US Environmental Protection Agency
Office of Pesticide Programs

Office of Pesticide Programs

Microbiology Laboratory

Environmental Science Center, Ft. Meade, MD

Standard Operating Procedure for
Performance Verification of Autoclaves

SOP Number: QC-13-09

Date Revised: 03-01-17


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SOP No. QC-13-09
Date Revised 03-01-17
Page 1 of 12

SOP Number

QC-13-09

Title

Performance Verification of Autoclaves

Scope

This protocol describes the procedures for verifying the performance
of autoclaves.

Application

Verification of autoclave performance is essential to maintaining the
quality and sterility of media and reagents, and to confirm the
inactivation of biohazardous waste.





Approval Date

SOP Developer:



Print Name:

SOP Reviewer



Print Name:

Quality Assurance Unit



Print Name:

Branch Chief



Print Name:





Date SOP issued:



Controlled copy
number:



Date SOP withdrawn:




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SOP No. QC-13-09
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TABLE OF CONTENTS
Contents	Page Number

1.

DEFINITIONS

3

2.

HEALTH AND SAFETY

3

3.

PERSONNEL QUALIFICATIONS AND TRAINING

3

4.

INSTRUMENT CALIBRATION

3

5.

SAMPLE HANDLING AND STORAGE

3

6.

QUALITY CONTROL

4

7.

INTERFERENCES

4

8. NON-CONFORMING DATA

4

9.

DATA MANAGEMENT

4

10.

CAUTIONS

4

11.

SPECIAL APPARATUS AND MATERIALS

5

12.

PROCEDURE AND ANALYSIS

6

13.

DATA ANALYSIS/CALCULATIONS

10

14.

FORMS AND DATA SHEETS

10

15.

REFERENCES

10


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1. Definitions

1.	A liquid cycle is a sterilization cycle in which steam is exhausted
slowly at the end of the cycle to allow the liquids to cool without
boiling over.

2.	A kill cycle is a liquid cycle with a duration of 180 minutes to sterilize
bio-hazardous waste.

3.	A gravity cycle is a sterilization cycle in which steam is rapidly
exhausted at the end of the cycle. Dry time may be added to the cycle.
A gravity cycle is used primarily for sterilization of dry laboratory
materials (e.g., glassware, plastic ware, carriers).

4.	Chemical indicator strips are engineered to integrate all 3 critical
parameters of sterilization (time, temperature and saturated steam) and
provide a distinct color change when exposed to the sterilization
process.

5.	Biological indicator ampules (sealed spore ampules containing spores
in liquid culture media) are intended for use as a challenge to steam
sterilization at 121°C and to confirm the inactivation of biohazardous
waste.

6.	Sterilization batch number. A distinct number accounting for the date
of sterilization, the autoclave used, and a counter for the number of
autoclave runs on a given day

7.	Additional abbreviations/definitions are provided in the text.

2. Health and
Safety

1.	Follow procedures specified in SOP MB-01, Laboratory Biosafety.

2.	Laboratory personnel are trained on the proper use of the autoclaves.
The autoclaves and materials being removed from the autoclaves are
very hot (often greater than 100°C). Lab personnel should wear lab
coats, eye protection and thermal gloves when handling materials
being removed from the autoclaves to prevent burns.

3. Personnel
Qualifications
and Training

Refer to SOP ADM-04, OPP Microbiology Laboratory Training.

4. Instrument
Calibration

Once a year, the laboratory's maximum registering thermometers are
verified at operating temperatures against a similar maximum registering
thermometer that has been certified by an ISO 17025 accredited vendor.
See EQ-02, Calibration of Thermometers.

5. Sample

Handling and
Storage

Biological indicator ampules must be stored according to manufacturer's
specifications to ensure shelf life. The biological indicator ampules must
be refrigerated upon receipt and until use.


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6. Quality
Control

1.	For quality control purposes, the required information is documented
on the appropriate form(s) (see section 14). Perform a quality control
assessment of the autoclaves monthly and record on the appropriate
form (see section 14).

2.	Record expiration dates of biological indicator ampules and chemical
indicator strips on the appropriate forms (see section 14).

3.	Quality control checks vary per type of autoclave run; refer to sections
12.3-12.5.

4.	If an autoclave undergoes repair, do not use the autoclave to sterilize
media until its performance is verified using the monthly verification
procedure for a short liquid cycle (refer to section 12.4).

a. If autoclave #4 in B202 undergoes repair when the laboratory is
commissioned for Select Agent work, do not use the autoclave
until its performance is verified using the monthly verification
procedure for a kill cycle (refer to section 12.5).

5.	Do not use autoclave #3 in B207 to sterilize media using a 15-minute
liquid cycle.

7. Interferences

1.	Maximum registering thermometers may provide inaccurate readings if
not used properly, as outlined in section 12.2b.

2.	The position of thermometers, chemical indicator strips, and biological
indicator ampules (when applicable) is critical to successful quality
control measurement. Refer to Attachments 1 and 2 for proper
placement of thermometers, indicator strips, and ampules.

3.	Certain media may require a lower (<121°C) sterilization temperature.
For those media, adjust the autoclave accordingly to ensure appropriate
sterilization.

4.	Changes in temperature and pressure within the autoclave but outside
the established tolerances may impact the quality and sterility of media
and reagents. It is therefore critical to ensure that the autoclaves are
operating within acceptable limits (see sections 15.1-15.2).

8. Non-
conforming
Data

Management of non-conforming data will be consistent with SOP ADM-
07, Non-Conformance Reports.

9. Data

Management

Data will be archived consistent with SOP ADM-03, Records and
Archives.

10. Cautions

1. Because autoclaves use high temperatures, it is necessary to exercise
extreme caution around the device and its associated plumbing. High-


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temperature surfaces can be encountered even when the device is not
in a sterilizing cycle.

2.	For autoclaves #1 and #2, a completed autoclave liquid cycle includes
the recommended 10-minute wait period (indicated on the LED screen
on the autoclave) once the door has been cracked open. When using
these autoclaves, it is recommended that the operator open the door
slowly (not greater than one inch) and wait at least 10 minutes prior to
unloading.

3.	Do not overload the autoclave. Allow for steam penetration between
materials. Avoid contact of load components with the walls of the
chamber.

11. Special

Apparatus and
Materials

1.	ProSpore Ampoule Biological Indicator (Mesa Labs; Lakewood, CO;
catalog no. PS-6-50). Each ampule is a hermetically sealed, type I
borosilicate glass ampule, filled with a modified Soybean Casein
Digest Broth containing bromocresol purple acid indicator and 106
spores of Geobacillus stearothermophilus. Biological ampules are
used to verify the 180-minute liquid cycle and the 45-minute gravity
cycle. See section 12.7a for a discussion of passing and failing results.

2.	Chemical Indicator Strips (SPS Medical; Rush, NY; catalog no. SSI-
100). See section 12.2c,i for a discussion of passing and failing results.

3.	Maximum Registering Thermometers (mercury-containing/teflon-
coated; scale range 80-135°C) are used to verify a maximum autoclave
temperature.

4.	Incubators with temperatures set at 36±1°C and 55±1°C.

5.	Autoclave #1 located in room B206, Amsco Eagle 3000 Scientific
Series, Model E3031-S-1, Serial No. 0105898-25.

6.	Autoclave #2 located in room B204, Amsco Eagle 3000 Scientific
Series, Model E3031-S-1, Serial No. 0108298-11.

7.	Autoclave #3 located in room B207, PRIMUS Model PSS5-AA-
MESD, Serial No. 18200.

a. Do not use this autoclave to sterilize media using a 15-minute
liquid cycle.

8.	Autoclave #4 located in room B202, Amsco Lab 250 Laboratory
Steam Sterilizer (20x20x38"), Model LG-250, Serial No. 0311511-10.

9.	Autoclave #5 located in room D122, Tuttnauer Prevacuum Steam
Heated Autoclave with Vertical Sliding Door and Steam Generator
(52x72x51"), Model 5596-EP-1V, Serial No. 2311036, when in use.


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12. Procedure and
Analysis

1. Refer to Attachment 1 for a summary of the performance verification
practices. Refer to Attachment 2 for photographs of quality control
indicator placement for monthly performance verification.

12.1 Sterilization
Batch
Number

a.	The sterilization batch number consists of two parts:

i.	The first seven digits represent the date the batch was
sterilized: S-MMDDYY where S=Sterilization,
MM=month, DD=day and YY=the last two digits of the
calendar year.

ii.	The suffix where the first digit after the dash indicates the
autoclave used and the next two digits act as a counter for
the number of preparations made on the same date.

b.	For example, the first batch sterilized on January 8, 2017 in
autoclave 1 (Room B206) would have the sterilization batch
number S-010817-101. The next batch sterilized on that same
day and same autoclave would have a suffix of -102, the third
batch sterilized would have a suffix of -103; etc.

c.	Record the sterilization batch number in the Daily Sterilization
Record Information Log Form (see section 14).

12.2 Verification
Per Cycle

Collect the following data for every autoclave cycle.

a.	Record the minimum and maximum temperatures achieved
during the "sterilize" portion of the cycle as indicated on the
autoclave printer readout on the appropriate form (see section 14).

i. The acceptable temperature range per cycle run is between
120-124°C, with the exception of certain media (e.g. CTA
stabs) which may require a lower sterilizing temperature.

b.	Use a maximum registering thermometer for each autoclave run.
Place the thermometer upright in a container and place the
container near the items to be processed.

i.	Record the results from the thermometer on the
appropriate form (see section 14). Hold thermometer in
an upright position for reading or a falsely high reading
will be obtained.

ii.	Reset the maximum registering thermometer prior to each
use by "shaking" the thermometer as would be done for a
fever thermometer. This will force the mercury through
the constriction located above the bulb. Shake the
thermometer until the column registers 110°C or lower.


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c.	Chemical Indicator Strip. Place the strip flat on top of the
container that holds the maximum registering thermometer.
Record the results from the chemical indicator strip on the
appropriate form (see section 14).

i. A chemical indicator strip is passing if the dark bar on the
strip reaches the "steam safe" section indicated at the end
of the strip. If the dark bar has not entered the "steam
safe" section of the strip, the chemical indicator strip is
failing.

d.	Failure of any of the quality control indicators (data on autoclave
printout, maximum registering thermometer, chemical indicator
strip, biological indicator ampule (when applicable), tryptic soy
broth (TSB) (when applicable)) results in a failed autoclave run.
Material in a failed autoclave run must either be re-autoclaved
(glassware or biohazardous waste) or remade (media or reagents).

i.	Verify that the maximum registering thermometer,
chemical indicator strip, biological indicator ampule
(when applicable), and TSB (when applicable) were
placed in the appropriate location as specified in
Attachments 1 and 2 and repeat the cycle.

ii.	If failure continues, call for service on the autoclave.

12.3 Monthly
Performance
Verification
of Short
Gravity
Cycles

a.	On a monthly basis, verify autoclave performance by running a
short gravity cycle in autoclaves #1, #2, #3, and #4. Use a
biological indicator ampule, maximum registering thermometer,
and chemical indicator strip.

b.	Place the biological indicator ampule and maximum registering
thermometer in an empty beaker in the bin holding the glassware.
Place the chemical indicator strip on top of the beaker containing
the ampule and thermometer. Run a short gravity cycle (45-
minute gravity cycle).

c.	Upon completion of the cycle, remove items from the autoclave
and record the minimum and maximum temperatures achieved
during the "sterilize" portion of the cycle from the autoclave
printer readout, the maximum registering thermometer reading,
and chemical indicator strip results (refer to section 12.2) on the
appropriate form (see section 14).

d.	Remove the ampule and label with autoclave #, cycle type (i.e.,
gravity cycle), and date of run.


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e.	Incubate the ampule as well as one control ampule that has not
been autoclaved at 55±1°C for 48-72 hours and record the results
on the appropriate form (see section 14).

f.	Refer to section 12.7a for interpretation of biological indicator
ampule results.

12.4 Monthly
Performance
Verification
of Short
Liquid Cycles

a.	On a monthly basis, verify autoclave performance by running a
short liquid cycle in autoclaves #1, #2, and #4. Use freshly
prepared tryptic soy broth (TSB), a maximum registering
thermometer, and chemical indicator strip.

b.	Prepare 1 L of TSB and dispense 500 mL into each of two 1 L
bottles; record preparation of TSB on the appropriate media
preparation sheet. In addition, prepare four 1 L bottles, each
containing 500 mL deionized water; no preparation sheet is
required for the water. Place the maximum registering
thermometer in an empty beaker or flask amongst the six 1 L
bottles. Place the chemical indicator strip on top of the beaker or
flask containing the thermometer. Run a short liquid cycle (15-
minute liquid cycle).

c.	Upon completion of the cycle, remove items from the autoclave
and record the minimum and maximum temperatures achieved
during the "sterilize" portion of the cycle from the autoclave
printer readout, the maximum registering thermometer reading,
and chemical indicator strip results (refer to section 12.2) on the
appropriate form (see section 14).

d.	Remove the two bottles of TSB and label with autoclave #, cycle
type (i.e., liquid cycle), and date of run.

e.	Incubate one bottle of TSB at 36±1°C and the other bottle at
55±1°C for 3-10 days and record the observations on the
appropriate form (see section 14). This preparation of TSB is
used only as a quality control check of the autoclave; discard after
incubation.

f.	Refer to section 12.7b for interpretation of TSB incubation
results.

12.5 Monthly
Performance
Verification
of Kill Cycles

a. On a monthly basis, verify autoclave performance by running a
kill cycle in autoclaves #2, #3, and #4. Performance verification
on autoclave #5 is only performed if the autoclave is anticipated
to be in use. Use a biological indicator ampule, maximum
registering thermometer, and chemical indicator strip.


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b.	Place the biological indicator ampule in the center of an autoclave
bag filled with solid waste. Place the maximum registering
thermometer in an empty beaker or flask in the bin with the bag.
Place the chemical indicator strip on top of the beaker or flask
containing the thermometer. Run a kill cycle (180-minute liquid
cycle).

c.	Upon completion of the cycle, remove items from the autoclave
and record the minimum and maximum temperatures achieved
during the "sterilize" portion of the cycle from the autoclave
printer readout, the maximum registering thermometer reading,
and chemical indicator strip results (refer to section 12.2) on the
appropriate form (see section 14).

d.	Remove the ampule from the autoclave bag and label with
autoclave #, cycle type (i.e., kill cycle), and date of run.

e.	Incubate the ampule as well as one control ampule that has not
been autoclaved at 55±1°C for 48-72 hours and record the results
on the appropriate form (see section 14).

f.	Refer to section 12.7a for biological indicator ampule results.

12.6 Performance
Verification
of Kill Cycles
with Select
Agent

a.	Prior to commissioning the laboratory, conduct the monthly
performance verification of a kill cycle on the autoclave in B202.

b.	Refer to the Select Agent Registration Biosafety Plan for Bacillus
anthracis, section 13 (Decontamination of Biohazardous Waste)
for quality control measures to decontaminate biohazardous waste
associated with select agent. These procedures include using a
biological spore ampule with each kill cycle.

c.	Spore ampule must indicate no growth, see section 12.7a.

12.7 Monthly
Performance
Results

a.	Biological indicator ampule results. The control ampule should
show growth: growth is evident by either turbidity and/or a color
change from a purple to or toward yellow. The autoclaved
ampule should not change color and be clear. If the autoclaved
ampule shows growth, repeat the performance verification,
verifying that the quality control indicators were placed
appropriately. If failure continues, call for service on the
autoclave.

b.	Observations after TSB incubation. After incubation, each bottle
of TSB should be free of growth with no turbidity. If either bottle
of the autoclaved TSB shows growth, repeat the performance
verification, verifying that the quality control indicators were


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placed appropriately. If failure continues, call for service on the
autoclave.

12.8 Autoclave
Alarms

1. An autoclave may go into alarm during a run.

a.	If the autoclave goes into alarm before the sterilization process
has begun and the cycle aborts (e.g., door alarm), attempt to
determine the cause of the alarm, resolve it, and restart the cycle.

b.	If the autoclave goes into alarm during the sterilization phase and
the cycle aborts, media and reagents must be discarded. Non-heat
sensitive reagents and glassware must be autoclaved again.

c.	If the autoclave goes into alarm after the conclusion of the
sterilization phase (e.g., exhaust phase), items may be used
provided that all quality control indicators pass.

c. If autoclave goes into alarm during subsequent runs, call for
service.

13. Data Analysis/
Calculations

None.

14. Forms and
Data Sheets

1.	Attachment 1: Performance Verification Practices for Autoclaves

2.	Attachment 2: Placement of Quality Control Indicators for Monthly
Performance Verification

3.	Test Sheets. Test sheets are stored separately from the SOP under the
following file names:

Daily Sterilization Record Log Form QC-13-09 Fl.docx
Monthly Sterilization Record Form QC-13-09 F2.docx
Recommended Media Sterilization Procedures QC-13-09_F3.docx

15. References

1.	Bordner, R.H., Winter, J.A., and Scarpino, P.V., eds. 1978.
Microbiological Methods for Monitoring the Environment, Water and
Wastes. EPA 600/8-78-017, Environmental Monitoring & Support
Lab., U.S. Environmental Protection Agency, Cincinnati, Ohio.

2.	Rice, E.W., Baird, R.B., Eaton, A.D. and Clesceri, L. S., 2012.
Standard Methods for the Examination of Water and Wastewater, 22nd
Edition. American Public Health Association, Washington, DC.

3.	Lee, C.H., Montville, T.J., and Sinskey, A.J., 1979. Comparison of
the efficacy of steam sterilization indicators. Appl. Environ.

Microbiol. 37(6): 113-117.


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Attachment 1: Performance Verification Practices for Autoclaves

Autoclave
ID



Performance Verification and Conditions

Room

Per Run

Monthly Performance Verification

* *



Short Gravity: 45 min gravity

Short Liquid: 15 min liquid

Kill Cycle: 180 min liquid

#1

B206



Ampule & thermometer
in empty beaker in the bin holding
the glassware, strip on top of
beaker containing ampule and
thermometer

TSB (0.5 L in two 1 L bottles), DI
water (0.5 L in four 1 L bottles).
Thermometer in a beaker/flask
amongst bottles, strip on top of
beaker/flask containing
thermometer

N/A

#2

B204

Thermometer/strip

Ampule & thermometer
in empty beaker in the bin holding
the glassware, strip on top of
beaker containing ampule and
thermometer

TSB (0.5 L in two 1 L bottles), DI
water (0.5 L in four 1 L bottles).
Thermometer in a beaker/flask
amongst bottles, strip on top of
beaker/flask containing
thermometer

Ampule in center of full waste bag,
thermometer in empty
beaker/flask, strip on top of

beaker/flask containing
thermometer; all inside a bin

#3

B207

located per

monthly
performance
verification
instructions

Ampule & thermometer
in empty beaker in the bin holding
the glassware, strip on top of
beaker containing ampule and
thermometer

N/A

Ampule in center of full waste bag,
thermometer in empty
beaker/flask, strip on top of

beaker/flask containing
thermometer; all inside a bin

#4

B202



Ampule & thermometer
in empty beaker in the bin holding
the glassware, strip on top of
beaker containing ampule and
thermometer

TSB (0.5 L in two 1 L bottles), DI
water (0.5 L in four 1 L bottles).
Thermometer in a beaker/flask
amongst bottles, strip on top of
beaker/flask containing
thermometer

Ampule in center of full waste bag,
thermometer in empty
beaker/flask, strip on top of

beaker/flask containing
thermometer; all inside a bin

#5*

D122



N/A

N/A

Ampule in center of full waste bag,
thermometer in empty
beaker/flask, strip on top of

beaker/flask containing
thermometer; all inside a bin

* Autoclave 5 is only verified when it is needed, otherwise it is shut down and not used. "Refer to Attachment 2 for photographs of quality control indicator placement.


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Attachment 2: Placement of Quality Control Indicators for Monthly Performance Verification
Short Gravity Cycle

Beaker containing
thermometer and
ampule with strip on
top, inside bin with
glassware.

Short Liquid Cycle

Beaker containing
thermometer with

strip on top, amongst
bottles of TSB (2)
and DI water (4).

Ampule in center of
autoclave waste bag.

Flask containing
thermometer with
strip on top.


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