1.

0 IDENTIFICATION OF CRITICAL CONTROL MONITORING PARAMETER

Before proceeding for Validation of Autoclave cum bung processor, following
parameters
To be Checked
Biological Indicator spore concentration must be 10 6.
Used media must be tested for its growth promoting test.
Calibration of Pressure Gauge
Calibration of Temperature Gauge
Calibration of PT -100 Sensors

2.0 VALIDATION PROCEDURE

Validation of an Autoclave will be considered qualified for consistent and reliable
performance (validated) on successful completion of the following
Vacuum Leak Test
Bowie-Dick Test
Heat Distribution Study
Heat Penetration Study
Biological Challenge Test
Bung Processing
Flip off Seal
To qualify these tests, the equipment should fulfill the acceptance criteria
described in the individual test procedures.

3.0 GENERAL CONSIDERATION/PREREQUISITE

A. Approved Standard operating procedure of the equipment shall be available
B. Approved analytical methods for testing the samples collected during the
processing
C. The impact analysis of the equipments shall be recorded in the summary
sheet.
D. The installation and operational qualification of the equipment shall be
successfully completed before the execution of the performance qualification.
E. All the deficiencies and discrepancies related to the equipment which affect
the product quality and corrective action taken shall be recorded in the
appropriate section of the protocol.
F. The analytical test results and other reports related with the equipment shall
be attached with the performance qualification of the equipment and finally
verified.

3.1 CALIBRATION DETAIL OF TEMPERATURE SENSORS

Equipment Equipment
Name

I.D

Data of

Due

Date of Pre

Date of

calibration Date calibration Post

performed

Done Checked
by

by

calibration
performed

3.2 CALIBRATION OF TEMPERATURE SENSORS:

Pre & Post Calibration of Temperature Sensors.
Pre & post calibration shall be carried out before starting and after completion
of heat distribution cycles as well as Heat penetration cycles.
PRE & POST CALIBRATION OF TEMPERATURE SENSORS:
PREPRATION OF ICE BATH:
Prepare a container with crushed ice and add enough purified water to ensure a
proper
Slush allow the temperature to stabilize ensure to add sufficient crushed ice to
maintain the equilibrium state of ice and water.
PROCEDURE :
Temperature sensors which are used for qualification study shall be calibrated in
ice bath at approximately 0oc and in high temperature reference block at
50,100,121,150oc prior to its usage in qualification.
Record the temperature of all the sensors while putting them in ice bath after
one minute of temperature stabilization. Put the individual sensor to the slot of
high temperature reference block which is stabilized at required
temperature. Record the temperature for five minute by data logger and attach
the report with print outs with reports.
ACCEPTANCE CRITERIA:
No temperature sensors vary by 1 oc in ice bath from the mean of temperatures
shown by the calibrated thermometer during the data logging period.
No temperature sensors vary by 1 oc in high temperature reference block from
the mean of temperatures Shown by the calibrated thermometer during the data
logging period.

4.0 RE-VALIDATION CRITERIA :

Replacement of major component / instrument.
Major modification in the existing equipment / utility.
During monitoring if system is found to be malfunctioning.
Shifting of the equipment/Instrument from one location to another

5.0 SYSTEM DESCRIPTION:

Autoclave cum Bung processor is designed to sterilize the components which are
used in the Aseptic area while processing the sterile products. Autoclave cum
Bung processor having the chamber size 9009001500mm and is made up of
SS 316L provided with 3 validation ports having 7 Nos of probes in Each
validation port. Argon welding is done in joints of piping. Vacuum Break filter 5
inches long Having 0.2 micron porosity is provided Bung & flip off carriage made
of SS 316L, 2 Nos. trolley made of SS 304L for keeping the components in
autoclave cum bung processor provided during sterilization. All the system is
controlled through PLC.

6.0 PERFORMANCE QUALIFICATION:

The Autoclave Cum Bung Processor will be qualified after validating (As per the
methods outlined in this Protocol) the equipment for desired performance and its
ability to sterilize different components and/or loads at the set parameters & set
loading patterns, repeatedly & consistently.
The Autoclave Cum Bung Processor will be considered qualified for consistent
and reliable performance (Validated) on successful completion of the following
tests.
Vacuum Leak test (3 Trials with probe)
Bowie Dick Test for Steam penetration (3 trials on 3 different days)
Steam qualification tests (3 trials on 3 different days)
Empty Chamber Heat Distribution studies (3 trials) with temperature mapping
probes at different locations of the sterilizer chamber.
Loaded Chamber heat penetration studies (3 trials) for each sterilization load of
fixed loading pattern, with temperature mapping probes inside the innermost
possible layer of the load subjected for sterilization.
Bio-Challenge studies using Bacillus stearothemophilus spore strips (containing
106 or more spores per strip) during the heat distribution & heat penetration
studies.
Estimation of the FO value achieved during the sterilization hold period at each
temperature-mapping probe.

 Quality of the steam condensate collected from the sampling point provided in
the sterilizer chamber condensate drain line for all loads.
Vacuum break filter integrity testing.
To qualify these tests the equipment should fulfill the acceptance criteria
described in the individual test procedures.

6.1 VACUUM LEAK TEST

Objective:
To verify the leakage in sterilization chamber during Vacuum Hold when the
sterilization chamber is empty.
Procedure:
Operate the equipment as per SOP .
Set the parameters in PLC.
PROCESS PARAMETERS:
PRE VACCUM

DELAY BEFORE HOLD

VACCUM HOLD TIME

CHAMBER PRESSURE

Record the vacuum Hold start pressure and vacuum Hold end pressure &
calculate the actual leakage by subtracting the former from later.
Three consecutive cycles shall be carried out as per above parameters and
procedure.
ACCEPTANCE CRITERIA
Vacuum leak rate should not be more than 0.013 BAR / 10 minute.
RESULTS:
Record the Observations in Annexure # 1

6.2 STEAM QUALITY TESTS

A continuous supply of saturated steam is required for steam sterilization. Too
high a level of non-condensable gases will prevent the attainment of sterilizing
conditions; too little moisture carried in suspension may allow the steam to
become superheated during expansion into the chamber, while excess moisture
may cause damp loads.

For all these tests, the steam should be sampled from the steam service pipe.
The measurements are taken during a period of maximum steam demand, when
steam is first admitted to the sterilizer chamber.
Presence of non-condensable gases more than the desired level might get
accumulated within the sterilization chamber in the form of air pockets, which
might affect steam penetration this leading to probable non-sterile unit.
NOTE: Silicone rubber tubing is porous to steam and should not be used to carry
steam in these tests.
6.2.1 Non-Condensable Gas Test
Objective
Objective of this test is to ensure that,
The pure steam supply of the High Pressure High Vacuum Steam Sterilizer does
not contain non-condensable gases more than the desire level (NMT 3.5%) when
measured on-line during the standard sterilization cycle.
Procedure
This test is used to demonstrate that the level of non-condensable gases in the
steam will not prevent the attainment of sterilization conditions in any part of the
load. The method described should be regarded not as measuring the exact level
of non-condensable gas, but a method by which the provision of acceptable
steam quality can be demonstrated. The apparatus is shown and described in
Figure No.1 (all sizes are nominal). Connect the needle valve to the steam
service pipe as shown in Figure No.1. Assemble the apparatus so that
condensate will drain freely from the long rubber tube into the sampling pipe. If
the tube is too short, copper or stainless steel tubing may also be used. Fill the
container with cold water until it overflows. Fill the burette and funnel with cold
water, invert them and place them in the container. Draw out any air that has
collected in the burette. With the steam sampling pipe out of the container, open
the needle valve and allow steam to purge the air form the pipe. Place the pipe
in the container, locate the end within the funnel, and add more cold water until
it flows through the overflow pipe. Place the empty measuring cylinder under the
container overflow. Adjust the needle valve to allow a continuous sample of
steam into the funnel sufficient to cause a small amount of Steam Hammer to
be heard. Ensure that all the steam is discharged into the funnel and does not
bubble out into the container. Note the setting of the needle valve. Close the
valve.
Ensure that the container is topped up with cold water and that the measuring
cylinder is empty. Draw out any air present in the burette. Ensure that the
sterilizer chamber is empty except for the usual chamber furniture. Select and
start the operating cycle. When the steam supply to the chamber first opens,
open the needle valve to the previously noted setting, allowing a continuous

sample of steam into the funnel sufficient to cause a small amount of steam
hammer to be heard. Allow the steam sample to condense in the funnel. Any
non-condensable gases will rise to the top of the burette. Overspill formed by the
condensate and the water displaced by the gases will collect in the measuring
cylinder.
When the temperature of the water in the container reaches 70-75 oc close the
needle valve. Note the volume of gas collected in the burette (Vb) and the
volume of water collected in the measuring cylinder (Vc).
Calculate the fraction of non-condensable gases as a percentage as follows.
When the temperature of the water in the container reaches 70-75 oc close the
needle valve. Note the volume of gas collected in the burette (Vb) and the
volume of water collected in the measuring cylinder (Vc).
Calculate the fraction of non-condensable gases as a percentage as
follows.
Fraction of non-condensable gases = 100 x (Vb/Vc).
The test should be considered satisfactory if the fraction of non-condensable
gases does not exceed 3.5 %.
The test should be done two more times to check consistency. If the results of
the three tests differ significantly, then the cause should be investigated before
proceeding further.
Figure No.1
Acceptance Criteria:
The measured non-condensable gases in the pure steam should not cross 3.5%
Observations & Results
Record the observations and results in formats enclosed as Attachement-2,
Annexure-1
6.2.2 Super Heat
Objective
This test is used to demonstrate that the amount of moisture in suspension with
steam from the service supply is sufficient to prevent the steam from becoming
superheated during expansion into the chamber.
The method described here uses a low-volume sample, continuously taken from
the center of the steam service pipe. The level of superheat determined by this
method cannot be regarded as indicative of the true dryness of the steam in the
pipe since condensate flowing along the inner surface is not collected. However,
devices designed to separate free condensate are incorporated into the steam
delivery system to the chamber and therefore the level determined by this

method is representative of steam conditions likely to prevail within the chamber

during the plateau period.
Procedure
This test should normally follow a satisfactory test for non-condensable gases.
This test, and the subsequent dryness value test, requires a pitot tube as shown
in figure No.2. The rest of the apparatus is shown and described in figure No.3.
All sizes are nominal. Fit the Pitot tube concentrically within the steam service
pipe as shown in figure No.3. Fit the sensor entry gland to the steam service
pipe. Insert one of the sensors through the gland and position the axis of the
pipe. Insert the second sensor through the gland in the expansion tube and
position it on the axis of the pipe. Wrap lagging around the expansion tube. Push
the tube on to the pitot.
Figure No.2.
Ensure that the sterilizer chamber is empty except for the usual chamber
furniture. Select and start the operating cycle.
From the measured temperatures, note the temperature in the steam service
pipe (for use in the dryness test) and in the expansion tube (Te) when the steam
supply to the chamber first opens. Calculate the superheat in oC from the
following equation:
Superheat = Te - To
Where:
To is the boiling point of water at local atmospheric pressure.
The test should be considered satisfactory if the superheat measured in the
expansion tube does not exceed 25oC.
Record the observations and results in formats enclosed as Attachement-2,
Annexure-2.
6.2.3 DRYNESS TEST
The accurate measurement of the percentage of moisture content in the steam is
difficult, and the traditional methods where constant steam flow is required are
not suitable for sterilizers. This test should be regarded not as measuring the
true content of moisture in the steam, but as a method by which the provision of
acceptable steam quality can be demonstrated.
6.2.4 Objective
This test is used to demonstrate that the dryness value is not less than 0.90 (if
metal loads be processed, the dryness value should not be less than 0.95);

throughout the operating cycle, the temperature measured in the steam service
pipe is within 30C of that measured during the superheat test.
6.2.5 Procedure
The test is conveniently carried out immediately after the superheat test.
This test requires a pitot tube as shown in Figure No.2. The apparatus is shown
and described in figure No.4. All sizes are nominal. A laboratory balance is also
required, capable of weighing a load up to 2 kg with an accuracy of 0.1g or
better. If it is not already fitted, fit the Pitot tube concentrically within the steam
service pipe as shown in figure No.3. If it is not already fitted, fit the sensor entry
gland to the steam service pipe. Insert a temperature sensor through the gland
and position it on the axis of the pipe.
Connect the rubber tube to the longer of the pipes in the stopper, place the
stopper in the neck of the vacuum flask, weigh the whole assembly and note the
mass (M1). Remove the stopper and tube assembly and pour 650 +50 ml of cold
water (below 27oC) in to the flask. Replace the stopper and tube assembly, weigh
the flask and record the mass (M2). Support the flask close to the pitot, and
ensure that the rubber tube and flask are protected from excess heat and
draughts, do not connect it to the pitot tube yet.
Introduce the second temperature sensor through the shorter of the two pipes in
the stopper and into the water in the flask. Note the temperature of the water in
the flask (To). Ensure that the sterilizer chamber is empty except for the usual
chamber furniture. Select and start the operating cycle.
When the steam supply to the chamber first opens, connect the rubber tube to
the pitot discharge and wrap lagging around it. Arrange the rubber tube to
permit condensate to drain freely into the flask. Not the temperature in the
steam service pipe (TS).
When the temperature of the water in the flask is approximately 80 oC, disconnect
the rubber tube from the pitot, agitate the flask so that the contents are
thoroughly mixed, and note the temperature of the water (T1).
Weigh the flask and stopper assembly and note the mass (M3).
The initial mass of water in the flask is given by Mw = M2 M1
The mass of condensate collected is given by MC = M3 - M2.
Calculate the dryness value of the steam from the following equation:
D= (T1-T0) (4.18MW + 0.24) / LMC - 4.18 (TS-T1) / L
Where:
T0 = Initial temperature of the water in the flask (oC);
T1 = Final temperature of the water and condensate in the flask ( oC);
TS = Average temperature of the steam delivered to the sterilizer ( oC);
MW = Initial mass of water in the flask (Kg);
MC= Mass of condensate collected (Kg);

L= latent heat of dry saturated steam at temperature TS (kJ Kg-1).

6.2.6 Acceptance Criteria
The test should be considered satisfactory if the following requirements are met.
A) The dryness value is not less than 0.90 (if metal loads are to be processed,
the dryness value should not be less than 0.95);
B) Throughout the operating cycle, the temperature measured in the steam
service pipe is within 3oC of that measured during the superheat test.
6.2.7 Observation and results
Record the observations and results in formats enclosed as Attachement-2,
Annexure-3 See next page for fig no.4.

6.3 BOWIE-DICK TEST

Objective:
To ensure that the vacuum pulses applied before the Sterilization hold period are
sufficient to remove the entrapped air or non condensable gases so as to
facilitate the event and rapid steam distribution into all parts of load and
maintaining these conditions for specified temperature holding time.
6.3.1 PROCEDURE
Operate the equipment as per SOP.
Set the following parameters in PLC.
PRE VACUUM
PRE PRESSURE
STERILIZATION HOLD TEMPERATURE
STERILIZATION HOLD TIME
Place one Bowie dick test kit pack in the center ( Near drain) of the sterilization
chamber supported approximately 100 to 200 mm above the sterilization
chamber base
The print outs taken during Bowie dick test cycle & bowie dick indicator shall
be attached as per exhibit
Compile the observation and take three consecutive cycles.
Acceptance Criteria
The Bowie Dick test indicator shows the uniform color change. No change, no
uniform change and / or Air entrapment (bubble) spot on the test pack indicate
inadequate air removal from the sterilization base chamber.
Results

Record

the

Observation

Related: Low Temperature Sterilization Process (115C)

6.4 HEAT DISTRIBUTION STUDY (EMPTY CHAMBER)

Objective:
Objective of this test is to verify that the temperature uniformity throughout the
chamber and to Locate the cold spot in empty Chamber.
The sterilizer is capable of attaining the temperature of 121.0 oc during the
sterilization
Hold period.
The profile point having the lowest temperature or slowest to heat is
designated as the cold spot.
Procedure:
Insert 16 nos. of temperature sensors inside the chamber through the validation
port of sterilizer .seal the port with silicone sealant to ensure that no steam
leakage during operation of sterilizer.
Fix the probe at the location at the location in the sterilizer so that sensors do not
touch the metallic surface of the chamber. Connect the temperature sensors to
the data logger which can scan and print the actual temperature at different
locations. Set the following parameters & operate the autoclave cum bung
processor and also start the Data logger to record the actual temperature.
SET PARAMETERS :
Sterilization Hold temp = 121.0oc
Sterilization Hold time = 30 min
Chamber high pressure = 1.5 BAR
After completion of sterilization collect thermograph from the multipoint
temperature recorder of the autoclave cum bung processor.
Download the data from data logger to the computer for data analysis & graph
prep.
Perform the three consecutive cycles to demonstrate the consistency of
sterilizer.
ACCEPTANCE CRITERIA:
1. Throughout the cycle time, all temperature measured in the chamber do not
fluctuate by more than 20C.
2. Throughout the cycle time, all temperatures measured in the chamber do not
differ from each other by more than 20C.
3. The interval of time between the attainment of the sterilization temperature
in the hottest and coldest part of the chamber does not exceed 60 seconds.
Results: Record the observations.
LOCATION OF TEMPERATURE SENSORS INSIDE THE CHAMBER

SENSOR NO. LOCATION OF SENSORS IN THE CHAMBER

S1

In the drain of autoclave chamber

S2

Lower left front corner of the non sterile side

S3

Upper left front corner of non sterile side

S4

Upper right front corner of non sterile side

S5

Lower left front corner of non sterile side

S6

Middle left side of the chamber

S7

Middle right side of the chamber`

S8

Middle front non sterile side of the chamber

S9

Middle back sterile side of the chamber

S10

Lower left sterile side of the chamber

S11

Upper left sterile side of the chamber

S12

Upper right sterile side of the chamber

S13

Lower right sterile side of the chamber

S14

Middle of upper most surface of the chamber

S15

Middle of lower surface of the chamber

6.5 HEAT PENETRATION STUDY:

Objective
Objective of this test is to ensure that, the steam is sufficiently penetrating into
the innermost portion of the load subjected for sterilization to achieve desired
temperature i.e. 121.40C during the sterilization cycle.
Heat penetration studies shall be carried out with the following different loads:
a.

Maximum garment load

b.

Minimum garment load

c.

Accessory load (filling machine parts load)

d.

Bung load

e.

Flip off seal

Temperature probe and biological indicator placement in the maximum garment

load
Load details
Sterile garments

- 24 pieces

Booties

- 48 Nos.

Head Gear

- 24Nos.

Gloves (7 inches) - 24 pairs

LOAD CONFIGURATION:
One Perforated box (C1) containing 8 pair of garments placed on lower front of
trolley (non sterile door side).

One Perforated box (C2) containing 8 pair of garments placed on lower back side
of trolley (sterile door side)
One Perforated box (C3) containing 8 pair of garments placed on upper front of
trolley (non sterile side)
One perforated box (C4) containing 24 pair of gloves wrapped in parchment
paper & 04 pair of garments placed on upper back side of trolley (sterile door
side)
Temperature Sensors with Chemical & Biological Indicator Placed In Maximum
Garment Load
Sensor no.

Location of sensors in the chamber

S1

In the drain of autoclave chamber

S2

Center of the garment pack located at the lower surface in C1

S3

Center of garment pack located in the middle of C1

S4

Center of the garment pack located at the upper surface in C1

S5

Center of the garment pack located at the lower surface in C2

S6

Center of the garment pack located in the middle of C2

S7

Center of the garment pack located at the lower surface in C2

S8

Center of the garment pack located at upper surface in C2

S9

Center of the garment pack located at lower surface in C3

S10

Center of the garment pack located in middle of C3

S11

Center of the garment pack located in middle of C3

S12

Center of the garment pack located at upper surface in C3

S13

Center of the garment pack located at lower surface of C4

S14

Center of the garment pack located at upper surface in C4

S15

Center of gloves pack in C4

Temperature probe and biological indicator placement in the minimum garment

load
Load Details
Sterile garments 6 pairs
Gloves (6 inches) 8 pairs
Load configuration:
One perforated box containing 6 pairs of garments & 8 pair gloves wrapped in
parchment paper placed on lower middle of chamber.
TEMPERATURE SENSORS WITH CHEMICAL & BIOLOGICAL
INDICATOR PLACEMENT IN PARTIAL GARMENT LOAD
SENSOR NO.
S1

LOCATION IN THE CHAMBER

In the drain of autoclave chamber

S2

Center of the garment pack located at the lower surface in C1

S3

Center of the garment pack located in the middle of C1

S4

Center of the garment pack located at the upper surface in C1

S5

Center of glove pack located at upper surface in C1

Temperature probe and biological indicator placement in the accessory

load (Filling machine parts)
Load details
Powder Hopper- 2 Nos.
Piston Wheel- (2 Nos.)
Piston- (32 Nos.)
Piston Tips- (36 Nos.)
Spoon-(2 Nos.)
Scoop-(2 Nos.)
Tubes (4 Nos.)
Bung Boxes- (2 Nos.)
Forceps- (4 Nos.)
Sterile Garments 12 pieces
Booties 12 pairs
Gloves (7 inches) 24 pairs
Load configuration:
12 sterile garments, 12 Booties & 24 Pairs Gloves (6 inches) in SS Perforated
Container placed on Top.(Non Sterile Side)
One Perforated box containing 2 Piston Wheel, 32 Nos. Piston and 36 Nos.
Piston Tips., Placed on Bottom.(Sterile Side)
One Perforated box containing 4 Nos. Tubes, 4 Nos. forceps, 2 Nos. Spoons, 2
Nos. Scoops, Placed on Top.(Sterile Side)
Powder Hopper 2 Nos. & 2 Nos. Bung Boxes Placed at Bottom ( Non Sterile
Side).
6.5.1 PROCEDURE
Conduct the study with loaded chamber cycles with temperature probes and
Biological Indicators. Transfer the load to sterilizer and connect the 15 probes &
Biological Indicators as per the locations. Connect the outputs of all the probes to
the temperature data logger and close the door of sterilizer. Switch ON the
MAINS of the control panel and set the parameters
Temperature

121.4C

Chamber Pressure High

1.5 BAR

Hold time

30 minutes

Simultaneously Insert new chart in chart recorder provided on the control panel
of an autoclave and adjust the start time and temperature of the instrument.

Now start the cycle as per SOP for Operating Instruction. After attaining
temperature 121.40C, record the chamber temperature and pressure for every
minute. Simultaneously start the recording with data logger and take print outs
At the end of the cycle Switch OFF the cycle. When pressure becomes -0.04 BAR,
open the door with the help of safety gloves. Remove the load. Repeat the same
cycle three times and compile the data.
6.5.2 Acceptance Criteria:
Throughout the cycle time all temperature measured in the chamber do not
fluctuate by more than 20C.
Throughout the cycle time, all temperatures measured in the chamber do not
differ from each other by more than 20C.
The calculated minimum F0 value should be more than biological F0 value for the
Biological indicator.
Each autoclaved Biological indicator gives the -ve test during the incubation.
Results:
Record the Observations.

6.6 Biological Challenge Test

Objective
To demonstrate the degree of Process Lethality provided by the Sterilization
Cycle.
Test Requirement
Spores of Geobacillus Stearothermophilus
Procedure
After determining the worst case items and worst locations i.e. cold spots,
challenge these items/locations with biological indicator (spore Geobacillus
Stearothermophilus)
Carry out the Microbial Challenge Studies concurrently with Loaded Chamber
Heat penetration studies.
Previously population validated biological ampoule indicators of specified
106 or more spores of Geobacillus Stearothermophilus should be issued for
validation as per procedure.
Place the biological indicators along with the probes at the same locations,
within each load type of the specified loading configuration and pattern, as in the
Loaded Chamber Heat penetration studies. Retain 2 biological indicators as
positive controls. Operate the Autoclave cum Bung Processor as per SOP.
Switch ON the Mains of the control panel and set the parameters
Temperature
Chamber Pressure High :

121.4C
1.5 BAR

Hold time

30 minutes

Simultaneously insert new chart recorder provided on the control panel of an

Autoclave and adjust the start time and temperature of the instrument.
Now start the cycle.
After attaining temperature 121.4C, record the chamber temperature and
pressure for every 60 seconds.
Simultaneously start the recording with data logger and take printouts. At the
end of the cycle Switch OFF the cycle. When pressure becomes -0.04 BAR, open
the door of an Autoclave with the help of safety gloves. After incubation observe
the indicator for growth. (+ve when purple color change to yellow color, -ve
when purple color remain as such) Conduct the test until a cycle time results in
three consecutive runs where the biological indicators show on growth.
Acceptance Criteria
1. Visually observe the ampoules, test +ve when purple color change to yellow
color, test -ve when purple color remain as such).
2. If no evidence of growth observed in any of the inoculated tube and growth
observed in positive control tube, the test meets the criteria to achieve the
Sterility Assurance Level (SAL) 10-6.

6.7 Bung Processing

Objective:
Objective of this test is to ensure that, the bung washing is proper and bung load
subjected for sterilization to achieve desired temperature i.e. 121.4C during the
sterilization cycle.
Load details
Maximum Bungs cycle (Approx 80000)
Minimum Bungs cycle (Approx 5000)
Load configuration
Bungs in Bung Carriage (Approx 80000)
6.7.1 Procedure
Load the bungs in Bungs Carriage cassettes. Approximately 8000 Bung Load in
each cassette.
Place one biological indicator in each cassette and transfer the load to
Autoclave cum Bung processor.
Switch ON the MAINS of the control panel and run cycle as per HPHV
Process Parameters.
Simultaneously Insert new chart in chart recorder provided on the control panel
of an autoclave and adjust the start time and temperature of the instrument
after attaining temperature 121.4C, record the chamber temperature and

pressure for every minute. At the end of washing cycle send WFI rinse sample to
QC for Analysis of Non Ionic Detergent Content. At the end of the cycle switch
OFF the cycle.
When pressure becomes -0.04 BAR, open the door with the help of safety gloves.
Remove the load. Repeat the same cycle three times and compile the data.
Acceptance criteria:
The calculated minimum F0 value should be more than biological F0 value for the
Biological indicator. Each autoclaved Biological indicator gives the -ve test
during the incubation. WFI rinse sample should be free from non ionic Detergent.
Results:
Record the Observations
7.0 F0 Calculation
The actual observations obtained during the heat penetration studies at different
temperature sensing locations were complied in the table and the observed
temperature were subjected for calculation of F0 values at that particular
location. The calculations are done by using the following formula and the
lethality factor computed (during the sterilization period) are given in the
following table.
F0=dt

10(T-121)/Z

__________ (a)

F0=dt (Sum of lethality factors)

Where,
Dt = The time interval between successive temperature measurements (1 min).
T = The observed temperature at that particular time (as per the actual
temperatures recoded
Z = The change in the heat resistance of Bacillus stearothermophilus spores as
temperature is Changed (10C)
The calculated minimum F0 value (by equation a) should be more than the
biological F0 value for the biological indicator strip exposed for the bio-challenge
study. The biological F0 value for biological indicator strip exposed during the
sterilization can be calculated as follows.
F0 = D121 (log A log B) (b)
Where,
D121

D value of the biological indicator at 1210C

Biological indicator concentration or spore

population
Desired level of sterility

8.0 INTERPRETATION OF RESULTS

All test results shall be compiled and evaluated. Compliance of all the results
with empty chamber and loaded chamber with different loads to the acceptance

criteria shall establish that the sterilization practices, control and facility are
suitable for various required loads.

9.0 REVALIDATION FREQUENCY

Schedule Revalidation
Twice in a year.
Unscheduled Revalidation
Unscheduled revalidation shall be carried out in following situation:
When major changes in the load is taken place.
After major maintenance of existing equipment.

10.0 DOCUMENTATION
Results and reports shall be compiled in a binder. Binder shall contain the
following sequentially:
Summary Report
Test Reports