Methods of Sterilization and Disinfection

Veterinary Microbiology (Unit-1)

Dr. Savita Kumari

Department of Veterinary Microbiology
Bihar Veterinary College, BASU, Patna
 Terms:
• Sterilization: Article, surface, or medium is freed of all living
microorganisms either in the vegetative or in the spore state

• Used only in absolute sense

• Disinfection: Destruction of microorganisms, especially potential

pathogens, on the surfaces of inanimate objects or in the
environment

• Reduce the microbial population, not bacterial endospores on

inanimate surfaces or in organic materials

• Antisepsis: Destruction or inhibition of microorganisms on living

tissues by chemicals (non-toxic and non-irritating)

• Chemical agents- Antiseptics

• 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

• Sanitization: Cleansing technique that mechanically

removes microorganisms to reduce the level of
contaminants

• Sanitizer- compound (e.g., soap or detergent)

 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
 METHODS
Sterilization and disinfection are done by :
(A). Physical methods
(B). Chemical methods

(a) Heat:
(i) Dry heat
(ii) Moist heat

(b) Radiation:
(i) Non-ionising radiation (Ultraviolet radiation)
(ii) Ionising radiation (X-ray, gamma ray)

(c) Filtration
(Berkfeld, Chamber land, Seitz, sintered glass, cellulose
membrane filters etc)
Heat
• Mostly used method

• Highly effective and most reliable process

• Two major methods:

• Dry heat and moist heat

• Dry heat induces:

• Denaturation of protein, oxidative damage and toxic effect
due to the high level of electrolytes
• Also damage the DNA of the microorganism

• As a result, the microorganism got killed

..

• Moist Heat:
kills the microorganisms by denaturation and
coagulation of proteins

• Temperature required to kill microbe by dry heat more

than moist heat

• Thermal death time-

Minimum time required to kill a suspension of
organisms at a predetermined temperature in a specified
environment
Factors affecting sterilization by heat
• Nature of heat: Moist heat is more effective

• Temperature and time: inversely proportional

• Number of microorganisms: More number- higher temperature or

longer duration

• Nature of microorganism: Species and strain, Spores highly resistant

• Type of material: heavily contaminated, higher

temperature/prolonged exposure

• Certain heat sensitive articles sterilized at lower temperature

• Presence of organic material: Organic materials (protein, sugars, oils

and fats) increase the time required
DRY HEAT:
• ..
• Red heat:
• sterilized by holding them in Bunsen flame
till they become red hot

• bacteriological loops, straight wires, tips of

forceps and searing spatulas

• limited to those articles that can be heated

to redness in flame

• Flaming:
• method of passing the article over a
Bunsen flame, but not heating it to redness

• e.g. scalpels, mouth of test tubes, flasks,

glass slides and cover

(Image source-Google)
Incineration:
• Method of destroying
contaminated material by
burning them in incinerator

• safely destroying infective

materials by burning them to
ashes

• e.g. soiled dressings; animal

carcasses, pathological material,
bedding

• Suitable only for those articles to

be disposed (Image source-Google)
Hot air oven:
• Introduced by Louis Pasteur

• Metallic instruments (like

forceps, scalpels, scissors)

• Glasswares (such as petri-

dishes, pipettes, flasks, all-glass
syringes)

• Swabs, oils, grease, petroleum

jelly and some pharmaceutical
products

• Unsuitable for rubber and

plastics
..
• Exposed to high temperature in an electrically heated oven

• Air poor conductor of heat, even distribution of heat throughout

the chamber by a fan

• fitted with a thermostat control, temperature indicator, meshed

shelves or trays

• 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

..

• 1600C for two hours, 1700C for 1 hour and 1800C for 30
minutes

• 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 60o C to prevent breakage of glassware

• To determine the efficacy of sterilization

- Thermocouples, chemical indicators, and bacteriological

spores of Bacillus subtilis as sterilization controls
Infra red rays:
• 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)
Sterilization by moist heat
• In the form of
• hot water
• boiling water
• steam (vaporized water)

• In practice, the temperature of moist heat usually ranges from 60 to 135°C

• Adjustment of pressure in a closed container- regulate the temperature of

steam

• kills microorganisms by denaturation and coagulation of proteins

• Sterilization by moist heat:

• temperature <100°C
• temperature of 100°C
• temperature >100°C
At temperature below 100o C
• Pasteurization:
• originally employed by Louis Pasteur

• employed in food and dairy industry

• Two methods of pasteurization

• holder method (heated at 63o C for 30 minutes)
• 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

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
Inspissation:
• Egg and serum containing media(Lowenstein- Jensen’s; Loeffler’s
serum)

• Inspissation means stiffening of protein without coagulation

• Placed in an inspissator and heated at 80-85o C for 30 minutes on

three successive days

• On the first day, the vegetative bacteria would die and those spores
that germinate by next day are then killed the following day

• The process depends on germination of spores in between

inspissation
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

• Syringes, forceps, scissors etc.

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
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:
• Sterilization by steam under pressure

• When a gas is compressed, its temperature rises in

direct relation to the amount of pressure

• 5 psi above normal atmospheric pressure- 109°C

• 10 psi above normal- 115°C
• 15 psi - 121°C

• Pressure–temperature combinations achieved with

special device “Autoclave”

• At 15 lb per sq. inch pressure, 121°C temperatures is

obtained, kept for 15 minutes for sterilization
Autoclave

(Image source-Google)
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 stearothermophilus
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

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 source-

sterilization 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:
• Does not kill microbes, it separates them out

• Membrane filters with pore sizes between 0.2-0.45 μm

• used to remove microbes from heat labile liquids such as serum,

antibiotic solutions, sugar solutions, urea solution

• For removing bacteria from ingredients of culture media,

preparing suspensions of viruses and phages

• Aided by using either positive or negative pressure using vacuum

pumps

• Older filters made of earthenware or asbestos- depth filters

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 (Image source: https://www.nlm.nih.gov)
..

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
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

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
Chemical Methods of Sterilization
• Several chemical agents- antiseptics/ disinfectants
Properties of Ideal Disinfectant
• Wide spectrum of antimicrobial activity

• Should act in the presence of organic matter

• Non-toxic, non-corrosive

• Stable upon storage, no chemical change

• Odorless or with a pleasant odor

• Soluble in water, lipids for penetration into microorganisms

• Effective in acidic as well as in alkaline media

• Speedy action, relatively inexpensive

Action of Disinfectants
• Damage to the cell wall and alter permeability of the cell membrane, resulting in
exposure, damage, or loss of the cellular contents

• Alter proteins and form protein salts or cause coagulation of proteins

• Inhibit enzyme action, nucleic acid synthesis or alter nucleic acid molecules

• Cause oxidation or hydrolysis

Factors Influencing Activity of Disinfectants

• Temperature: Increase in temp. increases the efficiency of disinfectants

• Type of microorganism: Vegetative cells more susceptible than spores. Spores may
be resistant

• Physiological state of cell: Young and metabolically active cells more sensitive

• Environment: Physical or chemical properties of medium or substance

e.g., pH of the medium and presence of extraneous materials
Types of Disinfectants
• (a) phenolic compounds

• (b) halogens

• (c) alcohols

• (d) aldehydes

• (e) gases

• (f) surface active agents

• (g) oxidizing agents

• (h) dyes

• (i) heavy metals

• (j) acids and alkalis

ALCOHOLS:
Mode of action:
• Alcohols dehydrate cells, disrupt membranes and cause coagulation of
protein

Examples:
Ethyl alcohol, isopropyl alcohol and methyl alcohol

Application:
• 70% ethyl alcohol (spirit) is used as antiseptic on skin
• Isopropyl alcohol is preferred to ethanol
• Also used to disinfect surfaces
• Used to disinfect clinical thermometers
• Methyl alcohol kills fungal spores, hence is useful in disinfecting
inoculation hoods

Disadvantages:
• Skin irritant, volatile (evaporates rapidly), inflammable
ALDEHYDES:
Mode of action:
Acts through alkylation of amino-, carboxyl- or hydroxyl group, damages nucleic acids.
It kills all microorganisms, including spores
Examples:
Formaldehyde, Glutaraldehyde

Application:
• Bactericidal, sporicidal, and also effective against viruses
• Can also be used as chemical sterilants

40% Formaldehyde (formalin): for surface disinfection and fumigation

• 10% formalin with 0.5% tetraborate sterilizes clean metal instruments

2% glutaraldehyde
• To disinfect hospital and laboratory equipments

• An exposure of at least 3 hours at alkaline pH is required for action by

glutaraldehyde

• Especially effective against tubercle bacilli, fungi, and viruses

PHENOL:
• Mode of action:
• Act by disruption of membranes, precipitation of proteins and inactivation
of enzymes

• Examples:
• 5% phenol, 1-5% Cresol, 5% Lysol, hexachlorophene, chlorhexidine,
chloroxylenol

• Applications:
• As disinfectants at high concentration and as antiseptics at low
concentrations

• Bactericidal, fungicidal, but are inactive against spores and most viruses

• Effective in the presence of organic material and remain active on surfaces

long after application
HALOGENS:
• Mode of action:
• Oxidizing agents and cause damage by oxidation of
essential sulfydryl groups of enzymes

• Chlorine reacts with water to form hypochlorous acid,

microbicidal

• Examples:
• Chlorine compounds (chlorine, bleach, hypochlorite) and
iodine compounds (tincture iodine, iodophores)

• Application:
• Effective disinfectants and antiseptics
• Microbicidal
• Also sporicidal with longer exposure
HEAVY METALS:
• Mode of action:
• Act by precipitation of proteins and oxidation of sulfydryl groups
• They are bacteriostatic

• Examples:
• Mercuric chloride, silver nitrate, copper sulfate, organic mercury
salts (e.g., mercurochrome, merthiolate)

• Applications:
• Silver compounds as antiseptics
• Silver sulfadiazine for burns
• Silver nitrate in eye infection
• Merthiolate in 1:10000 -preservation of serum
• Copper salts as a fungicide
SURFACE ACTIVE AGENTS:
• Mode of actions:
• Disrupt membrane resulting in leakage of cell constituents

• Examples:
• Soaps or detergents, anionic or cationic
• Anionic detergents- soaps and bile salts

• Cationic detergents are known as quaternary ammonium

compounds (or quat)
• Cetrimide and benzalkonium chloride act as cationic detergents

• Application:
• active against vegetative cells, Mycobacteria and enveloped viruses
• As disinfectants at dilution of 1-2% for domestic use and in hospitals
DYES:
• Acridine dyes, Aniline dyes

• Act by interfering with the synthesis of nucleic acids and proteins in

bacterial cells

• Acridine dyes such as acriflavin and aminacrine

• More effective against gram positive bacteria than gram negative bacteria
and are more bacteriostatic in action

• Aniline dyes (such as gentian violent, crystal violet, and malachite green)

• Also more active against Gram-positive bacteria than against Gram-

negative organisms

• Applications:
• Topically as antiseptics, on skin treat bacterial skin infections
• The dyes are used as selective agents in certain selective media
HYDROGEN PEROXIDE:
• Mode of action:
• Release of nascent oxygen, that damages proteins and DNA of microorganisms

• Application:
• 6% concentration to decontaminate the instruments, equipments such as
ventilators

• 3% for skin disinfection and deodorising wounds and ulcers

• Strong solutions are sporicidal

BETA-PROPIOLACTONE (BPL):
Mode of action:
• Acts through alkylation of carboxyl- and hydroxyl- groups

Application:
• Effective sporicidal agent, broad-spectrum activity, 0.2% to sterilize
biological products

• Carcinogen
ETHYLENE OXIDE (EO):
• Mode of action:
• by alkylating sulfydryl-, amino-, carboxyl- and hydroxyl- groups

• Application:
• Effective chemisterilant, capable of killing spores rapidly

• Highly flammable, used as 10% CO2+ 90% EO) or

dichlorodifluoromethane

• Used to sterilize heat labile articles such as bedding, textiles,

rubber, plastics, syringes, disposable petri dishes, respiratory and
dental equipments

• Highly toxic, irritating to eyes, skin, highly flammable, mutagenic

and carcinogenic