Sterilization 97105629971056299710562997.ppt

Course: Pharmaceutical Microbiology II
Course CODE: 203
COURSE TEACHER:ZARA SHEIKH

Difference between sterilization &
disinfection :
 Sterilization is defined as the process where all
the living microorganisms, including bacterial
spores are killed.
 Disinfection is the process of elimination of
most pathogenic microorganisms (excluding
bacterial spores) on inanimate (nonliving)
objects.
 Sterilization is an absolute condition while
disinfection is not. The two are not
synonymous.

Methods of sterilization:
 Sterilization can be achieved by physical, chemical and
physiochemical means.
 Physical methods include – heat (dry or moist heat)
- ultraviolet light
- ionising radiations
- filtration through a
bacteria proof filter
 Chemical methods involves the use of
- liquid and
- gaseous sterilizing agents.

Methods widely applied to Pharmaceutical Preparations:
 Heat
 Filtration
 Combined physical and chemical method involving heat
in the presence of a bactericide
Methods mainly used for Surgical materials and
Equipment :
 Ionising radiations
 Gaseous sterilization
Liquid sterilizing agents ----- > antiseptics and
disinfectants.

Methods for pharmaceutical
preparations :
The British Pharmacopoeia has five methods for
ensuring that injections are sterile :
 Dry heat
 Moist heat
 Moist heat in the presence of a bactericide
 Filtration through a bacteria-proof filter
 Aseptic technique during preparation

Pharmaceutical Importance of Sterilization :
 Moist heat sterilization is the most efficient biocidal agent. In the
pharmaceutical industry it is used for: Surgical dressings, Sheets, Surgical and
diagnostic equipment, Containers, Closures, Aqueous injections, Ophthalmic
preparations and Irrigation fluids etc.
 Dry heat sterilization can only be used for thermo stable, moisture sensitive or
moisture impermeable pharmaceutical and medicinal. These include products
like; Dry powdered drugs, Suspensions of drug in non aqueous solvents, Oils,
fats waxes, soft hard paraffin silicone, Oily injections, implants, ophthalmic
ointments and ointment bases etc.
 Gaseous sterilization is used for sterilizing thermolabile substances like;
hormones, proteins, various heat sensitive drugs etc.
 U.V light is perhaps the most lethal component in ordinary sunlight used in
sanitation of garments or utensils.
 Gamma-rays from Cobalt 60 are used to sterilize antibiotic, hormones, sutures,
plastics and catheters etc.
 Filtration sterilizations are used in the treatment of heat sensitive injections
and ophthalmic solutions, biological products, air and other gases for supply to
aseptic areas. They are also used in industry as part of the venting systems on
fermentors, centrifuges, autoclaves and freeze driers. Membrane filters are used
for sterility testing.

Physical method of sterilization:
HEAT
 Heat is considered to be most reliable method of
sterilization of articles that can withstand heat. Those
articles that cannot withstand high temperatures can
still be sterilized at lower temperature by prolonging the
duration of exposure.
 There are two types of heat – dry heat and moist heat.
 Moist heat is heat along with moisture.
 Dry heat kills microorganisms by oxidation.
 Moist heat kills microorganisms by coagulation of
proteins that leads to denaturation of proteins.

Sterilization by Heat
Factors affecting sterilization by heat are:
 Nature of heat: Moist heat is more effective than dry heat
 Temperature and time: temperature and time are inversely
proportional. As temperature increases the time taken decreases.
 Number of microorganisms: More the number of microorganisms,
higher the temperature or longer the duration required.
 Nature of microorganism: Depends on species and strain of
microorganism, sensitivity to heat may vary. Spores are highly resistant
to heat.
 Type of material: Articles that are heavily contaminated require higher
temperature or prolonged exposure. Certain heat sensitive articles
must be sterilized at lower temperature.
 Presence of organic material: Organic materials such as protein,
sugars, oils and fats on poorly cleaned equipments increase the time
required.
 Other factors: whether or not the devices /articles were properly loaded
into the sterilizer , whether or not the sterilizing agent is properly
delivered into the system , the sterilizer’s condition and maintenance
protocol and whether or not the correct sterilization method and cycle
were used.

Sterilization by Heat
Susceptibility of microorganisms to heat can be
expressed by :
 Thermal death point (TDP) is the lowest temperature at
which all the microorganisms in a particular liquid
suspension will be killed in 10 minutes.
 Thermal death time (TDT) is the minimum length of
time for all bacteria in a particular liquid culture to be
killed at a given temperature.
 Decimal reduction time (DRT or D-value) is the time in
minutes in which 90% of a population of bacteria at a given
temperature will be killed. It is related to bacterial heat
resistance.

Sterilization by DRY HEAT
Examples of Dry heat sterilization are:
1. Incineration -This is a method of destroying contaminated
material by burning them in incinerator. Articles such as soiled
dressings; animal carcasses, pathological material etc should be
subjected to incineration. This technique results in the loss of
the article, hence is suitable only for those articles that have to be
disposed.
2. Red heat- Articles such as bacteriological loops, straight wires,
tips of forceps and searing spatulas are sterilized by holding
them in Bunsen flame till they become red hot.
3. Flaming - This is a method of passing the article over a Bunsen
flame, but not heating it to redness. Articles such as
scalpels, mouth of test tubes, flasks, glass slides and cover slips
are passed through the flame a few times.
4. Hot air oven

 Sterilization by dry heat is usually carried out in an
apparatus known as hot-air oven in which heat is
transferred from its source to the load by radiation,
convection and to a lesser extent by conduction.
 The first stage in the design of a heat sterilization process is
choice of suitable temperatures and times.
 This depends on the need to obtain a sterile product which
is influenced by the stability of the material/preparation.
 Using dry heat for 1½ hours at 100°C will destroy all
vegetative bacteria, 3 hrs at 140°C for most resistant spores
and 1½ hours at 115°C for mould spores.

 Pharmacists must consider the stability of their
products and should not expose them to conditions
greatly in excess of those needed to produce sterility
e.g. the B.P. recommends 150°C for 1 hr for oily
solutions.
 There is no objection to the use of high temperatures
where harmful effects cannot result e.g for glass vessels
and containers and for these the B.P. specify not less
than 1 hr at not lower than 160°C.

HOT AIR OVEN
(Sterilization by dry heat)
The design of the oven must satisfy the following
requirements:
 Every article inside must receive the correct exposure,
wherever it is placed.
 The sterilizing temperature must be reached quickly and
maintained with little variation.
 Hot air oven: This method was introduced by Louis
Pasteur. Articles to be sterilized are exposed to high
temperature (160° C -180 ° C) for duration of 1 -2 hours
in an electrically heated oven. Since air is poor conductor of
heat, even distribution of heat throughout the chamber is
achieved by a fan. The heat is transferred to the article by
radiation, conduction and convection.

HOT AIR OVEN
 Dry-heat sterilization is accomplished by thermal
(heat) conduction, convection and radiation.
 Initially, heat is absorbed by the exterior surface of an
item and then passed to the next layer.
 Eventually, the entire object reaches the temperature
needed for sterilization.
 Death of microorganisms occurs with dry heat by
oxidation that leads to slow destruction of protein.

HOT AIR OVEN
Parts of a hot air oven :
i) An insulated chamber made of aluminium or stainless steel surrounded by
an outer case containing glass-fibre insulation and electric heaters.
ii) A fan (to allow circulation of hot air)
iii) Perforated Shelves ( perforated to allow circulation of hot air)
iv) Thermocouples
v) Temperature sensor
vi) Door locking controls with asbestos gasket that provides a tight seal.
vii) vents (on top of the oven).

HOT AIR OVEN
Operation of a hot air oven :
 i) Articles to be sterilized are first wrapped or enclosed in containers of
cardboard, paper or aluminum. Mouths of flasks, test tubes and both ends of
pipettes must be plugged with cotton wool. Articles such as petri dishes and
pipettes may be arranged inside metal canisters and then placed.
 ii) Then, the articles must be placed at sufficient distance so as to allow free
circulation of air in between them and to ensure uninterrupted air flow.
 iii) Oven may be pre-heated for materials with poor heat conductivity.
 iv) The door can then be shut and the heater and the fan is switched on.
 v) When the thermometer shoes that the oven air has reached sterilizing
temperature , heating is continued for the required period of time.
 vi) The temperature is allowed to fall to 400C, prior to removal of sterilized
material ; this prevents breakage of glasswares.

Pharmaceutical Applications of sterilization
by DRY HEAT
1. Glasswares – Glasswares that are regularly sterilized
by dry heat includes flasks, beakers, tubes,
containers (e.g ampoules), pipettes, petri dishes
and all glass syringes. At first they must be
thoroughly degreased by washing in hot water and
detergent and rinsing well, followed by a final three
rinses in apyrogenic distilled water. New or very dirty
articles should be soaked first in chromic cleaning
solution overnight. Then they are dried in a drying
oven at about 65°C.

by DRY HEAT
2. Other equipments include:
 some articles of porcelain (such as mortars, pestles, evaporating
basins and tiles) and
 Metals (such as beakers, dishes of stainless steel, scissors,
scalpels and ointment tubes).
3. Oils and similar anhydrous materials:
Dry heat sterilization is of particular importance when contact
with moisture must be avoided e.g.
 Powders
 Vehicles used for oily injections (e.g fixed oils, ethyl oleate and
other fatty acid esters)
 Ingredients of ointment bases (e.g liquid, soft and hard paraffins
,wool fat, wool alcohols and beeswax
 Medical lubricants (e.g. glycerol).

by DRY HEAT
4. Powders
 Dusting powders fall into two groups : medical and
surgical powders.
 Medical powder is used to treat superficial skin
conditions and so sterility is not essential.
 They must be free from dangerous pathogens and so
must be sterilized by maintaining the powder at not
less than 160°C for atleast an hour.
 Surgical powders must be sterile because they are used
in body cavities and major wounds or on burns.

by DRY HEAT
The following substances present special problems that
complicate their sterilization by heat :
 Starch – Starch does not flow easily because its particles tend to
stick together and this is made worse if the moisture content is
high. However, if it is dried at 100°C for about an hour and then
powdered (with other ingredients) before sterilization (at 150 °C
for 1 hr) its flow properties are enhanced.
 Sulphonamides – The main problem with sulphonamide which
is used as a diluent in penicillin is to produce a free-flowing
powder without discoloration. A number of factors are involved
such as particle size, moisture content, envelope paper and other
added substances (e.g Kaolin and zinc oxide gave a more free-
flowing powder). The usual method is to use crystals of suitable
fineness, to dry these in a thin layer at 100 °C, to pack preferably
in double paper envelopes and then to sterilize by maintaining at
150 °C for 1 hr.

by DRY HEAT
The following substances present special problems
that complicate their sterilization by heat :
 Lactose – Lactose has been used occasionally as a
diluent for penicillin. Since penicillin must be kept dry
to avoid decomposition, the lactose should be dried in
an oven at 105 °C, then sterilized and finally mixed
with the antibiotic. Paper envelopes do not give
sufficient protection against moisture and a well-
closed sifter vial should be used.

Advantages of dry-heat sterilization :
 It is an effective method of sterilization of heat stable
articles.
 It is the only method of sterilizing oils and powders.
 Provided sufficient time for penetration is allowed, it is
suitable for assembled equipment, e.g. all glass-syringes. In
moist-heat sterilization, steam or water must be in contact
with every surface and this is not always possible for e.g on
the closely fitted adjacent surfaces of the barrel and
plunger of an assembled syringe.
 It is less damaging to glass and metal equipment than
moist heat. Repeated exposure of glass to moisture at high
temperatures can produce clouding and alkali extraction
and rusting is a serious risk when instruments are sterilized
by wet methods.

Disadvantages of dry-heat sterilization:
 Due to high temperatures, long exposure and very
long heating up times, most of the medicaments,
rubbers and plastics are too much thermolabile for
sterilization by dry heat.
 It is unsuitable for surgical dressings.
 Dry-heat sterilization takes longer than steam
sterilization, because the moisture in the
steam sterilization process significantly
speeds up the penetration of heat and
shortens the time needed to kill
microorganisms.

Sterilization by moist heat :
Microorganisms can be exposed to moist heat by using
 Hot water
 Boiling water
 Steam at atmospheric pressure (steaming)
 Steam under pressure (autoclaving)
A long exposure at a relatively low temperature may cause
more damage to a pharmaceutical preparation than a
shorter treatment at a higher temperature. This explains
why boiling and steaming are not used for sterilization of
parenteral solutions.

Principles of sterilization by steam
under pressure (autoclaving)
 Pressure itself has no sterilising power.
 Steam under pressure can be used as a method of sterilisation as
it can provide temperatures high enough to destroy
microorganisms quickly.
 Steam for sterilisation are of two types:
1. Wet saturated steam
2. Dry saturated steam
 Wet saturated steam is produced in a portable boiler from
water present inside it and since this steam is in constant
contact with water, it will always contain water droplets and
thus known as wet saturated steam.
 Dry saturated steam is produced in a separate boiler and then
with the help of a pipe is transferred to another boiler or tank
where it can be used for sterilization purpose. This steam
practically contains no water droplets and is highly efficient
for sterilization of intra-venous fluids and surgical dressings.

 Steam is described as saturated when it is at a temperature
corresponding to liquid boiling point appropriate to its
pressure. Example: if appropriate equivalent temperature
is 115°C and the corresponding steam pressure is 1.7 bars,
that steam is saturated.
Steam pressure Approximate equivalent
(bars) temperature (°C)
1.7 115
2.0 121
2.4 126
3.0 134

 Draw the phase diagram by plotting a graph of temperature (on y axis)
against pressure (on x axis).
 The curve obtained is phase boundary curve.
 The phase boundary is obtained by joining points representing
saturated steam temperatures at different pressures e.g. 115°C at 1.7
bars, 121°C at 2 bars etc
 Consider the saturated steam represented by A.
 If it is cooled from A to B, it will deposit water provided that pressure
remains constant and same will happen is pressure is raised without
changing the temperature (A to C). B and C are not saturated steam.
 If the saturated steam A is isolated from water and heated without
change of pressure (A to D) or the pressure is lowered without change
of temperature (A to E), steam must become hotter because there is no
water from which further evaporation can occur.
 Therefore it is no longer at the temperature corresponding to the liquid
boiling point appropriate to its pressure and is called superheated
steam.
 Very slight cooling will not make superheated steam condense and so
before this can occur its temperature must be reduced to the
corresponding saturated steam point (e.g D to A or E to F).

Saturated Steam : an efficient
sterilising agent
Saturated steam is an efficient sterilising agent because-
1. A large percentage of its Heat energy is in the form of
Latent heat :
The heat energy in steam is in two forms which are
sensible heat and latent heat:
i) Sensible heat – The heat required to raise the
temperature of water from its freezing point (0° C) to
boiling point (100 ° C).
ii) Latent heat – Latent heat is the additional heat needed to
convert water from its boiling point (100 ° C) to steaming
point at the same temperature (100 ° C).

sterilising agent
 Whenever the saturated steam touches the cool surface of
an article inside, the saturated steam condenses and
liberates all its latent heat immediately.
 The large amount of latent heat is given to the article and
makes a major contribution in raising it to sterilisation
temperature.
 Since all the sensible heat is retained by the condensate
there is no fall of temperature in the surroundings.
 So, saturated steam is a much better heating agent than hot
air because the heat content of the hot air is small and the
heat transfer is slow and accompanied by a drop in air
temperature.

sterilising agent
2. It Condenses on Cooling :
 The protein coagulation by which moist heat kills
microorganisms occurs at lower temperatures if plenty of
moisture is available.
 A possible explanation is that the coagulation temperature of egg
albumen depends on the amount of water present –
Water (%) Coagulation Temperature (°C)
50 56
25 77
6 145
 If the level of water is low, high temperature is needed as
suggested from the above data.
 Therefore the lethal agent in steam sterilization is very hot water
and so the readiness of saturated steam to condense is a
tremendous advantage.

sterilising agent
3. When it Condenses it Contracts to an Extremely Small Volume.
 At 121°C only 1 ml of water of is produced from the condensation of
865ml of steam.
 As a result, a region of low pressure is created into which more steam
rapidly flows.
 This, in turn, condenses, gives up its latent heat and contracts, and the
cycle is repeated until the article has been raised to steam temperature.
 This property of saturated steam ensures quick penetration throughout
bulky porous materials such as surgical dressings.
 The inferiority of hot air in this respect can be explained which
compares the heating up times for a roll of flannel.
Hot air Saturated Steam
Air temperature 150°C Steam temperature 120°C
Temperature inside roll 80°C Temperature inside roll 117°C
after 3 hours after 10 minutes

sterilising agent
To summarise, the advantages of saturated steam as
sterilising agent are –
 It flows quickly to and if required into every article in
the load (volume contraction).
 It rapidly heats the load to sterilisation temperature
(liberation of latent heat).
 It provides, at high temperatures, the moisture
essential for killing microorganisms (production of
condensate).

Types of Steam
sterilizers/autoclaves
 The apparatus for sterilization by steam under pressure is called
an autoclave or steam sterilizer.
 They are of two types:
1. Portable sterilizer ––– > pressure-controlled and temperature
controlled sterilizer
2. Large sterilizer
 In a pressure-controlled type the pressure gauge is the sole
indicator of the internal conditions and therefore, all the air
must be removed before the sterilising exposure begins and it
is made of aluminium alloy.
 In the temperature-controlled type a thermometer or
thermostat is used to indicate or ensure respectively that the
exposure temperature has been reached and it is not essential
to expel the air. It is made of stainless steel.

Parts of an autoclave:
Autoclaves, or steam sterilizers essentially consist of following parts:
i) A cylindrical or rectangular chamber, with capacities ranging from
400 to 800 liters.
ii) Water heating system or steam generating system
iii) Steam outlet and inlet valves
iv) Single or double doors with locking mechanism.
v) Thermometer or temperature gauge
vi) Pressure gauges

Operation of an autoclave:
STEP 1: Decontaminate, clean and dry all instruments and other items to
be
sterilized.
STEP 2: All jointed instruments should be in the opened or unlocked
position, while instruments composed of more than one part or sliding parts
should be disassembled.
STEP 3: Instruments should not be held tightly together by rubber bands
or any other means that will prevent steam contact with all surfaces.
STEP 4: Arrange packs in the chamber to allow free circulation and
penetration of steam to all surfaces.
STEP 5: When using a steam sterilizer, it is best to wrap clean
instruments or
other clean items in a double thickness of muslin or newsprint. (Unwrapped
instruments must be used immediately after removal from the sterilizer,
unless kept in a covered, sterile container.)
STEP 6: Sterilize at 121°C for 30 minutes for wrapped items, 20
minutes for unwrapped items; time with a clock.

Operation of an autoclave:
STEP 7: Wait 20 to 30 minutes (or until the pressure gauge reads zero) to
permit the sterilizer to cool sufficiently. Then open the lid or door to allow
steam to escape. Allow instrument packs to dry completely before removal,
which may take up to 30 minutes. (Wet packs act like a wick drawing in
bacteria, viruses and fungi from the environment.) Wrapped instrument packs
are considered unacceptable if there are water droplets or visible moisture on
the package exterior when they are removed from the steam sterilizer chamber.
If using rigid containers (e.g., drums), close the gaskets.
STEP 8: To prevent condensation, when removing the packs from the
chamber, place sterile trays and packs on a surface padded with paper or
fabric.
STEP 9: After sterilizing, items wrapped in cloth or paper are considered
sterile as long as the pack remains clean, dry (including no water stains) and
intact. Unwrapped items must be used immediately or stored in covered,
sterile containers.

Advantages of pressure-controlled
autoclaves:
 The venting and constant escape of steam during
exposure ensures the absence of air.
 The lid is easier to fit and the method of fitting doesn’t
reduce the effective depth of the autoclave.
 The pressure regulator is simple and requires less
attention than a thermostat.

Advantages of temperature-
controlled autoclaves:
 The internal temperature is controlled and shown.
 The chief material of construction is stainless steel.
Alkaline solution will attack the aluminium alloy of
pressure-controlled autoclave.
 The method of closure is safer because the lid cannot
be removed while steam is at pressure inside.
Additional safety is provided by downward discharge
of the vent.
 The wire basket allows easy air drainage and is more
satisfactory than a solid chamber in which there is the
possibility of air layering or pocketing.

Large Sterilizers
There are two types of
large sterilizers:
• Surgical-dressings sterilizer
• Sterilizer for bottled fluids

Surgical-dressings sterilizer
The following stages are involved in the sterilization of
surgical dressings:
1. Suitably packed dressings are correctly loaded into the
chamber.
2. The door is closed and steam admitted to the jacket.
3. Air is partially or almost completely removed by the vacuum.
4. Dry saturated steam is admitted and if necessary may be used
to displace the rest of the air.
5. Heating-up and exposure are carried out; air (drained from the
dressings) and condensate are automatically discharged
meanwhile.
6. The supply steam is then cut off and the chamber vented.
7. The dressings are dried either by drawing a high vacuum or by
using a partial vacuum to suck warm sterile air through them.
8. When high vacuum drying has been used the vacuum is
broken by admitting sterile air.

Sterilizer for bottled fluids
The following stages are involved in the sterilization of
surgical dressings:
1. The bottles are loaded correctly.
2. The door is closed.
3. In some modern equipment, air is removed by high vacuum.
4. Dry saturated steam is admitted to displace the air.
5. Heating-up and exposure are carried out, air and condensate
being discharged meanwhile.
6. The supply steam is cut off.
7. Either (a) The chamber steam is allowed to vent slowly to
reduce the internal pressure to atmospheric OR
(b) A fine mist of cold water is sprayed over the bottles to
cool them to safely below 100 °C when they can be
removed immediately.

Applications of autoclaving:
 Some of the glass equipment used in aseptic
technique has rubber parts (e.g rubber closures) and
this must be autoclaved.
 Suitable exposures for glasswares and closures are
115°C for 30 minutes (British Pharmacopoeia) 0r 121°C
for 15 minutes.
 There are more than 100 official injections and the
majority is sterilized by autoclaving (B.P mentions
115 to 116°C for 30 minutes but also allows a shorter
time at a higher temperature (e.g 15 min at 121°C if
the medicament is sufficiently thermostable such as
sodium chloride).

Advantages of autoclaving :
 Autoclaving destroys microorganisms more efficiently than
dry heat and therefore a shorter exposure at a lower
temperature is possible.
 It can be used for a large proportion of the official
injections.
 In a sterilizer supplied with dry saturated steam porous
materials can be sterilized without damage.
 Equipment or components of rubber and certain plastics
such as nylon and P.V.C will withstand the conditions.
Disadvantages of autoclaving :
 It is unsuitable for anhydrous materials such as powders
and oils.
 It cannot be used for injections and articles such as some
plastics that deteriorate at 115°C.

Testing the efficiency of sterilizers
Indirect tests:
1. Instrumental test– Temperature, Pressure and Time are the
factors that affect instruments. Thermostat or thermometer are
used to check whether optimum temperature (high temperature
120°C) is maintained. High pressure (1.5 atm) is checked using a
pressure gauge. Whether a fixed time (20 mins) is maintained is
checked using a stopwatch.
2. Cultural test– The article is put into autoclave with
bacteriospore Bacillus subtilis. The limitation is that Bacillus
subtilis is mesophilic so when it reaches or comes into contact
with boiling water at 100°C it dies. Therefore either soil samples
or thermophilic bacterial spore such as Bacillus
stearothermophyllus is used.

Indirect tests:
3. Chemical test- i) Witness tube
ii) Klintex paper
iii) Test tablet
i) Witness tube: It is a sealed tube either containing
acetanilid (melts at 115°C) or benzoic acid (melts at 121°C).
These chemicals melts when sterilizing temperature is
reached. Depending upon the appearance of these
chemical, it is known whether sterilization is complete or
not. Apart from these chemicals, dye such as methylene
blue can be used. The article + benzoic acid will show one
colour before melting and article + benzoic acid + the dye
will show another colour after melting. The function of the
dye is recognition whether melting is complete.

ii) Klintex paper : When the Klintex paper is placed in
autoclave, the word autoclave is displayed in black on
the paper against a pale backgound which confirms
sterlization is complete.
iii) Test tablet : The tablet contains 75% starch, 24%
lactose and 1% magnesium trisilicate. Before
autoclaving, the appearance is white and hard. After
autoclaving, the appearance is brown and gelatinous.
This change occurs at 115°C after 24 minutes.

Sterility Testing
 A sterility test may be defined as — ‘a test that
critically assesses whether a sterilized
pharmaceutical product is free from
contaminating microorganisms’.
 Sterility testings are intended for detecting the presence
of viable forms of microorganisms in or on the
pharmacopoeal preparations.
 All the parenteral products of the B.P must comply
with the sterility testing required for that sample.

Preparations for which sterility
test is required:
1. Ready-made injections – it includes both solution and
suspension, both aqueous and oily.
2. Solids for injection – it includes materials from
biological sources e.g heparin, hyaluronidase and the
antibiotics.
3. Opthalmic products – eye drops, eye ointments, eye
lotions.
4. Water for injections
5. Human blood and human blood products
6. Immunological products – vaccines, antisera
7. Implants
8. Surgical sutures

Information given by a sterility test:
Sterility means freedom from living micro-organisms and
therefore it is not possible to claim that a batch of products is
sterile unless –
 The entire content of every container in the batch has been
tested and
 The test provides optimum conditions for the growth and
multiplication of every organism, vegetative or spore, healthy or
injured, that might be a contaminant.
Unfortunately, neither of these conditions can be satisfies
because –
 In sterility testing the article or preparation under test is either
destroyed (e.g. an injection solution) or made unusable (e.g. a
syringe); therefore only part of the batch is sampled.
 Even great care is taken to provide media and incubation
conditions satisfactory for most organisms it is impossible to
supply all the variations necessary to ensure that every type and
condition of contaminant will grow.

Information given by a sterility test:
Therefore,
 Sterility testing should not be used as the sole means of
controlling sterile processing. Heat sterilization methods
can be checked instrumentally and bacteriologically and
procedures involving asepsis may be controlled by careful
supervision of operatives, regular air sampling (within and
outside the screen) and full–scale runs using nutrient
broth.
 To obtain suitable samples from sterility tests it is
necessary to take sufficient samples, to use sensitive culture
media and during testing, to reduce accidental
contamination to a minimum.

Precautions against accidental
contamination:
 Ventilated aseptic room supplied with
bacteriologically cleaned air
 Highly trained staff
 Adequate control test should be performed at the
same time.
- In case of a negative result : sterility of the sample is
confirmed.
- In case of a positive result : contamination of the
sample.

CONTROL TESTS
Negative control – in these no growth is expected.
 A container of medium from each batch used for the test is
incubated at the same time as the test containers. This control
serves three purposes :
- It confirms that the medium is sterile.
- It shows that the oxidation-reduction qualities of the indicator-
containing anaerobic media are satisfactory. If they are not, the
colour quickly spreads down from the surface of the medium.
- It serves as a standard with which the corresponding test
container can be compared during and after incubation. A faint
turbidity is more easily detected if the suspect tube is examined
at the side of the control.
 Any substance, other than the sample, added to the test tube
should be proved sterile by incubating suitable amounts in
appropriate media. An example is penicillinase, a solution of
which has been used in testing certain penicillins.

CONTROL TESTS :
Positive Controls – in these growth is expected.
 The sensitivity of the media must be confirmed. Each type of medium
is inoculated with an appropriate organism (i.e. an exacting aerobe,
anaerobe or yeast) and after incubation under suitable conditions is
examined for growth. The European Pharmacopoeia suggests that
Staphylococcus aureus as the aerobe, Clostridium sphenoides as the
anaerobe and Candida albicans as the yeast.
 When a medium capable of detecting aerobes and anaerobes and fungi
is used for the test the different organisms should be added to a
separate control containers because if they are inoculated into the
same one it may not be possible to decide whether both have grown or
not.
 The medium must be shown capable of supporting the growth of
small numbers of bacteria in the presence of the sample. Eur.P. requires
atleast two tubes for each type of organism. The bacteria are incubated
at 30 to 32° C for 7 days and yeast 22 to 25 ° C for 2 days.

CONTROL TESTS :
Controls to check working conditions and operator’s
technique –
 General air sampling : This shows that the high quality
of the air supply to the room is being maintained.
 Air sampling at each working space : Settling plates
under and near the screen help to detect poor
technique and particularly excess movement.
 ‘Dummy runs’ : Tests are performed with materials
known to be sterile e.g. ampoules or bottles of Water
for Injections or sodium chloride that have been
sterilised for longer times and/ or higher temperatures
than normal.

Microbial Count
Air :
Air itself contain microorganism but it cannot produce
microorganism. Since air does not contain any nutrient
material and it should not allow microorganism to grow in
it. However, only those microorganisms that can tolerate
dessication and that can tolerate continuing dry state are
present in air.
Microorganisms that are present in air are
1. spore forming bacteria such as Bacillus and Clostridium
2. Nonspore forming bacteria such as corynebacterium,
staphylococcus, streptococcus
3. Certain moulds

Methods to determine the condition
of air :
 Exposure of a petri-dish containing nutrient agar to air
and then from the observation of the result (number
and shape and size of the colonies), conclusion can be
drawn whether air is contaminated or not.
 By air sampling machine – The air collected by air
sampling machine is taken on a petridish containing
nutrient agar or on plastic strip or in membrane filter.
In case of using plastic strip and membrane filter, these
should be innoculated in a media that contains
nutrient. Highly densed colonies represent more
contamination.

Principle of sterility testing:
 Sterility tests are exclusively based upon the
principle that in case the bacteria are strategically
placed in a specific medium that caters for the
requisite nutritive material and water, and maintained
at a favourable temperature (37 ± 2°C), the microbes
have a tendency to grow, and their legitimate
presence may be clearly indicated by the appearance
of a turbidity in the originally clear medium.

 Tests for sterility are adequately designed to reveal the
presence of microorganisms in the ‘samples’ used in
the tests.
 However, the interpretation of results is solely based
upon the assumption that the contents of each and
every container in the batch, had they been tested
actually, would have complied with the tests.
 As it is not practically possible to test every container,
a sufficient number of containers must be examined to
give a suitable degree of confidence in the ultimate
results obtained of the tests.

Aseptic Processing :
 Aseptic processing is concerned with the preparation of
those sterile products that cannot be subjected to a
terminal heating process because the medicaments they
contain are thermolabile.
 The most important example is Sterilization by
Filtration.
 Other examples include :
- the packaging of thermolabile solids for injection
- the preparation of injections from such solids
- the preparation of sterile dusting powders containing
thermolabile medicaments
- the preparation of eye ointments.

Sterilization by Filtration:
 Filtration process does not destroy but removes the
microorganisms. It is used for both the clarification and
sterilization of liquids and gases as it is capable of
preventing the passage of both viable and non viable
particles.
 The major mechanisms of filtration are sieving, adsorption
and trapping within the matrix of the filter material.
Sterilizing grade filters are used in the treatment of heat
sensitive injections and ophthalmic solutions, biological
products and air and other gases for supply to aseptic areas.
They are also used in industry as part of the venting
systems on fermentors, centrifuges, autoclaves and freeze
driers. Membrane filters are used for sterility testing.

 Application of filtration for sterilization of gases: HEPA
(High efficiency particulate air) filters can remove up to 99.97%
of particles >0.3 micrometer in diameter. Air is first passed
through prefilters to remove larger particles and then passed
through HEPA filters. The performance of HEPA filter is
monitored by pressure differential and airflow rate
measurements.
 Application of filtration for sterilization of liquids:
Membrane filters of 0.22 micrometer nominal pore diameter are
generally used, but sintered filters are used for corrosive liquids,
viscous fluids and organic solvents. The factors which affects the
performance of filter is the titre reduction value, which is the
ratio of the number of organism challenging the filter under
defined conditions to the number of organism penetrating it.
The other factors are the depth of the membrane, its charge and
the tortuosity of the channels.

 Filtration through a bacteria-proof filter is a suitable
method for the sterilization of injections containing
thermolabile medicaments.
 However, the solid or medicament must be stable in
solution or compatible with water.
 The process involves four stages :
1. Filtration of the solution through a bacteria-proof filter.
2. Aseptic distribution of the filtered solution into previously
sterilized containers.
3. Aseptic closure of the containers.
4. Testing of samples for sterility.

Classification of bacteria-proof
filter:
There are four classes:
1. Sintered ceramics – It is made from finely ground
porcelain. (may be used several times)
2. Fibrous pads – containing asbestos and wood
cellulose. (one time use)
3. Sintered glass – made from borosilicate glass (may be
used several times).
4. Microporous plastics – prepared from cellulose
esters, particularly the acetate or nitrate (one time
use).

Filtration techniques in sterility testing :
 Contaminants are removed from the sample by filtration
through a sterile bacteria-proof filter pad.
 Then bactericides and inhibitory medicaments are removed
from the organisms and filter by washing with a sterile solvent
and finally the whole of the pad incubated in a suitable culture
medium.
 Previously asbestos-cellulose filter pad was used which has been
replaced by a membrane filter because
- The membranes are so thin that the retention of inhibitory
substances is very small. Asbestos-cellulose pads are relatively
thick and fibrous and therefore may absorb and retain sufficient
inhibitor to cause bacteriostasis in the culture medium.
- Quick filtration.
- Oil pass through easily and quite quickly and there is no need to
dissolve them in an organic solvent first.

Advantages of sterilization by filtration :
 Wide application. They can be used for
- Solutions with or without inhibitory properties.
- Soluble solids with or without inhibitory properties.
- Insoluble solids without inhibitory properties.
- Oils
- Ointments, provided a non-inhibitory solvent or dispersing medium
can be found.
- Articles, such as syringes that can be rinsed with a sterile fluid.
 A very large volume can be tested with one pad. Therefore, the method
is applicable to the testing of poorly soluble solids.
 A much smaller volume of broth is required than for testing by direct
inoculation into the culture media.
 They are applicable to substances for which no satisfactory inactivators
are known e.g. many antibiotics.
 Some strongly adsorbed antibacterial agents such as the mercurials and
the quartenary ammonium compounds can be inactivated on the filter
by treatment with the appropriate neutralizing solution.
 Subculturing is often eliminated e.g for oils and oily preparations.

Disadvantages of sterilization by
filtration :
 Even with membrane filters the possibility of
adsorption of sufficient medicament cannot be
disregarded.
 Highly skilled staff and exceptionally good aseptic
techniques are necessary.

Tests for Sterility :
 Tests for sterility are carried out by two methods:
(a) Membrane Filtration Method
(b) Direct Transfer / Inoculation Method.
 The Membrane Filtration Method is used as the
method of choice wherever feasible.
Media used in Sterility Testing :
 Fluid Thioglycollate Medium (Medium 1) and
Soybean-Casein Digest Medium (Medium 2) are the
two media generally used for tests for sterility.

Method of Membrane Filtration
Procedure
 The filter should be a membrane filter disc of cellulose esters or other
suitable plastics, having a nominal average pore diameter not
exceeding 0.45 μm. The membrane should be held firmly in a filtration
unit which consists of a supporting base for the membrane, a
receptacle for the fluid to be tested, a collecting reservoir for the
filtered fluid, and the necessary tubes or connections. The apparatus is
so designed that the solution to be filtered can be introduced and
filtered under aseptic conditions. It permits the aseptic removal of the
membrane for transfer to medium or it is suitable for carrying out the
incubation after adding the medium to the apparatus itself.
 Cellulose nitrate filters are recommended for aqueous, oily and weakly
alcoholic solutions and cellulose acetate filters for strongly alcoholic
solutions. The entire unit should be sterilized by appropriate means
with the membrane filter and sterile airways in place. The method of
sterilization should not be deleterious to the membrane, e.g, weaken it
or change the nominal average pore diameter.

Method of Direct Transfer
Procedure :
Liquids and soluble or dispersible solids:
 Appropriate quantities of the preparation to be examined are added
directly into Medium 1 and Medium 2. Approximately equal quantities
of the preparation should be added to each vessel of medium. The test
vessels of Medium 1 is incubated at 30 - 35°C and the vessels of Medium
2 is incubated at 20- 25°C.
 The volume of Medium 1 should be such that the air space above the
medium in the container is minimized. The volume of Medium 2
should be such that sufficient air space is left above the medium to
provide conditions that permit the growth of obligate aerobes. Unless
otherwise prescribed, in no case should the volume of material under
test be greater than 10% of the volume of the medium alone, i.e. 90%
medium and 10% product. If a large volume of product is to be tested it
may be preferable to use concentrated media, prepared so as to take the
subsequent dilution into account.

 Where appropriate the concentrated medium may be added directly to
the product in its container. Wherever possible solid articles such as
devices should be tested by immersion in or filling with culture media.
Immerse all parts of each article in sufficient medium contained in one
vessel to completely cover all parts. The volume of Medium 1 should be
such that the air space above the medium in the container is
minimized. The volume of Medium 2 should be such that sufficient air
space is left above the medium to provide conditions that permit the
growth of obligate aerobes. Place half the articles into Medium 1 and
the remaining half into Medium 2. Incubate the test vessels of Medium
1 at 30 - 35°C and the vessels of Medium 2 at 20 - 25°C.
Ointments and oily preparations: Ointments and oily preparations
may be tested by the method of Direct Transfer if testing by the
method of Membrane Filtration is not feasible, i.e. when a suitable
solvent is not available .

Incubation and examination of sterility tests:
 All test vessels of Medium 1 are incubated at 30 - 35°C. The vessels of
Medium 2 are incubated at 20 - 25°C. All test and control vessels, other
than the subcultured vessels referred to below, must be incubated for at
least 14 days unless microbial contamination is detected at an earlier
time.
 If turbidity, precipitate, or other evidence of microbial growth during
incubation is seen: the suspected growth is examined microscopically
by Gram stain; colonies of each type of micro-organism present are
examined for colonial morphology and cellular morphology by Gram
stain.
Interpretation of the test results:
 If microbial growth is not evident in any of the vessels inoculated with
the product, the sample tested complies with the test for sterility, if
microbial growth is evident the product does not comply with the test
for sterility unless it can be clearly demonstrated that the test was
invalid for causes unrelated to the product being examined.

Sterilization by Gas:
Sterilization by gas involves sterilization with a chemical in the
gaseous state.
 The chemically reactive gases such as formaldehyde,
(methanol, H.CHO) and ethylene oxide (CH2) 2O possess
biocidal activity. Ethylene oxide is a colorless, odorless, and
flammable gas.
 The mechanism of antimicrobial action of the two gases is
assumed to be through alkylations of sulphydryl, amino,
hydroxyl and carboxyl groups on proteins and amino groups of
nucleic acids. The concentration ranges (weight of gas per unit
chamber volume) are usually in range of 800-1200 mg/L for
ethylene oxide and 15-100 mg/L for formaldehyde with operating
temperatures of 45-63°C and 70-75°C respectively.
 Both of these gases being alkylating agents are potentially
mutagenic and carcinogenic. They also produce acute toxicity
including irritation of the skin, conjunctiva and nasal mucosa.

Sterilization by Gas:
Ethylene oxide sterilizer:
 An ethylene oxide sterilizer consists of a chamber of 100-300-
Litre capacity and surrounded by a water jacket. Air is removed
from sterilizer by evacuation, humidification and conditioning
of the load is done by passing sub-atmospheric pressure steam,
then evacuation is done again and preheated vaporized ethylene
oxide is passed. After treatment, the gases are evacuated either
directly to the outside atmosphere or through a special exhaust
system.
 Ethylene oxide gas has been used widely to process heat-
sensitive devices, but the aeration times needed at the end of the
cycle to eliminate the gas made this method slow.
Low temperature steam formaldehyde (LTSF) sterilizer:
 An LTSF sterilizer operates with sub atmospheric pressure steam.
At first, air is removed by evacuation and steam is admitted to
the chamber.

Plasma Sterilization
Hydrogen Peroxide Sterilization:
 This method disperses a hydrogen peroxide solution in a vacuum
chamber, creating a plasma cloud.
 This agent sterilizes by oxidizing key cellular components, which
inactivates the microorganisms.
 The plasma cloud exists only while the energy source is turned on.
 When the energy source is turned off, water vapor and oxygen are
formed, resulting in no toxic residues and harmful emissions.
 The temperature of this sterilization method is maintained in the 40-
50°C range, which makes it particularly well-suited for use with heat-
sensitive and moisture-sensitive medical devices.
 The instruments are wrapped prior to sterilization, and can either be
stored or used immediately.
 An advantage of the plasma method is the possibility, under
appropriate conditions, of achieving such a process at relatively low
temperatures (≤50 °C), preserving the integrity of polymer-based
instruments, which cannot be subjected to autoclaves and ovens.
Furthermore, plasma sterilization is safe, both for the operator and the
patient, in contrast to EtO.

Sterilization by Radiation:
Two types of radiation are used:
ionizing and non-ionizing.
Non-ionizing rays are low energy rays with
poor penetrative power while ionizing rays are
high-energy rays with good penetrative power.
Since radiation does not generate heat, it is
termed "cold sterilization".

Non-ionizing rays:
 Rays of wavelength longer than the visible light are non-ionizing.
Microbicidal wavelength of UV rays lie in the range of 200-280 nm,
with 260 nm being most effective. UV rays are generated using a high-
pressure mercury vapor lamp. It is at this wavelength that the
absorption by the microorganisms is at its maximum, which results in
the germicidal effect. UV rays induce formation of thymine-thymine
dimers, which ultimately inhibits DNA replication.
 UV readily induces mutations in cells irradiated with a non-lethal dose.
Microorganisms such as bacteria, viruses, yeast, etc. that are exposed to
the effective UV radiation are inactivated within seconds. Since UV rays
don’t kill spores, they are considered to be of use in surface
disinfection. Disadvantages of using UV rays include low penetrative
power, limited life of the UV bulb, some bacteria have DNA repair
enzymes that can overcome damage caused by UV rays, organic matter
and dust prevents its reach, rays are harmful to skin and eyes. It doesn't
penetrate glass, paper or plastic.

Ionizing rays:
Ionizing rays are of two types, particulate and electromagnetic rays.
 Electron beams are particulate in nature while gamma rays are
electromagnetic in nature. High-speed electrons are produced by a
linear accelerator from a heated cathode. Electron beams are employed
to sterilize articles like syringes, gloves, dressing packs, foods and
pharmaceuticals. Sterilization is accomplished in few seconds. Unlike
electromagnetic rays, the instruments can be switched off.
Disadvantage includes poor penetrative power and requirement of
sophisticated equipment.
 Electromagnetic rays such as gamma rays emanate from nuclear
disintegration of certain radioactive isotopes (Co 60, Cs 137). They have
more penetrative power than electron beam but require longer time of
exposure. These high-energy radiations damage the nucleic acid of the
microorganism. A dosage of 2.5 megarads kills all bacteria, fungi,
viruses and spores. It is used commercially to sterilize disposable petri
dishes, plastic syringes, antibiotics, vitamins, hormones, glasswares
and fabrics. Disadvantages include; unlike electron beams, they can’t
be switched off, glasswares tend to become brownish, loss of tensile
strength in fabric. Bacillus pumilus E601 is used to evaluate sterilization
process.

References :
 Cooper and Gunn’s Dispensing for Pharmaceutical
students, Twelfth Edition.
 Microbiology – An Introduction by Tortora, Funke and
Case, Eighth Edition.

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