1.0 IDENTIFICATION OF CRITICAL CONTROL MONITORING PARAMETERBefore proceeding for Validation of Autoclave cum bung processor, following parametersTo be Checked Biological Indicator spore concentration must be 106. Used media must be tested for its growth promoting test. Calibration of Pressure Gauge Calibration of Temperature Gauge Calibration of PT -100 Sensors2.0 VALIDATION PROCEDUREValidation 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 SealTo qualify these tests, the equipment should fulfill the acceptance criteria described in the individual test procedures.3.0 GENERAL CONSIDERATION/PREREQUISITEA.Approved Standard operating procedure of the equipment shall be availableB.Approved analytical methods for testing the samples collected during the processingC.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 SENSORSEquipment NameEquipment I.DData of calibrationDue DateDate of Precalibrationperformed Date of PostcalibrationperformedDone byChecked by

3.2CALIBRATION 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 ICEBATH:Prepare a container with crushed ice and add enough purified water to ensure a properSlush 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 50,100,121,150ocprior 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 stabilizedat 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 1oc in ice bath from the mean of temperatures shown by the calibrated thermometer during the data logging period.No temperature sensors vary by 1oc 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 another5.0 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) BowieDick 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 106or more spores per strip) during the heat distribution & heat penetration studies. Estimation of the FOvalue 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.6.1VACUUM LEAK TESTObjective: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.PROCESSPARAMETERS: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 CRITERIAVacuum leak rate should not be more than 0.013 BAR/10 minute.RESULTS:Record the Observations in Annexure # 16.2 STEAM QUALITY TESTSA 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 TestObjectiveObjective 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.ProcedureThis 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 70-75occlose 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 70-75occlose 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 & ResultsRecord the observations and results in formats enclosed as Attachement-2, Annexure-16.2.2 Super HeatObjectiveThis 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.ProcedureThis 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 inoC from the following equation:Superheat = Te - ToWhere: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 TESTThe 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 ObjectiveThis 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 ProcedureThe 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 80oC, 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 M1The 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) / LWhere: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 CriteriaThe 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 resultsRecord the observations and results in formats enclosed as Attachement-2, Annexure-3 See next page for fig no.4.6.3 BOWIE-DICK TESTObjective: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.6.3.1PROCEDURE Operate the equipment as per SOP. Set the following parameters in PLC.PRE VACUUMPRE PRESSURESTERILIZATION HOLD TEMPERATURESTERILIZATION HOLD TIME Place oneBowiedick 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 duringBowiedick test cycle &bowiedick indicator shall be attached as per exhibit Compile the observation and take three consecutive cycles.Acceptance CriteriaThe 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.ResultsRecord the Observation

Related:Low Temperature Sterilization Process (115C)6.4HEAT DISTRIBUTIONSTUDY (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 121.0oc during the sterilizationHold 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 = 121.0ocSterilization Hold time = 30 minChamber 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 CHAMBERSENSOR NO.LOCATION OF SENSORS IN THE CHAMBER

S1In the drain of autoclave chamber

S2Lower left front corner of the non sterile side

S3Upper left front corner of non sterile side

S4Upper right front corner of non sterile side

S5Lower left front corner of non sterile side

S6Middle left side of the chamber

S7Middle right side of the chamber`

S8Middle front non sterile side of the chamber

S9Middle back sterile side of the chamber

S10Lower left sterile side of the chamber

S11Upper left sterile side of the chamber

S12Upper right sterile side of the chamber

S13Lower right sterile side of the chamber

S14Middle of upper most surface of the chamber

S15Middle of lower surface of the chamber

6.5 HEAT PENETRATION STUDY:ObjectiveObjective 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 loadb.Minimum garment loadc.Accessory load (filling machine parts load)d.Bung loade.Flip off sealTemperature probe and biological indicator placement in the maximum garment loadLoad details Sterile garments - 24 piecesBooties - 48 Nos.Head Gear - 24Nos.Gloves (7 inches) - 24 pairsLOAD 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 LoadSensor no.Location of sensors in the chamber

S1In the drain of autoclave chamber

S2Center of the garment pack located at the lower surface in C1

S3Center of garment pack located in the middle of C1

S4Center of the garment pack located at the upper surface in C1

S5Center of the garment pack located at the lower surface in C2

S6Center of the garment pack located in the middle of C2

S7Center of the garment pack located at the lower surface in C2

S8Center of the garment pack located at upper surface in C2

S9Center of the garment pack located at lower surface in C3

S10Center of the garment pack located in middle of C3

S11Center of the garment pack located in middle of C3

S12Center of the garment pack located at upper surface in C3

S13Center of the garment pack located at lower surface of C4

S14Center of the garment pack located at upper surface in C4

S15Center of gloves pack in C4

Temperature probe and biological indicatorplacement in the minimum garment loadLoad DetailsSterile garments 6 pairsGloves (6 inches) 8 pairsLoad 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 INDICATORPLACEMENT IN PARTIAL GARMENT LOAD SENSOR NO.LOCATION IN THE CHAMBER

S1In the drain of autoclave chamber

S2Center of the garment pack located at the lower surface in C1

S3Center of the garment pack located in the middle of C1

S4Center of the garment pack located at the upper surface in C1

S5Center 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 pairsLoad 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 PROCEDUREConduct 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 thelocations. 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 : 121.4CChamber Pressure High : 1.5 BARHold time : 30 minutesSimultaneously 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 outsAt 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 cyclethreetimes 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 F0value should be more than biological F0value for the Biological indicator.Each autoclaved Biological indicator gives the -ve test during the incubation.Results:Record the Observations.6.6 Biological Challenge TestObjectiveTo demonstrate the degree of Process Lethality provided by the Sterilization Cycle.Test RequirementSpores of Geobacillus StearothermophilusProcedureAfter determining the worst case items and worst locations i.e. cold spots, challenge these items/locations with biological indicator (sporeGeobacillus Stearothermophilus)Carry out the Microbial Challenge Studies concurrently with Loaded Chamber Heat penetration studies.Previously population validated biological ampoule indicators of specified 106ormore spores ofGeobacillus Stearothermophilusshould be issued for validation as per procedure.Place the biological indicators along with the probes at the same locations, withineach 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 : 121.4CChamber Pressure High : 1.5 BARHold time : 30 minutesSimultaneously 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 Criteria1. 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 ProcessingObjective: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 ProcessParameters.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 sendWFIrinse 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 F0value should be more than biological F0value 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 Observations7.0F0CalculationThe 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 F0values 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 recodedZ =The change in the heat resistance ofBacillus stearothermophilusspores as temperature is Changed (10C)The calculated minimum F0value (by equation a) should be more than the biological F0value for the biological indicator strip exposed for the bio-challenge study. The biological F0value for biological indicator strip exposed during the sterilization can be calculated as follows.F0= D121(log A log B) (b)Where,D121D value of the biological indicator at 1210C

ABiological indicator concentration or spore population

BDesired level of sterility

8.0 INTERPRETATION OF RESULTSAll 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 FREQUENCYSchedule RevalidationTwice in a year.Unscheduled RevalidationUnscheduled 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 DOCUMENTATIONResults and reports shall be compiled in a binder. Binder shall contain the following sequentially:Summary ReportTest Reports