STERILIZATION AND

DISINFECTION
Terminology Relating to the Control of Microbial Growth
• Sterilization
• Complete elimination of all forms of microbial life including
the spores
• It is an absolute term

• Disinfection
• Destruction of all pathogens or organisms capable of producing
infections but not necessarily spores.
All organisms may not be killed but the number is reduced to a
level that is no longer harmful to health.
Antiseptics
Chemical disinfectants which can be safely applied to living tissues and
are used to prevent infection by inhibiting the growth of
microorganisms
Asepsis
The technique of preventing the occurrence of infection into an
uninfected tissue is prevented.
Sanitization
The process of lowering microbial counts to safe public health levels
and minimize the chances of transmission from one person to another
Terminology Relating to the Control of Microbial Growth
• Sunlight
• Possesses appreciable bactericidal activity due to presence of
ultraviolet & heat rays

• Its sterilising power varies according to circumstances under

natural condition

• Natural method of sterilisation of water in tanks, rivers and

lakes
 Heat
• Common & best method of sterilization
• Use other methods only if there is a good reason not to use it
e.g. damage to material
• Different microbes have different capacities in terms of heat
resistance.
• Thus, these differences can be expressed through the concepts
of:
• Thermal death point (TDP)
• thermal death time(TDT)
• Decimal reduction time (DRT),
TDP: the lowest temp at which all the microbes in a particular
liquid suspension are killed in 10 minutes.

• TDT: the minimal length of time for all bacteria in a particular

liquid culture to be killed at a given temperature

• TDP and TDT are used to indicate the severity of treatment

required to kill a given population of bacteria.

• DRT or D value is the time in minutes, in which 90% of a

population of bacteria at a given temperature will be killed
The factors influencing sterilisation by heat are:

Nature of heat
Temperature and time
Number of microorganisms present
Characteristics of the organisms, such as species, strain and
sporing capacity
Type of material from which the organisms have to be
eradicated
• Mechanism of action of heat sterilisation

• Dry heat Kills organisms by protein denaturation,

oxidative damage and toxic effects of elevated levels of
electrolytes

• Moist heat Kills microorganisms by coagulation and

denaturation of their enzymes and structural proteins
 Dry heat
•Dry heat methods include:
• Incineration,
• Flaming,
• Hot air oven,
• Red heat
 Incineration:
• Materials are reduced to ashes by burning.
• Instrument used is incinerator to burn
• Soiled dressings
• Animal carcasses
• Bedding
• Pathological material
 Flaming
• Materials are passed through the flame of a Bunsen
burner without allowing them to become red hot.
• Glass slides
• scalpels
• Mouths of culture tubes and bottles
 Red heat
• Materials are held in the flame of a Bunsen burner till
they become red hot.
• Inoculating wires or loops
• Tips of forceps
• Surface of searing spatulae
• Needles
 Hot-air oven
• Most widely used method of sterilisation by dry heat

• It is used to process materials which can withstand high

temperatures, but which are likely to be affected by contact
with steam

• It is a method of choice for sterilisation of glassware, forceps,

scissors, scalpels, swab sticks packed in test tubes & sealed
• Materials such as oils, jellies and powders which are
impervious to steam are sterilised by hot air oven

• Hot air oven is electrically heated and is fitted with a

thermostat that maintains the chamber air at a chosen
temperature

• Fitted with a fan that distributes hot air in the chamber

Tests of efficiency
• Browne tube,
• living spore suspension
Hot air oven
 Holding temperature & time

• General purpose Temperature and time:

• 1600C for 2 hours
• 1700C for 1 hour
• 1800C for 30 minutes
• Cutting instruments such as those used in ophthalmic surgery,
Should be sterilized at 1500C for 2 hours
• Oils, glycerol and dusting powder should be sterilised at 1500C
for 1 hour
 Precautions
• Should not be overloaded
• Arranged in a manner which allows free circulation of air
• Material to be sterilized should be perfectly dry.
• Test tubes, flasks etc. should be fitted with cotton plugs.
• Petri dishes and pipettes should be wrapped in Al foil.
• No rubber and inflammable materials inside
• The oven must be allowed to cool for two hours before
opening, since glass ware may crack by sudden cooling.
 Moist heat
• More effective than dry heat

• Vegetative organisms die in a few minutes at 70oC

• Moist heat is divided into three forms
• Temperature below 1000C
• Temperature of 1000C
• Temperature above 1000C
 Temperatures below 1000C

• Pasteurization
• Vaccine bath
• Water bath
• Inspissation
• Low temperature steam-formaldehyde (LTSF)
sterilization
 Pasteurization
• Milk is sterilised by this method; Two methods
• Holder method (630C for 30 min followed by rapid cooling to 130C or
lower)
• Flash method (720C for 15-20 seconds followed by rapid cooling to
130C or lower)
• The dairy industry sometimes uses ultra high temperature
(UHT)sterilization (140 to 1500C for 1-3 seconds followed by rapid
cooling to 130C or lower)
• All nonsporing pathogens such as mycobacteria, brucellae and
salmonellae are destroyed by these processes
• Coxiella burnetii is relatively heat resistant and may survive the holder
method
Inspissation
• Media containing egg or serum such as Lowenstein-Jensen and
Loeffler’s serum slope are rendered sterile by heating at 80-850C for
30 min on three successive days

• This process is called inspissation and instrument used is called

inspissator

LTSF sterilization
• Used for sterilizing items which cannot withstand the temperature of
1000C

• In this method steam at 750C with formaldehyde vapor is used

 Autoclave
• Steam under pressure in a chamber where air is replaced by pure
steam

• Pressure 15lbs/sq inch – 121oC. Spores are killed in 15 mins

30lbs/sq inch - 134oC. Spores are killed in 15 mins

Higher pressure, higher temp, less time is needed for

autoclaving
• Large industrial autoclaves are called retorts
 Autoclave cycle
• Purge Phase: Steam flows through the sterilizer and starts to
displace the air; temperature and pressure ramp slightly to a
continuous flow purge.

• Exposure (Sterilization) Phase: in this phase, autoclave’s control

system is programmed to close the exhaust valve, interior temp. and
pressure increase to desired set point and hold the temp till desire
time.

• Exhaust Phase: Pressure is released from the chamber through an

exhaust valve to restore ambient interior pressure
Simple autoclave
https://www.youtube.com/watch?v=j23s_cxwxSE
https://www.youtube.com/watch?v=j23s_cxwxSE
 Autoclave 2
• Action – saturated steam condenses on the object, this raises
the temp

• Checks of function
• Thermocouple
• Spore strip
• Browne’s tube
• Autoclave tape
 Autoclave
• Simple autoclave disadvantages
• Inefficient air removal
• Wet load
• Trapped air – low temp

Steam jacketed autoclave/ porous load autoclave

pack, extract air, steam enters, holding time, dry load, remove
Steam-jacketed autoclave
 Autoclave
• New autoclaves
• Automatic process, start button & cycle runs
• Graphic record of every cycle

• Bowie Dick test

• Uniform intensity of strips shows adequate air removal
If test fails call engineers
 Flash autoclave

• High temp
• Unwrapped items
• 134oC for 3 mins
• Instruments are available quickly for use
 Uses

• Surgical instruments
• Dressings
• Culture media

• All with autoclave tape attached to show efficiency

 Filtration
• Passage of a liquid or gas through a screenlike material
with pores small enough to retain microorganisms.
• Removes dead and live microbes from:
• Solutions- media, enzymes, vaccines & antibiotic solutions.
• Air- operating theaters and rooms occupied by burn patients
• Other gases
 Filters
• Different types
• Asbestos
• Sintered glass
• Membrane filters (made of cellulose esters or plastic
polymers) are of different pore sizes
• pore size 0.22-mm and 0.45-mm are intended for bacteria.
• Millipore membranes <0.1µm or less can hold back viruses and
large proteins
 Applications in lab
• Remove bacteria from viral culture media
• Sterilization of heat sensitive substances
• Growth medium
• Serum
• Drugs
• Separate bacteria from toxins etc
 Applications
Water
• Home
• Industry
Air – High-efficiency particulate air (HEPA) filters
• remove almost all microorganisms larger than about 0.3 mm
in diameter
• Safety cabinets
• Laminar flow rooms –operation, isolation, clean rooms in
industry eg pharmaceuticals
 Radiation
• Non-ionizing (UV light) long wave lengths >1nm
• Causes genetic damage(thymine dimerisation), less penetrating
• Surfaces – safety cabinet

Ionizing
• has wave length < 1nm
• produced from radioactive isotopes, highly penetrating e.g. ɣ-rays,
X-rays, high energy electron beams
• Ionizes H2O, which forms highly reactive hydroxyl radicals
• radicals kill organisms by reacting with organic cellular
components, especially DNA
 Industrial uses

• Instruments – syringes, needles, gloves, IV lines, catheters

• Sterilise containers

• Pharmaceuticals products – vaccines,

• Food
 Chemicals
• Ethylene oxide – colourless explosive gas

• Uses
• Industry for sterile substances

• Disadvantages
• High cost
• Hazardous, need special equipment
 Chemical 2
• Gluteraldehyde (Cidex)
• 2% alkaline solution is cidal for
• Bacteria
• Fungi
• Viruses

Use to sterilize equipment destroyed by heat e.g.

endoscopes (contain lenses)
 Formaldehyde
• Irritant water soluble gas
Use to sterilse
• Rooms and furniture
• Fabrics, blankets, wood, leather

• Formaldehyde/steam 80oC
• Heat sensitive equipment
 Hypochlorites
• Examples
• Household bleach
• Parazone

• Cheap
• Can be diluted
• 1:10– for spilt blood
• 1:100 -- decontamination
• 1:1000 – for wiping floors
 Disinfection

• Theoretically disinfected objects could transmit infection

• But the possibility is highly reduced

 Heat disinfection
• Low temperature steam 71 – 75oC

• Pasteurization 55-75oC
• Removes vegetative organisms but not spores

• Milk, serum, vaccines

 Boiling(heat at 100oC)

• Clean well before boiling

• Forceps, scissors, tubings

Steaming at 100oC – atmospheric pressure

Tyndallization – intermittent exposure at 20-45 mins on 3

consecutive days
sugar containing media
 Conditions affecting Chemical disinfection
• Satisfactory contact- grease, protein
• Concentration- wetting agent eg alcohols
• Neutralization – hard water, soap
• Stability – dilution ,deterioration
• Speed of action – some act slowly
• Range of action – all bact/G-ve,G+ve
• Cost – savlon very expensive for environmental use
 Indications for Chemical use
• Skin and mucous membrane

• Instruments which are damaged by heat

• Decontamination of surfaces

• To make items safe to handle

 Types of disinfectants
• Phenolics
• Halogens
• Alcohols and aldehydes
• Detergents
• Metallic salts
 Phenolics
•E.g. lysol, cresol
•Strong smell

•For decontamination of environment,

not food preparation areas
•For toilets etc
 Halogens--Cl, I,
• Inexpensive & fast acting
• Active against all organisms
• High conc. Very toxic and corrosive
• After dilution it can loose potency

• Eg- Cl = household bleach, milton

• I = tincture of iodine(2% I in 70% alcohol)

• povidone iodine = surgical scrub
 Alcohols
• Evaporates quickly and leaves dry surface
• E.g. ethyl alcohol
• isopropyl alcohol
• 70% in water is a skin
disinfectant
• disinfect surfaces, hands,
thermometers
 Aldehydes

• Formaldehyde – cidal for all

• Potential carcinogen
• Embalming agent

• Gluteraldehyde– causes eye, skin irritation

• High level disinfectant
• Endoscopes, anaesthetic equipment
 Others

• Hydrogen peroxide 3%
• Soft contact lenses

• Virkon

• Irgasan – antiseptic soaps

Detergents & others
• E.g. cetavlon, cetrimide
• Some G-ve like Pseudomonas can grow in
it

• Metallic compounds – mercuric chloride,

silver nitrate
• Dyes-crystal violet
• brilliant green
• acridine orange
 Rational approach to sterilization and disinfection
• Critical items at high risk of infection should be sterilised

• Semi critical
In contact with mucous membrane & skin (not intact)
• High level disinfection- endoscopes
• Gluteraldehyde, H2O2, Chlorine

• Non-critical
• Contact with skin but not mucous membrane, eg bed pans,
furniture – Phenolics, alcohols
• What is the principal mechanisms of action of the following physical
methods of microbial control?
• Boiling and flowing steam, pasteurization, autoclaving, flaming , red
heating, hot air sterilization, incineration, refrigeration, desiccation,
filtration, deep freezing, lyophilization, high pressure, osmotic
pressure, ionising radiation non-ionizing