperature by 10 degrees shortens the sterilizing time

by 50 percent
 The hot air oven must not be opened until the temperature inside has
fallen below 60ºC to prevent breakage of glassware
 To determine the efficacy of sterilization - Thermocouples,
chemical indicators, and bacteriological spores of Bacillus subtilis
as sterilization controls
HOT AIR OVEN (cont..)
Advantages:
 It is an effective method of sterilization of heat stable articles.
The articles remain dry after sterilization.
 This is the only method of sterilizing oils and powders.
Disadvantages:
 Hot air has poor penetration
 Cotton wool and paper may get slightly charred.
 Glasses may become smoky.
 Takes longer time compared to autoclave.
INFRARED STERILIZATION

 Sterilization by generation of heat

 Articles placed in a moving conveyer belt and
passed through a tunnel that is heated by
infrared radiators to a temperature of 180o C •
 The articles are exposed to that temperature for
a period of 7.5 minutes • Articles sterilized:
metallic instruments and glassware
 Requires special equipment
 Efficiency can be checked using Browne’s
tube No.4 (blue spot)
Moist Heat
 Moist heat invariably kills microbes by coagulation of proteins
which is caused by Hydrogen bond cleavage
 It has high penetration power
 Moist heat sterilization is achieved by following methods:
1. At temperature below 100º C
2. At temperature 100º C
3. At temperature above 100º C
At temperature below 100º C
Pasteurization:
• originally employed by Louis Pasteur
• employed in food and dairy industry
• Two methods of pasteurization
1. • holder method (heated at 63o C for 30 minutes)
2. • flash method (heated at 72o C for 15 seconds) followed by quickly cooling to 13º C
• Suitable to destroy most milk borne pathogens (Mycobacteria, Streptococci, Staphylococci, Brucella
etc.)
• Inactivates most viruses and destroys the vegetative stages of 97–99% of bacteria, fungi, does not kill
endospores or thermoduric species
• Efficacy tested by phosphatase test and methylene blue test
• Newer techniques: Ultra-High Temperature (UHT), 134°C for 1–2 second
At temperature below 100º C

Vaccine bath:
 The contaminating bacteria in a vaccine preparation can be inactivated by heating in a
water bath at 60o C for one hr
 Only vegetative bacteria are killed and spores survive
Serum bath:
 The contaminating bacteria in a serum preparation can be inactivated by heating in a
water bath at 56o C for one hour on several successive days
 Proteins in the serum will coagulate at higher temperature
 Only vegetative bacteria are killed and spores survive
At temperature 100º C

Boiling
 Boiling water (100o C) for 10–30 minutes kills most vegetative bacteria and
viruses
 Certain bacterial toxins such as Staphylococcal enterotoxin heat resistant
 Some bacterial spores are resistant to boiling and survive
 Not a substitute for sterilization
 The killing activity can be enhanced by addition of 2% sodium bicarbonate
 Certain metal articles and glasswares disinfected by placing them in boiling
water for 10-20 minutes
 The lid of the boiler must not be opened during the period
At temperature 100º C

Steam at 100º C
 Subjected to free steam at 100o C
 Traditionally Arnold’s and Koch’s steamers were used
 A steamer is a metal cabinet with perforated trays to hold the articles and a
conical lid • The bottom of steamer is filled with water and heated
 The steam generated sterilizes the articles when exposed for a period of 90
minutes
 Media such as TCBS, DCA and Selenite broth are sterilized by steaming
At temperature 100º C
 Tyndallisation:
 Heat labile media like those containing sugar, milk, gelatin
 Tyndallisation (after John Tyndall)/ fractional sterilization/ intermittent
sterilization
 Steaming at 100°C is done in steam sterilizer for 20 minutes followed by
incubation at 37°C overnight
 Repeated for another 2 successive days
 The vegetative bacteria are killed in the first exposure and the spores that
germinate by next day are killed in subsequent days
 The success of process depends on the germination of spores
At temperature above 100º C
Autoclave:
 Sterilization can be effectively achieved at a temperature above 100º C
using an autoclave.
 In an autoclave the water is boiled in a closed chamber.
 At a pressure of 15 lbs inside the autoclave, the temperature is 121º C.
 Exposure of articles to this temperature for 15 minutes sterilizes them.
 To destroy the infective agents associated with spongiform
encephalopathies (prions), higher temperatures or longer times are used;
135o C or 121o C for at least one hour are recommended.
 At temperature above 100º C
Advantages of steam:
 It has more penetrative power than dry air, it moistens the spores
(moisture is essential for coagulation of proteins), condensation of
steam on cooler surface releases latent heat, condensation of steam
draws in fresh steam.
 Different types of autoclave:
 Simple “pressure-cooker type” laboratory autoclave, Steam jacketed
downward displacement laboratory autoclave and high pressure pre-
vacuum autoclave
 At temperature above 100º C

Autoclave
Vertical or horizontal cylindrical body

Articles sterilized: Culture media, dressings, certain equipment, linen, rubber (gloves), heat-resistant plastics,
liquids etc. Ineffective for sterilizing substances that repel moisture (oils, waxes, or powders)

Kills all the vegetative as well as spore forms of bacteria

Sterilization controls: (a) Thermocouples (b) Chemical indicators- Brown’s tube No.1 (black spot) (c)
Bacteriological spores- Bacillus stearothermophilu
 Autoclave
 Vertical or horizontal cylindrical body
 Articles sterilized: Culture media, dressings, certain
equipment, linen, rubber (gloves), heat-resistant
plastics, liquids etc.
 Ineffective for sterilizing substances that repel
moisture (oils, waxes, or powders)
 Kills all the vegetative as well as spore forms of
bacteria
 Sterilization controls: (a) Thermocouples (b)
Chemical indicators- Brown’s tube No.1 (black spot)
(c) Bacteriological spores- Bacillus
stearothermophilu
Autoclave

Advantage:
 Very effective way of sterilization, quicker than hot air oven.
Disadvantages:
Drenching and wetting or articles may occur, trapped air may reduce the efficacy, takes long
time to cool
RADIATION:
 Ionizing and non-ionizing
Non-ionizing rays
 Low energy rays, poor penetrative power
 Rays of wavelength longer than visible light
 Microbicidal
 Wavelength of UV rays: 200-280 nm, 260 nm most effective
 UV rays induce formation of thymine-thymine dimers, ultimately inhibits DNA replication
 UV readily induces mutations in cells
 Don’t kill spores
Uses of UV radiation:

 Disinfection of closed areas in microbiology laboratory, inoculation hoods,

laminar flow, and operating theaters
 Harmful to skin and eyes
 Doesn't penetrate glass, paper or plastic
 Of use in surface disinfection
 Generated using a high-pressure mercury vapor lamp
RADIATION (cont..):

Ionizing rays
 High-energy rays, good penetrative power
 Radiation does not generate heat- "cold sterilization“
 e.g. (a) X-rays, (b) gamma rays, and (c) cosmic rays
 Gamma radiation from cobalt-60 sourcesterilization of antibiotics, hormones, vitamins,
sutures, catheters, animal feeds, metal foils, and plastic disposables, such as syringes, petri
dishes
 A dosage of 2.5 megarads kills all bacteria, fungi, viruses and spores
 Also used for meat and other food items
 Damage the nucleic acid of the microorganism
 Filtration
 It is the process of removing particles from a solution by allowing the liquid or
gas through a membrane or particle barrier.
 Serum, antibiotic solutions, sugar solutions, urea solution are sterilized by
filtration.
 Two types of filtration are employed
 High Efficiency Particulate Air
 It is used to purify environmental air such as ICU, Burns unit, Industrial sterile
zones etc.
 Membrane Filtration
 Cellulose esters
 Plastic Polymers
 It is employed to sterilize heat sensitive substances, culture media, vaccines,
enzymes and several antibiotic solutions
Types of filters

 1. Earthenware filters:
 Made up of diatomaceous earth or porcelain
 usually baked into the shape of candle
 a. Pasteur-Chamberland filter:
 Candle filters from France, of porcelain
 Various porosities, graded as L1, L1a, L2, L3, L5, L7, L9 and L11
 Similar filter from Britain is Doulton; P2, P5 and P11
Types of filters

 b. Berkefeld filter:
 made of Kieselguhr, a fossilized diatomaceous earth found in Germany
 Three grades depending on their porosity (pore size)
 V (veil), N (normal) and W (wenig)
 c. Mandler filter:
 from America
 made of kieselguhr, asbestos and plaster of Paris
Types of filters

 Asbestos filters:
 Made up of asbestos such as magnesium silicate
 Examples- Seitz and Sterimat filters
 Disposable, single-use discs in different grades
 Tend to alkalinize the filtered fluid
 Use limited, carcinogenic potential of asbestos Sintered glass filters:
 Made up of finely powdered glass particles, which are fused together
 Available in different pore sizes
Types of filters

 Membrane filters:
 made up of
 (a) Cellulose acetate
 (b) Cellulose nitrate,
 (c) Polycarbonate
 (d)Polyvinylidene fluoride,
 (e) Other synthetic materials
 Widely used, circular porous membranes, usually 0.1 mm thick
 Variety of pore sizes (0.015–12 μm)
 Membranes with pores about 0.2 μm are used, smaller than the size of bacteria
 Uses of membrane filters
 Sterilize pharmaceutical substances, ophthalmic
solutions, liquid culture media, oils, antibiotics, and
other heat-sensitive solutions

 To obtain bacterial free filtrates of clinical specimens

for virus isolation

 To separate toxins and bacteriophages from bacteria

 DESICCATION

 Microbes require water for their optimum growth

 Desiccation is a process to expel or extract out the water content.
 When applied to in sterilization, desiccation can temporarily retard or
restrict the growth and reproduction by disrupting metabolism but it
does not completely kill them.
 It is usually applied with Lyophilization
 OSMOTIC PRESSURE
 Osmosis is another important parameter required for optimum growth
and reproduction.
 The sterility is provided by plasmolysis
 Osmotic pressure is the pressure that develops when two solutions are
separated by semi-permeable membrane.
 This process is applied in food industry for preservation using salt or
sugar solutions to retard growth and reproduction of microbes
 CHEMICAL STERILIZATION
 Different chemicals are used to manage and control usual growth of
microorganisms
 Chemical sterilants/ disinfactants should have following properties:
 wide spectrum of activity
 able to destroy microbes within practical period of time
 active in the presence of organic matter
 effective contact and be wettable
 active in any pH
 Stable, speedy, and long shelf-life
 high penetrating power
 non-toxic, non-allergenic, non-irritative or non-corrosive
 Effective Disinfection
 Disinfectant
 Substance that prevents infection by killing an organism
 Selection of disinfectant to be an effective sterilizing agent, following factors must be taken into
consideration:
 The concentration of disinfectant determines the effectiveness
 Disinfectant should be diluted as instructed by manufacturer on the label
 Diluted solutions may act as bacteriostatic rather than a bactericidal
 Nature of the material must be taken into account whether it is organic substance or inorganic substance and the
pH of that disinfectant
 Accessibility of microbes must be taken into consideration
 Higher the temperature used for actual application of disinfectant higher would be its effectiveness or versatility
CLASSIFICATION OF DISINFECTANTS:

 1. Based on consistency
 a. Liquid (E.g., Alcohols, Phenols)
 b. Gaseous (Formaldehyde vapor, Ethylene oxide)
 2. Based on spectrum of activity
 a. High level
 b. Intermediate level
 c. Low level
 3. Based on mechanism of action
 a. Action on membrane (E.g., Alcohol, detergent)
 b. Denaturation of cellular proteins (E.g., Alcohol, Phenol)
 c. Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2, Halogens)
 d. Alkylation of amino-, carboxyl- and hydroxyl group (E.g., Ethylene Oxide, Formaldehyde)
 e. Damage to nucleic acids (Ethylene Oxide, Formaldehyde)
DISINFECTANT VARIANTS