EQUIPMENT FOR
AUTOCLAVING
BY
D.NARENDAR
M. Pharm-II sem
DEPARTMENT OF PHARMACEUTICS
UNIVERSITY COLLEGE OF PHARMACEUTICAL
SCIENCES
KAKATIYA UNIVERSITY, WARANGAL
CONTENTS










INTRODUCTION
ADVANTAGES
DISADVANTAGES
WHAT CAN BE AUTOCLAVED
EQUIPMENT
TYPYS AND OPERATION
VERIFICATION
VALIDATION
CONCLUSION
REFERENCE
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.
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.
DISADVANTAGES


Unsuitable for anhydrous materials such
as powders and oils.
Not used for injections, articles such as
some plastics, that deteriorate at 115°C
What can be autoclaved

Surgical Instruments

Glassware

Plastic tubes and pipette tips

Solutions and water

Animal food and bedding

Waste
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 autoclave



Steam penetrates objects in the autoclave
Condensation creates negative pressure
and draws in additional steam
Moist heat kills microorganisms via
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
• exhaust
Conditioning:
• 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
Monitoring of the temperature and pressure within the chamber
gives indication that the process has been properly carried out.
Verification of autoclaving by
٠ Instrumental method
٠ Bacteriological methods
٠ chemical indicators
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.
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 - 115°C, acetanilide - 116°C, succinic anhydride - 120°C,
benzoic acid -121°C.

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 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.

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:







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.



















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 party’s calibration certificates.
Acceptance criteria:
The variation between the temperature of thermocouples and the standard
thermometer found to be within the acceptance criteria.





4.3 Heat Distribution Studies
Carry out heat distribution studies by using a multipoint 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 121-124°C and
pressure must be within 15 to 18 lbs for 15min cycle.
Diagram-1 Probe to 12 inside the chamber

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, Pipette10ml=10nos,Pipette 2ml=10nos,
Pipette 5ml=10nos, Pipette 1ml = 10nos,100ml
bottles=20nos ,Filtering unit=10nos, Test tubes =25nos
Minimum Load
Load with all the glassware and media required for a day’s 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 1ml=10nos,
Bottles=10nos, Test tubes - 25nos.
Record the temperature and lag time

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 121°C to 124°C
and pressure must be within 15 to 18 lbs. for 15 min
cycle.








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 – 25°C 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 121°C 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.















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
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.
REFERENCE












Ansel’s – Pharmaceutical dosage forms and drug delivery
systems.
Bentle’s – Text book of pharmaceutics.
Cooper Gunn’s – 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