Medical Device Sterility Assurance Tests

Once a sterilization method has been validated for a particular product, and the product is being manufactured, routine medical device sterility testing must be performed. These included bioburden tests, quarterly dose audits, cleaning and disinfection, and environmental monitoring, among others.


  1. Sample Item Portion (SIP) Preparation
  2. Bioburden Method Validation
  3. Bioburden Enumeration
  4. Bacteriostasis/Fungistasis Test
  5. AAMI/ISO Dose Audit
  6. Sterility Testing
  7. Microbial Environmental Monitoring
  8. Package Intergrity Testing
  9. Bacterial Endotoxins (LAL) Test

Sample Item Portion (SIP) Preparation

A sample item portion is a specially prepared portion of a medical device that is used in AAMI/ISO dose setting procedures. Some large or complex devices cannot practically be tested in their entirety. Using nonsterile samples, a defined portion of the device is aseptically removed and packaged. This portion (SIP) is used for the bioburden and verification dose studies.

For some complex devices, it may be necessary to disassemble or cut up the device so that it can fit into rinse fluid and media containers used for bioburden and verification dose testing. This disassembly or cutting must be done aseptically so that the natural bioburden of the product is not affected. It also must not reduce the challenge to the sterilizing process.

The adequacy of the SIP must be demonstrated by means of a sterility test of 20 non-sterile SIP samples. The SIP is considered adequate if at least 17 of the samples test positive in the sterility test.

Please submit a product sample to Pacific BioLabs for evaluation so that an appropriate SIP preparation procedure can be determined.

Bioburden Method Validation

ANSI/AAMI/ISO Guideline 11737-1, Sterilization of Medical Devices – Microbiological Methods, Part 1: Determination of a Population of Microorganisms on Products, requires that the bioburden test method be validated for each medical device. The purpose of this validation is to insure that the bioburden test method which will be used to determine the product bioburden level, is effective in a) recovering microorganisms that are present on the product and b) does not inhibit growth of the recovered microorganisms. Insufficient recovery or inhibition would result in underestimation of a product’s true bioburden and could lead to an inadequate sterilization cycle or dose. The recovery data from validation testing will indicate if a recovery factor should be applied to results obtained by routine bioburden testing.

The bioburden method validation must be performed prior to proceeding with actual bioburden testing of the product. If any changes affecting materials, assembly or configuration are made to the product, the bioburden method should be revalidated.

Repetitive (Exhaustive) Recovery

This method uses the naturally occurring bioburden of the product. A bioburden test is performed on the same device three or more times. The counts obtained from the replicate extractions are used to calculate a percent recovery. Pacific BioLabs recommends this method for devices which have a moderate to high bioburden level and do not contain absorbent materials.

Product Inoculation (Simulated) Recovery

This method simulates a product bioburden by inoculating a known amount of spores onto a sterile device. The device is then tested for bioburden with the same method proposed for use in routine analysis. The recovered level is compared to the known inoculation level and the percent recovery is calculated. Pacific BioLabs recommends this method for devices with low bioburden levels or complex configurations. It also may be used for devices which absorb the rinsing fluid used in bioburden tests. The method does have limitations, because the spore inoculation may not reproduce the adherence properties of the natural bioburden. Please note that sterile samples are usually required for simulated recovery tests.

Screening for Release of Substances Adversely Affecting Bioburden Estimates

Some medical devices are manufactured with materials that adversely affect the bioburden testing of that device. These materials, or substances that are extracted from these materials during bioburden testing, have bacteriostatic, bacteriocidal, fungistatic and/or fungicidal properties which damage, inhibit the growth of, or kill microorganisms removed from the product during the extraction phase of the bioburden test. ANSI/AAMI/ISO Guideline 11737-1 includes a method that screens for the presence of these adverse substances. This method requires the inoculation of a known population of test organisms into a test container with the product and the bioburden extraction fluid. Following a holding period equivalent to that which occurs during routine bioburden testing, the test organisms are enumerated and compared to the initial population. If the counts are not similar, modification of the bioburden procedure is necessary.

This screening test should be performed when:

  1. The product contains materials which could have biocidal or biostatic effects.
  2. The repetitive or product inoculation results indicate low recovery.
  3. The product is liquid, gel or powder.

Bioburden Enumeration

Bioburden is the population of microorganisms on a raw material, product component or finished medical device just prior to sterilization. For finished medical devices, the bioburden test data is used to establish parameters for an effective sterilization process. To insure the ongoing safety of the sterilization process, it is necessary to verify that the bioburden level remains consistent over time. There are two important aspects of product bioburden control – maintaining consistency from lot to lot and avoiding spikes within a single lot. A bioburden spike occurs when the bioburden for an individual product is 2 or more times greater than the group average. A significant increase in the device bioburden would reduce the sterility assurance level of the sterilization process.

For new medical devices, ten randomly selected samples from three separate newly manufactured lots should be tested for bioburden. It is useful to track samples by date and time of assembly or packaging to determine when and where any inconsistencies may be occurring. After initial data is generated, bioburden tests should be conducted monthly to quarterly, depending on frequency and volume of production, as part of an ongoing environmental monitoring program. It is also important to check bioburden levels whenever any changes are made in packaging locations, manufacturing processes, raw material vendors, or personnel involved with production. If bioburden data increases significantly or shows extreme variability, the manufacturing process should be investigated so that corrective measures can be implemented.

Bioburden studies are also used to monitor microorganism levels on materials that could affect the bioburden of the finished device, such as product components, manufacturing fluids and product packaging. The bioburden test data may provide useful information in the investigation of bioburden spikes and dose audit failures. Bioburden testing can also be used as a material qualifications tool. For additional information refer to Sterilization of medical devices—Microbiological methods, Part 3: Guidance on evaluation and interpretation of bioburden data (ANSI/AAMI/ISO 11737-3:2004).

To perform a bioburden test, a sample is aseptically transferred to an appropriate volume of extraction fluid and then mechanically agitated to remove microorganisms. Membrane filtration is the preferred method for the culturing and microbial enumeration of the extraction fluid. Pacific BioLabs uses this method for products with filterable extraction fluid. When the extraction fluid cannot be filtered, the plate count method is used. Bioburden results are reported on individual samples showing total aerobic count with a breakdown of bacteria and fungi. Anaerobic bioburden and heatshocking methods for enumeration of spores are also available upon request.

Bioburden Test Method Validation

  1. Recovery Study – Repetitive Treatment (3 to 5 extractions/sample)
  2. Recovery Study – Simulated  (spore inoculation of products)
  3. Screening for the Release of Adverse Substance Affecting Bioburden Estimates

Bioburden – Total Aerobic Bacteria and Fungi by Filtration Method

  1. Small Devices (low level bioburden)
  2. Medium Devices (moderate level bioburden)
  3. Large Devices, Small Kits, Papers, Fabrics (higher level bioburden)

Bioburden – Total Anaerobic Bacteria Bioburden – Total Aerobic Spores

Bioburden – Process Fluids (Aerobic Bacteria by Filtration Method)

Bacteriostasis/Fungistasis Test

The bacteriostasis/fungistasis test is designed to validate the procedure used to test a product for sterility by demonstrating that microorganisms present on the product will be detected in the course of the sterility test. The USP (and FDA) requires this test because some products contain substances that inhibit the growth of microorganisms. Although a product may harbor microorganisms and be nonsterile, the presence of growth inhibition substances can cause a falsely negative sterility test.

The test is conducted by performing a simulated sterility test, then adding low levels of selected bacteria and fungi as challenge microorganisms to the culture media. The organisms will remain viable, grow and be detectable in the culture media if the product does not exert a bacteriostatic or fungistatic effect. If the product is found to be bacteriostatic or fungistatic, the sterility test procedure must be modified and another bacteriostasis/fungistasis test must be performed. This testing should be performed on all new products and when any significant changes are made in the manufacturing or materials of an existing product. Pacific BioLabs strongly recommends repeating the B/F test biannually to account for any possible changes to the product or manufacturing process. For medical devices, three to six sterile samples are required for the B/F test.

Bacteriostasis/Fungistasis Test – Direct Transfer Method

  1. 3 organisms in SCDM (Radiation Dose Audits)
  2. 6 organisms – 3 in FTM and 3 in SCDM (USP; EtO sterilized products)

AAMI/ISO Dose Audit

A dose audit is a sterility test of samples which have been irradiated at a defined kGy level determined as part of an AAMI/ISO dose validation study. The AAMI/ISO standard requires that dose audits be performed quarterly (or with each lot if production is less frequent than quarterly) for all devices that been validated according to ANSI/AAMI/ISO 11137 Method 1 or AAMI TIR 33 VDmax. (The dose audit interval may be extended to semiannually or annually if it is demonstrated over time that the product bioburden is stable with respect to levels, microorganism types, and microorganism resistance.) All samples are tested using Soybean Casein Digest Medium and incubated for 14 days at 30º±2ºC.

The information below may be used to determine a likely dose audit fee. Compare the sample measurements with the container dimensions. The cost of a dose audit is usually based on the amount of medium required to test the sample. Samples which are difficult to aseptically handle will incur an additional charge. Please contact Pacific BioLabs to request a quote. Samples which can be easily cut with scissors can be aseptically divided and possibly tested in smaller containers. To minimize the chance of a sample being compromised in sterility testing, it is highly desirable to minimize sample manipulation. We prefer to use the smallest container and volume which will allow the test sample to be submerged in media (although it is not always possible to completely submerge all samples). An appropriate media volume must be validated for each product by bacteriostasis-fungistasis testing. If the product cannot be cut to fit into any of the following containers, SIP preparation will be required.

Media containers used for Dose Audits

Media ContainerDimensionsOrifice I.D.Media Volume
Culture Tube25 mm D x 150 mm H25 mm40 mL
Culture Tube25 mm D x 250 mm H25 mm80 mL
Culture Tube38 mm D x 200 mm H38 mm100 – 120 mL
0.9 L Glass Jar90 mm D x 165 mm H75 mm500 mL
1.8 L Glass Jar135 mm D x 150 mm H95 mm1000 mL
Nalgene Jug150 mm Square x 250mm H82 mm1000 mL
Nalgene Jug150 mm Square x 250mm H82 mm2000 mL
D = Diameter, H = Height

In a Nalgene jug, 1000 mL of media is about 65 mm deep; 2000 mL is about 130 mm deep. Some large or long samples and SIP preparations will only fit in the Nalgene jugs. The higher fees reflect increased costs for media, significantly greater time for media production and media quality control, extended time for sterility testing and significantly greater incubator space requirements.

Notes: To expedite processing of your dose audit samples, please call us prior to sending samples so that we can schedule your tests. Some samples are difficult to aseptically cut and transfer to media containers. If possible, please include 3 to 5 additional samples with your dose audit sample submission.

Sterility Testing

Sterility testing of products and/or biological indicators (i.e. spore strips) exposed to a sterilization process is an important part of all sterility assurance programs. Most manufacturers of EO sterilized medical devices monitor their validated EO sterilization loads with B. atrophaeus spore strips and release their products for distribution based on negative sterility test results of the spore strips. The spore strips may be placed inside or outside the product depending on the type of spore strip testing performed during the validation. The spore strips should be distributed in locations throughout the sterilization chamber. A positive control (i.e. unprocessed spore strip) should be included with all spore strip sterility tests. These routine sterility tests must be supplemented periodically with more extensive cycle validations.

Routine lot release of terminally sterilized medical devices by means of end product sterility testing is not recommended for several reasons. The bioburden of most medical devices generally is a lesser challenge to the sterilization process than biological indicators, and overkill cannot be demonstrated. Statistically, the probability that a sterility test of 20 or 40 product samples will detect nonsterile samples among a much larger number of products is very limited. Also, it is generally recognized that the process of sterility testing has a significantly lower sterility assurance level than most validated terminal sterilization processes. However, end product sterility testing of medical devices is occasionally performed as part of investigations or to support other information in making a lot release decision.

Spore Strips Only

This test is appropriate for devices sterilized by steam or ETO in a validated cycle. Generally 10 to 20 spore strips are used to monitor a cycle.  Spore strips are cultured in SCDM and then usually incubated for 7 days. Shorter incubation times can be validated.

Product with Spore Strips

This test is generally used for fractional and half cycles in ethylene oxide and steam sterilization validations. Spore strips are normally cultured in SCDM and incubated for at least 7 days. Products are usually tested in SCDM and FTM and incubated for 14 days.

Product Only – Direct Transfer or Fluid Path

Entire devices or portions of devices are rinsed with or submerged in Soybean Casein Digest Medium and Fluid Thioglycolate Medium. Usually 40 product samples are required, unless a product is large such that it can be divided to provide for each medium type, in which case 20 samples are required. Incubation time is 14 days.

Inoculated Product

For this test, product samples which have been inoculated with a microorganism that is resistant to the sterilization process are used as biological indicators. This test is used most frequently to verify steam or EtO penetration into an area of a medical device that is too small to be monitored with a spore strip.  It is often used as part of the sterilization validation of a reusable medical device. Ten or more samples are usually required. Incubation time is 7 days.

Microbial Environmental Monitoring

Medical device manufacturers use microbial environmental monitoring programs to evaluate the effectiveness of cleaning and disinfection procedures and to assess the overall microbial cleanliness of their manufacturing environment. An effective program to control microorganism levels in the manufacturing environment is essential to minimize the bioburden on the medical device being manufactured and reduce potential for bioburden spikes. Spikes in the bioburden of finished medical devices can cause a reduction in the sterility assurance level for the product.

Air and surface samples are taken during routine production operations to obtain a microbiological profile of the manufacturing environment.  Observation of work practices are made during the survey. Test data and other information are evaluated to determine what actions can be taken to reduce or stabilize the bioburden of the medical device. Once an intensive survey has been conducted and strategic sampling locations are determined, samples can be taken by the manufacturer’s personnel on a regular schedule. Pacific BioLabs can provide the necessary supplies for microbiological sampling. Exposed materials are returned to Pacific BioLabs for enumeration and reporting.

If any major changes are made at the facility or in the manufacturing process, or if product bioburden levels increase significantly, a re-evaluation of environmental conditions should be conducted. For additional information about environmental monitoring, refer to USP general chapter <1116> Microbiological Evaluation of Clean Rooms and other Controlled Environments or PDA TR 13 (revised 2001) Fundamentals of a Microbiological Environmental Monitoring Program.

Pacific BioLabs works with many of its clients to establish and maintain cost-effective programs to meet FDA requirements for monitoring the microbial cleanliness of their manufacturing environments and assessing the efficacy of production area disinfection procedures. Our microbiologists can train quality assurance and manufacturing personnel to collect microbial environmental samples. Following is an overview of aspects of a microbial environmental monitoring program. Please call the Microbiology/Sterility Assurance departments at any of our facilities to order supplies or for more information.

Microbial Environmental Monitoring Plan for Production Areas

    1. Review manufacturing procedures
      1. movement of materials
      2. personnel practices
      3. cleaning and disinfection procedures
    2. Visit production and packaging areas. Observe manufacturing process.
    3. Evaluate possible sources of microorganisms and potential to impact product bioburden.
      1. water and ancillary fluid used in production
      2. HVAC systems
      3. manufacturing equipment
      4. manufacturing personnel
    4. Formulate sampling plan.
      1. prepare a production area schematic
      2. identify sites for air and surface samples
      3. determine if water or other process fluid sampling is required
    5. Collect environmental samples.
      1. identify each with a site code and the date and time collected
    6. Ship samples for next day delivery to Pacific BioLabs.
    7. Pacific BioLabs incubates samples at temperatures appropriate for the various environmental samples.
    8. Following incubation, bacteria and mold colonies are enumerated.
      1. microorganisms will be characterized or identified if desired
    9. Results are reported to client.
    10. Results are evaluated by a Pacific BioLabs microbiology manager and the client.
    11. Appropriate follow-up action is recommended.

Materials used for Microbial Environmental Monitoring

BiotestTM Air Sampler: A mechanical instrument which pulls in a preset volume of air, impacting microorganisms onto a strip filled with a nutrient agar.

Contact Plate: A petri dish with an elevated convex surface of nutrient agar. It is used for taking samples of flat surfaces. The lid of the dish is removed. The agar surface is pressed lightly against the surface to be sampled. The lid is then immediately replaced on the dish.

Fallout PlateA  petri dish containing a nutrient agar. It is used for semi-quantitative air sampling. The dish is placed in the desired location, the lid is removed and the agar is exposed for a defined amount of time, usually for 30 minutes to 2 hours. Organisms falling from the air settle on the surface of the agar.

DE Neutralizing AgarA neutralizing agar formulated with Tween 80 (a surfactant), lecithin (a general purpose neutralizer), sodium thiosulfate and sodium thioglycollate. This combination is used to neutralize chlorine, glutaraldehyde, iodophor, phenolic and quat based disinfectants.This agar is used to culture many types of bacteria and some fungi.

Letheen AgarA neutralizing agar formulated with Tween 80 and lecithin. This combination is used to neutralize alcohol, phenolic and quaternary ammonium chloride based disinfectants.

Rose Bengal Agar: A neutral pH agar which contains stain that inhibits bacterial growth. It is used to select for yeast and molds.
Sabouraud Dextrose Agar: A high sugar, low pH agar used to select for yeast and molds.

Sodium Thiosulfate: A chemical added to agar to neutralize halogen based disinfectants, such as bleach.

Sterile Buffer Solution with SwabThe buffer solution is supplied in a 10 mL screw cap test tube. The sterile swab is provided in a paper pouch. Swabs are used for taking samples of non-flat surfaces. The swab is moistened by dipping it in the sterile buffer solution. The surface to be sampled is swabbed. The tip of the swab is then cut or broken into the test tube of solution and the cap is replaced on the test tube.

Tryptic Soy AgarA general purpose nutrient agar used to culture many bacteria and some fungi.

Microbial Environmental Services and Supplies

    1. Microbiological Environmental Survey
    2. Microbial Samples Enumeration and Report: Microbial Samples Enumeration and Report:
      1. Fallout Plate Count
      2. Contact Plate Count
      3. Biotest Air Sampler Strip Count
      4. Buffer Solution – Aerobic Count (Membrane Filtration)
    3. Microbial Environmental Monitoring Supplies
      1. Fallout Plate, 100 mm, Tryptic Soy Agar (TSA)
      2. Fallout Plate, 100 mm, Sabouraud Dextrose Agar (SDA)
      3. Contact Plate, DE Neutralizing Agar
      4. Contact Plate, Tryptic Soy Agar (TSA)
      5. Contact Plate, Sabouraud Dextrose Agar (SDA)
      6. Biotest Air Sampler Strip, Tryptic Soy Agar (TSA)
      7. Biotest Air Sampler Strip, Sabouraud Dextrose Agar (SDA) or Rose Bengal Agar
      8. Biotest Centrifugal Air Sampler – Daily Rental
      9. Sterile Buffer Solution with Dacron Swab
      10. Sterile Specimen Cups, Screw Cap, 120 m

Package Intergrity Testing

Package integrity tests are used to detect packaging problems that could adversely affect the sterility of a medical device. Sterile products are subjected to an environmental stress intended to simulate extreme conditions that a product might encounter in shipping or storage. The product packaging is then subjected to microbial challenge or dye penetration testing to determine if it has retained its properties as a microbial barrier. Thirteen samples are recommended for this test.

  1. Package Integrity by Microbial Challenge (13 products/test station are recommended)
  2. Package Integrity by Dye Penetration (13 products/test station are recommended)

Bacterial Endotoxins (LAL) Test

A pyrogen is the product of the action of heat on an organic substance and, in medical terms, is frequently described as a fever producing substance. The most potent pyrogens originate from gram negative bacteria, which are common water-borne organisms. Although not entirely accurate, the terms pyrogen and endotoxin are often used interchangeably.

Detection of bacterial endotoxin contamination is essential to insure the safety of certain medical devices. The Bacterial Endotoxins Test using Limulus Amebocyte Lysate (LAL) is recommended for the detection of endotoxins in medical devices. Any product that is labeled as nonpyrogenic must be tested to verify that claim. Medical devices with bloodstream or cerebrospinal fluid contact must also be tested for the presence of bacterial endotoxins.

Whether or not a device is considered pyrogenic is based on the amount of endotoxin the device contains in correlation to the accepted human tolerance of 5 endotoxin units (EU) per kilogram of body weight. Nonpyrogenic water is used to extract medical devices. Historically, the USP rabbit pyrogen test was used to test the extract. It specified a 40 mL extract per device with a 10 mL/kg injection volume. This is equivalent to 40 mL tested against 0.5 EU/mL LAL reagent sensitivity, and an allowable limit of 20 EU per device.

The LAL test is usually performed on a composite of the extracts of 10 samples. It is possible that one device of the composite could contain > 20 EU, when others in the composite would contain < 20 EU, and the composite test would pass. The most endotoxin one device could contain, if all others in a 10 sample composite contained zero endotoxin, would be 200 EU. This is still below the human tolerance of 350 EU, based on an average human body weight of 70 kg (70 kg x 5 EU/kg = 350 EU). In a composite test, 20 EU is the average endotoxin limit for most medical devices. However, the limit for the devices which contact cerebrospinal fluid is 2.15 EU per device. The maximum allowable extraction volume is calculated to insure that the average endotoxin burden per device in the LAL test does not exceed these limits.

FDA has published guidelines outlining validation procedures for endotoxin testing of finished products using the LAL test. This document is called Guideline on the Validation of the Limulus Amebocyte Lysate Test for Human and Animal Parenteral Drugs, Biological Products and Medical Devices, December 1987. A more current AAMI reference document is ANSI/AAMI ST72 Bacterial endotoxins – Test methodologies, routine monitoring, and alternatives to batch testing, 2011. For medical devices, the following sampling strategy is recommended for the LAL test: 2 samples for lot sizes less than 30, 3 samples for lot sizes 30-100, and 3% of the lot not to exceed 10 samples for lot sizes equal to or greater than 101. One test is performed on a composite of the test samples. Because water is a source of pyrogens, it is important to routinely monitor water systems using the bacterial endotoxins test.  For process water samples, the amount of sample required is 10 mL.

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