How To Calibrate Your Autoclave

How To Calibrate Your Autoclave (Includes 11 Step Sample Procedure)

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Written by: Scott Mechler

BS Mechanical Engineering, Mechanical Engineer

“Calibration” is a word that is frequently used in the steam sterilization industry. This blog post will explore what it is, what is involved in doing it right, alternatives, and the potential effects on an existing sterilization process.

What is Calibration?

At its very basic premise, calibration is bringing the response of a sensor (e.g. transducer, thermocouple, etc.) to within a specified range relative to a primary reference standard. This is done by comparing the autoclave sensor’s response to a previously calibrated device (referred to hereafter as “standard”) whose response is traceable to a national reference standard, maintained in the United States by the National Institute of Standards and Technology (NIST).

As a rule of thumb, the standard should be five times as accurate as the device being calibrated. Therefore, when calibrating a temperature probe with a desired accuracy of ±0.5°C, the calibration standard should be accurate to ±0.1°C.

Why Calibrate?

The short answer is calibration ensures consistent results from a process. Steam sterilization efficacy is highly dependent upon actual temperature. For example, if a steam autoclave is running at 120°C for 15 minutes, the theoretical lethality of that cycle is only 82% of that of a cycle running at 122°C for the same amount of exposure time.

Since most laboratory autoclaves do not require temperature to be accurate to better than ±1°C, this variability can be more common than one would think. If your temperature transducers are calibrated, this problem will diminish.

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Equipment Required to Calibrate

The proper way to calibrate an autoclave is with the use of a NIST-traceable device (standard) such as a dry block, oil bath, or temperature probe. If using a dry block or an oil bath (pricing starts at approximately $1,250) make sure it is designed to control to a constant temperature (±0.1°C). If the dry block or oil bath is not NIST-traceable, or its calibration has expired, then use a NIST-traceable temperature probe (cost is approximately $500) with ±0.1°C minimum accuracy.

If the above-recommended equipment is not available one could use boiling water to help calibrate the autoclave’s temperature sensors. Boiling water can act as a constant-temperature (i.e. 100°C/212°F) bath that is somewhat near sterilization temperature. However, if the facility isn’t exactly at sea level then boiling water isn’t necessarily going to be 100°C/212°F. Check the atmospheric pressure in your area (obtained online at or use an absolute pressure manometer (mercury column or electronic) to obtain the exact pressure reading, then calculate the actual “pressure corrected” boiling temperature of water by using this steam table: ( If you place your sensor into boiling water (not in contact with the bottom of the vessel holding the water) and it isn’t within 1°C of the theoretical temperature then you will need to carry out a calibration.

Calibration Methods

Calibration instructions can vary by the number of calibration points measured (i.e. 1-point, 2-point, or 3-point). What is the difference?

Single Point Calibration

A single point calibration is valid only within the accuracy at that specific point. When sterilizing at only one temperature, say 121°C/250°F, this is not too much of a problem, although you have no idea what is happening in any process excursions to higher or lower temperatures.

Two Point Calibration

Some calibration instructions recommend taking two measurements and calculating the slope (gain) and y-intercept (zero offset). Using two points for calibration is relatively fast and convenient; however, two points define a straight line and reveal nothing about any non-linearity in the probe’s reading. Also, any errors in the two measurements are not going to be evident.

Multipoint Calibration

A multipoint (more than two point) calibration will indicate if the probe is behaving in a nonlinear manner, which could be a good reason to replace it, and also allows any measurement errors to be averaged out over the greater number of points. These instructions suggest taking three or more measurements and performing a linear regression to get the slope and y-intercept. Any multipoint calibration should be done with points both outside the working range of the sterilization cycle(s) you will use. For example, a lab running at 121°C (and only 121°C) should calibrate at 116, 121, and 126°C or at 116 and 126°C for a two point calibration. This allows the response of the measurement and control system to be taken into account. If the lab is running cycles over a greater range, calibration should start and finish 5°C below the minimum temperature and 5°C above the maximum temperature used. A good rule of thumb is to calibrate against at least the number of decades in °C plus one. So if you run cycles at three different temperatures (e.g. 115°C, 121°C, and 134°C), you would make calibration measurements at four points (e.g. 110°C, 120°C, 130°C, and 140°C.

Sample Calibration Procedure for an Autoclave

  1. Record the as-found calibration data (zero and gain) for each sensor to be calibrated.
  2. Using appropriate caution (shut the steam off and wait for the pressure to go to zero!), remove the sensors to be calibrated from the sterilizer, leaving their cables connected to the control system.
  3. Set the zero and gain to 0 and 1, respectively.
  4. If using a NIST-traceable dry block or oil bath place the sensor in the dry block or oil bath.
  5. If using a NIST-traceable temperature probe, place the temperature probe into a central position in the dry block or oil bath and the sensors as close to it as possible.
  6. Measure at the selected temperatures and record the standard and transducer data. You should wait for at least one minute at each temperature to allow the measurement to stabilize. Don’t rush this step.
  7. Do a linear regression of the data collected. This is straightforward in Microsoft Excel [intercept() and slope() functions] with the standard’s data on the y-axis and the sensor’s data on the x-axis.
  8. Do a correlation as well [correl() function]. It should be at least 0.999999 (i.e. a very straight line).
  9. Enter the zero and gain values to the sterilizer controller to enter the calibration.
  10. Verify the calibration using at least one point like your process temperature. If more than one process temperature, then verify at each one. Then you will have an exact statement of the accuracy of the sensors.
  11. Re-install the sensors in the sterilizer.

If you have additional calibration questions or any questions related to autoclaves in general, please contact us for more information.

Written by: Jonathan A. Wilder, Ph.D., Stericert div. of H & W Technology

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