Autoclave Cycle Development, Validation and Verification · University of Saskatchewan Autoclave...

2019 Safety Resources

Autoclave Cycle Development, Validation and Verification

Table of Contents

1 INTRODUCTION ........................................................................................................................ 1

2 DEFINITIONS AND ACRONYMS .................................................................................................. 1

3 SAFETY ...................................................................................................................................... 2

4 EQUIPMENT OR MATERIALS REQUIRED .................................................................................... 2

5 PROCEDURE .............................................................................................................................. 4

5.1 For Cycle Development, Validation, and Verification of New Waste Loads ...................................................... 4

5.2 Verification of Established Cycles ..................................................................................................................... 6

5.3 Annual Validation for Facility Re-Certification ................................................................................................. 7

6 HIGHLIGHTS/CRITICAL CONTROL POINTS.................................................................................. 7

7 REPORTING ............................................................................................................................... 7

8 REGULATORY/STANDARDS ....................................................................................................... 7

10 TROUBLE SHOOTING ................................................................................................................ 8

11 REFERENCES ............................................................................................................................. 8

APPENDIX 1: AUTOCLAVE CYCLE VALIDATION LOG .......................................................................... 9

APPENDIX 2: AUTOCLAVE BIOLOGICAL INDICATOR USE LOG ......................................................... 10

APPENDIX 3: AUTOCLAVE CYCLE VERIFICATION – HOLD FOR RESULTS .......................................... 11

University of Saskatchewan Autoclave Cycle Development, Validation, and Verification SOP Page 1

1 Introduction

This document describes the establishment of standard autoclave loads as well as determining

effective time and temperature for each cycle, based on efficacy testing using biological

indicators. This document describes the validation of autoclave cycle parameters during the initial

commissioning of an autoclave, the annual validation, the verification of autoclave loads used on

campus, and after a repair or modification to the autoclave.

Cycle verification and validation must be carried out annually on each autoclave for representative

mock loads.

2 Definitions and Acronyms

Biological

Indicators (BIs)

Self-Contained BI

Paper strip impregnated with Geobacillus stearothermophilus spores, packaged in a container with cap, along with growth media within a crushable glass ampoule.

Just before incubation, the cap is sealed and the ampoule is crushed and mixed to suspend the spores in the growth media.

Requires a 10 - 48 hour incubation period at 55C (time depends on the make of the BI; consult manufacturer’s instructions). Growth of spores results in pH change and resultant color change of media from purple to yellow and/or turbidity

Self-Contained Ampoule BI

Sealed borosilicate glass ampoule containing broth, pH indicator and a population of Geobacillus stearothermophilus spores.

Used to test steam sterilization of liquid or non-liquid loads

Requires a 48-hour incubation at 55C.

Growth of spores results in a pH change and color change of media from purple to yellow and/or turbidity.

BSC Biological Safety Cabinet

CFIA Canadian Food Inspection Agency

CL2 Containment Level 2

CL2-Ag Containment Level 2 – Agriculture (Large Animal)

Personal

Protective

Equipment

(PPE)

Refers to protective clothing designed to protect the wearer’s body

from injury or infection due to hazards which are physical, electrical,

chemical, produce heat, biohazardous, and airborne particulate

matter.

PHAC Public Health Agency of Canada

Validation The act of confirming that a method achieves its objective by

observing that specific parameters have been met (e.g. using

biological indicators or independent temperature loggers to confirm

that a given autoclave cycle can decontaminate a representative load


of waste). (CBS, 2nd Ed. 2015; and CBH, 2nd Ed., 2016). Autoclave

cycles are validated annually.

Verification The routine monitoring of equipment and processes to ensure

continued efficacy between validations. Autoclave cycles must be

routinely are verified by using biological indicators or independent

temperature loggers. The waste cycle programmed in the autoclave

is verified using biological indicators and is dependent on its use.

Refer to the U of S Monitoring Autoclave Efficiency Guideline (2017).

3 Safety

1. All mock loads must consist of non-contaminated materials (non-infectious), which is comprised of representative materials and waste anticipated to be generated within the CL2 and/or CL2-Ag spaces.

2. A maximum number of bags and/or autoclave pans can be accommodated in the autoclave chambers. The number of bags or pans depends on the load and the type and amount of material in each autoclave bag, which must be described during cycle development. Place pans and bags into chamber such that steam flow is not obstructed. If data logger results show temperature deficit in the chamber or load, or BI results are positive for growth, reduce the amount of material in the bag, increase the temperature, increase the number of conditioning pulses, or increase the time of exposure at temperature. If using a temperature logger, use data to help guide these decisions. Continue until negative BI results are attained and/or temperature logger results record set temperature attained in the load and held for required length of time.

3. Wear autoclave gloves when removing loads from autoclave, and when handling hot

BIs and data loggers.

4. Store and handle BIs according to manufacturer’s instructions.

5. Do not use expired BIs.

6. Inspect BIs closely for damage before and after processing; inspect for missing or damaged components or any signs of ampoule breakage (wet spore strip, wet or dried residual media inside vial, or apparent lack of media in ampoule).

7. Ensure the required incubation time has elapsed before checking for bacterial growth

in the BI.

4 Equipment or Materials Required

Autoclave Site-specific to the facility


BIs Minimum population of 1.0x105 spores (e.g. 3M

ATTEST, Raven Prospore, Getinge Biosign, etc.). See

U of S Monitoring Autoclave Efficiency Guideline

(2017).

Incubator for BIs e.g. 3M ATTEST, Raven Prospore, Getinge Biosign,

etc.

Autoclave bags with

biohazardous symbol

Used for biohazardous waste collection (e.g. rated for 134°C, 2ml polypropylene).

Autoclave bags without

biohazardous symbol

e.g. Clear, rated for 135°C

Twist ties, string, marker

Forceps

String (non-plastic)

Autoclave pans

Autoclave tape

Autoclave transfer carriage and loading car

Dependent on model of autoclave, if applicable.

Materials for mock-loads* Dependent on the unit/facility in which the autoclave is located (NOTE: a waste assessment would be required to define the mock load prior to initiation of verification and validation process).

Autoclave Cycle Validation Log Appendix 1

Autoclave Biological Indicator Use Log

Appendix 2

Autoclave Cycle Verification – Hold for Results

Appendix 3

* Examples of Mock Loads:

Mixed lab waste, (e.g. gloves, paper towels, disposable gowns, plastic tips and boxes, plastic bottles, cardboard, dry absorbent pads or wet with water (to simulate material from spill cleanup))

Plastic petri dishes with agar: 40 to 60 dishes per bag, double bag and place in pan

Linens, i.e. scrubs, coveralls, towels, surgical drapes; make autoclave bag 2/3 full with linens (number depends on the type of article of clothing; scrub sets take up less volume than coveralls)

Animal bedding – used bedding from non-infectious animals, approximately equal to


50 cage changes, or 35L of clean bedding from a 70L bag of rodent bedding + 0.5L water

Rodent cages, lids, and feeders with feed

Liquid media – glass bottles and flasks (e.g. culture media, buffer solutions, sterile water)

5 Procedure

5.1 For Cycle Development, Validation, and Verification of New Waste Loads

1. Print a copy of the Autoclave Cycle Validation Log (Appendix 1).

2. Determine the maximum quantity of material that may be run at one time for each load

type (refer to sections 3(2) and 4 for set-up and load-type examples). This quantity

should be representative of the maximum amount of material typically autoclaved and

should never be exceeded. If exceeded, the validation is no longer valid, and must be

redone (e.g. two autoclave bags of mixed waste 2/3 full).

3. Place autoclave tape on the side of the bag.

4. Obtain the required number and type of BIs. Inspect BIs closely for damage and

ensure that expiry date has not passed. If damaged or expired, discard BI into sharps

waste container.

5. Label the BI with the date and cycle to be used.

6. Tie a long string to the BI vial and insert it into the middle of the load to be sterilized

(the string will allow easy removal after autoclaving).

For example:

Mixed Lab Waste Loads: Place the BI centrally in the bag.

Plastic Petri Dishes with Agar Loads: This load is recommended to be double bagged and placed on an autoclave pan. If using a glass ampoule type BI, wrap ampoule with cardboard or paper to protect it from melted plastic and make retrieval easier.

Animal Bedding or Linens Loads: Place BI inside the bag with the densest material – rodent bedding and feed pellets have proven to be the most difficult to achieve temperature, bags of scrubs and coveralls are the second most difficult.

Animal Cages Loads: Place BI into the empty cage.


Liquid Loads: Place the BI into an empty glass bottle or flask.

7. Fold inner bags when double-bagging and close outer bags loosely with twist tie and

label each bag with a number using a marker. The bag should be tied loosely, leaving

the top open a few inches to allow steam to circulate within the bag.

8. Record on the Autoclave Cycle Validation Log (Appendix 1) the following:

Cycle type and maximum load number for the cycle;

Bag numbers and positions on the load car;

Bag contents, including any BI inside; and

Lot number of BIs and make.

9. Place mock loads into the autoclave and close the door.

10. Select cycle as per manufacturer’s instructions and wait for the autoclave cycle to finish.

11. When the cycle is complete, don gloves and safety glasses, and open autoclave door

12. Allow materials to cool slightly before handling. Referring to the Autoclave Cycle Validation Log (Appendix 1), open the relevant bags and retrieve the BIs. If necessary, wipe moisture or agar off of BI with paper towel. Cool slightly. Close bags again with twist tie.

13. Transfer autoclave bags into a storage area (e.g. shelf, bin), and print off the Autoclave Cycle Verification – Hold for Results” signage (Appendix 3), and post signage by the loads. Record all relevant information on the signage.

14. Close autoclave door and remove PPE.

15. Take processed BIs to area in which BIs will be incubated at 55C.

16. Handle BIs with care and handle only by the cap, if applicable. When cool, remove string or twist tie (if applicable). Ensure load information on label is still intact. Re-label if necessary. Label with date and run number.

17. Obtain a non-autoclaved BI from the same lot number as the autoclaved BI, to use as a positive control for incubation temperature and media to promote bacterial growth, and to ensure that storage and handling has not affected the viability of the BI. Label control BI with run number, date, and the letters “CON” to depict control.

18. Depress the lids of the autoclaved and non-autoclaved BIs to seal the top of the tube (if applicable). Crush the ampoules and incubate the autoclaved and the non-autoclaved BI in the incubator according to manufacturer’s instructions. Record BI information in the Autoclave Biological Indicator Log (Appendix 2).

19. After the required incubation time, check BIs and record results on Autoclave Cycle


Validation Log (Appendix 1) and Autoclave Biological Indicator Log (Appendix 2).

20. Discard incubated self-contained BIs (autoclaved and non-autoclaved) into sharps container.

21. In the event of sterilization failure, repeat the process until the necessary loading configuration and sterilization time have been determined.

22. Run three mock loads for each new cycle.

23. Failure to achieve sterilization may be due to:

Improper loading or overloading of the autoclave so that the center of the load failed to achieve sterilization temperature; or

Insufficient sterilization time in order to attain a 4 - 6 log10 reduction of G.

stearothermophilus spores.

24. Retain printout copies for each run and attach to Autoclave Cycle Validation Log (Appendix 1) for records.

25. Place Autoclave Cycle Validation Log (Appendix 1) and all attached files into binder

stored or store electronically. If using a binder, ensure it is divided into sections for each autoclave.

26. Effective time and load configuration must be used for all subsequent cycles for that

type of load.

5.2 Verification of Established Cycles

1. Identify which established cycle needs to be verified. Load the autoclave with a load of biohazardous waste with a BI. Place the BI on the outside of the bag with autoclave tape during the verification, for safety (for further information refer to the University of Saskatchewan Monitoring Autoclave Efficiency Guideline (2017)). Once done placing the BI on the bag, run the autoclave in accordance to manufacturer’s instructions.

2. When cycle is complete, remove materials as previously described in section 5.1.

3. Print a copy of the “Autoclave Cycle Verification – Hold for Results” (Appendix 3) and

record relevant information; place on storage area or bin containing the autoclaved

waste.

4. Process as described in section 5(17-22). 5. Retain printout from autoclave and attach to Autoclave Cycle Validation Log

(Appendix 1); place log sheet and attached documents in the autoclave cycle


verification binder or electronically file. 6. If BIs indicate failure to attain sterilization parameters, reload waste into

autoclave and run cycle again. Contact Biosafety Officer (306-966-8496) for any assistance.

7. If BIs indicate successful sterilization, complete and sign Autoclave Cycle

Verification – Hold for Results form (Appendix 3) and place in autoclave verification binder. Deface biohazard symbol on autoclave bag or place sterile waste into black garbage bag and tie with a knot and place into Loraas bin.

5.3 Annual Validation for Facility Re-Certification

1. Each load type must be validated annually. Validate established cycles with BIs placed into mock loads as described in Section 5.1.

2. If the most difficult load to decontaminate was determined during initial validation,

(e.g. the one load type that failed when tested with all load types), subsequent annual validations can use the longer run time used to decontaminate the most difficult load. Otherwise, each load type must be validated annually.

6 Highlights/Critical Control Points

1. Three mock loads for each new cycle must be run before the specific cycle parameters for that load are deemed effective. One mock load of established cycles must be verified annually, testing the materials most difficult to sterilize.

2. Ensure BIs have not expired.

7 Reporting

1. Printout copies from autoclave and BI results for each waste run must be stored and available to the Biosafety Officer and/or PHAC upon request.

8 Regulatory/Standards

1. BIs must be used to verify cycles run at 121°C. A non-autoclaved BI must be incubated as a positive control along with the autoclaved indicator. Ensure the same lot number is used for test and positive control BIs. Record Lot # and Results in Autoclave Cycle Validation Log (Appendix 1).


i. Retain printout copies for each run and attach to Autoclave Cycle

Validation Log (Appendix 1) for records.

ii. Place Autoclave Cycle Validation Log (Appendix 1) and all attached files into binder stored or store electronically. If using a binder, ensure it is divided into sections for each autoclave.

iii. Effective time and load configuration must be used for all subsequent

cycles for that type of load.

10 Trouble Shooting

Problem Action

BI showed growth after incubation 1. Autoclave was overloaded or loaded improperly. Adjust load size and retest.

2. Sterilization time was inadequate.

Increase time of cycle and retest.

11 References

Public Health Agency of Canada. Canadian Biosafety Handbook. 2nd Edition, 2016.

Public Health Agency of Canada. Canadian Biosafety Standard. 2nd Edition, 2015.

University of Saskatchewan Monitoring Autoclave Efficiency Guideline (2017)


Appendix 1: Autoclave Cycle Validation Log

Date:

Autoclave Name:

Program Number:

Cycle Parametres:

Exposure Temperature:

Exposure Time:

Notes:

Load Description (list load type – autoclave

bag contents) (e.g. mixed lab waste, bag

2/3 full)

Bag #

BI – Make, Lot# and BI Placement

BI Result (Pass/Fail)

Comments

Required Time x Temperature Conditions Met? (Circle)

YES NO Signature and Date:

Printouts (Tape below):


Appendix 2: Autoclave Biological Indicator Use Log

Date Autoclave BI # BI

Model

BI Lot #

Time in at

Incubator Date

Time/Date out

Result Initials

Result: NG (no growth) = PASS; G (growth) = FAIL


Appendix 3: Autoclave Cycle Verification – Hold for Results

Date:

Autoclave Name:

Program #:

Operator Name:

Notes:

Autoclaved load can be released for disposal: Yes No

Signature: _________________________ Date: ____________________

Place completed, signed form into autoclave documents binder or store electronically.

THIS BIN CONTAINS AUTOCLAVED MATERIALS

AWAITING VERIFICATION RESULTS – MAY TAKE

UP TO 48 HOURS

BI Model and Lot #: __________________________________

Positive Control BI Lot #: ______________________________

BI Results (Circle one): No Growth = PASS; Growth – FAIL; N/A

Positive Control BI Results (Circle one): Growth; No Growth; N/A

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