STERILISATION

Definition;
Sterilization is the freeing of an article
from all living organisms, including
bacteria and their spores.
Definition cont…
• The sterilization of culture media, containers and
instruments is essential in bacteriological work
for the isolation and maintenance of pure
cultures.
• In surgery and medicine, the sterilization of
instruments, drugs and other supplies is
important for the prevention of infections.
• The efficiency and efficacy of sterilization in the
hospital lab and elsewhere may from time to
time be confirmed by biological testing.
 METHODS OF STERILISATION
• Sterilization can be effected in a variety of
ways which can be conveniently categorized
as follows.
1.Physical methods
a)Heat
i) Dry heat
ii) Moist heat
b) Radiation.
 Methods cont…
i) Ultra-violet radiation.
ii) Ionizing radiation
c) Filtration
2. Chemical methods
Sterilization cont….
• Heat is often used, since it is generally the
simplest and most reliable means of sterilization.
• Bacteria-stopping filters are used to sterilize
liquids that would be spoiled by heat, e.g. blood
serum, antibiotic solutions.
• Chemical agents are generally less reliable than
heat; they are used mainly for disinfecting the
skin, floors, furniture and other articles that
cannot be heated effectively without damage.
 Sterilization cont…
• Once articles have been rendered sterile they
must be protected from contact with unsterile
objects and from exposure to airborne dust or
else they will become recontaminated. To
ensure maintenance of sterility, articles are
usually enclosed in a dust-proof container or
wrapping before being sterilized.
Subsequently they remain protected by it until
required for use.
 STERILIZATION BY HEAT
• Heat can be applied in two forms:
-Dry heat
-Moist heat
Moist heat is more effective than dry heat. It kills
microorganisms by coagulating and denaturing
their enzymes and structural proteins, a process
in which water participates. Sterilization of most
resistant spores, generally requires moist heat at
121oc. For 15-30 minutes. Culture media since
they contain water, must be sterilized by moist
heat.
 Heat sterilization cont….
• Dry heat kills microorganisms by oxidative
destruction of essential cell constituents.
Killing of the most resistant spores by dry heat
requires a temperature of about 160oc. For 60
minutes.
• Dry heat is mainly employed for glassware,
syringes, metal instruments and are required
dry.
 Heat sterilization cont…
• It can also be used to sterilize anhydrous fats,
oils and powders which are impermeable to
moisture and thus cannot be sterilized by
moist heat.
 Factors Influencing Sterilization by
Heat.
1. The Temperature and Time of exposure
2. The number of microorganisms and spores
present
3. The species, strains and spore-forming ability
of microorganisms
4. The nature of the material that contains the
microorganisms.
 Factors cont….
1. Temperature and time of exposure. The
temp and time for killing microorganisms is
inversely related. i.e. shorter times require
higher temperatures.
• For surgical and bacteriological sterilization, a
10 – 12 minute exposure of microorganisms to
moist heat at 1210c is sufficient.
Note: When articles are being sterilized by
exposure to heat, the total duration of the
 Factors cont…
exposure must include time for the article to
become heated up to the sterilizing
temperature, in addition to the recommended
minimal sterilizing time at that temp.
2. The number of microorganisms and spores
affects the rapidity of sterilization. The
number of microorganisms surviving
diminishes exponentially with the duration of
heating, and the time of complete sterilization
 Factors cont…
increases with the number initially present. In
practice, it’s advantageous to minimize
bacterial contamination by cleaning
procedures before applying heat for the
purpose of sterilization.
3. The species, strain and spore-forming ability
of the microbe greatly affect it’s susceptibility
to heat. The amount of heat required to kill a
given variety is normally stated in terms of the
 Factors cont…
temp and time of exposure, either as the
thermal death point (the lowest temp to give
complete killing in an aqueous suspension) or
as thermal death time (i.e. the shortest time
for complete killing at a stated temperature)
e.g. the thermal death point of some strains of
staphylococcus aureus is about 62oc for half
an hour where as Bacillus anthracis spores will
resist moist heat for 5-10 minutes at 100oc.
 Susceptibility to moist heat
- Vegetative forms of most bacteria, yeasts and
fungi are killed in 10 min at 50oc -65oc
- Thermophillic bacilli e.g. B.stearothermophilus
can have their vegetative forms grow at temp
of up to 80oc.
- Poliomyelitis requires heating at 75oc for 30
min to die.
 Susceptibility to moist heat cont…
- Many bacteriophages are more resistant than
their host bacterium. Heat at 60oc for 15-30
min will kill the bacterium without affecting
the phage.
- The spores of bacterial species are killed by
moist heat in 10 min at temp between 100oc-
121oc.
 Susceptibility to dry heat.

- For vegetative bacteria, dry heat at 100oc for

60 min is required to kill strains that would
succumb to moist heat at 60oc in 30 min.
- Fungal spores are killed in hot air at 115oc
within 60 min and bacterial spores are killed
at temp between 120oc-160oc.
 Factors cont…
4. The nature of material in which the organisms
are killed may affect the rate of killing. A high
content of organic substances generally tends
to protect spores and vegetative organisms
against the lethal action of heat. E.g. proteins,
sugars, starch, nucleic acids, fats and oils.
- The effect of fats and oils is greatest with
moist heat since they prevent access of
moisture to the microbes.
 Factors cont…
- The pH is important; heat resistance of spores is
greatest in neutral media and reduces with
increasing acidity or alkalinity.

Practical example; Spores of cl. tetani whose

killing required moist heat at 100oc for 29 min
at pH 7.2, were killed in only 11 min at pH 10.2.
 Factors cont…
• Boiling of metal instruments in water
containing 2% Na2Co3 gives as effective killing
in 10 min as boiling in plain water for several
hours.
 STERILISATION BY DRY HEAT
• Under dry heat, the following methods can be
employed;
i) Red Heat
ii)Flaming
iii)Hot-Air Oven
iv)Incineration.
• Red Heat. Used for sterilizing inoculating
wires, points of forceps, surfaces of spatulas.
 Sterilization by dry heat cont…
The articles to be sterilized are held over the
Bunsen burner or gas flame until they are
seen to be red hot. The article should be
allowed to cool before use.
• Flaming. This method is used for sterilizing
scalpels, needles, mouths of culture tubes,
glass slides and cover slips. It involves passing
the article through the Bunsen flame without
allowing it to become red hot.
 Sterilization by dry heat cont…
Needles, scalpels are sometimes treated by
immersing them in methylated spirit and
burning off the spirit. However, this does not
produce a sufficiently high temp for
sterilization.
• Hot-Air Oven. This is the main means of
sterilization by dry heat. It’s best for sterilizing
loads that can’t be easily penetrated by steam
 Sterilization by dry heat cont…
and can tolerate high temp of 160oc-180oc
like dry glassware such as test-tubes, Petri-
dishes, flasks, pipettes and instruments such
as forceps, scalpels, scissors and glass
syringes.
• Before sterilization, test-tubes and flasks
should be plugged with cotton wool and other
glass-ware like pipettes, may be wrapped in
Kraft paper.
 Sterilization by dry heat cont…
• The Hot-Air Oven is also used for sterilizing
dry materials in sealed containers, and
powders, fats, oils and greases that are
impermeable to moisture.
• The Holding period of 1 hour at 160oc is timed
when the thermometer first shows that the
oven air has reached 160oc. Finally, the oven is
allowed to cool gradually before the door is
opened.
 Sterilization by dry heat cont…
• Description of a Hot-Air Oven.
- It has a thermostat that maintains the
chamber air constantly at the chosen temp.
- Has a fan (turbo-blower) to assist the
circulation of air and so ensures rapid,
uniform heating of the load.
- It has an open mesh shelving on to which
articles are put.
 Sterilization by dry heat cont…
- Has double walls made of copper or steel with
glass wool or asbestos wool which is a poor
conductor of heat. This avoids heat loss by
conduction.
- Has a temperature recorder and a port for
thermocouples.
- May have door interlocks to ensure that
heating starts only when the door is properly
shut and opens when the temp have dropped
 Sterilization by dry heat cont…
to below 60oc.
Sterilization cycle of a Hot-Air oven.
1. Sterilization Hold Time. This starts when the
oven chamber reaches the chosen temp and
when further period of time has been allowed
for all parts of the load to be raised up to that
temp. Commonly used time-temp relations for
sterilization hold time is 30 min (20 min) at
180oc, 40 min at 170oc, 60 min at 160oc.
 Sterilization by dry heat cont…
2. Cooling. This is facilitated by a
thermostatically controlled cooling fun which
circulates air from outside into the chamber
through a HEPA-filter (HEPA; High Efficiency
particle Arrestor) or an external cooling coil.
• Testing the efficiency of the Hot-Air Oven.
(controls used in a Hot-Air Oven)
- Browne’s tubes type III or Iv. Include the
Browne’s tube in a load and observe for color
 Sterilization by dry heat cont…
change. Type III changes from red to Green at
160oc for 60 min and type Iv changes from red
to blue at 180oc for 30 min.
- Use of biological tests e.g. spores of Bacillus
subtilis. Types NCTC ( National Collection Type
Culture) and ATCC (America Type Collection
Culture)
- Use of Thermocouples
- -Use of Sulphur metals which melt at 155oc.
 Principle

• Hot air oven is usually heated by electricity

and has a thermostat that maintains the
chamber air constantly at a chosen
temperature.
• It has a fan and a turbo blower to assist in air
circulation and to ensure rapid uniform
heating of the load.
• Exposure at temperature of 160°C for one
hour is generally sufficient for sterilization.
 Operation

• Load the oven with dry glassware. Wet

glassware is not suitable because it is easily
cracked therefore, it should first be dried in a
dry oven in about 100°C.
• The sterilisation oven must not be overloaded
and space must be left for circulation of air
through the load.
 Cont….
• It may be cold or warm when loading and is
then heated up to sterilization temperature.
• When the required temperature has been
attained e.g. 160°C, then time for 1 hour. This
is called the holding time, while time allowed
for the temperature to reach 160°C, is called
the heating time.
 Cont….
• The holding time for 1 hour at 160°C is timed
at the beginning when the thermometer first
shows that the hot air oven has reached
160°C.
• Finally, the oven is allowed to cool gradually
for about 2 hours before the door is opened
since the glassware may be cracked by sudden
or uneven cooling.
 Dry heat sterilizer
(Hot air oven)
• After cooling, the materials are removed and
kept in a sterile area for use.
 Sterilization by dry heat cont…
4. Incineration. This method is used to destroy
materials or articles which are not required for
use again. The machine INCINERATOR is used
where materials are put on fire for destruction.
• Materials are burnt to ashes at temp of 870-
980oC.
• Toxic air emissions and presence of heavy
metals have limited the use of incineration in
most developed countries.
 STERILIZATION BY MOIST HEAT
• Sterilization by moist heat can be employed;
i) At temp below 100oc
ii)At temp of 100oc
iii)At temp. above 100oc ( in saturated steam
under increased pressure).
Moist heat at temp. below 100oc.
1.Pasteurization. This is used for sterilizing
serum, body fluids, milk, cream and certain
 Cont…..
beverages e.g. wine and beer. Examples of
organisms destroyed by this method include;
mycobacterium Tuberclosis which is
destroyed within 15 minutes at 60oC and
Rickettsia organisms which are destroyed at
72oC for 20 min.
• The water bath is the equipment used in
pasteurisation.
 Cont….
Types of pasteurization.
a) Holder method. Here milk is heated for 30 min
at 63-66oc for 30 min.
b)Flash method. Where milk is heated for 20 min
at 72oC.
2. At 56oC.
This is used to sterilize sera or body fluids
containing coagulable proteins. It is done at
56oC for 30-60 min.
 Cont….
3. At 60oC.
• This is used for sterilization of vaccines
prepared from cultures of non-sporing
bacteria.
Note: To ensure complete destruction of
vegetative bacteria, sterilization of body fluids
and vaccines is done on several successive
days.
 Cont….
4. At 75-85oC.
• This is used for sterilizing Lowenstein Jensen
(LJ) and Loffler's media for 1-2 hours in a
water bath.
Moist heat at temp. of 100oc.
1.Steaming at 100oC
• The boiling water produces free steam at
normal atmospheric pressure.
 Cont….
• Saturated steam is a more efficient sterilizing
agent than the hot air oven because;
I. Provides greater lethal action
II.It is quicker in heating up the exposed article
III.It is quicker in penetrating porous materials.
IV.Bottles with rubber linings and culture media
can be sterilized by this method. These would
otherwise be damaged by dry heat.
 Cont…..
• The heat generated is good for sterilizing
culture media like Nutrient Agar, Nutrient
broth, can also be used for dissolving agar,
sterilizing heat labile culture media like
TCBS,DCA and Selenite F broth.
• The equipment used is called Koch and
Anold’s steamer.
• The steamer consists of a verticle metal
 Cont….
cylinder with a removable lid having an opening
for escape of steam while preventing inlet of
air.
• The water which is just below the perforated
tray is boiled by an electrical heater or
element.
• The perforated tray situated above the water
level holds the articles.
 Cont…
• Sterilization with Koch and Anold’s steamer may
be effected in two ways;
a) By single exposure at 100oC for 90 minutes
• The time for 90 minutes includes; the time
required for the tubes and bottles to be heated
up from room temperature to 100°C.
b) By intermittent exposure at 100°C
• Also called Tyndalization or fractional
sterilization.
 Cont….
• This method involves an intermittent
exposure at 100oC for a period of 30 min on 3
successive days and having an incubation in
between at room temp.
Principle.
Exposure on the first day sufficiently kills the
vegetative organisms between the heating.
Incubation for overnight at room temperature
to encourage the germination of endospores.
 Cont…
• The vegetative forms which arise from spores
that survive, are killed during subsequent
heating on the second and third days.
• This method is used for media containing
sugars that may be decomposed at high
temperatures and for gelatin media, which
after prolonged heating fails to solidify on
cooling.
 Disadvantages of the method
• Thermophilic organisms or spore bearing
whose spores may not germinate in the
particular media may escape being killed.
• Anaerobic organisms may not be killed unless
incubated anaerobically.
2. Boiling at 100°C
• This is done using a water bath or fish-kettle
type of water bath
 Cont…
• The fish-kettle has a removable tray provided
so that the sterilized material can be raised
above the water for taking.
• The method is mostly used on the ward for
sterilizing dressing instruments and at times
surgical instrument.
• The article to be sterilized is boiled at 100oC
for 10 min. This is enough to kill non-sporing
organisms but may not kill the sporing
 Cont…
organisms.
• Therefore, this method does not ensure sterility
but has been found satisfactory for certain
purposes where complete sterility is not
essential or where better methods are not
available.
Moist heat at temp. above 100oc (autoclaving)
• This is used for sterilizing cultures and culture
media plus other lab supplies.
 Cont…..
• The materials to be sterilized should be water
containing, permeable or wet able and should
not be liable to damage by this process.
Principle.
• Water boils until its vapour pressure equals
the pressure of the surrounding atmospheres.
This occurs at 100°C for the normal
atmospheric pressure (760mmHg).
 Cont….
But when water is boiled within a closed
vessel at increased pressure, the temperature
at which it boils and that of the steam it
forms, will rise above 100°C. This extra heat
that causes the water to boil is latent heat.
When it condenses on a cool surface, it rises
its temperature to a lethal level and at the
temperature of 121°C and pressure of 15psi
for 15 minutes is sufficient for sterilization.
 Description of a portable
autoclave.
• Its consists of a vertical cylinder having a lid with
a gasket to make it tightly fitted and it is fastened
by means of screw clamps (wing nuts).
• The cylinder is filled with water up to the
perforated tray. The water is heated by means of
electricity or in some cases, gas which is carried
into the water by a metal(element) which is a
good conductor of electricity or heat.
 Cont….
• The element usually consist of copper.
• The bottles, tubes or infected culture media or
any other items to be sterilized are placed on
the perforated tray situated above the level of
the water.
• The apparatus also consist of a draw off lock
which allows excess water to flow off, air
outlet valve for the escape of air from the
apparatus.
 Cont…
• It is important that all air from the autoclave is
removed so that the articles loaded are
exposed to pure steam during the period of
sterilization.
Reasons for complete discharge of air from
the autoclave.
1.The mixture of air with steam results in lower
temp being achieved at the chosen pressure.
 Cont….
2. The air hinders penetration of steam into the
article of porous materials and the narrow
openings of containers.
3. The air being denser than steam tend to form
a separate and cooler layer in the lower part
of the autoclave and so prevents the
adequate heating of the articles inside.
An autoclave
Front-loading autoclaves
 Operation of an autoclave

• Put in a correct volume of water, load the

articles to be sterilized in the autoclave but do
not overload to allow free circulation of steam
(caps on tubes, bottles should be loosened).
• Put controls, close the autoclave and tighten
the wing nuts.
• Switch on power and allow to heat. As the
water boils, air and steam will match through
the discharge valve.
 Cont…..
• When all the water droplets have been
expelled and only steam is emerging through
the discharge valve, wait for one minute and
then close the discharge valve. This will cause
the pressure and temperature of the steam to
rise .
• NB: It is essential to expel all the air from the
autoclave to ensure the correct temperature
will be reached at set pressure.
 Cont….
• Let the pressure build up to 15psi at
temperature of 121°C.
• When the required pressure has been reached
which shows on the pressure gauge and
excess steam begin to be released on the
safety valve, reduce the heat and begin timing
. (holding time, which is the time it takes to
penetrate and sterilize the load).
 Cont….
• The holding time for 15 minutes at 15psi and
temperature of 121°C is sufficient to sterilize
most culture media. Longer times are required
when sterilizing large volumes of media (over
200mls especially agar media).
• At the end of the sterilizing time, turn off the
heat and allow the autoclave to cool naturally.
This usually takes few hours.
 Cont….
• Check that the pressure gauge is showing zero
and then open the discharge valve. Wait for a
few minutes before opening the lid to allow
time of the autoclave to become fully vented.
• Then open and unload the autoclave. Tighten
the bottle cups of the media sterilized.
• Store the article in a sterile place for use.
 Failures in Autoclaving

1. Over-packing of the articles will not allow free

steam to circulate.
2. Faulty of the pressure gauge.
3. Failure to release air from the packing
chamber.
4. Incorrect timing of the holding time.
5. Attempt to sterilize articles that are
incompatible i.e. sterilize powders, oils, and
fats using steam
 Cont…
• NB: Under-autoclaving results to unsterile
media while over-autoclaving causes
precipitation and lowering the PH, which can
seriously affect the performance of the media
therefore, ensure use of correct holding time.
Controls used in autoclaving.
 Controls and sterilization
indicators cont…
ii) Bowie dick autoclave tape tests for steam
penetration and when used in conjunction
with other tests yields valuable information.
5. Spore indicators/biological indicators. A
preparation of dried bacterial spores is
placed within the load in the autoclave and
after autoclaving is tested for viability. E.g.
Bacillus stearothermophilus, a thermophile
requires to grown at 55o-60oc is a suitable
 Controls and sterilization
indicators cont…
organism. It’s spores are killed within 12 min
at 121oc. The dried spores on filter papers are
sealed in small envelopes which are placed in
different parts of the load before autoclaving.
After autoclaving, the envelope is cut with
sterile scissors and transferred to a recovery
media like CMB (cooked meat broth),
thioglycollate broth etc, incubated for up to 7
days and then examined for growth. Growth
 Controls and sterilization
indicators cont…
of the sterilized spores would indicate
incomplete sterilization and so the procedure
has to be repeated.
 STERILIZATION BY RADIATION
• Radiation is a form of energy transmitted from
atomic activities and dispensed at high velocity
through matter or space. There are 2 types of
radiation;
1. Ionizing type of radiation e.g. Gamma rays and
X-rays and cathode rays
2. Non-ionizing type of radiation e.g. ultra-violet
rays and infra-red rays.
 Sterilization by Radiation
MECHANISM OF INTERACTION OF RADIATION
WITH MATTER.
1. The ionizing type of radiation pull electrons
from molecules and therefore ionizing them.
When they are passed through cells, they
create free hydrogen and hydroxyl radicals
and some peroxides which cause intra-
cellular damage.
• The ionizing radiation is also referred to as
cold sterilization because it sterilizes in the
absence of heat.
2. The non-ionizing type do not ionize but excite
electrons and raise the to higher energy levels
therefore creating new chemical species
which interfere with the chemical reactions of
organisms.
• As UV radiation passes through a cell, it is
initially absorbed by DNA. Specific nmolecular
damage occurs on the pyrimidine bases (thy
and cyt), which form abnormal linkages with
each other (pyrimidine dimers). These bonds
occur between adjacent bases on the same
DNA strand and interfere with normal DNA
replication and transcription.
 Sterilization by Radiation
The result are inhibition of growth and cellular
death.
USES OF RADIATION
1. Gamma radiation.
• It has a high penetrating and microbial effect
and it’s used for sterilizing materials of
considerable thickness or volume e.g. packed
foods, plastic syringes, culture plates, catheters,
oils, grease, fabrics etc.
 Sterilization by Radiation
2. X-rays
• This method has high energy and penetrating
power.
• It’s lethal to microorganisms and higher
forms of life.
• It’s used for sterilizing similar equipment as
mentioned under gamma radiation.
• Sterilization is achieved by the use of high
 Sterilization by Radiation
speed electrons from a machine such as a
linear accelerator.
• Efficiency of ionizing methods of radiation is
tested by the use of spores of Bacillus
pumillus.
Non-ionizing types of radiation
1.U.V rays
• The ability of sunlight to kill bacteria is mainly
 Sterilization by Radiation
due to the ultra violet (U.V) rays that it
contains
• Has a low penetration power.
• Mercury vapour lamps also emit U.V light
within the region of 250 to 260nm as
opposed to 290nm of sunlight. Therefore the
U.V lamps are only used after disinfection of
the surfaces.
 Sterilization by Radiation
• U.V rays should be shielded from the eyes and
the skin and should not be put on while the
cabinet is being used.
• It should be noted that daylight passing
through ordinary window glass has a
significant effect in shortening the survival of
pathogenic organisms in the dust on the floor
and furniture in rooms.
 Sterilization by Radiation
• Glassware tend to develop a brown colour
and so should not be sterilized by this
method.
• The source of radiation is cobalt 60 having a
photon energy of 1.3mu and cesium 137 with
a photon energy of 0.6mu.
• A dose of 2.5mrads is adequate for producing
sterility for a few weeks.
 Sterilization by Radiation
2. Infra-red radiation.
• This is a very convenient method for
sterilizing a large number of syringes in a
comparatively short period of time.
• The articles to be strilised are put in small
metal containers and are loaded in metal
conveyor belts.
• They are passed through an insulated
 Sterilization by Radiation
• chamber and exposed to infra-red radiation
where they attain a temp. of 190oc for a
period of 10 min. Including the time taken for
the material to heat up and cool after
sterilizing, the whole process is completed
within 30 min.
Controls used for infra-red radiation.
i) Browne’s tubes types 3 and 4 which change
from red to green after 12 min at 180oc or 6
 Sterilization by Radiation
min at 190oc respectively.
ii)Thermocouple
iii)Spores of Bacillus pumillus.
 STERILIZATION BY FILTRATION
• It is possible to render fluids, including
bacterial cultures, free from bacteria by
passing them through special filters.
• The method is useful in sterilizing liquids that
would be damaged by heat such as serum,
antibiotics, enzymes, vitamins etc.
• These filters have pores so small that ordinary
bacteria are arrested. An efficient filter should
be able to retain serratia marcescens. This
 Filtration cont….
indicates an average pore diameter of 0.75u
or less.
TYPES OF FILTERS
There are various types of filters used in
bacteriological work and these include;
1.Earthenware candles e.g.. Berkefeld,
chamberland.
2.Asbestos and asbestos-paper disks, e.g. seitz
 Filtration cont….
3. Sintered glass filters.
4. Cellulose membrane filters.
Berkfeld Filters
• These are made out of a material known as
Kieselguhr which is a fossil diatomaceous
earth found in deposits of German.
• Filters made from this material are coarse i.e.
have relatively large pores
 Filtration cont….
• They are made in three grades of porosity i.e.;
- V (viel) the coarsest
- W (wenig) the finest
- N (normal) the intermediate.
• Of these, the Berkefeld W is the one usually
employed, and it should not pass a small
organism such as serratia marcescens also
called Bacillus prodigiosum
 Filtration cont….
• A similar type to the Berkefeld is the Mandler
filter.
• These filters are made in form of hollow
candles open at one end. A metal nozzle is
inserted and cemented in the open end and
the fluids to be sterilized are poured in the
candle.
 Filtration cont….
Chamberland filter
• These are made of porcelain and are produced
in various grades of porosity. The finer grade
will not allow the passage of bacteria but may
allow the minute viruses to pass such as the
viruses of foot and mouth disease.
• It is made of various porosities which include
L1, L1a, L2 and L3.
 Filtration cont….
• The L1, allows many organisms to pass hence
it’s merely a clarifying filter.
• L1a, L2, and L3 are compared to Berkefeld V, N,
and W candle filters respectively.
• The filter is made in form of a hollow candle
and fluid is forced by suction or pressure from
the inside to the outside or the vice versa.
 Filtration cont….
Method of use.
• The whole set up of the apparatus must be
clean.
• Cotton wool plug or any other suitable plug is
removed before the infected fluid is poured
into the candle.
• The rubber tubing of the flask is connected to
the suction stroke pump. The clip is loosened
and the pump is set going.
 Filtration cont….
• Vacuum is created in the flask and the pressure
will pull out the fluid into the flask. It is
important that the fluid is not filtered out
completely, otherwise pressure will force
bacteria through the candle.
Cleaning and sterilization of the filters
- The filter is autoclaved after use.
- It is then washed well in soapy water and
scrubed with a test tube brush.
 Filtration cont….
- A 2% sodium hydroxide solution is then passed
through the filter in a reverse direction to that
it was last used. This is followed by 1N HCL
until the filtrate has a pH of 7.4.
- It is then washed through distilled water,
drained and dried.
- The instrument is then wrapped with Kraft
paper and autoclaved, after which it is kept in
a sterile condition for subsequent use.
 Filtration cont….
Diagram of Berkefeld filter
 Filtration cont….
• When candle filters are clogged with organic
matter, they are heated to redness and then
allowed to cool slowly.
2.SINTERED GLASS FILTER
• These are made of finely ground glass fused
sufficiently to make the small particles adhere.
• They are in 5 grades.
 Filtration cont….
Grade 1: Coarse filter
2: Medium filter
3: Fine precipitation
4: very fine precipitation
5: Sterilizing filter
• The filter is supplied in form of a disc fussed
into a glass funnel.
 Filtration cont….
• To clean the filter, first disinfect in a suitable
disinfectant, run water through the filter in a
reverse direction and then pass warm
sulphuric acid with potassium nitrate through
it.
SEITZ FILTERS
• This consist of a disc of an asbestos supported
on a perforated metal disc within a metal
funnel.
 Filtration cont….
• After use the asbestos disk is discarded and a new
one employed for each filtration.
• The discs are supplied in three grades;
- FCB or K which is a clarifying filter
- Normal or N which is sterilizing filter
- GS or EK which is also sterilizing
• The normal and EK grades do not allow the
ordinary test bacteria e.g. serratia mersescens to
pass.
 Filtration cont….
• The filter is loosely assembled with the
asbestos disk in position, wrapped in Kraft
paper and sterilized in the autoclave.
Method of use
• The fluid to be sterilized is poured into the cap
of a sterile assembled apparatus and the
vacuum pump is set working.
• The air in flask is sucked all out causing a
vacuum inside the flask.
 Filtration cont….
NOTE: i) The fluid should not all be sucked
completely otherwise, some organisms maybe
forced into the filtrate.
ii) When using seitz filters it is
advisable to moisten the disk with sterile
saline and then screw down tightly the upper
part of the metal on softened asbestos before
pouring in the liquid to be filtered
• When a little fluid is remaining, the pump is
 Filtration cont….
switched off and the rubber bung tightened.
• The sterile fluid in the flask is then taken away
and aseptically transferred into other sterile
containers.
• After use, the metal mount together with
asbestos discs are autoclaved. The discs are
removed from the autoclave and thrown
away.
 Filtration cont….
• The metal mount is then cleaned by washing in
soapy water followed by plenty of tap water
and distilled water and then made dry in the
Oven.
HEMMING’S FILTER.
• Small amounts of fluid can be sterilized by this
method.
• The filter consist of a filter pad fitted between
two bijou bottles.
 Filtration cont….
• The bottles are centrifuged and the liquid is
forced into the second bottle through the pad
and the filtrate is sterile.
 Filtration cont….
MEMBRANE FILTERS
• They are made from a variety of polymeric
materials such as cellulose nitrate, cellulose
diacetate, polycarbonate and polyester.
• They are made in form of disks varying from 13-
293mm in diameter and with porosities from
0.015-12um.
• They are used in syringe filters, pressure filters,
air filters .e.g. HEPA filters.
 Filtration cont….
• After use, the membrane is discarded and a
fresh membrane is inserted. It is also possible
to use the membrane for identification of
bacteria where the membrane is placed on a
culture medium, incubated and after, visible
colonies are seen to develop.
 CHEMICAL METHODS OF
STERILIZATION
• Joseph Lister was a pioneer of
antiseptic surgery