Autoclaving Guidelines

Requirements for Decontamination by Autoclaving
Summary: Steam sterilization (autoclaving) is one of the most effective methods for decontaminating
biohazardous material. Autoclaves use saturated steam under high pressure to decontaminate
infectious materials (i.e., cultures, cells, contaminated glassware, pipettes, etc.) and to sterilize media,
lab ware and other items. Biohazardous material and full sharps containers generated within research
laboratories are placed in Contaminated Materials Containers (CMCs) and treated at the hospital
autoclave. However, some laboratories that generate biohazardous material may use local laboratory
(or departmental) autoclaves. When using laboratory or departmental autoclaves to decontaminate
biohazardous materials, the Principal Investigator (or Department Head) is responsible for meeting
the following mandatory requirements:
1. Maintaining a log book in accordance with Section 4a below.
2. Autoclaving for a minimum of 30 minutes (see Section 4b) unless validation data demonstrates
that less time is sufficient for decontamination.
3. Documenting proper validation techniques when decontaminating materials (see Section 8).
The recommendations below serve as guidelines to help autoclave operators ensure a safe and
effective process. For questions concerning autoclave requirements or guidelines and safety, contact
the Office of Environmental Health and Safety Office at 434-982-4911.
What to do
1
Select appropriate containers/bags for

autoclaving.
How to do it
For biohazardous dry materials:
a. Use polypropylene (clear) bags or reusable
autoclavable (Nalgene) containers. Make sure your
plastic bag or container is suitable, since not all plastics
can be autoclaved (i.e., polyethylene or HDPE). They
can melt and ruin the autoclave chamber.
b.
Ensure that contaminated materials are free of

sharp objects that may puncture bags. Autoclave bags
are tear resistant, but can be punctured or burst in the
autoclave.
c.
Fill bags only 2/3 full. Ensure adequate steam

penetration by closing bags loosely, leaving a small
opening and closing with autoclave indicator tape.
d.
Sharps containers EHS recommends that these be

picked up by EHS.
For biohazardous liquids (or non-biohazardous liquids):

e. Fill liquid containers only 1/2 full. Loosen caps or use
vented closures.
f.
Never put sealed containers in an autoclave. They

can explode. Large bottles with narrow necks may also
explode if filled too full of liquid.
For non-biohazardous glassware/labware:

g. Cap Pyrex bottles loosely whether empty or filled to
prevent explosions due to expansion.
h.
Revised 4-3-2012 EHS Biosafety
Cover bottles that are not made of safety glass (e.g., not
Pyrex) with aluminum foil.
Place clear autoclave bag or other

container in a SECONDARY container
labeled with a biohazard symbol/sticker.
Select the correct autoclave cycle for

your materials and check the drain
screen.
Fill out the autoclave logbook and be

aware of required cycle times.
a.
Make sure your plastic secondary container is

suitable for autoclaving. Not all plastics can be
autoclaved (i.e., polyethylene or HDPE).
b.
Polypropylene, polycarbonate or stainless steel

tubs are typically used for secondary containment. See
Nalgene Labware's Autoclaving Web page for additional
plastic considerations.
c.
Select a container with the lowest sides and widest

diameter possible for the autoclave. This will promote
penetration of steam and will collect any leakage or
overflow of liquids.
d.
Leave space between items/bags to allow steam

circulation. Water can also be added to plastic pans at
a depth of 2.5 cm or into bags with solid materials.
e.
Ensure secondary container is labeled with the

universal Biohazard symbol so that BSL2 waste can
be easily identified.
a.
Review the manufacturers operating manual and any

laboratory Standard Operating Procedures (SOP) for
the autoclave unit. Training should be provided for any
new autoclave operators.
b.
Slow Exhaust is the recommended cycle for

biohazardous dry goods and liquids.
c.
Check the drain screen at the bottom of the chamber

before using the autoclave. Clean out any debris.
d.
For efficient heat transfer, steam must flush the air out
of the autoclave chamber. If the drain screen is blocked
with debris, a layer of air may form at the bottom of the
autoclave and prevent proper operation.
e.
Autoclave the load immediately after preparation.

Do not leave unprocessed items in the autoclave
overnight.
PLEASE NOTE THE FOLLOWING REQUIREMENTS (a-b):

a. Each unit used to decontaminate must have an
autoclave log where the operator records the date,
name, cycle time, and monthly biological indicator
results.
For both dry and liquid biohazardous materials:
b. Cycle time must be set for a minimum of 30 minutes @
0
1
121 C, 15 psi unless decontamination in less time can
be demonstrated (see section 8 for more information on
validation procedures).
c.
Liquids and large loads containing many bags may

require more time.
For sterilization of non-biohazardous dry goods (i.e.,

glassware) or liquids (i.e., broth or media):
d. Cycle time for sterilization will vary and does not require
validation. For larger volumes allow extra time and for

dry goods, allow approximately 20 minutes of drying
time. Dry time may need to be increased for enclosed
items such as pipette tips or bottles with lids.
Follow these precautions when the

autoclave cycle is finished.
Properly dispose of materials.
Materials NOT appropriate for

autoclaving:
a.
Wear personal protection equipment:

a. Lab coat
b. Eye protection
c. Closed-toe shoes
d. Heat-resistant gloves to remove items,
especially hot glassware
b.
Never open an autoclave set for "slow exhaust"

until the cycle is complete. Superheated liquids can
boil over and damage the autoclave and injure the
operator.
c.
Open the door cautiously. Stand behind the door and

slowly open it. Allow all steam to escape before
reaching inside.
d.
Let liquids stand another 1020 minutes after the

autoclave is opened to avoid any movement that could
cause them to boil.
e.
Do not override autoclave's built-in safety control

features under any circumstance (contact your
autoclave repair representative for assistance).
a.
Biohazardous materials that are autoclaved should

be discarded as non-contaminated laboratory waste and
placed in the regular trash or outside dumpster.
b.
Biohazardous materials not autoclaved (that either

cannot be autoclaved or when the laboratory chooses
not to autoclave themselves) must be placed in a
Contaminated Materials Container (CMC).
a.
Never put materials containing solvents, corrosives

or radioactive materials in the autoclave (e.g.,
2
phenol, chloroform, pyridine, or bleach ).
b.
Pathological laboratory materials including

pathological specimens (i.e., whole cadavers,
recognizable human body parts, animal carcasses or
tissues) must not be autoclaved. Contact EHS
Biosafety (982-4911) for disposal instruction.
Monitor and perform validation tests
REQUIRED VALIDATION TEST (MONTHLY):

a. Biological Indicators (BI) are the most accepted
means of monitoring the sterilization process because
they directly determine whether the most resistant
microorganisms (e.g., Geobacillus or Bacillus species)
are present rather than merely determine whether the
physical and chemical conditions necessary for
3
sterilization are met.
b.
For the BI test, ampoules of B. stearothermophilus

are autoclaved along with a load of waste. Upon
completion of the cycle and following manufacturers
instructions, ampoules are incubated for 24-48 hours
and then observed for any sign of growth that would
indicate that autoclave is not sterilizing properly.
Record results and keep in the autoclave log book.
c.
BI tests performed on a monthly basis and:
Whenever a new type of packaging material or

tray is used.
After training new sterilization personnel.
After a sterilizer has been repaired.
After any change in the sterilizer loading

procedures.
Note: 3M Attest Biological Indicator Test kits

available through the Hospital Storeroom.
d. Other types of indicators such as Mechanical

Indicators (gauges and digital displays) or Chemical
Indicators (autoclave tape or strips) do not ensure
sterilization, but can be used to show that autoclaving
has been performed. Additionally, mechanical
indicators can identify when a problem has occurred
with the sterilization cycle.
9 Perform preventative maintenance
a.
Autoclaves should be maintained on a regular

service contract. Contact the unit manufacturer or the
Biosafety Office for more information.
b.
Calibration should be performed annually

(requirement of the ASME Boiler and Pressure Vessel
Code and Public Law PL 104-113) and can be
performed by the manufacturer or other qualified third
party autoclave service technician as part of an annual
maintenance contract.
c.
If you suspect there is a problem with your

autoclaves performance, contact your autoclave repair
representative for assistance.
References
1
Le, R.N., et al (2005) Autoclave Testing in a University Setting. Applied Biosafety, 10(4), 248-252.
Trace amounts of bleach used to decontaminate laboratory materials during procedures (e.g., decontaminating
pipette tips or other labware) may be autoclaved.
3
Centers for Disease Control and Prevention, Oral Health Resources Sterilization Monitoring FAQs, April
2005. www.cdc.gov/oralhealth/infectioncontrol/faq/sterilization_monitoring.htm
4
ASME Boiler and Pressure Vessel Code and Public Law PL 104-113.
2
This page was adapted from the UCSD EH&S website

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Requirements for Decontamination by Autoclaving

Select appropriate containers/bags for

Ensure that contaminated materials are free of

Fill bags only 2/3 full. Ensure adequate steam

Sharps containers EHS recommends that these be

For biohazardous liquids (or non-biohazardous liquids):

Never put sealed containers in an autoclave. They

For non-biohazardous glassware/labware:

Revised 4-3-2012 EHS Biosafety

Place clear autoclave bag or other

Select the correct autoclave cycle for

Fill out the autoclave logbook and be

Make sure your plastic secondary container is

Polypropylene, polycarbonate or stainless steel

Select a container with the lowest sides and widest

Leave space between items/bags to allow steam

Ensure secondary container is labeled with the

Review the manufacturers operating manual and any

Slow Exhaust is the recommended cycle for

Check the drain screen at the bottom of the chamber

Autoclave the load immediately after preparation.

PLEASE NOTE THE FOLLOWING REQUIREMENTS (a-b):

Liquids and large loads containing many bags may

For sterilization of non-biohazardous dry goods (i.e.,

Revised 4-3-2012 EHS Biosafety

validation. For larger volumes allow extra time and for

Follow these precautions when the

Properly dispose of materials.

Materials NOT appropriate for

Revised 4-3-2012 EHS Biosafety

Wear personal protection equipment:

Never open an autoclave set for "slow exhaust"

Open the door cautiously. Stand behind the door and

Let liquids stand another 1020 minutes after the

Do not override autoclave's built-in safety control

Biohazardous materials that are autoclaved should

Biohazardous materials not autoclaved (that either

Never put materials containing solvents, corrosives

Pathological laboratory materials including

Monitor and perform validation tests

REQUIRED VALIDATION TEST (MONTHLY):

For the BI test, ampoules of B. stearothermophilus

BI tests performed on a monthly basis and:

Whenever a new type of packaging material or

After training new sterilization personnel.

After a sterilizer has been repaired.

After any change in the sterilizer loading

Note: 3M Attest Biological Indicator Test kits

d. Other types of indicators such as Mechanical

9 Perform preventative maintenance

Autoclaves should be maintained on a regular

Calibration should be performed annually

If you suspect there is a problem with your

This page was adapted from the UCSD EH&S website

Revised 4-3-2012 EHS Biosafety

Revised 4-3-2012 EHS Biosafety

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