STERILIZATION AND DISINFECTION

STERILIZATION: Process by which an article, surface or medium is freed

of all microorganisms either in vegetative or spore
state

DISINFECTION: Destruction of all the pathogenic organisms (organisms

that are capable of giving rise to infection)

DISINFECTANT: Chemicals used for disinfection

ANTISEPSIS: Prevention of infection, usually by inhibiting the

growth of bacteria of wounds or tissues

ANTISEPTIC: Chemical disinfectant that can be used safely on

mucous membrane or skin to prevent infection
 METHODS
PHYSICAL CHEMICAL
1. Sunlight 1. Alcohols
2. Drying - Ethyl alcohol, methyl
3. Dry heat alcohol, isopropyl alcohol
- Flaming 2. Aldehydes
- Incineration - Formaldehyde,
- Hot air oven glutaraldehyde
4. Moist heat 3. Dyes
- Below 1000C - Aniline, acridine
~ Pasteurization 4. Halogens
~ Vaccine bath 5. Phenols
~ Inspissation 6. Surface active agents
- At 1000C 7. Metallic salts
~ Boiling 8. Gases
~ Steam under normal - Ethylene oxide
pressure - Formaldehyde
~ Tyndallization - BPL
- Above 1000C
~ autoclave
5. Filtration
- Sintered glass filters
- Candle
- Asbestos pad
- Membrane
6. Radiation
- Non-ionizing
- Ionizing
FACTORS INFLUENCING STERILIZATION
1. Nature of heat – dry or moist
2. Time and temperature
3. Number of organisms and character (i.e. species, strain, sporing, etc)
4. Type of material to be sterilized
5. Organic load

MECHANISM OF ACTION:
1. Protein denaturation
2. Oxidative damage
3. Toxic effects of elevated electrolytes

THERMAL DEATH TIME:

Minimum time required to kill suspension of organisms at a pre-
determined temperature in a specified environment
STERILIZATION BY DRY HEAT

1. FLAMING Item to be sterilized is held in a flame till it becomes

‘RED HOT’
Inoculating loops, wire, tip of forceps, etc
If infected, they should be dipped in chemical
disinfectant and then flamed
2. INCINERATION Excellent method – rapid destruction
Soiled dressing, animal carcasses
Bedding, pathological materials

3. HOT AIR OVEN

- Most widely used method
- It is double walled chamber of inner copper plate and outer asbestos.
Chamber has several adjustable racks.
- It is used for sterilization by dry heat. It kills all vegetative bacteria
and spores
- Prerequisites:
o Should not be over crowded
o Items should be arranged to ensure free flow of air between
materials
o Glassware – perfectly dry
o Testtube and flasks – plugged with cotton
o Petri dishes and pipettes – wrapped in kraft paper
- Sterilization temperature and holding time:
o 1600C for 1 hour
o 1800C for ½ hour
- It should be allowed to cool slowly and then the door is opened, if not,
the glassware gets cracked
- Uses:
o Sterilization of glassware (testtubes, flasks, petriplates, pipettes,
measuring cylinders)
o Sterilization of oils, grease, powders, liquid paraffin
- Sterilization Controls:
o Biological: spores of non-toxigenic strain of Clostridium tetani
o Physical: Thermocouples
o Chemical: Browne’s tube
 MOIST HEAT
BELOW 1000C

A. PASTEURIZATION:
- Holder method : 630C for 30 min
- Flash method : 720C for 15-20 s followed by rapid cooling to 130C or
lower
- All non-sporing bacteria like Mycobacteria, Brucella and Salmonella
are destroyed

B. VACCINE BATH
- 560C for 1 hour
- Used for sterilization of vaccines, sera, body fluids

C. SERUM INSPISSATOR
- This is a chamber made up of copper plates with an outer water jacket.
- The water jacket contains holes in chamber at a level higher than that
of water so that water keeps the medium moist.
- The chamber is closed with a glass lid and temperature is regulated
automatically
- Tubes containing media are heated at 80-850C for 30 mins daily for 3
successive days
- Uses:
Certain media containing protein like Loeffler’s serum slope, Dorset’s
egg medium, Lowenstein Jensen’s medium cannot be exposed to high
temperatures (proteins get denatured) for sterilization. Such media are
sterilized by inspissation
 AT 1000C

B. BOILING
- Vegetative bacteria are destroying at 90 – 1000C almost immediately
- Spore bearing bacteria require longer time (not a reliable method for
these)
- Hard water should not be used
- 2% sodium bicarbonate – enhances the effect
- Cases were boiling is considered adequate, material should be
immersed in water and boiled for 10 – 30 minutes

C. STEAM AT ATMOSPHERIC PRESSURE

i. Koch’s steamer
ii. Arnold steamer
- Koch or Arnold steam sterilizer consists of cylinder with perforated
tray through which free steam passes and then escapes through an
opening at the top.
- It may be gas or electrically operated
- The articles to be sterilized are exposed to steam at atmospheric
pressure. The boiling point of water and temperature of such steam is
1000C, the exposure time is 90 minutes. A single exposure of 90
minutes usually ensures sterilization,
However, for media containing sugars or gelatin, an exposure of 1000C
for 20 minutes for 3 successive days is used. This is known as
tyndallization or intermittent sterilization.
- Most vegetative forms are killed by these methods except spores of
certain anaerobes and thermophiles.
- Uses:
It is used to sterilize media (containing sugar and gelatin) which will
decompose if subjected to higher temperatures
 ABOVE 1000C

AUTOCLAVE (STEAM UNDER PRESSURE)

- It is a double walled chamber, outer layer made up of gun metal or hard
stainless steel. It has a steam tap at top and pressure gauge with safety
valve.
- It is used for moist heat sterilization above 1000C.
- It kills all forms of life including spores.
- Principle:
• Normally water boils at 1000C at atmospheric pressure and forms
steam
• When water is boiled in a closed vessel, the enclosed space
becomes saturated with steam and the pressure inside increases
slowly.
• As the pressure increases, temperature at which water boils and
temperature of steam also increases
• Steam at higher temperature has higher penetrative power. When
it comes in contact with cooler surface, it condenses into water
giving its latent heat to that surface.
• This process continues till the temperature of that surface equals
that of steam
- Sterilization temperature: 1210C
- Sterilization time: 15 mins
- Pressure used: 15 pounds
- Uses:
i. Sterilization of culture media, surgical dressing, gloves, OT
instruments, rubber articles.
D. Sterilization Controls:
- Biological: spores of Bacillus stearothermophilus
- Physical: Thermocouples
- Chemical: Browne’s tube
 FILTRATION
- Used for heat labile liquids – Antibiotic solution, media containing
sugars, sera, hydatid fluid, vaccines, etc
- To get bacteria free filtrate of toxins and viruses
- Earthenware filters widely used for purification of water
- To separate microorganisms present in scanty numbers in large volumes
of liquid
- Under normal sterilization grades, filters will not hold mycoplasma and
viruses, hence not suitable for clinical use

- TYPES:
1. Earthenware candle filters
a. Barkefeld candle filter (made of diatomaceous earth)
b. Chamberland candle filter (made of unglazed porcelain)

2. Asbestos disc filters

a. Seitz filter
Carcinogenic potential of asbestos has discouraged its use

3. Sintered glass filter

- Prepared by heat fusing finely powdered glass particles of
graded sizes

4. Colladion or membranous filters

- The pore size of filters is around 0.75 ùm in diameter which
retains bacteria. Membrane filters with smaller pore size are
also available which can retain some viruses too.
 RADIATION

1. IONIZING
- X-ray, gamma rays, cosmic rays
- Lethal to DNA and vital cell constituents
- High penetrating power
- No increase in temperature (a/k/a cold sterilization)
- GAMMA RAYS:
Used for sterilization of plastic, syringes, swabs, culture plates,
catheters, rubber, fabric, greases, oil, animal feeds, etc

2. NON-IONIZING
- Electromagnetic waves of wavelength more than visible light –
absorbed as heat
- IR – hot air sterilization
- Mass sterilization of syringes, catheters
- UV rays – enclosed areas like entry ways, hospital wards, operation
theatres, labs, etc