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-13-05
Date Revised: 11-07-11

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Initiated By:
Technical Review:
QA Review:
Approved By:
SOP No. QC-13-05
Date Revised 11-07-11
Page 1 of 12
EPA/OPP MICROBIOLOGY LABORATORY
ESC, Ft. Meade, MD
Standard Operating Procedure
for
Performance Verification of Autoclaves
SOP Number: QC-13-05
Date Revised: 11-07-11
Print Name:
Print Name:
Technical Staff
Print Name:
QA Officer
Print Name:
Branch Chief
Date: / /
Date: / /
Date: / /
Date: / /
Effective Date:
/ /
Controlled Copy No.:
Withdrawn By:
Date: / /

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SOP No. QC-13-05
Date Revised 11-07-11
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TABLE OF CONTENTS
Contents
Page Number
1.0	SCOPE AND APPLICATION	3
2.0	DEFINITIONS	3
3.0	HEALTH AND SAFETY	3
4.0	CAUTIONS	4
5.0	INTERFERENCES	4
6.0	PERSONNEL QUALIFICATIONS	4
7.0	SPECIAL APPARATUS AND MATERIALS	4
8.0	INSTRUMENT OR METHOD CALIBRATION	5
9.0	SAMPLE HANDLING AND STORAGE	5
10.0	PROCEDURE AM) ANALYSIS	5
11.0	DATA ANALYSIS/CALCULATIONS	8
12.0	DATA MANAGEMENT/RECORDS MANAGEMENT	8
13.0	QUALITY CONTROL	9
14.0	NONCONFORMANCE AND CORRECTIVE ACTION	9
15.0	REFERENCES	9
16.0	FORMS AND DATA SHEETS	9

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SOP No. QC-13-05
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1.0 SCOPE AND APPLICATION:
1.1 This protocol describes procedures for verifying the performance of the
autoclaves. Small changes in temperature within the autoclave produce a large
effect on the time required to achieve the predetermined process parameters. It is
therefore critical to ensure that the autoclaves are operating within acceptable
limits (see ref. 15.1 and 15.2). This protocol also describes the procedure for
verifying that the autoclave KILL cycle used to sterilize biohazardous waste is
operating within expected parameters.
2.0 DEFINITIONS:
2.1	NIST = National Institute of Standards and Technology
2.2	Completed Liquid Cycle (for autoclave #s 1 and 2) = a complete 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 (not greater than
one inch). During this time, the steam is allowed to escape and the contents
allowed to cool and become acclimated to the ambient temperature to minimize
thermal shock, especially to liquids in glass containers.
2.3	A Kill cycle is a liquid cycle with a duration of 180 minutes to sterilize
biohazardous waste.
2.4	A gravity cycle is a dry cycle used for sterilization of dry laboratory materials
(e.g., glassware, carriers).
2.5	Chemical Indicator Strips = STEAM/7.s Steam Sterilization Integrator strips
from SPS Medical are engineered to integrate all 3 critical parameters of
sterilization (time, temperature and saturated steam) and are certified to perform
equal to a biological indicator plus an added safety factor.
2.6	Biological Indicator Ampule = Raven Biological PROSPORE Biological
Indicator is a hermetically sealed, type I borosilicate glass ampule. The ampule is
filled with a modified Soybean Casein Digest Broth containing bromocresol
purple acid indicator. Each ampule also contains a population of Geobacillus
stearothermophilus spores. Growth is evident by either turbidity and/or a color
change from a purple to or toward yellow.
3.0 HEALTH AND SAFETY:
3.1 Laboratory personnel have been trained on the proper use of the autoclaves. The

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SOP No. QC-13-05
Date Revised 11-07-11
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autoclaves and materials being removed from the autoclaves are very hot (often
greater than 100C). Lab personnel should wear lab coats, eye protection and
thermal gloves when handling materials being removed from the autoclaves to
prevent burns.
4.0 CAUTIONS:
4.1 Since autoclaves use high temperatures, it is necessary to exercise extreme
caution around the device and its associated plumbing. High-temperature
surfaces can be encountered even when the device is not in a sterilizing cycle.
5.0 INTERFERENCES:
5.1	The maximum registering thermometers should be reset prior to each use as
described in 10.2.2.1.
5.2	Shake the thermometer until the column registers 110C or lower.
5.3	The thermometer should be allowed to cool to ambient temperature before it is
read. Hold thermometer in an upright position for reading, and only after it has
cooled to ambient temperature, or you will obtain a falsely high reading.
6.0 PERSONNEL QUALIFICATIONS:
6.1 Personnel are required to be knowledgeable of the procedures in this SOP.
Documentation of training and familiarization with this SOP can be found in the
training file for each employee.
7.0 SPECIAL APPARATUS AND MATERIALS:
7.1	Raven Biological Laboratories ProSpore Biological Indicator Ampules with 106
spores of G. stearothermophilus (ATCC #7953) per unit
7.2	SPS Medical Chemical Indicator Strips
7.3	Incubator with temperature set at 55C  1C
7.4	Autoclave #1 located in room B206, Amsco Eagle 3000 Scientific Series, Model
E3031-S-1, Serial No. 0105898-25
7.5	Autoclave #2 located in room B204, Amsco Eagle 3000 Scientific Series, Model

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SOP No. QC-13-05
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E3031-S-1, Serial No. 0108298-11
7.6	Autoclave #3 located in room B207, Amsco Century Series Scientific Gravity
Sterilizer (16x16x26"), Model SG-116, Serial No. 0121198-15
7.7	Autoclave #4 located in room B202, Amsco Lab 250 Laboratory Steam Sterilizer
(20x20x38"), Model LG-250, Serial No. 0311511-10
7.8	Autoclave # 5 located in room D122, Tuttnauer Prevacuum Steam Heated
Autoclave with Vertical Sliding Door and Steam Generator (52 x 72 x 51"),
Model 5596-EP-1V, Serial No. 2311036
7.9	Certified Maximum Registering Thermometers (scale range 80-135C)
8.0 INSTRUMENT OR METHOD CALIBRATION:
8.1 Maximum registering thermometers must be calibrated against a NIST traceable
thermometer and certified annually (see SOP EQ-02, Thermometers).
9.0 SAMPLE HANDLING AND STORAGE:
9.1 Biological indicator ampules (sealed spore ampules containing spores in liquid
culture media) must be stored according to manufacturer's specifications to insure
shelf life. Upon receipt, the biological indicators ampules must be placed in the
refrigerator.
10.0 PROCEDURE AND ANALYSIS:
10.1 Summary.
Autoclave performance will be verified per run, monthly for short gravity cycles,
monthly for short liquid cycles, and monthly for kill cycles as summarized below.
Per run Verification (see 10.2): With every sterilization cycle, verify the
performance by examining the autoclave printout, a maximum registering
thermometer, and a chemical indicator strip.
Monthly Verification of Short Gravity Cycles and Short Liquid Cycles (see 10.3):
On a monthly basis, short gravity cycles and short liquid cycles are verified on
autoclaves #1, #3, and #4. Per run verification and biological ampules are used.
Monthly Verification of Kill Cycles (see 10.4): On a monthly basis, kill cycles

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SOP No. QC-13-05
Date Revised 11-07-11
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(used to demonstrate that the autoclave cycle used to sterilize biohazardous waste
is operating within expected parameters) are verified on autoclaves #2, #3, #4,
and #5. Per run verification and biological ampules are used.
Refer to Table 1 for a summary of the performance verification practices.
Table 1: Performance Verification Practices for Autoclaves and the Drying Oven	
Quality Control
Unit
Location
Use
Per Run
Short

Kill



V
Gravity
Liquid

Autoclave #1
B206
Media
V
V

Autoclave #2
B204
Kill
V


V
Autoclave #3
B207
Media, kill
V
V
V
V
Autoclave #4
B202
Media, kill
V
V
V
V
Autoclave #5
D122
Kill
V


V
10.2 Performance Verification of Daily Runs (Per Run Verifications). The following
data are collected for every autoclave cycle.
10.2.1 Autoclave Printout. For each run, 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 16.0).
10.2.2 Maximum Registering Thermometer. A maximum registering
thermometer is used for each daily autoclave run. Place the thermometer
upright in a container and place the container in the autoclave pan along
with the items to be processed.
10.2.2.1 Reset the maximum registering thermometer prior to each
use by "shaking" the thermometer as you would a fever
thermometer. This will force the mercury through the
constriction located above the bulb (see 14.1).
10.2.2.2 Record the results from the thermometer on the appropriate
form (see 16.0). The maximum registering thermometer
acceptable temperature range per cycle run is between 120-
124C, with the exception of certain media (e.g. CTA stabs)
which may require a lower sterilizing temperature. The
thermometer should be allowed to cool to ambient
temperature before it is read (see 5.3).

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SOP No. QC-13-05
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10.2.3 Chemical Indicator Strip. Place the strip flat on top of the container that
holds the maximum registering thermometer.
10.2.3.1 Record the results from the chemical indicator strips on the
appropriate form (see 16.0). A passing chemical indicator
strip must reach the "steam safe" section indicated at the
end of the strip. A failing indicator strip does not reach the
"steam safe" section at the end of the indictor strip.
10.3 Monthly Performance Verification of Short Gravity and Liquid Cycles. On a
monthly basis, performance verification is conducted by running a short gravity
cycle and a short liquid cycle in Autoclaves 1, 3, and 4. For Autoclave 5, only the
short liquid cycle is run. In addition to recording Per Run Verification* data,
biological ampules are used.
* For Autoclave #1, use two Maximum Registering Thermometers, two
chemical indicator strips, and two biological indicator ampules. One set of
thermometer/strip/ampule is placed in the center of the top shelf, and the
other set is placed in the center of the bottom shelf.
10.3.1	Place a biological indicator ampule into a test tube containing an
appropriate volume of liquid (-10 mL for 20x 150 mm test tubes and -20
mL for 25 x 150 mm test tubes). Place the test tube containing the ampule
in a test tube rack containing 39 other similarly filled test tubes (each rack
holds 40 test tubes).
10.3.2	Place the tube with the biological indicator ampule as close to the center
of the rack as possible. Place this rack into an autoclave pan along with a
maximum registering thermometer, and a chemical indicator strip. Place
the chemical indicator strip in the test tube rack near the tube with the
biological indicator ampule.
10.3.3	For Autoclave #1, place two pans, each containing a rack with an ampule,
thermometer, and strip, as described in 10.3.2, into the unit, one in the
center of the top shelf and one in the center of the bottom shelf.
10.3.4	For Autoclave #3, #4 and #5, place one pan containing an ampule,
thermometer, and strip, as described in 10.3.2 in the center of the bottom
shelf.
10.3.5
Run a gravity cycle for Autoclave #1, #3 and #4 (not #5) for the minimum
time necessary to kill all spores as indicated with the QC documentation

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SOP No. QC-13-05
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that accompanies each lot of biological ampules (typically from 15 to 20
minutes).
10.3.5.1	Immediately upon completion of the cycle, remove the
pans from the autoclave.
10.3.5.2	Remove the ampule from the test tube and label with:
location in autoclave (i.e., top or bottom rack, if
applicable), cycle type (i.e., gravity cycle, liquid cycle, kill
cycle), and date of run). Incubate the ampule as well as one
control ampule that has not been autoclaved at 55C  1C
for 48  2 hours and record the results on the appropriate
form (see 16.0). See section 14.2 for non-conformance and
corrective action.
10.3.5.3 Repeat the process for a liquid cycle for Autoclave #1, #3,
#4 and #5 also set for the minimum time necessary to kill
all spores as indicated in 10.3.5.
10.4 Monthly Performance Verification of Kill Cycles. On a monthly basis,
performance verification is conducted by running a KILL cycle in Autoclaves 2,
3, 4, and 5. In addition to recording Per Run Verification data, biological ampules
are used.
10.4.1 To verify KILL cycles, place a biological indicator ampule in the center of
an autoclave bag filled with solid waste. Run a standard kill load (3 hour
liquid cycle). After completion of the cycle, recover and label the ampule
and incubate for 48  2 hours at 55C  1C along with a control ampule
that has not been autoclaved. Record the results on the appropriate form
(see 16.0).
11.0 DATA ANALYSIS/CALCULATIONS: None
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT:
12.1 Data will be recorded promptly, legibly, and in indelible ink. Completed forms
are archived in notebooks kept in secured file cabinets in file room D217. Only
authorized personnel have access to the secured files. Archived data is subject to
OPP's official retention schedule contained in SOP ADM-03, Records and
Archives.
12.2 The autoclave printouts will be collected annually and stored in room D217.

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SOP No. QC-13-05
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13.0 QUALITY CONTROL:
13.1 NIST traceable Maximum registering thermometers are calibrated annually. See
EQ-02, Calibration of Thermometers.
14.0 NONCONFORMANCE AND CORRECTIVE ACTION:
14.1 Refer to Table 2 for a complete discussion of nonconformance and corrective
action scenarios. If a processed biological indicator ampule fails (i.e., growth as
indicated by ampule color changing from purple to yellow after incubation at
55C 1C for 48  2 hours), another ampule should be processed with the same
parameters. If the nonconformance persists, service should be scheduled and the
autoclave should not be used.
15.0 REFERENCES:
15.1	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.
15.2	Eaton, A. D., Clesceri, L. S., Rice, E. W., Greenberg, A. E. and Franson, M. A. H.
eds. 2005. Standard Methods for the Examination of Water and Wastewater, 21st
Edition. American Public Health Association, Washington, DC.
15.3	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.
16.0 FORMS AND DATA SHEETS:
16.1	Daily Sterilization Record Log Form
16.2	Monthly Sterilization Record Form

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SOP No. QC-13-05
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Table 2: Nonconformance and Corrective Action Scenarios
Temperature
Chem
Strip
Nonconformance and Corrective Action*
Max
Unit
P
P
F
Cheek the location of the Cliem Strip . Check that the clicm strip for the next cycle
passes. If the nonconformance pattern persists, run a cycle and process a biological
indicator. Incubate and record the results of the biological indicator (see 14.2).
Check sterility of media and use if it passes. Repeat the cycle for glassware/dry
goods.
F
P
P
If a maximum registering thermometer deviates from the sterilize set temperature by
greater than 3EC, the cycle is considered to be in nonconformance. Repeat the cycle
to verify the nonconformance. If the nonconformance pattern persists, verily the
nonconformance again using a different maximum registering thermometer. If the
nonconformance persists, run a cycle and process a biological indicator (see 14.2).
Call for service. Check sterility of media and use if it passes. Repeat the cycle for
glassware/dry goods.
F
P
F
Check the location of the Chem Strip1. Repeat the cycle to verily the
nonconformance. If the nonconformance pattern persists, verify the nonconformance
again using a different maximum registering thermometer. If the nonconformance
persists, run a cycle and process a biological indicator (see 14.2). Check sterility of
media and use if it passes. Repeat the cycle for glassware/dry goods.
P
F
F
The autoclave alarms have been set to trip when the sterilize temperature falls below
the sterilize set point (121EC) by more than 1EC. The under temperature alarms will
occur during the sterilize phase of the cycle whenever the chamber drain temperature
falls below 120EC. If the alarm occurs, an under temperature message will be
printed. If the chamber drain temperature rises above the alarm set point (120EC),
the alarm will silence and another message will be printed. The autoclaves are set to
resume the sterilize phase once the temperature falls within acceptable limits.
Consider the unit temperature to have passed and see the appropriate
nonconformance if the Maximum registering thermometer or the Chem strip fail. If
the total sterilize phase lasts longer than the set time by more than 15 minutes, the
Too Long in Sterilize alarm will sound and the cycle will be aborted. A message will
be printed and the LED readout will Hash AComponent Failure. 0 Call for service. If
the autoclave goes into alarm but the cycle resumes and is completed successfully,
the media should be checked for sterility and performance and can be used if it
passes. Repeat the cycle for glassware/dry goods. If the AToo Long in Sterilize @
alarm is tripped, media should be discarded and glassware/dry goods should be
autoclaved again.
P
F
P
F
F
F
F
F
P
* Italics indicate corrective action to be taken in regards to media being processed at the time the original nonconformance occurred.
1 The Chem Strip should be placed in a location that is fully exposed to the steam. It should not be placed within the beaker that holds the Max. registering
thermometer. If the load includes racks and tubes of media, the Chem Strip should be placed within the rack between tubes.
P = Pass
F = Fail

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16.1 Daily Sterilization Record Log Form
OPP Microbiology Laboratory	

Cycle1
Unit Temp.2
Max. Thermometer3
Chemical Indicator Strips
Items Processed
Sterilization No.5
Init.
Type
Time
Min.
Max.
Temp.
Serial #
Lot No.
Exp. Date
Result4








































































































































































1	Record the cycle as G = gravity, L = liquid under Type and the duration of the cycle in minutes under Time.
2	Record the maximum and minimum temperature achieved during the sterilize phase of the cycle as indicated by the autoclave printout (Unit).
3	Record the corrected value for the maximum registering thermometer (Max.) and the serial number of the thermometer.
4	Record the results of the chemical indicator strips as P for pass or F for fail.
5	The Sterilization No. indicates the date as well as the unit location and the run number.

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16.2 Monthly Sterilization Record Form
OPP Microbiology Laboratory
STERILIZER ID AND INFORMATION
Chem. Strip LotNo./Exp. Date
Room and
Unit ID
Cycle1
Biological Ampule
Results2
Record the cycle as G= gravity, L = liquid under Type and the duration of the cycle in minutes under Time.
Record the results of the ampules after incubation as growth (+) or no growth (0) for the autoclaved ampule (Growth) and unautoclaved control.
Record the maximum and minimum temperature achieved during the sterilization phase as indicated by the autoclave printer (Unit)
Record the corrected value for the maximum registering thermometer and the serial number of the thermometer.
The Sterilization No. indicates the date as well as the unit location and the run number.
Record the results for the analysis of kill loads (see 10.3.4.8). If not performed, put an ND, not done.

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