Department of Biochemistry Doc.

No: SOP-BCH-LCP-004
Yong Loo Lin School of Medicine, NUS
Standard Operating Procedure Version No: 001
Issue date: 30 Sept 2010
Title: Operation of Autoclaves
Page: 1 of 4

Dr. Lim Cheh Peng A/Prof. Tang Bor Luen

-
Mr. Wong Veen Senn A/Prof. Caroline Lee
Prepared by Approved by Review Date

1. OBJECTIVE

This SOP outlines procedures to provide guidance on the safe operation of autoclaves in
the laboratories.

2. SCOPE

This SOP is applicable to all users of the autoclave in the department.

3. RESPONSIBILITY

3.1 Principal Investigator

The PI is to ensure that:
a. All users under his/her charge are adequately trained, by receiving appropriate
training on the hazards and safe use of the autoclave.
b. Record books are maintained where users log the use of the autoclave.
c. Annual sterilization monitoring procedure were performed.
d. Make necessary arrangements for repair works to the autoclave.
e. Report to the Safety Committee of unsafe practices by autoclave users.

3.2 Designated Person

a. Arrange for annual safety certification.

3.3 Users
a. Autoclave users shall operate the autoclave for the purpose of sterilization
only. Autoclave users shall not autoclave any biohazard wastes unless
authorized to do so, such as in the case of Pathogen Labs. For Biological
Waste Disposal, please follow the procedure outlined in SOP-BCH-WVS-001.
b. Autoclave users must receive adequate training prior to the use of the
autoclaves.
c. Autoclave users shall report any defects, breakdowns or quality control issues
to their PI.
d. Autoclave users shall report any injuries to their PI and Safety Committee.
Prepare a written report of the accident to the PI or lab supervisor, and file it in
the lab’s Accident/Incident Register. Report the incident to OSHE online at
https://staffweb.nus.edu.sg/oshe/submit_airs.htm

4. DEFINITION

Autoclaves: Mechanical devices using pressurized steam for sterilization of laboratory

glassware, media, and reagents. When authorized to do so, it is also used to destroy
microorganisms for decontamination of laboratory waste, such as in the case of Pathogen
Labs.

5. PROCEDURES

5.1 Preparation of materials

a. Use only autoclavable type of materials such as polycarbonate, PTFE
("Teflon"), polypropylene, borosilicate (Pyrex) glass or stainless steel.
 Department of Biochemistry Doc. No: SOP-BCH-LCP-004
Yong Loo Lin School of Medicine, NUS
Standard Operating Procedure Version No: 001
Issue date: 30 Sept 2010
Title: Operation of Autoclaves
Page: 2 of 4

b. Ensure that the materials do not contain incompatible materials for use in an
autoclave such as solvents and water-sensitive chemicals. Oils, waxes, some
plastics, flammable materials, radioactive materials, and samples containing
solvents or substances that may emit toxic fumes should not be autoclaved.
c. Items should be tagged with autoclave indicator tape to provide immediate
identification of processed items.
d. Liquid containers should be filled to about half of their contents.
e. All container caps must be loosened or containers must have vents for release
of pressure.

5.2 Loading of materials

a. Open the chamber lid and ensure that the drain screen is clear of debris to
allow proper circulation of steam. Also, check inside the autoclave for any
items left by the previous user that could pose a hazard (e.g. sharps).
b. Check the water level of the chamber. The water level should reach the bottom
plate but not submerging the plate. Add distilled/RO water as required.
c. Place materials to be sterilized into the basket/autoclave bin. Do not overload.
Ensure that there is adequate space between items to allow for steam
circulation.
d. Close the chamber lid and turn the hand-wheel clockwise to firmly tighten it. Do
not overturn the lid handle.

5.3 Operating Autoclave (TOMY)

a. Check the water level in the exhaust bottle. It should be within the lower and
upper limit as indicated on the bottle. Fill with distilled water or decant excess
water accordingly.
b. Turn on the POWER switch.
c. The control panel will display programmed Temperature and Time, and the
Mode/Process indicator will display “READY”.
d. Choose the appropriate cycle, and set the appropriate time and temperature
for the cycle. Please refer to the user’s manual. A typical autoclaving cycle is
121oC for 20 min.
e. Turn the EXHAUST knot to CLOSE if it is not in CLOSE position.
f. Press the START key to put the autoclave into operation.
g. Do not attempt to open the lid while autoclave is operating.
h. When the cycle is completed, the unit will beep 3 times. When the temperature
of the chamber falls to 60oC, the unit will beep 10 times and the LED display
will flash “COMPLETE”.

5.4 Removing the load

a. Wear lab coat, eye protection, heat-insulating gloves and covered-toed shoes.
b. Ensure that the pressure of the chamber is 0 kPa and the temperature in the
chamber is below 50°C before opening the lid.
c. Turn locking wheel to unlock the chamber lid. Always stand behind the lid,
using it as a shield. Open the lid partially and beware of the rush of steam. It
should open easily. Do not force the lid to open.
d. Allow liquids to cool down to ambient conditions before handling them.
e. Wearing heat-insulating gloves, remove the basket/autoclave bin from the
chamber. Avoid contact with walls of the autoclave chamber.
 Department of Biochemistry Doc. No: SOP-BCH-LCP-004
Yong Loo Lin School of Medicine, NUS
Standard Operating Procedure Version No: 001
Issue date: 30 Sept 2010
Title: Operation of Autoclaves
Page: 3 of 4

5.6 Monitoring & control

a. The stripes on the autoclave indicator tape should turn black after exposure to
steam sterilization conditions to provide immediate identification of processed
items.
b. Sterilization monitoring procedure should be conducted annually to ensure
quality assurance. The following tests are to be used:
i. A chemical indicator (such as Emulating Indicator Class 6, code 3760,
Albert Browne Ltd) to indicate that the temperature reaches 121°C for
20 min.

ii. A biological indicator (such as 3MTM ESPE AttestTM Biological

Monitoring System, code 1262P) containing heat-resistant spores of
Geobacillus stearothermophilus that are killed by exposure to 121°C for
20 min.

Both types of tests should be placed well down in the center of the bag or
container, at the point slowest to heat.
c. In the case of Pathogen Labs, autoclaves used to decontaminate laboratory
waste should be tested periodically to assure effectiveness. The Geobacillus
stearothermophilus spore test should be done at least once a month.
d. If any of the tests fail, inform the PI immediately.

6. SAFETY PRECAUTIONS
Autoclaves are sterilizers using high pressure and high temperature steam. The potential
safety risks for the operators are:
6.1 Heat burns – from hot materials and autoclave chamber walls and lid.
6.2 Steam burns – from residual steam coming out from autoclave and materials on
completion of cycle.
6.3 Hot fluid scalds – from boiling liquids and spillage in autoclave.
6.4 Hand and arm injuries when closing the chamber lid.
6.5 Body injury if there is an explosion.
6.6 An authorized training session must be successfully completed prior to the use of
autoclaves.
6.7 It is the supervisor’s responsibility to ensure his/her employees are trained before
operating any autoclave unit.
 Department of Biochemistry Doc. No: SOP-BCH-LCP-004
Yong Loo Lin School of Medicine, NUS
Standard Operating Procedure Version No: 001
Issue date: 30 Sept 2010
Title: Operation of Autoclaves
Page: 4 of 4

6.8 Procedural and instructional documents must be followed.

6.9 Personal protective clothing and equipment, such as closed-toed shoes and heat-
insulating gloves, must be worn when loading and unloading the autoclave.

7. MAINTENANCE

7.1 Autoclaves must be subjected to annual safety (mechanical) inspections by MOM

authorized boiler inspectors.

7.2 A current certificate bearing the autoclave number and the Ministry of Manpower
(MOM) registration number must be displayed on the autoclave. The certificate
must display the test, approval and expiry dates.

8. RECORDS

8.1 Certificates of inspections by authorized inspectors are kept by Designated

Person.

8.2 The label of inspection should be displayed at a conspicuous location on the

autoclave. Only labels approved by MOM are to be posted. The following must be
written clearly on the label:
a. C.I.F. Registration No.
b. Autoclave serial number.
c. Test pressure/load.
d. Test date.
e. SWP/SWL.
f. Approval date.
g. Cert expiry date.
h. Inspector No.

8.3 A record book to log the details of users and use of each autoclave.

8.4 Records of chemical and biological indicator tests should be kept in the record
book.

8.5 Record any accident/incident in lab’s Accident/Incident Register and report in

OSHE’s Accident/Incident Reporting System.

9. REFERENCES

9.1 Biological Waste Disposal (SOP-BCH-WVS-001).

9.2 Incident/Accident Reporting and Investigation procedure (OSHE/SOP/U/02).

9.3 Recommended Template for Incident/Accident Report.

https://share.nus.edu.sg/corporate/procedures/safety_and_health/General/Templat
e-for-incident-report.pdf

9.4 OSHE’s online Accident/Incident Reporting System.

https://staffweb.nus.edu.sg/oshe/submit_airs.htm