procedure for operation calibration and temperature mapping of horizontal and vertical autoclave

1.0       Purpose: 

To lay down the procedure for operation, calibration and temperature mapping of horizontal and vertical autoclave.

2.0       Scope:

This procedure applies to lab horizontal and vertical autoclave of the following:           

3.0       Responsibility:

3.1       The responsibility of operation and calibration of horizontal and vertical autoclave lies with the microbiologist.

4.0       Definitions/Abbreviations: Nil

5.0       Procedure:

5.1       Operating Procedure for Horizontal Autoclave:

5.1.1    Open the door of the autoclave manually by rotating lever at non-sterile end.

5.1.2The chamber of the autoclave is cleaned properly with purified water.

5.1.3Open the water inlet valve and start the water supply. When the water level rises up to ¾^th height of the gauge of the glass tube, then close the water inlet valve. 

5.1.4Put the main switch in ON position, which will be indicated by glowing of the red bulb.

5.1.5Switch on the heater, which will be indicated by glowing of the yellow bulb.

5.1.6When jacket steam pressure reaches from 0 to approx.1.2 to 1.4 Kg/cm2. 

5.1.7Load the autoclave according to the standard validated load pattern and doors are closed properly indicating by the lamps.

5.1.8Switch ON circular chart recorder and note the load identification number.

5.1.9Pre Vacuum Cycle:

5.1.9.1Before the standard sterilization cycle the vacuum shall be applied.

5.1.9.2The chamber steam exhaust valve shall be closed and open the vacuum valve connecting to the sterilizer.

5.1.9.3Open the water inlet valve to the condenser of vacuum pump.

5.1.9.4Keep the condenser overflow valve crack open.

5.1.9.5Keep the steam inlet valve in close position.

5.1.9.6Switch ON vacuum pump.

5.1.9.7Observe the degree of vacuum being created in chamber, which is indicated in the compound gauge.

5.1.9.8After attaining the vacuum (10 to 20 of Hg), close the vacuum line valve and stop the vacuum pump.

5.1.9.9Close the water inlet valve to the vacuum pump condenser.

5.1.9.10 Follow the above mentioned procedure for 2 more times.

5.1.10 Set the Temperature and time on the panel (Soak Timer) with the help of the keys.

5.1.11  Press the ‘PGM’ Key and hold it for 3-4 seconds, the set temperature will be displayed.

5.1.12 The temperature shall be changed by using the increment, decrement keys and then press the   Enter/PGM Key.

5.1.13 Then the Time parameter will be displayed.

5.1.14  Set Time shall be changed similar to the temperature and then press the Enter / PGM key.

5.1.15  When the jacket steam pressure shown in the gauge reaches the operating pressure 1.2 to   1.4 Kg/cm², then press the Start Key on the panel (Soak Timer). Or

5.1.16  Then open the valve for steam inlet from jacket to chamber, which allows the steam into the chamber from jacket. 

5.1.17  The steam entered into the chamber is spread through out the chamber.

5.1.18  As soon as the set temperature and pressure (Example: 121°C / 1.2 Kg/ cm²) attains,sterilization timer will start the count down which will be indicated by the display on timer.

5.1.19  After the sterilization time is over, the Buzzer will sound indicating end of sterilization cycle and press ‘ACK’ Key.

5.1.20  To check for effectiveness of sterilization place one chemical indicator (class-6) along with each load.

5.1.21  Record the time and maintain the sterilization cycle as given below for different loads.  

5.1.22  After completion of the sterilization cycle, switch OFF the heaters, open exhaust slowly and  allow the chamber pressure to come down to zero kg/cm².

5.1.23  If required then create the post-vacuum cycle for drying as per the procedure given in the prevacuum cycle.

5.1.24  Three pulses of pre vacuum cycle shall be created only to filtration units, garments load and sample bottles.

5.1.25  For drying of the garments load after sterilization, post vacuum shall be created for 10 minutes and ( If required) 0.2µ filtered air is allowed in the chamber for vacuum break.

5.1.26  Open the door of the autoclave by manually when the chamber pressure reaches to zero lbs and unload the contents at sterile end / Non-sterile end.

5.1.27   Record the details in autoclave logbook as shown in the form No.xxxxxx

5.2       Operating Procedure for Vertical Autoclave:

5.2.1    Open the top lid of the autoclave by unscrewing the nuts and lift the lid by using handle.

5.2.2The chamber of the autoclave is cleaned with Purified Water.

5.2.3Open the water inlet valve and start the water supply. When the water level rises up to ¾^th height of the gauge of the glass tube, then close the water inlet valve. 

5.2.4Put the main switch in ON position, which will be indicated by glowing of the red bulb.

5.2.5Switch ON the heaters, which will be indicated by glowing of the amber bulb.

5.2.6Keep the required articles to be sterilized in the chamber of the autoclave, close the top lid, place all the locking bolts in position, and tighten all the Bakelite knobs.

5.2.7Set the Temperature and Time on the panel (Soak Timer) with the help of the keys at 121°C / 30 minutes.

5.2.8Press the ‘PGM’ Key and hold it for 3-4 seconds, the Set Temperature will be displayed.

5.2.9The Temperature shall be changed by using the Up arrow key or Down arrow key and then press the Enter / PGM Key.

5.2.10Then the Time parameter will be displayed. 

5.2.11Set Time shall be changed similar to the temperature and then press the Enter / PGM key.

5.2.12When the jacket steam pressure shown in the gauge has reaches the operating pressure 1.2 kg/cm^{2 (}17 psi), then press the start key on the panel (SOAK TIMER).

5.2.13Then open the steam inlet valve from jacket to chamber, which allows the steam from jacket to chamber. 

5.2.14  The steam entered into the chamber is spread through out the chamber.

5.2.15  To check for proper sterilization place one chemical indicator (class-6) along with the load.

5.2.16  Sterilization cycle starts, when the temperature in the chamber at 121°C.

5.2.17When the sterilization time is over, then the buzzer will sound indicating the end of   sterilization cycle.

5.2.18Record the time and maintain the sterilization cycle as given below.

5.2.19After completion of the sterilization cycle, switch OFF the heaters, open exhaust slowly and allow the berressure to come down to 0 kg/cm².

5.2.20Open the top lid of the autoclave by unscrewing bakelite knobs and lift the lid by using   handle.

5.2.21Unload the contents from the chamber of the autoclave

5.2.22Record the details in autoclave logbook as shown in the Form No.: xxxxx

5.3       Calibration of Horizontal / Vertical Autoclave:

5.3.1The calibration of horizontal or vertical autoclave shall be carried out as per each calibration schedule.

5.3.2The procedure for operation of horizontal or vertical autoclave shall be same as above mentioned in ‘Operation’ of each.

5.3.3The calibration of horizontal or vertical autoclave shall be carried out for Empty chamber,    Media load and Material load.

5.3.3.1 F₀ Value Determination: The number of minutes to kill a specified number of  microorganisms with a Z-Value of 10 °C(50°F) at a temperature of 121.1 °C(250°F) can beachieved  with bio burden spore strips having 10⁶ population in Empty chamber, Media load  and Material load is based  on the sterility of the spore strips. Positive control having 10⁶

            population. After sterilization of spore strips and incubated for 7 days, if there is no turbidity in the medium mean that F₀                   Value is achieved.

5.3.4Record details of the materials used in the calibration of the horizontal or vertical autoclave in the Format xxxxx

5.3.5    Empty chamber:

 5.3.5.1    Arrange the 5 spore’s strips of bacillus stearothermophilus in the empty chamber of the autoclave systematically at different locations with each having chemical indicator (class-6) except 6^th location . Each strip is cover with butter paper.

 5.3.5.2    One Bacillus stearothermophilus strip shall be used for positive control. 

 5.3.5.3    Set the temperature and time for 121ºc and 30 minutes respectively on the control panel. 

 5.3.5.4    Close the door tightly and the sterilization cycle shall be run at 121º-124ºc /15lbs pressure.

5.3.5.5     Record the temperature for every 5mins, in the observation report Format, xxxxx.

5.3.5.6     After completion of the sterilization cycle collect all the spore strips from the autoclave.

5.3.5.7    The sterility of the spore strips shall be tested by direct inoculation method.

5.3.5.8    Inoculate the each sterilized spore strips in 30ml of sterile soya bean casein digest medium    tubes in side of                 the LAF bench. Ensure that the spore strips should immerse in the medium.

5.3.5.9    Positive and Negative controls shall be run along with test samples. For positive control inoculate spore strip in              30ml of sterile soya bean casein digest medium tubes. For negative control incubate 30ml of sterile soya bean                casein digest medium tube without inoculating the spore strips.

5.3.5.10  Incubate at 55º -60º c for 7 days.

5.3.5.11Observe the each tube every day and record the results in the test report Format xxxxx.

5.3.5.12If early and copious growth is conformed remove the tubes.

5.3.5.13Positive control must be “positive”, Negative control must be “negative”.Sterilized spore’s strips must be “negative’.

5.3.6    Media load:

5.3.6.1  Load the chamber with media load (any media).

5.3.6.2Arrange the 5 spore’s strips of bacillus stearothermophilus in the   chamber of the autoclave systematically at the different locations with each having chemical indicators (class-6) . Set time shall be 15 minutes.

5.3.6.3One Bacillus stearothermophilus strip shall be used for positive control.

5.3.6.4Set the temperature and time for 121ºc and 15 minutes respectively on the control panel.

5.3.6.5Close the door tightly and the sterilization cycle shall be run at 121º-124º c for 15 minutes at 15lbs pressure.

5.3.6.6Record the temperature for every 5mins, in the observation report Format xxxxx.

5.3.6.7After completion of the sterilization cycle collect all the spore strips from the autoclave.

5.3.6.8The sterility of the spore strips shall be tested by direct inoculation method.

5.3.6.9Inoculate the each sterilized spore strips in 30ml of sterile soya bean casein digest medium tubes in side of the LAF bench. Ensure that the spore strips should immerse in the medium.

5.3.6.10Positive and Negative controls shall be run along with test samples. For positive control inoculate spore strip in 30ml of sterile soya bean casein digest medium tubes. For negative control incubate 30ml of sterile soya bean casein digest medium tube without inoculating the spore strips.

5.3.6.11Incubate at 55º -60ºc for 7 days.

5.3.6.12Observe the each tube every day and record the results in the test report Format xxxxx.

5.3.6.13If early and copious growth is conformed remove the tubes.

5.3.6.14Positive control must be “positive” and Negative control must be “negative”. Sterilized spore’s strips must be “negative’.

5.3.7    Material load:

5.3.7.1  Load the chamber with materials ( any sterilizable material garments,filters etc.).

5.3.7.2Arrange the 5 spore’s strips of bacillus stearothermophilus in the   material chamber load of the autoclave systematically at the different locations with each having chemical indicator (class-6).

5.3.7.3One Bacillus stearothermophilus strip shall be used for positive control.

5.3.7.4Set the temperature and time for 121ºc and 30 minutes respectively on the control panel.

5.3.7.5Close the door tightly and the sterilization cycle shall be run at 121º- 124ºc for 30 minutes at 15lbs pressure. 

5.3.7.6Record the temperature for every 5mins, in the observation report Format xxxxx.

5.3.7.7After completion of the sterilization cycle collect all the spore strips from the autoclave.

5.3.7.8The sterility of the spore strips shall be tested by direct inoculation method.

5.3.7.9Inoculate the each sterilized spore strips in 30ml of sterile soya bean casein digest medium tubes in side of the LAF bench.  Ensure that the spore strips should immerse in the medium.

5.3.7.10Positive and Negative controls shall be run along with test samples. For positive control inoculate spore strip in 30ml of sterile soya bean casein digest medium tubes.  For negative control incubate 30ml of sterile soya bean casein digest medium tube without inoculating the spore strips.

5.3.7.11Incubate at 55º -60ºc for 7 days.

5.3.7.12Observe the each tube every day and record the results in the test report Format xxxxx.

5.3.7.13If early and copious growth is conformed remove the tubes.

5.3.7.14Positive control must be “positive” and Negative control must be “negative”.Sterilized spore’s strips must be “negative’.

5.3.7.15If all the results of biological indicators, chemical indicators and temperature readings on the control panel are found satisfactory then the process of horizontal or vertical autoclave is acceptable.  If any of the results are not found satisfactory label the instrument as’ UNDER BREAKDOWN’ and call the service engineer.

5.3.7.16Frequency: Calibration of horizontal or vertical autoclave shall be done in every 6 months with ± 7 days.

5.4   Temperature mapping of horizontal or vertical autoclave: 

5.4.1    Temperature mapping of horizontal or vertical autoclave shall be carried out as per each schedule and done by using data logger with temperature sensor probes.

5.4.2    Arrange the 6 no’s of probes in horizontal or vertical autoclave chamber as systematically shown positions.

5.4.3Close the door tightly and set the temperature of horizontal autoclave to 121º c .

5.4.4After reaching the temperature of data logger and autoclave to 121 º c maintained the sterilization cycle for 30 minutes at 15 lbs pressure. Acceptance temperature range is 121-   124 º c.

5.4.5Record the temperature readings of autoclave for every 5 minutes in format  xxxxx.

5.4.6The data logger recording intervals are every 1 minute. 

5.4.7If all the results of temperature readings of data logger and digital display of the autoclave are found acceptable then the temperature mapping of horizontal or vertical autoclave is satisfactory. If any of the results are not found satisfactory label the instrument as ‘UNDER BREAKDOWN’ and call the service engineer.

5.4.8Frequency: Temperature mapping of horizontal or vertical autoclave shall be done once in a year with ± 15 days by external agency / Internal.

5.5       Bowie-Dick Test: To confirm there is no air gaps in the vacuum cycle by placing Bowie-Dick test kit. Bowie-Dick test kit colour changes from blue to black if there is no air gaps.Frequency: Once in 3 months ± 7 days.

6.0       Related Documents:  Nil.

Sterilization cycle, microbiology, horizontal, vertical autoclave, temperature