EQUIPMENT FOR EQUIPMENT FOR AUTOCLAVINGAUTOCLAVING

BYBY D.NARENDARD.NARENDAR

M. Pharm-II semM. Pharm-II sem

DEPARTMENT OF PHARMACEUTICSDEPARTMENT OF PHARMACEUTICSUNIVERSITY COLLEGE OF UNIVERSITY COLLEGE OF

PHARMACEUTICAL SCIENCESPHARMACEUTICAL SCIENCESKAKATIYA UNIVERSITY, WARANGALKAKATIYA UNIVERSITY, WARANGAL

CONTENTSCONTENTS

INTRODUCTIONINTRODUCTION ADVANTAGESADVANTAGES DISADVANTAGESDISADVANTAGES WHAT CAN BE AUTOCLAVEDWHAT CAN BE AUTOCLAVED EQUIPMENT EQUIPMENT TYPYS AND OPERATIONTYPYS AND OPERATION VERIFICATIONVERIFICATION VALIDATIONVALIDATION CONCLUSIONCONCLUSION REFERENCEREFERENCE

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.

ADVANTAGESADVANTAGES

Destroys micro-organisms more efficiently Destroys micro-organisms more efficiently than dry heat and therefore a shorter than dry heat and therefore a shorter exposure at a lower temperature is possible.exposure at a lower temperature is possible.

Used for large proportion of official injections.Used for large proportion of official injections. Porous materials can be sterilized without Porous materials can be sterilized without

damage.damage. Equipment or components of rubber and Equipment or components of rubber and

certain plastics such as nylon and pvc.certain plastics such as nylon and pvc.

DISADVANTAGESDISADVANTAGES Unsuitable for anhydrous materials Unsuitable for anhydrous materials

such as powders and oils.such as powders and oils. Not used for injections, articles such Not used for injections, articles such

as some plastics, that deteriorate at as some plastics, that deteriorate at 115115°C°C

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

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.  

Principle of autoclavePrinciple of autoclave

Steam penetrates objects in the Steam penetrates objects in the autoclaveautoclave

Condensation creates negative Condensation creates negative pressurepressure and draws in additional and draws in additional steamsteam

Moist heat kills microorganisms viaMoist heat kills microorganisms via

Coagulation of proteins.Coagulation of proteins.

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

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

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

Autoclave cycle:

Involves three phases. • conditioning • exposure • exhaustConditioning: • first phase

• steam enters chamber and conditions load • air displaced through chamber drain

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

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.

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.

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.

VERIFICATION TESTS VERIFICATION TESTS Monitoring of the temperature and pressure within the Monitoring of the temperature and pressure within the

chamberchamber

gives indication that the process has been properly gives indication that the process has been properly carried out.carried out.

Verification of autoclaving byVerification of autoclaving by

٠٠ Instrumental method Instrumental method

٠٠ Bacteriological methodsBacteriological methods

٠٠ chemical indicatorschemical indicators

Instrumental methodInstrumental method

Temperature conditions within the load can be sensed Temperature conditions within the load can be sensed by thermocouples or thermisters inserted into it by thermocouples or thermisters inserted into it various places.various places.

In case of liquids, the probe is placed in representative In case of liquids, the probe is placed in representative bottles and in case of dressings inserted well into bottles and in case of dressings inserted well into specimen tanks.specimen tanks.

Autoclave fitted with ports and connecting to Autoclave fitted with ports and connecting to recorders which give a continuous tracing.recorders which 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 as Existence of superheating within porous loads such as

surgical dressings.surgical dressings.

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 Consisting of a crystalline substances of known melting point

contained in a glass tubes .contained in a glass tubes . E.g. sulphur - 115°C, acetanilide - 116°C, succinic anhydride - E.g. sulphur - 115°C, acetanilide - 116°C, succinic anhydride -

120°C, benzoic acid -121°C.120°C, benzoic acid -121°C. A dye could be included to show more clearly that the crystal had A dye could be included to show more clearly that the crystal had

melted.melted. Brown’s tubes method – a controlled chemical reaction is involved Brown’s tubes method – a controlled chemical reaction is involved

in the change of colour of a red liquid through amber to green.in the 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 and Type-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 Chemical methods also available in the colour of adhesive tape(3M

Ltd)Ltd) or sheets of paper marked with sensitized strips(E.S.& A Robinson or sheets of paper marked with sensitized strips(E.S.& A Robinson

Ltd).Ltd). These are useful when a high autoclaving temperature 134C These are useful when a high autoclaving temperature 134C

applied with applied with holding holding time of not more than 3.5 min. time of not more than 3.5 min.

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

VALIDATION TEAM:VALIDATION TEAM:

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 Connections and safety features of the

systems/equipment which need to be documented.systems/equipment which need to be documented.

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 kept in 4.1 Verify the following as per instrument operating procedure and calibration certificate kept in

place before validation.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 standard Calibrate 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 thermometer found to be The variation between the temperature of thermocouples and the standard thermometer found to be

within the acceptance criteria.within the acceptance criteria.

4.3 Heat Distribution Studies4.3 Heat Distribution Studies

Carry out heat distribution studies by using a multi-Carry out heat distribution studies by using a multi-point data logger and maintain holding time for 15 point data logger and maintain holding time for 15 minutes at 15 lbs. by fixing all the 12 probes as per minutes at 15 lbs. by fixing all the 12 probes as per diagram. Record the temperature and lag time of diagram. Record the temperature and lag time of each probe.each probe.

Acceptance criteriaAcceptance criteria All probes must reach temperature 121-124°C and All probes must reach temperature 121-124°C and

pressure must be within 15 to 18 lbs for 15min pressure must be within 15 to 18 lbs for 15min cycle.cycle.

Diagram-1 Probe to 12 inside the chamberDiagram-1 Probe to 12 inside the chamber

Load PatternLoad Pattern

Maximum LoadMaximum Load Load with all the glassware and media filled upto 70%, of the chamber Load with all the glassware and media filled upto 70%, of the chamber

and the details are as follows.and the details are as follows. Test-1 : 250ml Conical flasks = 12 Nos with media, 13 Nos without media, Test-1 : 250ml Conical flasks = 12 Nos with media, 13 Nos without media,

500ml Conical flasks with media = 4nos, 1000ml Conical flasks with media 500ml Conical flasks with media = 4nos, 1000ml Conical flasks with media =4nos, Pipette10ml=10nos,Pipette 2ml=10nos, Pipette 5ml=10nos, =4nos, Pipette10ml=10nos,Pipette 2ml=10nos, Pipette 5ml=10nos, Pipette 1ml = 10nos,100mlPipette 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 analysis Load with all the glassware and media required for a day’s analysis

(average).(average). 250ml Conical flasks with media = 6nos, 500ml Conical flask with media – 250ml Conical flasks with media = 6nos, 500ml Conical flask with media –

3 nos., 1000ml Conical flask - 1 Pipette 10ml= 10nos, Pipette 2ml= 10nos, 3 nos., 1000ml Conical flask - 1 Pipette 10ml= 10nos, Pipette 2ml= 10nos, Pipette 5ml= 10nos, Pipette 1ml=10nos, Bottles=10nos, Test tubes - Pipette 5ml= 10nos, Pipette 1ml=10nos, Bottles=10nos, Test tubes - 25nos.25nos.

Record the temperature and lag time Record the temperature and lag time

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 by Carry 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°C and All 12 probes must reach temperature 121°C to 124°C and

pressure must be within 15 to 18 lbs. for 15 min cycle.pressure must be within 15 to 18 lbs. for 15 min cycle.

4.2 Microbial limit test :4.2 Microbial limit test : Incubate the sterilized media flask or tubes from any one Maximum Incubate the sterilized media flask or tubes from any one Maximum

and Minimum load of heat penetration studies and observe for and Minimum load of heat penetration studies and observe for nutritive properties. Bacteria: 30 – 35 °C for 72 hrs, Fungi : 20 – 25°C nutritive properties. Bacteria: 30 – 35 °C for 72 hrs, Fungi : 20 – 25°C for 120 hrsfor 120 hrs

Acceptance criteria:Acceptance criteria: No microbial growth should be observed i.e. Negative control and No microbial growth should be observed i.e. Negative control and

Nutritive properties of media must passNutritive properties of media must pass 4.3 Microbial challenge test :4.3 Microbial challenge test : Keep ampoules containing spores suspension of Bacillus Keep ampoules containing spores suspension of Bacillus

stearothermophilus 106 population at various location of the stearothermophilus 106 population at various location of the autoclave along with probes and maintain the sterilization autoclave along with probes and maintain the sterilization temperature at 15psi and 121°C during the heat penetration studies, temperature at 15psi and 121°C during the heat penetration studies, once on the maximum load.once on the maximum load.

Acceptance criteria:Acceptance criteria: Autoclaved ampoules containing Bacillus stearothermophilus spores Autoclaved ampoules containing Bacillus stearothermophilus spores

suspension ampoules should not show any colour change after five suspension ampoules should not show any colour change after five days of incubation.days of incubation.

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 from the All temperature readings for autoclave being validated should be collected from the

approved external agency.approved external agency. Validation report with observed any error, statement of calibration and next Validation 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, the Once in a year until and unless no change in autoclave. In case of any change, the

autoclave must be revalidatedautoclave must be revalidated 8.08.0 CONCLUSION :CONCLUSION : Finally conclusion should be drawn based on the results Finally conclusion should be drawn based on the results

of above tests and documentedof above tests and documented

conclusionconclusion

Autoclaving are used to render items Autoclaving are used to render items (such as waste) sterile or free of any living(such as waste) sterile or free of any livingorganisms. To accomplish this, autoclaves organisms. To accomplish this, autoclaves

use extreme heat, steam and pressure.use extreme heat, steam and pressure.Once autoclaved, waste can be disposed Once autoclaved, waste can be disposed of in the regular trash as it is no longerof in the regular trash as it is no longer

hazardous.hazardous.

REFERENCEREFERENCE Ansel’s – Pharmaceutical dosage forms and drug delivery systems.Ansel’s – Pharmaceutical dosage forms and drug delivery 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. 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 of pharmaceutical James swabrick, James C. Boylan, Encyclopedia of pharmaceutical

technology, Edition-3, Volume-Itechnology, Edition-3, Volume-I