EQUIPMENT FOR AUTOCLAVING

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EQUIPMENT FOR AUTOCLAVING

• BY
• D.NARENDAR
• M. Pharm-II sem
• DEPARTMENT OF PHARMACEUTICS
• UNIVERSITY COLLEGE OF PHARMACEUTICAL SCIENCES
• KAKATIYA UNIVERSITY, WARANGAL

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CONTENTS

• INTRODUCTION
• ADVANTAGES
• DISADVANTAGES
• WHAT CAN BE AUTOCLAVED
• EQUIPMENT
• TYPYS AND OPERATION
• VERIFICATION
• VALIDATION
• CONCLUSION
• REFERENCE

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INTRODUCTION

• Sterilization Killing or removing all forms of
  microbial life (including endospores) in a
  material or an object. 
• Autoclaving is the process of moist heat
  sterilization, in which micro-organisms are
  exposed to steam under saturated pressure.

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ADVANTAGES

• Destroys micro-organisms more efficiently than
  dry heat and therefore a shorter exposure at a
  lower temperature is possible.
• Used for large proportion of official injections.
• Porous materials can be sterilized without
  damage.
• Equipment or components of rubber and certain
  plastics such as nylon and pvc.

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DISADVANTAGES

• Unsuitable for anhydrous materials such as
  powders and oils.
• Not used for injections, articles such as some
  plastics, that deteriorate at 115C

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What can be autoclaved

• Surgical Instruments
• Glassware
• Plastic tubes and pipette tips
• Solutions and water
• Animal food and bedding
• Waste

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Equipment

• Autoclave Chamber which is filled with hot steam
  under pressure.  Preferred method of
  sterilization, unless material is damaged by
  heat, moisture, or high pressure.
• Temperature of steam reaches 121C at twice
  atmospheric pressure.
• Most effective when organisms contact steam
  directly or are contained in a small volume of
  liquid.
• All organisms and endospores are killed within 15
  minutes.
• Require more time to reach center of solid or
  large volumes of liquid.  

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Principle of autoclave

• Steam penetrates objects in the autoclave
• Condensation creates negative pressure and draws
  in additional steam
• Moist heat kills microorganisms via
• Coagulation of proteins.

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Working of autoclave

• Steam enters the chamber jacket, pass through an
  operating valve
• Enters the rear of the chamber behind a baffle
  plate
• It flows forward and down ward through the
  chamber and load, existing at the front bottom.
• Pressure regulator maintains pressure in the
  chamber and in jacket at a minimum of 15psi,the
  pressure required for steam to reach 121C
• Conditions inside are thermostsstically
  controlled

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Types

• Two types of autoclaves
• gravity displacement
• vacuum assisted
• gravity
• most common
• steam pumped into top of chamber
• air forced out bottom during conditioning
  phase

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Pre-Vacuum

• Uses a vacuum pump to remove air from chamber
  prior to sterilization
• Steam able to penetrate all surfaces within the
  chamber
• Pot-vacuum drying phase ensures dryness
• Based on amount sterilized autoclaves are two
  types
• portable type
• horizontal type

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Autoclave cycle

• Involves three phases.
• conditioning
• exposure
• exhaust
• Conditioning
• first phase
  steam
  enters chamber and conditions load
  air displaced through chamber drain

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Autoclave cycle (contd)

• Exposure
• Steam processes load at selected time and
  temperature.
• Effects kills of infectious agents
• Exhaust
• Steam is removed from chamber and pressure is
  released
• Load is dried, if drying option is selected

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Other methods of autoclaving

• Pasteurization Developed by Louis Pasteur to
  prevent the spoilage of beverages.  Used to
  reduce microbes responsible for spoilage of beer,
  milk, wine, juices, etc.
• Classic Method of Pasteurization Milk was
  exposed to 65C for 30 minutes.
• High Temperature Short Time Pasteurization
  (HTST) Used today.  Milk is exposed to 72C for
  15 seconds.
• Ultra High Temperature Pasteurization (UHT) Milk
  is treated at 140C for 3 seconds and then cooled
  very quickly in a vacuum chamber.
•  Advantage  Milk can be stored at room
  temperature for several months.

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Other methods of autoclaving

• Tyndallization
• - heating the material at 80C for 1hr or
  at 100C for less on three successive days.
• - vegetative cells killing in the first
  heating,
• spores will germinate before the next, when
  they also will be killed.
• Heating with a bactericide
• - heating the injection solutions in their
  final containers in boiling water or in a steamer
  at 98 to 100C for 30 mins, the permitted
  bactericides being o.2 chlorocresol and o.002
  phenylmercuricnitrate.
• - it is not used for intrathecal,
  intracysternal and iv injections greater than 15
  ml in volume.

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Other Autoclaving methods

• Low pressure method
• Steam is admitted to a previously evacuated
  autoclave to give a negative pressure of about
  360mm of Hg, resulting in a temperature of 80C.
• It maintained for 10 mins kill all vegetative
  bacteria.
• Effectiveness of process increased by adding
  formaldehyde vapour to the steam and spores can
  be killed if conditions are held for 2 hr.

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VERIFICATION TESTS

• Monitoring of the temperature and pressure within
  the chamber
• gives indication that the process has been
  properly carried out.
• Verification of autoclaving by
• ? Instrumental method
• ? Bacteriological methods
• ? chemical indicators

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Instrumental method

• Temperature conditions within the load can be
  sensed by thermocouples or thermisters inserted
  into it various places.
• In case of liquids, the probe is placed in
  representative bottles and in case of dressings
  inserted well into specimen tanks.
• Autoclave fitted with ports and connecting to
  recorders which give a continuous tracing.
• Disadvantages
• Not give information on the humidity conditions.
  
• Existence of superheating within porous loads
  such as surgical dressings.

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Chemical indicators

• Previously called as witness tubes method.
• Consisting of a crystalline substances of known
  melting point contained in a glass tubes .
• E.g. sulphur - 115C, acetanilide - 116C,
  succinic anhydride - 120C, benzoic acid -121C.
• A dye could be included to show more clearly that
  the crystal had melted.
• Browns tubes method a controlled chemical
  reaction is involved in the change of colour of a
  red liquid through amber to green.
• For autoclaving two types are available
• Type-I -- changes to full
  green in about 16 min at 120C and in
  10 min at 125C.
• Type-II -- changes in about 10
  min at 120C.
• ? Chemical methods also available in the
  colour of adhesive tape(3M Ltd)
• or sheets of paper marked with sensitized
  strips(E.S. A Robinson Ltd).
• These are useful when a high autoclaving
  temperature 134C applied with holding time
  of not more than 3.5 min.

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VALIDATION PROTOCOL

• Validation of the Autoclave is classified into
  the following
• 1.0 IQ _Installation Qualification
• 2.0 OQ Operational Qualification
• 3.0 PQ Performance Qualification

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• VALIDATION TEAM

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• Installation Qualification
• Name
• Description
• Model and identification no.
• The location
• Utility requirements
• Connections and safety features of the
  systems/equipment which need to be documented.

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• OPERATIONAL QUALIFICATION PROTOCOL (OQ)
• 1.0 PURPOSE
• To demonstrate and document that the operations
• of the Autoclave take place as specified .
• 2.0 SCOPE
• Autoclave xxxxx will be qualified to meet OQ.
• 3.0 RESPONSIBILITY
• Microbiologist, Manager Q.C
• 4.0 PROCEDURE
• This should be performed by external agency.
• 4.1 Verify the following as per instrument
  operating procedure and calibration certificate
  kept in place before validation.
• 4.1.1 Temperature display of Autoclave.
• 4.1.2 Compound pressure gauge of Autoclave.
• Acceptance criteria
• All calibration data found to be within the
  acceptable norms of calibration certificate.
• 4.2 Calibration of Thermocouples
• Calibrate all the thermocouples of data logger
  before and after the validation using standard
  thermometer and also made available partys
  calibration certificates.
• Acceptance criteria

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• 4.3 Heat Distribution Studies
• Carry out heat distribution studies by using a
  multi-point data logger and maintain holding time
  for 15 minutes at 15 lbs. by fixing all the 12
  probes as per diagram. Record the temperature and
  lag time of each probe.
• Acceptance criteria
• All probes must reach temperature 121-124C and
  pressure must be within 15 to 18 lbs for 15min
  cycle.
• Diagram-1 Probe to 12 inside the chamber

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• Load Pattern
• Maximum Load
• Load with all the glassware and media filled upto
  70, of the chamber and the details are as
  follows.
• Test-1 250ml Conical flasks 12 Nos with
  media, 13 Nos without media, 500ml Conical flasks
  with media 4nos, 1000ml Conical flasks with
  media 4nos, Pipette10ml10nos,Pipette 2ml10nos,
  Pipette 5ml10nos, Pipette 1ml 10nos,100ml
• bottles20nos ,Filtering unit10nos, Test tubes
  25nos
• Minimum Load
• Load with all the glassware and media required
  for a days analysis (average).
• 250ml Conical flasks with media 6nos, 500ml
  Conical flask with media 3 nos., 1000ml Conical
  flask - 1 Pipette 10ml 10nos, Pipette 2ml
  10nos, Pipette 5ml 10nos, Pipette 1ml10nos,
  Bottles10nos, Test tubes - 25nos.
• Record the temperature and lag time

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• PERFORMANCE QUALIFICATION PROTOCOL (PQ)
• 1.0 PURPOSE
• To provide a performance qualification protocol
  for Autoclave.
• 2.0 SCOPE
• Specified to Autoclave xxxxx.
• 3.0 RESPONSIBILITY
• Microbiologist, Manager Q.C
• 4.0 PROCEDURE
• 4.1 Heat Penetration Studies Carry out the heat
  penetration studies by using a multi point data
  logger for the following loads mentioned. Record
  the temperature and lag time.
• Acceptance criteria
• All 12 probes must reach temperature 121C to
  124C and pressure must be within 15 to 18 lbs.
  for 15 min cycle.

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• 4.2 Microbial limit test
• Incubate the sterilized media flask or tubes from
  any one Maximum and Minimum load of heat
  penetration studies and observe for nutritive
  properties. Bacteria 30 35 C for 72 hrs,
  Fungi 20 25C for 120 hrs
• Acceptance criteria
• No microbial growth should be observed i.e.
  Negative control and Nutritive properties of
  media must pass
• 4.3 Microbial challenge test
• Keep ampoules containing spores suspension of
  Bacillus stearothermophilus 106 population at
  various location of the autoclave along with
  probes and maintain the sterilization temperature
  at 15psi and 121C during the heat penetration
  studies, once on the maximum load.
• Acceptance criteria
• Autoclaved ampoules containing Bacillus
  stearothermophilus spores suspension ampoules
  should not show any colour change after five days
  of incubation.

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• 5.0 DOCUMENTATION
• 5.1
• Master Instrument used for validation of
  autoclave
• Institutes name and address carrying out
  calibration.
• Standard calibrating instrument name and number.
• Instrument certified against (Instrument of
  national or international standards)
• Date of calibration and validity period of
  calibration.
• Training certificate of persons (External agency)
  carrying out validation.
• 6.2 Autoclave being calibrated
• All temperature readings for autoclave being
  validated should be collected from the approved
  external agency.
• Validation report with observed any error,
  statement of calibration and next validation due.
• 7.0 FREQUENCY
• Once in a year until and unless no change in
  autoclave. In case of any change, the autoclave
  must be revalidated
• 8.0
• CONCLUSION Finally conclusion should be drawn
  based on the results of above tests and documented

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conclusion

• Autoclaving are used to render items (such as
  waste) sterile or free of any living
• organisms. To accomplish this, autoclaves use
  extreme heat, steam and pressure.
• Once autoclaved, waste can be disposed of in the
  regular trash as it is no longer
• hazardous.

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REFERENCE

• Ansels Pharmaceutical dosage forms and drug
  delivery systems.
• Bentles Text book of pharmaceutics.
• Cooper Gunns Dispensing for pharmaceutical
  students.
• www. google. com
• www. Autoclaving. com
• www. Validation of autoclave. Com
• www. Moist heat sterilization. co.in
• www. Pharma E text book. com
• Lachmann Theory and practice of industrial
  pharmacy
• Ananthnarayana Text book of microbiology
• www. Wikipedia. Com
• James swabrick, James C. Boylan, Encyclopedia of
  pharmaceutical technology, Edition-3, Volume-I

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