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STERILISATION
Mr. Dipak B. Bhingardeve
Asst. Professor
Department of Pharmaceutics
Dr.Shivajirao kadam college of Pharmacy,kasabe
digraj,Sangli
STERILISATION
 Sterilisation is the process of complete destruction of all
microorganisms present in a system.
 The product free from living microorganisms are called
‘sterile product.’
 Antiseptic : A substance that prevents the growth of
microorganisms by inhibiting their activity without destroying
them is called antiseptic.
 Bactericide : The substance that kills bacteria is called
bactericide.
 Bacteriostatic : The substance that arrest or retards the
growth of bacteria is known as bacteriostatic.
 Disinfection : A process that removes the infection potential
by destroying microorganisms but not generally bacterial
spores.
CONT..
 Germicide: A substance that kills pathogenic bacteria but
not necessarily bacterial spores.
 Viricidie : A substance that kills viruses.
 Sterility : The absence of viable microorganism is called
‘sterility’ and preparation free from viable microorganisms
are called ‘sterile’.
THERMAL RESISTANCE OF MICROORGANISMS
 The microorganism show varying resistance to different
Methods of sterilisation.
1. Thermal death time : It may be defined as the time
required to kills a specific types of microorganisms at a
given temperature under specific conditions.
1. Death rate of microorganism : There is no direct method
to determine when the sterility will be achieved.
1. Decimal reduction time (D value):
It is defined as the time in minutes required to reduce the
number of viable organism by 90%.
CONT..
 The order of death of microorganisms can be
calculated from equation:
 K = 1/t (log No.— log N)
 Where,
 K= constant which depends on organism ,temperature and
medium.
 t = time of exposure in minutes.
 No= number of organisms viable at the beginning of time
interval.
 N=Number of organism viable at the end of the time
interval.
 K= 1/t bcz it is after 90% reduction in microorganism.
 Time t is defined as the decimal reduction time which
is called the D value.
 D= 1/K
METHODS OF STERILISATION
 A) Physical Methods
1) dry heat sterilisation
2) Moist heat sterilisation
3) Radiation sterilisation
a) use of ultra violet rays
b) Ionising radiation
 B) Chemical methods :
1. Sterilisation by heating with bactericide.
2. Gaseous Sterilisation.
C) MECHANICAL METHODS:
 Sterilisation involves the filtration of parenteral
preparation through the following bacteria proof
filters:
1. Ceramic filters
2. Seitz filter
3. Sintered glass filters
4. Sintered metal filters
5. Membrane filters
A) STERILISATION BY PHYSICAL METHODS
I) Dry Heat Sterilisation :
 All microorganisms including bacterial spores can be
destroyed by heat.
 Dry heating at 100ºC kills all vegetative bacteria in one hour
but it does not kill spores.
 According to pharmacopoeia ,sterilisation by dry heating is
effected by heating at temperature of 160 º C for two hours.
HOT AIR OVEN :
 It is used for sterilisation of pharmaceutical products and other
materials.
 It is double walled chamber made of steel.
 Insulation material ,such as glass fibres or asbestos is filled
between the two walls of the oven to avoid heat loss.
 Two or three perforated shelves are fixed inside the oven.
 An electric fan is also fitted to ensure the uniform circulation
of hot air in the oven in order to maintain the required
temperature in all the shelves.
 A thermometer is fitted in the oven to note down the
temperature inside the oven.
HOT AIR OVEN
PRECAUTIONS TO BE TAKEN WHILE PLACING MATERIAL
MEANT FOR STERILISATION.
 1.Glass apparatus must be wrapped with clean cloth or filter
paper and container must be plugged with non-absorbent
cotton wool.
 2.The article & substance which are to be sterilised should
not be placed at the floor of the oven as it receives direct heat
and becomes much hotter.
 3.The oven should not be overloaded with the material.
ADVANTAGES
 It is used for sterilisation of those substance which get
spoiled during moist heat sterilisation e.g. oily material
and powders.
 The method is suitable for glass syringes.
 Disadvantages :
 This method is not suitable for surgical dressing.
 This method is not suitable for most of the medicaments,
rubber and plastic goods.
APPLICATIONS :
 It is mainly used for sterilisation of glass wares, such as,
pestle and mortar, petridishes, flasks ,pipettes, bottles, test
tube etc.
 Used for sterilise powder such as, sulphacetamide,
sulphadiazine , kaolin , talc, zinc oxide ,starch etc.
 Also scissors, spatula, blades and glass syringes , scalpels.
II)MOIST HEAT STERILISATION
 Moist heat sterilisation is more effective than dry heat
method.
 Bcz steam has more penetration power than dry heat and
thermal capacity of steam is more than thermal capacity of
dry heat.
 The method is very useful for killing of bacterial spores.
CONDITIONS FOR HOLDING PERIOD OF
STERILISATION
Sr.
No.
Holding
temperature
Minimum
Holding time in
minute
1 115 º to 118 º 30
2 121 º to 124 º 15
3 126º to 129º 10
4 134º to 134º 5
AUTOCLAVE :
 Moist heating is done in an “autoclave”.
 It consist of strong metallic chamber usually made of
stainless steel.
 It has cover fitted with a steam vent, a pressure gauze and a
safety valve.
 Rubber gasket is fitted on the inner side of lid, in order to
make autoclave air tight.
 The cover is closed with wing nuts and bolts.
 The electrically heated element is fitted at the bottom to
heat the water to convert into steam.
 A sufficient quantity of water is poured into chamber after
removing the perforated chamber.
 The level of the water is adjusted in such a way that it does
not touch the bottom of perforated chamber.
CONT..
 The material is packed in the perforated chamber.
 The lid is then closed with wing nuts and bolts.
 WORKING :
 The autoclave is switched on to heat the water.
 The vent is opened and safety valve is set at the required
pressure .
 When steam starts coming out from the vent and it continues
for 5 minutes, it is then closed.
 The steam pressure starts raising and it comes to desired
pressure i.e. 10 lbs /sq.inch with corresponding temperature
115º c or 15 lbs/sq.inch with corresponding temperature 121º c.
 After stated period , switch off the autoclave .
 Allow it to cool to about 40 ºc before opening the vent.
CONT..
 When whole of the steam inside the autoclave is removed , the
lid is opened and the sterilised material is taken out.
 Advantages :
 1.Autoclaving destroys microorganisms more efficiently than
dry heat and hence the material is exposed to a lower
temperature for shorter period.
 It is used for sterilisation of a large number of official
injections.
 Disadvantages :
 Unsuitable for sterilisation of powders and oils.
 Cannot be used for sterilisation of injection & articles ,
 Such as ,plastics which get spoiled at 115-116 º c for 30 min.
OTHER METHODS OF STERILISATION BY MOIST HEAT
 1.Tyndallisation
 2. Pasteurisation
 3.Sterilisation of vaccines.
 1.Tyndallisation method:
 This is fractional sterilisation method.
 The method was official in B.P 1932, for sterilisation of
medicaments.
 The method was used for sterilisation of culture media.
 THE method was deleted from B.P. 1932 by 4th
addendum(1941) and replaced by Heating with a
Bactericide.
CONT..
 Solution to be sterilised is packed & sealed in its final
container an heated at 80º c for one hour on each of three
successive days.
 The first heating destroys the vegetative cells but not the
bacterial spores.
 These bacterial spores germinate into the vegetative forms in
the interval between first and second heating and are killed
in the second heating.
 The third heating provides a safeguard against any spores
which may not germinate until the second interval.
2. PASTEURISATION
 It is partial sterilisation method which is used to make milk
safe and also to improve its keeping properties.
 The process kills only 97 to 99 percent microorganism but it
does not kill bacterial spores.
 The methods used for pasteurisation of milk are as under:
1) Holder method:
 Milk is heated at 62.8 ºc for 30 min in a steam jacketed
stainless steel tank containing agitators.
 This provides correct exposure throughout the milk and
prevent skin formation.
 Clean dry steams is blown to the space above the liquid to
prevent the formation of skin and foam.
 The method kills all types of bacteria including
mycobacterium tuberculosis.
II) FLASH METHOD
 The milk is heated to 71.6 º c for 15 sec and then quickly
cooled.
 The milk is heated by passing through narrow horizontal
pipes inside large ones through which water passes in the
opposite direction.
 The method is commonly used by most of the firms bcz it is
less time consuming process.
 It also needs less floor space and it is a continuous process.
3.STERILISATION OF VACCINES:
 The suspension of microorganism is prepared in normal
saline solution and transferred into a sealed container.
 Sterilisation is carried out by immersing the container in a
thermostatically controlled water bath at a temperature
between 55 ºC to 60º C for one hour.
 The strick aseptic precautions are observed to exclude any
possibility of contamination.
 The vaccine is tested for sterility to confirm the proper
sterilisation of vaccine.
III) RADIATION STERILISATION :
 Two types
 1. Sterilisation by ultra violet light
 2.Sterilisation by ionising radiation.
 1. Sterilisation by ultra by violet light:
 Direct sunlight can destroy microorganism on account of
its ultra violet rays of long wavelength.
 The antimicrobial activity of U.V. Light depends on its
wavelength.
 It is maximum at 265 nm wave length.
 Ultra violet rays for sterilisation is produced by passing a
low current at high voltage through mercury vapour in an
evacuated glass tube.
APPLICATIONS:
 It is used for sterilisation of air to prevent cross infection in
hospitals.
 Used for sterilisation and maintenance of aseptic area in the
pharmaceutical industry.
 It is used for sterilisation of thermolabile substances before
packing.
 Disadvantages :
 Ultra violet rays have a low penetration power, so it is not
applicable to the sterilisation of packed pharmaceuticals.
 U.V. rays are less effective against organisms in the
atmosphere .
 U.V light is harmful for worker. Eyes & skin should be
protected from direct ultra violet rays.
STERILISATION BY IONISING RADIATION
 Ionising radiations are X-rays and Gamma rays.
 These are lethal to bacterial cell and destroy the nuclei of the
cell.
 Gamma rays are produced from radio-isotonic source such as
cobalt -60 or cesium -137.
 The material to be sterilised is packed in the final container
and then exposed to ionising radiation.
 Ionising radiations are much more efficient as a sterilising
agent than Ultra violet rays.
APPLICATIONS:
 The method is mainly used for sterilisation of plastic syringe
,hypodermic needles, surgical blades and adhesive dressings.
 Also used for sterilisation of bone and tissue transplant,
plastic tubing, sutures.
 Used for sterilisation of thermolabile medicaments.
B)CHEMICAL METHODS OF STERILISATION
 Two types:
 1. Sterilisation by heating with a bactericide
 2. Gaseous Sterilisation
 1. Sterilisation by heating with a bactericide:
 This method used for sterilising aqueous preparations which
are unstable at higher temperature attained in moist heat
sterilisation processes.
 In this process the medicament is dissolved or suspended in a
suitable solution of bactericides so as to achieve the
concentration of bactericides.
 The preparation is then transferred into the final container
which is then sealed so as to exclude microorganisms and
heated at 98-100º C for 30 minute by heating it in boiling
water.
PROPERTIES OF BACTERICIDES
 1.It should be non- toxic.
 2.Compatible with medicament.
 3.Stable and active at various pH.
 4.Stable during heating and storage.
 Application :
 Used for sterilisation of injections
2.GASEOUS STERILISATION
 Sterilisation is done with a chemical in gaseous state.
 In olden days, formaldehyde was very commonly used, but
now it has been replaced by ethylene oxide.
 Beta propiolactone is new chemical which is also used as
sterilising agent.
 A) Formaldehyde:
 It is alkylating agent.
 Nowadays, it is used for the fumigation of empty rooms
after infectious diseases.
 Formaldehyde is inferior to ethylene oxide due to
following reasons:
CONT..
 It has weak penetration power.
 It is difficult to maintain a high concentration in the
atmosphere.
 It requires a high humidity to be effective sterilising agent.
 It is irritating to respiratory tract.
 B) Ethylene oxide :
 It is colourless gas at room temperature.
 It can be liquefied easily and the liquid boil at appro.10.8ºC.
 It is readily dissolved in water and organic solvents.
 It is highly inflammable so it is used by:
1.MIXING ETHYLENE OXIDE WITH SOME INERT GAS IN
FIXED RATIO
 i) Ethylene oxide = 1 part
Carbon dioxide = 9 part
 ii) Ethylene oxide = 11% w/v
Trichlorofluromethane =79%w/w
Dichlorofluromethane = 10% w/w
2.Using ethylene oxide in the absence of air:
 Pure ethylene oxide or a mixture containing 90% ethylene
oxide and 10 % carbon dioxide is used as sterilising agent.
 Ethylene oxide kills microorganisms by alkylation of protein
molecules.
 It is active against all types of microorganisms.
FACTORS INFLUENCING EFFICIENCY OF ETHYLENE OXIDE
 Temperature – At room temp, bt effective at 40ºC
 Concentration- Conc between 200mg to 1 g/litre.
 Relative humidity- 40-50% moisture is necessary
 Power of penetration- high penetration power, can penetrate thr
paper ,fabrics.
 Absorption – Amt & rate of absorption depends on the nature ,thickness,
surface area of article.
 Advantage:
 It is suitable for heat sensitive substance bcz sterilisation is
effective at room temperature.
 Ethylene oxide has a good penetration power .
 The method is useful for sterilisation of moist sensitive
substance and equipment bcz only a low humidity is
required.
APPLICATIONS:
 Used for sterilisation of thermolabile materials, such as
rubber and plastic items.
 Used for sterilisation of delicate instrument.
 Applicable for sterilisation of powders.
 Suitable for sterilisation of syringes and needles.
 C) Beta –Propiolactone-
 It is largest sterilising agent.
 It has low vapour pressure and is a liquid at room
temperature.
 Its boiling point is 160º C hence it is non-flammable.
 It has less penetration power than ethylene oxide.
 So it is mainly used for sterilisation of operation theatre and
aseptic rooms etc.
 It is carcinogenic.
C) STERILISATION BY MECHANICAL METHODS
 The solution containing thermolabile medicament can be
sterilised by filtration through bacteria proof filters.
 These filters retain the bacteria and the sterile filtrate is
collected in sterilised receiver.
 For Successful sterilisation by filtration ,the following
points should be observed :
 1.The whole apparatus must be sterile.
 2.An aseptic technique should be followed in order to
minimize the risk of contamination.
TYPES OF BACTERIA PROOF FILTERS
 1. Ceramic filters:
 These are also called filter candles.
 These are made of porcelain or kieselguhr and are available in
a range of pore size.
 These candles are numbered according to its pore size.
 The candle is placed in the solution to be sterilised and its
opening is attached to the vacuum system.
 When vacuum is applied pressure inside the candle is
decreased.
 Due to the difference in pressure between outside & inside of
the candle, the solution moves into candle.
 The filtrate is collected in sterile container.
 Main disadvantage is its tendency to absorb materials from
aqueous solution.
2.SEITZ FILTER:
 It consist of two parts.
 The lower part holds a perforated disc, on it compressed
asbestos sheet is placed.
 Two parts are joined together with the help of nuts.
 The asbestos sheet is made up of asbestos fibres but may also
contain cellulose and alkaline earth such as magnesium
compounds.
 The asbestos pads are used once only and then discarded.
 Asbestos pads may yield alkali and cause precipitation of
alkaloids from aqueous solution of their salts.
 And also absorbs drugs from solution.
 Hence, a few millilitres of filtrate should always be rejected and
sintered glass disc may also be fixed in the filtration unit
immediately after seitz filter.
3. SINTERED GLASS FILTERS:
 These are made from borosilicate glass.
 The glass is finely powdered and particles of the required
size are separated and is then packed into disc moulds.
 These moulds are heated until a suitable adhesion has taken
place between the granules.
 These discs are fused to funnels of suitable shape and size.
 Sintered glass filters are available in different pore size and
are numbered accordingly.
 For bacteria proof filtration number 5 or 3 must be used.
 The filtration is carried out under a reduced pressure .
CONT..
 Sintered glass filter do not absorb the medicaments from
the solution.
 These filters are made from borosilicate glass, so it does
not change the pH of the solution.
 Sintered glass filter are cleaned after use by passing water
in the reverse direction.
 Organic matter may be removed by passing strong
sulphuric acid with 1 % sodium nitrate through the
filter.
 4. Sintered metal filters:
 These are the metallic counter part of sintered glass filters.
 These are usually made from stainless steel, and having
greater mechanical strength.
5. MEMBRANE FILTER :
 These are made of cellulose acetate or cellulose nitrate.
 These are fixed in metallic holders similar to those used with
asbestos pads.
 The pore size in the membrane lies in the range of 100- 150 u.
 They are also called millipore filters.
 Membrane filters are suitable for sterilising aqueous and oily
solution but are not suitable for organic solvent, such as
alcohols, ketones, esters or chloroform.
STERILISATION BY FILTRATION INVOLVES FOUR
BASIC STEPS:
 1.Filtration of the solution through one of the bacteria –
proof filters which have been described above.
 2.Aseptic distribution of the filtered solution into the
previously sterilised final containers.
 3.Aseptic closure of the containers.
 4.Performing the sterility test.
 Advantages :
 Suitable for sterilisation of blood products, insulin and
enzymes.
 All types of bacteria are removed from preparation.
 Both clarification and sterilisation are done side by side.
DISADVANTAGES:
 The method is not reliable one and therefore a sterility test
is necessary.
 The suspension and oily preparations cannot be sterilised by
this method.
 They are chances of absorption of medicament from
solution by the filter.
 Units may leak if carelessly handled.
 Aseptic technique is necessary.
 Highly trained staff is required.
 Applications :
 Method is useful for sterilisation of parenteral solutions
containing thermolabile medicaments without any
decomposition
 E.g Insulin , blood serum .
TESTS FOR STERILITY
 These tests are based upon the principle that if bacteria or
fungi are placed in a medium which provides nutritive
material, moisture , the desire pH and are kept at a
favourable temperature, the organisms will grow and their
presence can be indicated by the turbidity in the originally
clear medium.
 The tests for sterility are done by detecting the presence of
viable forms of bacteria , fungi and yeast in or on
pharmacopoeial preparations.
 The tests must be carried out under strict aseptic technique.
CULTURE MEDIA :
 Culture media is required for aerobic and anaerobic bacteria
and for fungi.
 The media used should comply with the tests, such as, sterility,
nutritive properties, effective of media as per details given in
I.P.
 Methods of testing :
 1. Membrane filtration method
 2. Direct inoculation method
 1. Membrane filtration method :
 The method is preferred in the following cases.
 A) An oil or oily preparation.
 B) An ointment that can be put into solution.
 C)A soluble powder or a liquid that possess bacteriostatic and
fungistatic properties.
CONT,,,
 D) Liquid products where the volume in a container is 100
ml or more.
 The method involves filtration of sample under test
through a membrane filter having normal porosity of 0.45 µ
and a diameter of approx 47 mm.
 After filtration, the membrane is removed aseptically from
the metallic holder & divided into two halves.
 The first half is transferred into 100 ml of culture media
meant for fungi & incubated at 20ºC to 25ºC for NLT 7 days
.
 The other half is transferred into 100ml of fluid
thioglycollate medium and incubated at 30ºC to 35 ºC for
NLT 7 days. & Observe the growth in media.
2.DIRECT INNOCULATION METHOD
 In this method the specified quantity of sample under test is
drawn aseptically from the container and transferred into a
vessel of culture medium.
 Mix the liquid with medium and incubate for NLT 14 days.
 Observe the growth of microorganism in the medium.
 Observations :
 1. No evidence of growth; hence the preparation being
examined passes the test for sterility.
 2.There is evidence of growth.
 So, re-testing is performed using the same number of
sample , volumes to be tested and media as in the original test.
 If no evidence of microbial growth is then found, the
preparation being examined passes the tests for sterility.
CONT..
 3. There is evidence of microbial growth.
 So, isolate and identify the organism.
ASEPTIC TECHNIQUES
 The methods which are used to prevent the access of
microorganism during the preparation of parenteral product
and their testing are called ‘Aseptic Techniques’.
 The source of contamination are:
 Atmosphere , which is contaminated with dust , droplet and
droplet nuclei becomes the breeding ground of
microorganism.
 The hands are a major means of transmitting infection.
 Coughing ,sneezing and spitting can cause contamination at a
considerable distance.
 The cloth which absorb dust particles.
 A handkerchief is the richest source of contamination.
 The hair .
DESIGN OF AN ASEPTIC ROOM.
 For performing various techniques, it is desirable to
maintain sterile conditions in the place of working .
 So, the following points must be taken before designing an
aseptic room:
 1. Site -Site selected for aseptic room.
 2. Size –Size of aseptic room
 3. Windows- To provide good light glass panes should used.
 4. Doors- Air lock double door.
 5. Surface material-Floor,walls& bench tops must be smooth,
easily cleanable.
 6. Services – Ventilation, electricity ,gas supply, vacuum
arrangement.
 7. Furniture- Working benches, chairs, trolleys, and screen.
STERILISATION OF SURGICAL DRESSING.
 The material like cotton-wool balls, gauze swabs, ribbon gauze,
bandages etc.are used in surgery and in wards.
 These are commonly known as materials of surgical dressing.
 Stages :
 1. Packing or wrapping of the unsterilised surgical dressing into
a suitable container or packing material.
 For this purpose metal drums are used.
 2. Packing should be done in such a way that it ensure steam
penetration and air removal.
 The heavy material should not be sterilised with soft material.
 3.The steriliser is closed and air inside the steriliser is replaced by
steam.
 4.The fabric pack(surgical dressing) is exposed for 30 -45 min at
121º C to sterilise the surgical dressings in it.
 5.Switch off steriliser and condense the steam inside it. Contents
of steriliser are then removed.
 6.The containers are labelled indicating date of sterilisation so as
to prevent overlong storage.
Sterilisation

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Sterilisation

  • 1. STERILISATION Mr. Dipak B. Bhingardeve Asst. Professor Department of Pharmaceutics Dr.Shivajirao kadam college of Pharmacy,kasabe digraj,Sangli
  • 2. STERILISATION  Sterilisation is the process of complete destruction of all microorganisms present in a system.  The product free from living microorganisms are called ‘sterile product.’  Antiseptic : A substance that prevents the growth of microorganisms by inhibiting their activity without destroying them is called antiseptic.  Bactericide : The substance that kills bacteria is called bactericide.  Bacteriostatic : The substance that arrest or retards the growth of bacteria is known as bacteriostatic.  Disinfection : A process that removes the infection potential by destroying microorganisms but not generally bacterial spores.
  • 3. CONT..  Germicide: A substance that kills pathogenic bacteria but not necessarily bacterial spores.  Viricidie : A substance that kills viruses.  Sterility : The absence of viable microorganism is called ‘sterility’ and preparation free from viable microorganisms are called ‘sterile’.
  • 4. THERMAL RESISTANCE OF MICROORGANISMS  The microorganism show varying resistance to different Methods of sterilisation. 1. Thermal death time : It may be defined as the time required to kills a specific types of microorganisms at a given temperature under specific conditions. 1. Death rate of microorganism : There is no direct method to determine when the sterility will be achieved. 1. Decimal reduction time (D value): It is defined as the time in minutes required to reduce the number of viable organism by 90%.
  • 5. CONT..  The order of death of microorganisms can be calculated from equation:  K = 1/t (log No.— log N)  Where,  K= constant which depends on organism ,temperature and medium.  t = time of exposure in minutes.  No= number of organisms viable at the beginning of time interval.  N=Number of organism viable at the end of the time interval.  K= 1/t bcz it is after 90% reduction in microorganism.  Time t is defined as the decimal reduction time which is called the D value.  D= 1/K
  • 6. METHODS OF STERILISATION  A) Physical Methods 1) dry heat sterilisation 2) Moist heat sterilisation 3) Radiation sterilisation a) use of ultra violet rays b) Ionising radiation  B) Chemical methods : 1. Sterilisation by heating with bactericide. 2. Gaseous Sterilisation.
  • 7. C) MECHANICAL METHODS:  Sterilisation involves the filtration of parenteral preparation through the following bacteria proof filters: 1. Ceramic filters 2. Seitz filter 3. Sintered glass filters 4. Sintered metal filters 5. Membrane filters
  • 8. A) STERILISATION BY PHYSICAL METHODS I) Dry Heat Sterilisation :  All microorganisms including bacterial spores can be destroyed by heat.  Dry heating at 100ºC kills all vegetative bacteria in one hour but it does not kill spores.  According to pharmacopoeia ,sterilisation by dry heating is effected by heating at temperature of 160 º C for two hours.
  • 9. HOT AIR OVEN :  It is used for sterilisation of pharmaceutical products and other materials.  It is double walled chamber made of steel.  Insulation material ,such as glass fibres or asbestos is filled between the two walls of the oven to avoid heat loss.  Two or three perforated shelves are fixed inside the oven.  An electric fan is also fitted to ensure the uniform circulation of hot air in the oven in order to maintain the required temperature in all the shelves.  A thermometer is fitted in the oven to note down the temperature inside the oven.
  • 11. PRECAUTIONS TO BE TAKEN WHILE PLACING MATERIAL MEANT FOR STERILISATION.  1.Glass apparatus must be wrapped with clean cloth or filter paper and container must be plugged with non-absorbent cotton wool.  2.The article & substance which are to be sterilised should not be placed at the floor of the oven as it receives direct heat and becomes much hotter.  3.The oven should not be overloaded with the material.
  • 12. ADVANTAGES  It is used for sterilisation of those substance which get spoiled during moist heat sterilisation e.g. oily material and powders.  The method is suitable for glass syringes.  Disadvantages :  This method is not suitable for surgical dressing.  This method is not suitable for most of the medicaments, rubber and plastic goods.
  • 13. APPLICATIONS :  It is mainly used for sterilisation of glass wares, such as, pestle and mortar, petridishes, flasks ,pipettes, bottles, test tube etc.  Used for sterilise powder such as, sulphacetamide, sulphadiazine , kaolin , talc, zinc oxide ,starch etc.  Also scissors, spatula, blades and glass syringes , scalpels.
  • 14. II)MOIST HEAT STERILISATION  Moist heat sterilisation is more effective than dry heat method.  Bcz steam has more penetration power than dry heat and thermal capacity of steam is more than thermal capacity of dry heat.  The method is very useful for killing of bacterial spores.
  • 15. CONDITIONS FOR HOLDING PERIOD OF STERILISATION Sr. No. Holding temperature Minimum Holding time in minute 1 115 º to 118 º 30 2 121 º to 124 º 15 3 126º to 129º 10 4 134º to 134º 5
  • 16. AUTOCLAVE :  Moist heating is done in an “autoclave”.  It consist of strong metallic chamber usually made of stainless steel.  It has cover fitted with a steam vent, a pressure gauze and a safety valve.  Rubber gasket is fitted on the inner side of lid, in order to make autoclave air tight.  The cover is closed with wing nuts and bolts.  The electrically heated element is fitted at the bottom to heat the water to convert into steam.  A sufficient quantity of water is poured into chamber after removing the perforated chamber.  The level of the water is adjusted in such a way that it does not touch the bottom of perforated chamber.
  • 17.
  • 18. CONT..  The material is packed in the perforated chamber.  The lid is then closed with wing nuts and bolts.  WORKING :  The autoclave is switched on to heat the water.  The vent is opened and safety valve is set at the required pressure .  When steam starts coming out from the vent and it continues for 5 minutes, it is then closed.  The steam pressure starts raising and it comes to desired pressure i.e. 10 lbs /sq.inch with corresponding temperature 115º c or 15 lbs/sq.inch with corresponding temperature 121º c.  After stated period , switch off the autoclave .  Allow it to cool to about 40 ºc before opening the vent.
  • 19. CONT..  When whole of the steam inside the autoclave is removed , the lid is opened and the sterilised material is taken out.  Advantages :  1.Autoclaving destroys microorganisms more efficiently than dry heat and hence the material is exposed to a lower temperature for shorter period.  It is used for sterilisation of a large number of official injections.  Disadvantages :  Unsuitable for sterilisation of powders and oils.  Cannot be used for sterilisation of injection & articles ,  Such as ,plastics which get spoiled at 115-116 º c for 30 min.
  • 20. OTHER METHODS OF STERILISATION BY MOIST HEAT  1.Tyndallisation  2. Pasteurisation  3.Sterilisation of vaccines.  1.Tyndallisation method:  This is fractional sterilisation method.  The method was official in B.P 1932, for sterilisation of medicaments.  The method was used for sterilisation of culture media.  THE method was deleted from B.P. 1932 by 4th addendum(1941) and replaced by Heating with a Bactericide.
  • 21. CONT..  Solution to be sterilised is packed & sealed in its final container an heated at 80º c for one hour on each of three successive days.  The first heating destroys the vegetative cells but not the bacterial spores.  These bacterial spores germinate into the vegetative forms in the interval between first and second heating and are killed in the second heating.  The third heating provides a safeguard against any spores which may not germinate until the second interval.
  • 22. 2. PASTEURISATION  It is partial sterilisation method which is used to make milk safe and also to improve its keeping properties.  The process kills only 97 to 99 percent microorganism but it does not kill bacterial spores.  The methods used for pasteurisation of milk are as under: 1) Holder method:  Milk is heated at 62.8 ºc for 30 min in a steam jacketed stainless steel tank containing agitators.  This provides correct exposure throughout the milk and prevent skin formation.  Clean dry steams is blown to the space above the liquid to prevent the formation of skin and foam.  The method kills all types of bacteria including mycobacterium tuberculosis.
  • 23. II) FLASH METHOD  The milk is heated to 71.6 º c for 15 sec and then quickly cooled.  The milk is heated by passing through narrow horizontal pipes inside large ones through which water passes in the opposite direction.  The method is commonly used by most of the firms bcz it is less time consuming process.  It also needs less floor space and it is a continuous process.
  • 24. 3.STERILISATION OF VACCINES:  The suspension of microorganism is prepared in normal saline solution and transferred into a sealed container.  Sterilisation is carried out by immersing the container in a thermostatically controlled water bath at a temperature between 55 ºC to 60º C for one hour.  The strick aseptic precautions are observed to exclude any possibility of contamination.  The vaccine is tested for sterility to confirm the proper sterilisation of vaccine.
  • 25. III) RADIATION STERILISATION :  Two types  1. Sterilisation by ultra violet light  2.Sterilisation by ionising radiation.  1. Sterilisation by ultra by violet light:  Direct sunlight can destroy microorganism on account of its ultra violet rays of long wavelength.  The antimicrobial activity of U.V. Light depends on its wavelength.  It is maximum at 265 nm wave length.  Ultra violet rays for sterilisation is produced by passing a low current at high voltage through mercury vapour in an evacuated glass tube.
  • 26. APPLICATIONS:  It is used for sterilisation of air to prevent cross infection in hospitals.  Used for sterilisation and maintenance of aseptic area in the pharmaceutical industry.  It is used for sterilisation of thermolabile substances before packing.  Disadvantages :  Ultra violet rays have a low penetration power, so it is not applicable to the sterilisation of packed pharmaceuticals.  U.V. rays are less effective against organisms in the atmosphere .  U.V light is harmful for worker. Eyes & skin should be protected from direct ultra violet rays.
  • 27. STERILISATION BY IONISING RADIATION  Ionising radiations are X-rays and Gamma rays.  These are lethal to bacterial cell and destroy the nuclei of the cell.  Gamma rays are produced from radio-isotonic source such as cobalt -60 or cesium -137.  The material to be sterilised is packed in the final container and then exposed to ionising radiation.  Ionising radiations are much more efficient as a sterilising agent than Ultra violet rays.
  • 28. APPLICATIONS:  The method is mainly used for sterilisation of plastic syringe ,hypodermic needles, surgical blades and adhesive dressings.  Also used for sterilisation of bone and tissue transplant, plastic tubing, sutures.  Used for sterilisation of thermolabile medicaments.
  • 29. B)CHEMICAL METHODS OF STERILISATION  Two types:  1. Sterilisation by heating with a bactericide  2. Gaseous Sterilisation  1. Sterilisation by heating with a bactericide:  This method used for sterilising aqueous preparations which are unstable at higher temperature attained in moist heat sterilisation processes.  In this process the medicament is dissolved or suspended in a suitable solution of bactericides so as to achieve the concentration of bactericides.  The preparation is then transferred into the final container which is then sealed so as to exclude microorganisms and heated at 98-100º C for 30 minute by heating it in boiling water.
  • 30. PROPERTIES OF BACTERICIDES  1.It should be non- toxic.  2.Compatible with medicament.  3.Stable and active at various pH.  4.Stable during heating and storage.  Application :  Used for sterilisation of injections
  • 31. 2.GASEOUS STERILISATION  Sterilisation is done with a chemical in gaseous state.  In olden days, formaldehyde was very commonly used, but now it has been replaced by ethylene oxide.  Beta propiolactone is new chemical which is also used as sterilising agent.  A) Formaldehyde:  It is alkylating agent.  Nowadays, it is used for the fumigation of empty rooms after infectious diseases.  Formaldehyde is inferior to ethylene oxide due to following reasons:
  • 32. CONT..  It has weak penetration power.  It is difficult to maintain a high concentration in the atmosphere.  It requires a high humidity to be effective sterilising agent.  It is irritating to respiratory tract.  B) Ethylene oxide :  It is colourless gas at room temperature.  It can be liquefied easily and the liquid boil at appro.10.8ºC.  It is readily dissolved in water and organic solvents.  It is highly inflammable so it is used by:
  • 33. 1.MIXING ETHYLENE OXIDE WITH SOME INERT GAS IN FIXED RATIO  i) Ethylene oxide = 1 part Carbon dioxide = 9 part  ii) Ethylene oxide = 11% w/v Trichlorofluromethane =79%w/w Dichlorofluromethane = 10% w/w 2.Using ethylene oxide in the absence of air:  Pure ethylene oxide or a mixture containing 90% ethylene oxide and 10 % carbon dioxide is used as sterilising agent.  Ethylene oxide kills microorganisms by alkylation of protein molecules.  It is active against all types of microorganisms.
  • 34. FACTORS INFLUENCING EFFICIENCY OF ETHYLENE OXIDE  Temperature – At room temp, bt effective at 40ºC  Concentration- Conc between 200mg to 1 g/litre.  Relative humidity- 40-50% moisture is necessary  Power of penetration- high penetration power, can penetrate thr paper ,fabrics.  Absorption – Amt & rate of absorption depends on the nature ,thickness, surface area of article.  Advantage:  It is suitable for heat sensitive substance bcz sterilisation is effective at room temperature.  Ethylene oxide has a good penetration power .  The method is useful for sterilisation of moist sensitive substance and equipment bcz only a low humidity is required.
  • 35. APPLICATIONS:  Used for sterilisation of thermolabile materials, such as rubber and plastic items.  Used for sterilisation of delicate instrument.  Applicable for sterilisation of powders.  Suitable for sterilisation of syringes and needles.  C) Beta –Propiolactone-  It is largest sterilising agent.  It has low vapour pressure and is a liquid at room temperature.  Its boiling point is 160º C hence it is non-flammable.  It has less penetration power than ethylene oxide.  So it is mainly used for sterilisation of operation theatre and aseptic rooms etc.  It is carcinogenic.
  • 36. C) STERILISATION BY MECHANICAL METHODS  The solution containing thermolabile medicament can be sterilised by filtration through bacteria proof filters.  These filters retain the bacteria and the sterile filtrate is collected in sterilised receiver.  For Successful sterilisation by filtration ,the following points should be observed :  1.The whole apparatus must be sterile.  2.An aseptic technique should be followed in order to minimize the risk of contamination.
  • 37. TYPES OF BACTERIA PROOF FILTERS  1. Ceramic filters:  These are also called filter candles.  These are made of porcelain or kieselguhr and are available in a range of pore size.  These candles are numbered according to its pore size.  The candle is placed in the solution to be sterilised and its opening is attached to the vacuum system.  When vacuum is applied pressure inside the candle is decreased.  Due to the difference in pressure between outside & inside of the candle, the solution moves into candle.  The filtrate is collected in sterile container.  Main disadvantage is its tendency to absorb materials from aqueous solution.
  • 38.
  • 39. 2.SEITZ FILTER:  It consist of two parts.  The lower part holds a perforated disc, on it compressed asbestos sheet is placed.  Two parts are joined together with the help of nuts.  The asbestos sheet is made up of asbestos fibres but may also contain cellulose and alkaline earth such as magnesium compounds.  The asbestos pads are used once only and then discarded.  Asbestos pads may yield alkali and cause precipitation of alkaloids from aqueous solution of their salts.  And also absorbs drugs from solution.  Hence, a few millilitres of filtrate should always be rejected and sintered glass disc may also be fixed in the filtration unit immediately after seitz filter.
  • 40.
  • 41. 3. SINTERED GLASS FILTERS:  These are made from borosilicate glass.  The glass is finely powdered and particles of the required size are separated and is then packed into disc moulds.  These moulds are heated until a suitable adhesion has taken place between the granules.  These discs are fused to funnels of suitable shape and size.  Sintered glass filters are available in different pore size and are numbered accordingly.  For bacteria proof filtration number 5 or 3 must be used.  The filtration is carried out under a reduced pressure .
  • 42.
  • 43. CONT..  Sintered glass filter do not absorb the medicaments from the solution.  These filters are made from borosilicate glass, so it does not change the pH of the solution.  Sintered glass filter are cleaned after use by passing water in the reverse direction.  Organic matter may be removed by passing strong sulphuric acid with 1 % sodium nitrate through the filter.  4. Sintered metal filters:  These are the metallic counter part of sintered glass filters.  These are usually made from stainless steel, and having greater mechanical strength.
  • 44. 5. MEMBRANE FILTER :  These are made of cellulose acetate or cellulose nitrate.  These are fixed in metallic holders similar to those used with asbestos pads.  The pore size in the membrane lies in the range of 100- 150 u.  They are also called millipore filters.  Membrane filters are suitable for sterilising aqueous and oily solution but are not suitable for organic solvent, such as alcohols, ketones, esters or chloroform.
  • 45.
  • 46. STERILISATION BY FILTRATION INVOLVES FOUR BASIC STEPS:  1.Filtration of the solution through one of the bacteria – proof filters which have been described above.  2.Aseptic distribution of the filtered solution into the previously sterilised final containers.  3.Aseptic closure of the containers.  4.Performing the sterility test.  Advantages :  Suitable for sterilisation of blood products, insulin and enzymes.  All types of bacteria are removed from preparation.  Both clarification and sterilisation are done side by side.
  • 47. DISADVANTAGES:  The method is not reliable one and therefore a sterility test is necessary.  The suspension and oily preparations cannot be sterilised by this method.  They are chances of absorption of medicament from solution by the filter.  Units may leak if carelessly handled.  Aseptic technique is necessary.  Highly trained staff is required.  Applications :  Method is useful for sterilisation of parenteral solutions containing thermolabile medicaments without any decomposition  E.g Insulin , blood serum .
  • 48. TESTS FOR STERILITY  These tests are based upon the principle that if bacteria or fungi are placed in a medium which provides nutritive material, moisture , the desire pH and are kept at a favourable temperature, the organisms will grow and their presence can be indicated by the turbidity in the originally clear medium.  The tests for sterility are done by detecting the presence of viable forms of bacteria , fungi and yeast in or on pharmacopoeial preparations.  The tests must be carried out under strict aseptic technique.
  • 49. CULTURE MEDIA :  Culture media is required for aerobic and anaerobic bacteria and for fungi.  The media used should comply with the tests, such as, sterility, nutritive properties, effective of media as per details given in I.P.  Methods of testing :  1. Membrane filtration method  2. Direct inoculation method  1. Membrane filtration method :  The method is preferred in the following cases.  A) An oil or oily preparation.  B) An ointment that can be put into solution.  C)A soluble powder or a liquid that possess bacteriostatic and fungistatic properties.
  • 50. CONT,,,  D) Liquid products where the volume in a container is 100 ml or more.  The method involves filtration of sample under test through a membrane filter having normal porosity of 0.45 µ and a diameter of approx 47 mm.  After filtration, the membrane is removed aseptically from the metallic holder & divided into two halves.  The first half is transferred into 100 ml of culture media meant for fungi & incubated at 20ºC to 25ºC for NLT 7 days .  The other half is transferred into 100ml of fluid thioglycollate medium and incubated at 30ºC to 35 ºC for NLT 7 days. & Observe the growth in media.
  • 51. 2.DIRECT INNOCULATION METHOD  In this method the specified quantity of sample under test is drawn aseptically from the container and transferred into a vessel of culture medium.  Mix the liquid with medium and incubate for NLT 14 days.  Observe the growth of microorganism in the medium.  Observations :  1. No evidence of growth; hence the preparation being examined passes the test for sterility.  2.There is evidence of growth.  So, re-testing is performed using the same number of sample , volumes to be tested and media as in the original test.  If no evidence of microbial growth is then found, the preparation being examined passes the tests for sterility.
  • 52. CONT..  3. There is evidence of microbial growth.  So, isolate and identify the organism.
  • 53. ASEPTIC TECHNIQUES  The methods which are used to prevent the access of microorganism during the preparation of parenteral product and their testing are called ‘Aseptic Techniques’.  The source of contamination are:  Atmosphere , which is contaminated with dust , droplet and droplet nuclei becomes the breeding ground of microorganism.  The hands are a major means of transmitting infection.  Coughing ,sneezing and spitting can cause contamination at a considerable distance.  The cloth which absorb dust particles.  A handkerchief is the richest source of contamination.  The hair .
  • 54. DESIGN OF AN ASEPTIC ROOM.  For performing various techniques, it is desirable to maintain sterile conditions in the place of working .  So, the following points must be taken before designing an aseptic room:  1. Site -Site selected for aseptic room.  2. Size –Size of aseptic room  3. Windows- To provide good light glass panes should used.  4. Doors- Air lock double door.  5. Surface material-Floor,walls& bench tops must be smooth, easily cleanable.  6. Services – Ventilation, electricity ,gas supply, vacuum arrangement.  7. Furniture- Working benches, chairs, trolleys, and screen.
  • 55. STERILISATION OF SURGICAL DRESSING.  The material like cotton-wool balls, gauze swabs, ribbon gauze, bandages etc.are used in surgery and in wards.  These are commonly known as materials of surgical dressing.  Stages :  1. Packing or wrapping of the unsterilised surgical dressing into a suitable container or packing material.  For this purpose metal drums are used.  2. Packing should be done in such a way that it ensure steam penetration and air removal.  The heavy material should not be sterilised with soft material.
  • 56.  3.The steriliser is closed and air inside the steriliser is replaced by steam.  4.The fabric pack(surgical dressing) is exposed for 30 -45 min at 121º C to sterilise the surgical dressings in it.  5.Switch off steriliser and condense the steam inside it. Contents of steriliser are then removed.  6.The containers are labelled indicating date of sterilisation so as to prevent overlong storage.