This document provides information on autoclaving equipment and processes. It discusses the types of autoclaves, how they work, parameters for sterilization cycles, and methods for verification. Different loads and cycle conditions are tested to ensure proper heat distribution and sterilization throughout the chamber. Validation protocols outline installation, operational and performance qualification tests to demonstrate that the autoclave functions as intended.

1. EQUIPMENT FOREQUIPMENT FOR
AUTOCLAVINGAUTOCLAVING
BYBY
Sardar Liaqat AliSardar Liaqat Ali
Chemistry Department Hazara UniversityChemistry Department Hazara University
MansehraMansehra

2. CONTENTSCONTENTS
 INTRODUCTIONINTRODUCTION
 ADVANTAGESADVANTAGES
 DISADVANTAGESDISADVANTAGES
 WHAT CAN BE AUTOCLAVEDWHAT CAN BE AUTOCLAVED
 EQUIPMENTEQUIPMENT
 TYPYS AND OPERATIONTYPYS AND OPERATION
 VERIFICATIONVERIFICATION
 VALIDATIONVALIDATION
 CONCLUSIONCONCLUSION
 REFERENCEREFERENCE

3. 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.

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

5. DISADVANTAGESDISADVANTAGES
 Unsuitable for anhydrous materials suchUnsuitable for anhydrous materials such
as powders and oils.as powders and oils.
 Not used for injections, articles such asNot used for injections, articles such as
some plastics, that deteriorate at 115some plastics, that deteriorate at 115°C°C

6. What can be autoclavedWhat can be autoclaved
 Surgical InstrumentsSurgical Instruments
 GlasswareGlassware
 Plastic tubes and pipette tipsPlastic tubes and pipette tips
 Solutions and waterSolutions and water
 Animal food and beddingAnimal food and bedding
 WasteWaste

7. 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 121°C 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.

8. Principle of autoclavePrinciple of autoclave
 Steam penetrates objects in the autoclaveSteam penetrates objects in the autoclave
 Condensation creates negative pressureCondensation creates negative pressure
and draws in additional steamand draws in additional steam
 Moist heat kills microorganisms viaMoist heat kills microorganisms via
Coagulation of proteins.Coagulation of proteins.

10. 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 121°C
 Conditions inside are thermostsstically controlled

12. 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

13. 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

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

15. 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

16. 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
65°C for 30 minutes.
 High Temperature Short Time Pasteurization (HTST):
Used today. Milk is exposed to 72°C for 15 seconds.
 Ultra High Temperature Pasteurization (UHT): Milk is
treated at 140°C for 3 seconds and then cooled very quickly in a
vacuum chamber.
 Advantage: Milk can be stored at room temperature for
several months.

17. Other methods of autoclaving:
 Tyndallization:
- heating the material at 80°C for 1hr or at 100°C 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 100°C 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.

18. 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
80°C.
 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.

19. VERIFICATION TESTSVERIFICATION TESTS
Monitoring of the temperature and pressure within the chamberMonitoring of the temperature and pressure within the chamber
gives indication that the process has been properly carried out.gives indication that the process has been properly carried out.
Verification of autoclaving byVerification of autoclaving by
٠٠ Instrumental methodInstrumental method
٠٠ Bacteriological methodsBacteriological methods
٠٠ chemical indicatorschemical indicators

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

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

26. 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

27.  VALIDATION TEAM:VALIDATION TEAM:

28.  Installation Qualification:Installation Qualification:
 NameName
 DescriptionDescription
 Model and identification no.Model and identification no.
 The locationThe location
 Utility requirementsUtility requirements
 Connections and safety features of the systems/equipmentConnections and safety features of the systems/equipment
which need to be documented.which need to be documented.

29.  OPERATIONAL QUALIFICATION PROTOCOL (OQOPERATIONAL QUALIFICATION PROTOCOL (OQ))
 1.0 PURPOSE :1.0 PURPOSE :
 To demonstrate and document that the operationsTo demonstrate and document that the operations
 of the Autoclave take place as specified .of the Autoclave take place as specified .
 2.0 SCOPE :2.0 SCOPE :
 Autoclave xxxxx will be qualified to meet OQ.Autoclave xxxxx will be qualified to meet OQ.
 3.0 RESPONSIBILITY :3.0 RESPONSIBILITY :
 Microbiologist, Manager Q.CMicrobiologist, Manager Q.C
 4.0 PROCEDURE :4.0 PROCEDURE :
 This should be performed by external agency.This should be performed by external agency.
 4.1 Verify the following as per instrument operating procedure and calibration certificate kept4.1 Verify the following as per instrument operating procedure and calibration certificate kept
in place before validation.in place before validation.
 4.1.1 : Temperature display of Autoclave.4.1.1 : Temperature display of Autoclave.
 4.1.2 : Compound pressure gauge of Autoclave.4.1.2 : Compound pressure gauge of Autoclave.
 Acceptance criteria :Acceptance criteria :
 All calibration data found to be within the acceptable norms of calibration certificate.All calibration data found to be within the acceptable norms of calibration certificate.
 4.2 Calibration of Thermocouples :4.2 Calibration of Thermocouples :
 Calibrate all the thermocouples of data logger before and after the validation using standardCalibrate all the thermocouples of data logger before and after the validation using standard
thermometer and also made available party’s calibration certificates.thermometer and also made available party’s calibration certificates.
 Acceptance criteria:Acceptance criteria:
 The variation between the temperature of thermocouples and the standard thermometerThe variation between the temperature of thermocouples and the standard thermometer
found to be within the acceptance criteria.found to be within the acceptance criteria.

30.  4.3 Heat Distribution Studies4.3 Heat Distribution Studies
 Carry out heat distribution studies by using a multi-pointCarry out heat distribution studies by using a multi-point
data logger and maintain holding time for 15 minutes atdata logger and maintain holding time for 15 minutes at
15 lbs. by fixing all the 12 probes as per diagram.15 lbs. by fixing all the 12 probes as per diagram.
Record the temperature and lag time of each probe.Record the temperature and lag time of each probe.
 Acceptance criteriaAcceptance criteria
 All probes must reach temperature 121-124°C andAll probes must reach temperature 121-124°C and
pressure must be within 15 to 18 lbs for 15min cycle.pressure must be within 15 to 18 lbs for 15min cycle.
 Diagram-1 Probe to 12 inside the chamberDiagram-1 Probe to 12 inside the chamber

31.  Load PatternLoad Pattern
 Maximum LoadMaximum Load
 Load with all the glassware and media filled upto 70%, of theLoad with all the glassware and media filled upto 70%, of the
chamber and the details are as follows.chamber and the details are as follows.
 Test-1 : 250ml Conical flasks = 12 Nos with media, 13 Nos withoutTest-1 : 250ml Conical flasks = 12 Nos with media, 13 Nos without
media, 500ml Conical flasks with media = 4nos, 1000ml Conicalmedia, 500ml Conical flasks with media = 4nos, 1000ml Conical
flasks with media =4nos, Pipette10ml=10nos,Pipette 2ml=10nos,flasks with media =4nos, Pipette10ml=10nos,Pipette 2ml=10nos,
Pipette 5ml=10nos, Pipette 1ml = 10nos,100mlPipette 5ml=10nos, Pipette 1ml = 10nos,100ml
 bottles=20nos ,Filtering unit=10nos, Test tubes =25nosbottles=20nos ,Filtering unit=10nos, Test tubes =25nos
 Minimum LoadMinimum Load
 Load with all the glassware and media required for a day’s analysisLoad with all the glassware and media required for a day’s analysis
(average).(average).
 250ml Conical flasks with media = 6nos, 500ml Conical flask with250ml Conical flasks with media = 6nos, 500ml Conical flask with
media – 3 nos., 1000ml Conical flask - 1 Pipette 10ml= 10nos,media – 3 nos., 1000ml Conical flask - 1 Pipette 10ml= 10nos,
Pipette 2ml= 10nos, Pipette 5ml= 10nos, Pipette 1ml=10nos,Pipette 2ml= 10nos, Pipette 5ml= 10nos, Pipette 1ml=10nos,
Bottles=10nos, Test tubes - 25nos.Bottles=10nos, Test tubes - 25nos.
 Record the temperature and lag timeRecord the temperature and lag time

32.  PERFORMANCE QUALIFICATION PROTOCOL (PQ)PERFORMANCE QUALIFICATION PROTOCOL (PQ)
 1.0 PURPOSE :1.0 PURPOSE :
 To provide a performance qualification protocol for Autoclave.To provide a performance qualification protocol for Autoclave.
 2.0 SCOPE :2.0 SCOPE :
 Specified to Autoclave xxxxx.Specified to Autoclave xxxxx.
 3.0 RESPONSIBILITY :3.0 RESPONSIBILITY :
 Microbiologist, Manager Q.CMicrobiologist, Manager Q.C

4.0 PROCEDURE4.0 PROCEDURE ::
 4.1 Heat Penetration Studies:4.1 Heat Penetration Studies: Carry out the heat penetration studies byCarry out the heat penetration studies by
using a multi point data logger for the following loads mentioned.using a multi point data logger for the following loads mentioned.
Record the temperature and lag time.Record the temperature and lag time.

 Acceptance criteria:Acceptance criteria:
 All 12 probes must reach temperature 121°C to 124°CAll 12 probes must reach temperature 121°C to 124°C
and pressure must be within 15 to 18 lbs. for 15 minand pressure must be within 15 to 18 lbs. for 15 min
cycle.cycle.

33.  4.2 Microbial limit test :4.2 Microbial limit test :
 Incubate the sterilized media flask or tubes from any one Maximum andIncubate the sterilized media flask or tubes from any one Maximum and
Minimum load of heat penetration studies and observe for nutritiveMinimum load of heat penetration studies and observe for nutritive
properties. Bacteria: 30 – 35 °C for 72 hrs, Fungi : 20 – 25°C for 120 hrsproperties. Bacteria: 30 – 35 °C for 72 hrs, Fungi : 20 – 25°C for 120 hrs
 Acceptance criteria:Acceptance criteria:
 No microbial growth should be observed i.e. Negative control and NutritiveNo microbial growth should be observed i.e. Negative control and Nutritive
properties of media must passproperties of media must pass
 4.3 Microbial challenge test :4.3 Microbial challenge test :
 Keep ampoules containing spores suspension of BacillusKeep ampoules containing spores suspension of Bacillus
stearothermophilus 106 population at various location of the autoclave alongstearothermophilus 106 population at various location of the autoclave along
with probes and maintain the sterilization temperature at 15psi and 121°Cwith probes and maintain the sterilization temperature at 15psi and 121°C
during the heat penetration studies, once on the maximum load.during the heat penetration studies, once on the maximum load.
 Acceptance criteria:Acceptance criteria:
 Autoclaved ampoules containing Bacillus stearothermophilus sporesAutoclaved ampoules containing Bacillus stearothermophilus spores
suspension ampoules should not show any colour change after five days ofsuspension ampoules should not show any colour change after five days of
incubation.incubation.

35.  5.0 DOCUMENTATION :5.0 DOCUMENTATION :
 5.15.1
 Master Instrument used for validation of autoclaveMaster Instrument used for validation of autoclave
 Institutes name and address carrying out calibration.Institutes name and address carrying out calibration.
 Standard calibrating instrument name and number.Standard calibrating instrument name and number.
 Instrument certified against (Instrument of national or international standards)Instrument certified against (Instrument of national or international standards)
 Date of calibration and validity period of calibration.Date of calibration and validity period of calibration.
 Training certificate of persons (External agency) carrying out validation.Training certificate of persons (External agency) carrying out validation.
 6.2 Autoclave being calibrated :6.2 Autoclave being calibrated :
 All temperature readings for autoclave being validated should be collected fromAll temperature readings for autoclave being validated should be collected from
the approved external agency.the approved external agency.
 Validation report with observed any error, statement of calibration and nextValidation report with observed any error, statement of calibration and next
validation due.validation due.
 7.0 FREQUENCY :7.0 FREQUENCY :
 Once in a year until and unless no change in autoclave. In case of any change,Once in a year until and unless no change in autoclave. In case of any change,
the autoclave must be revalidatedthe autoclave must be revalidated
 8.08.0
 CONCLUSION :CONCLUSION : Finally conclusion should be drawn based on theFinally conclusion should be drawn based on the
results of above tests and documentedresults of above tests and documented

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

37. REFERENCEREFERENCE
 Ansel’s – Pharmaceutical dosage forms and drug deliveryAnsel’s – Pharmaceutical dosage forms and drug delivery
systems.systems.
 Bentle’s – Text book of pharmaceutics.Bentle’s – Text book of pharmaceutics.
 Cooper Gunn’s – Dispensing for pharmaceutical students.Cooper Gunn’s – Dispensing for pharmaceutical students.
 www. google. comwww. google. com
 www. Autoclaving. comwww. Autoclaving. com
 www. Validation of autoclave. Comwww. Validation of autoclave. Com
 www. Moist heat sterilization. co.inwww. Moist heat sterilization. co.in
 www. Pharma E text book. comwww. Pharma E text book. com
 Lachmann – Theory and practice of industrial pharmacyLachmann – Theory and practice of industrial pharmacy
 Ananthnarayana – Text book of microbiologyAnanthnarayana – Text book of microbiology
 www. Wikipedia. Comwww. Wikipedia. Com
 James swabrick, James C. Boylan, Encyclopedia ofJames swabrick, James C. Boylan, Encyclopedia of
pharmaceutical technology, Edition-3, Volume-Ipharmaceutical technology, Edition-3, Volume-I