Sterilization 2

Objectives
 Understand and utilize correct sterilization and

disinfection techniques
 Distinguish between sterilization and disinfection
 List the characteristics of an ideal antiseptic
 Describe sterilizing agents and rank their
effectiveness
 Discuss the time/temperature relationship in
destroying microorganisms
STERILIZATION
Definition:
•The freeing of an article from all living
organisms, including viruses, bacteria ,
spores & fungi
•Pathogenic & non-pathogenic.
Uses:
Sterilization is used for :
1.Microbiology : Culture media,
suspending fluids, reagents,
containers & equipment
2.Hospital : medical & surgical
instruments , surgical operations,
I.V. infusions, hypodermic
injections , diagnostic aspirations.
Methods:
Physical or chemical:-
(1)Physical Methods:
* Heat * Filtration * Irradiation
(2) Chemical Methods:
• Strong disinfectants:
*Formaldehyde* Ethylene
oxide
Sterilization By Heat
Moist Heat:
@ Most effective and efficient.
@ Kills organisms by denaturing their
enzymes & proteins
@ Spores are killed by exposure to moist
heat at 121°C for 10-30 minutes.
Dry Heat:
@ Kills organisms by oxidative
destruction of cell constituents.
@ Spores killed at 160°C for 1 hour
Factors influencing Sterilization
by Heat:
1.Temperature & exposure time
Higher temperature, shorter
time and vice versa.
2.No.of Vegetative cells+spores
@ Plenty of organisms , make
sterilization less efficient .
@ So, clean before sterilizing
3. Nature of organism:
Vegetative cells & spores affect susceptibility
of organism to heat.
4. Nature of material
containing organisms:
@ Efficiency of sterilization is reduced by
presence of organic matter that protect
organisms from lethal action of heat
(protein, gelatin, sugar, nucleic acid, fats, oil)
@ Presence of a disinfectant enhances the kill.
@ Spores best killed in acidity or in alkalinity.
The Holding Time:
Time required for killing organism.
@ Not including heating - up time.
The Thermal Death Point:

Lowest temperature to give complete
killing in aqueous suspension within 10
min. at standard conditions.
The Thermal Death Time:
Shortest time for complete killing at a
stated temperature under standard
conditions.
The Decimal Reduction Time
(D-value):
Time in minutes required to achieve a ten
fold reduction in viability of a bacterial
population at a given temperature under
standard conditions.
Methods of Sterilization By
Dry Heat:
1.Red Heat:
Article is held on flame until red hot
(inoculating wires, forceps, spatulas)
2.Flaming:
Burning an article in spirit or gas flame
(scalpels, needles)
Method will not produce sterilization.
 Direct flame
 Can damage the exposed object
3. Hot-air oven:
@ Oven has a thermostat,& a fan to
circulate hot air.
@ Works at 160˚C for 1 hour, to sterilize
glassware, metals , swabs, oils, powder
4. Infra-red Radiation:
@ Given by an electrically heated element
@ To sterilize glass syringes at 160°C &
surgical instruments above 200°C in
vacuum chamber in which N2 is passed
during cooling time to avoid oxidation.
Methods of Sterilization
by Moist Heat:
@ Employed at a temp. below
100°C ,at 100˚C,and above 100°C.
@ First two are disinfection methods,
third is a sterilization method.
Temperature Below 100°C:
@ This is the pasteurization process.
@ Used for milk, vaccines & utensils.
Milk:
@ Pasteurized by 2 methods:-
1. Holder method: at 63°–66°C
for 30 minutes.
2. Flash method: at 72°C for 20
seconds.
@ Both methods destroy only
milk-borne organisms (Myco.TB,
Brucella, Salmonella)
Pasteurization is tested by :-
@ Phosphatase test : testing
for presence of phosphatase
enzyme found in milk &
destroyed by pasteurization.
@ Methylene blue test : To
indicate that bacteria present
in milk is destroyed.
Coxiella burneti is destroyed by flash method.
Vaccines:
Inactivated in the vaccine
water bath, at 60°C for 1 hr
House articles:
Utensils, clothing, bedding,
are disinfected at 70°–80°C
for several minutes.
Temperature at 100°C:
Boiling at 100°C:
@ Kills non- sporing organisms
within 5 – 10 min.
@ Used to disinfect blades,
syringes,
@ Dry article on removing from
boiler (sterilizer) to prevent
contamination
Steaming at 100°C:
@ This is done by the
steamer (Koch Steamer).
@ It uses steam of boiling
water at 100°C and at
atmospheric pressure.
@ Steamer is used in two ways:-
1. Single exposure at 100°C for 90
minutes.
@ Time includes heating-up
time.
@ Thermophilic & mesophilic
spores survive this treatment .
2. Tyndallization: Exposure at 100°C
for 20 – 45 minutes for 3 successive days.
@ Used for sterilizing sugars & gelatin
@ First steaming kills vegetative bacteria
& spores germinating following day are
killed by subsequent heating and so on.
@ Draw-back of tyndallization : spores
not germinating in medium sterilized +
thermophilic & anaerobic bacteria will
escape killing.
Temperature above 100˚C:
@ Uses saturated steam - better
than dry hot air because:-
1. Lethal action of moist heat is more
Quicker in :
• heating up exposed particles
• penetrating cotton stoppers, paper
wrappers, surgical linen and hollow
apparatus.
How Does it Act?
@ Saturated steam when meets
an article, it condenses to a small
volume of water & liberates its
latent heat to article surface.
@ Avoid presence of air that
prevents steam penetration into
article.
What is the apparatus Used?
@ Autoclave : provides
sterilization by dry saturated
steam (steam at point of
condensing to water)
@ Steam is under pressure
higher than atmospheric.
Importance of Air Discharge:
@ Air is removed from autoclave:

a) Mixture of steam + air lower
the temperature.
b) Air hinders penetration of
steam in the load .
@ Air denser than steam, sinks
down & makes a layer over load
@ Sealed bottles containing
solutions are autoclaved
although air is present in them
Because water present in
these solutions is heated up to
steam temp. and so performing
same work as moist heat.
Types of Autoclaves:
1. Simple Autoclave:
@ Pressure-cooker type, simple, not
jacketed, & used in laboratory.
@ Consists of a cylinder for water to
be heated, articles placed on a tray
@ Has got a discharge valve, +
pressure & temperature gauges.
@ Autoclave is opened at
right time because:
* If opened still under

pressure, an explosion occurs
* If opened below atmos.
pressure, evaporation of
aqueous materials occurs.
Simple Autoclave Deficiencies :
1. Lacks control of air discharge
(nothing to show that

discharge is complete)
2. Lacks means of drying load
after sterilization.
@ Suitable for load wrapped in
paper to prevent contamination.
2. Steam-Jacketed
Autoclave:
@ Has got an automatic air
& condensate discharge.
@ Load is dried by steam
circulating in jacket, &
vacuum in autoclave.
3. High Pre-Vacuum Sterilizers:
@ Have electric pumps creating a
vacuum area in chamber.
@ First a vacuum is drawn, steam is
admitted to chamber, load is
heated very rapidly.
@ Temp. used 135˚C for 3 minutes
at 30 lb pressure & load dried by
exhaustion of chamber
Advantages of high pre-
vacuum sterilizers :-
1.Operation time is shortened.
2. Damage to heat sensitive

materials (sugars) is avoided.
Autoclave Control and
Sterilization Indicators:-
1. Automatic process control,
* Advantages are:-
@ Saving time of an operator.
@ Safeguard against errors
due to negligence.
2. Recording Thermometer,
@ Draws graphic records of

temperature changes inside
chamber discharge channel.
@ Helps to avoid errors in

timing the holding period.
3. Thermocouple load temp.
measurement
@ Thermocouple is inserted
deeply inside load, its wire is
carried to a potentiometer
which reads temperature
inside the load .
4. Chemical indicators.
a)Browne’s Tubes, containing a red
fluid that turns green on heating:
* at 115˚C for 25 minutes (Type 1)
* at 115˚C for 15 minutes (Type 2)
* at 160˚ C for 60 minutes (Type 3)
@ Tubes are stored below 20˚C to
avoid change of colour .
@ Satisfactory for routine work .
b) Bowies-Dick tape,
@ Used to test efficiency of high
pre-vacuum sterilizers & high-
pressure autoclaves
@ An adhesive tape is applied around
load in the shape of (X).
@ After sterilization, tape changes
colour all over .
5. Spore indicators,
@ B. Stearothermophilus
spores are destroyed at
121˚C for 12 minutes.
@Spores placed within load
& cultured after sterilizing
Disadvantages of Chemical
and Spore Indicators:
@ Will not give a perfect efficiency of
sterilization :
( heating may be inadequate in a site away
from of indicator)
* To solve problem, autoclave is correctly
operated & controlled by a
thermometer & not by pressure gauge
alone
Sterilization by Radiation
1. Ultra-violet Radiation:
@ U.V. rays induce thymine
diamers in cell DNA, to destroy
bacterial cell .
@ Produced by mercury vapour
lamps, used to sterilize plastics
2. Ionizing Radiation:
@ High-speed electrons, X-ray,
gamma rays, using cobalt 60
@ Produces free DNA radicals that
destroy bacteria .
Indicator for Radiation:
Micrococcus radiodurans that
resists radiation (has efficient
DNA repair)
Ultraviolet irradiation 
Destroys microorganisms in air, liquid, and 

surface
Germicidal UV radiation is generated by 
passing electricity through mercury vapor in
special glass tubes
UV lamps sterilize most effectively in still air at 
room temperature
Cold sterilization 
Gamma radiation 
Kill microorganisms without a rise in temperature 
Highly successful in sterilizing 

Syringes 
Stitching (sutures) materials 
Containers 
Ethylene oxide 
Makes possible the use of low cost, plastic 
materials for sterile, disposable medical
instruments
Primary gas used in hospitals and clinics to 
sterilize items that cannot withstand steam
sterilization
Vapors are hazardous to people 
Filtration 
Complete removal of microorganisms and 
particles of a certain size from liquid or
gas
Used by medical personnel in producing 
sterile and particle-free fluids, such as
intravenous fluids
Sterilization by Filtration
@ Used to sterilize toxins,
serum, antibiotics
@ Uses a filter of pore less than
0.75 µm for bacteria, & much
smaller for viruses.
@ Bacterial filters allow viruses,
& Mycoplasma to pass through.
Test of Filter:
@ Retain Serratia marcescens
from broth culture.
Type of Filters:
1. Asbestos disk (Seitz):
@ Consists of an upper cylinder
and a lower funnel with an
asbestos pad in between.
Grades of filter pads :
1.Rapid clarification of fluids.

2. General clarification.
3. Clarifying viscous fluids.
4. General sterilization.
5. Removing pyrogenes.
6. Liquids sensitive to alkalis.
7. Removing small organisms.
8. Sterilizing serum.
Disadvantage :
Asbestos pads absorb some of
filtrate.
2. Sintered glass filters ;
Made up of ground glass, fused to make
glass granules adhere together .
3. Cellulose membrane filters :
@ Less absorptive, has got a greater
rate of filtration than Seitz filter.
@ Used to separate viruses.
* Two types of membrane filters:
a) Cellulose nitrate( Gradocol membrane) .

b) Cellulose acetate: commonly used now.
@ Membrane is 120 µm thick,
placed in two layers.
@ Bacteria are trapped in upper
layer :better than Seitz filter
* Micro-filters:
@ To filter small fluid volumes @
May be membrane, asbestos @
May be syringe filter or
centrifuge filter.

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Objectives

 Understand and utilize correct sterilization and

The Thermal Death Point:

@ Air is removed from autoclave:

* If opened still under

(nothing to show that

1.Operation time is shortened.

2. Damage to heat sensitive

@ Draws graphic records of

@ Helps to avoid errors in

Destroys microorganisms in air, liquid, and 

Highly successful in sterilizing 

1.Rapid clarification of fluids.

a) Cellulose nitrate( Gradocol membrane) .

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