Bioburden, aerobic bacteria only

To determine the total number of viable microorganisms in or on a medical device, container or component after completion of all in-process steps before sterilization.

To act as an early warning system for possible production problems which could lead to inadequate sterilization or possible product recall.

To calculate the necessary dose for effective radiation sterilization, and to monitor product routinely to ensure adequate dosing.

Study Outline

DEFINITIONS:

Bioburden: Population of viable microorganisms on a product and/or package.

Bioburden estimate (theoretical bioburden): Value established for the number of microorganisms comprising the bioburden by applying to a viable count or presterilization count a factor compensating for the recovery efficiency.

Recovery efficiency: Measure of the ability of a specified technique to remove microorganisms from product.

Validation: Documented procedure for obtaining, recording and interpreting the results needed to show that a process will consistently yield a product complying with predetermined specifications. The validation of a technique for bioburden estimation consists of a series of investigations to determine the effectiveness and reproducibility of the test method.

I. TEST METHODS:

   A. Extraction Procedures
      1. Stomaching 
      2. Ultrasonicating
      3. Shaking 
      4. Flushing
      5. Other methods 

   B. Enumeration Procedures
      1. Membrane filtration 
      2. Pour plating
      3. Spread plates
      4. Most Probable Number (MPN) 
      5. Other methods

   C. Incubation Conditions
      1. Aerobic bacteria and spores:  30-35 °C for 2 to 5 days
      2. Fungi (mold and yeast):  20-25 °C for 5 to 7 days 
      3. Anaerobic bacteria:  30-35 °C for 3 to 5 days

BIOBURDEN DISCUSSION

All Manufacturers of medical devices should perform routine, random bioburden testing. The appropriate frequency and number of samples varies depending upon the sterility assurance level [SAL], type of product, type of sterilization used, environmental control, and the process controls of the manufacturer.

The AAMI Radiation Sterilization guideline (AAMI/ANSI/ISO 11137) mandates testing ten samples for bioburden with each quarterly audit. Products sterilized by radiation for shipment to many countries in Europe must be dosed based on the actual bioburden for that lot. Manufacturers pursuing the use of radiation sterilization for products for shipment to Japan must perform routine environmental and product bioburden tests and identify the isolates.

The type and frequency of bioburden tests performed on products validated using the AAMI Industrial Ethylene Oxide Sterilization Guideline (AAMI/ANSI/ISO 11135) will also vary with the method used in the initial validation. Products validated using the absolute bioburden method require frequent and significant bioburden testing. Even products validated with the BI overkill method should be tested periodically (at least quarterly) for bioburden.

Bioburden tests may include aerobic bacteria, spores, aerobic fungi, and anaerobes. Many factors enter into the choice of the type of tests most appropriate for your device. Generally, at least the aerobic bacteria and fungi should be counted.

Manufacturers who wish to validate product families instead of individual products for sterilization will find identification of isolates from bioburden tests not only helpful but necessary. The AAMI guidelines do not clearly describe validation of components as a family by using the master product concept. Blind classification of products into families based on materials of construction or area of production is inadequate for establishing a family. We believe that the assignment of a product to a family should be based on bioburden counts and types [genus and species or resistance to sterilization] as well.

BIOBURDEN IDENTIFICATION

We have added significant new technology to the laboratory to permit rapid and inexpensive identification of bioburden and environmental isolates to aid the manufacturers in making necessary decisions. The Biolog™ system permits us to identify even unusual isolates, using a computer based 95 chemical metabolic profile system. Unknown organisms are inoculated into the 95 wells of the Biolog™ system and the metabolic profile is entered into a computer. The computer then searches a large data base for a similar pattern. The system also permits us to recognize the same organism in the future, alerting us of a reoccurrence of an isolate. The MIDI system relies on qualitative and quantitative analysis of the fatty acid composition of organisms. The fatty acid profile provides a fingerprint of the organism. The organism to be identified is grown on controlled culture medium under controlled time and temperature of incubation before comparing the fatty acid compositions with the Sherlock Microbial Identification System (MIS) standard libraries. This system also permits us to recognize the same organism in the future, alerting us of a reoccurrence of an isolate.

EXTRACTION EFFICIENCY

Nelson Laboratories also performs extraction efficiency tests on bioburden samples. If you send ten or more samples at one time for bioburden tests, we will perform an extraction efficiency test at no charge. Extraction efficiency tests are performed by repeatedly extracting the same sample until the recovery count approaches or is zero. The number of organisms recovered in the initial extraction is divided by the total number from all extractions. Multiplying this result by 100 yields a percentage. This value is very important for radiation dose setting studies since low extraction efficiencies can result in verification failures.

You may choose to plot bioburden data in graphic format. Plotted data permits rapid and simple assessment of trends and also satisfies the AAMI Radiation Validation guideline for evaluation of bioburden data on a quarterly basis, and FDA's requirement for trend analysis data.

BIOBURDEN SPIKES

The definition of a bioburden spike is an individual result in a set of bioburden results that is two or more times greater than the average. Spikes are not necessarily an indication of quality problems and should not be automatically viewed as such. The ideal situation is to reduce or eliminate spikes as much as possible by operational and quality controls.

Some individuals have suggested and recommended the use of bioburden spikes in establishing or verifying sterilization doses. The use of bioburden spikes was originally incorporated in the ISO 13409 document as a standard practice in the validation of a dose, but this has since been revised. It is currently, or will soon be, under review by a working group of AAMI.

The decision to use a spike should be made only after careful evaluation of the current and historical bioburden data as well as other data pertinent to the sterilization and validation processes. Due to the diversity of products and the unique nature of bioburden testing and results, the data should always be evaluated by a microbiologist experienced in medical device testing, dose-setting methods, and regulatory requirements.

Sample Requirements

GENERAL SAMPLING STRATEGIES

SAMPLING:

The method of obtaining samples for either bioburden or sterility tests might influence the test results observed. The preferred method for evaluating products or processes is to obtain random samples. These samples may be selected from routine production which should include products produced at different times during the production of a single lot. The production lot selected to be sampled needs to be representative of typical processing and conditions. Samples may be obtained from in-process rejected materials if those materials have been subjected to the same processing conditions used for the remainder of the lot.

ASEPTIC:

Be as clean as possible when sampling. Samples for sterility testing should be double bagged before they are sent to be dosed. This provides an extra sterile barrier to take into the clean room and can cut down on the number of false positives.

RANDOM:

Random samples are pulled throughout the production lot, not at the beginning or the end of the production day. Random samples are taken from each area of the production room and not from one specific location. Random samples are pulled from various individuals and are not taken from one technician alone.

REPRESENTATIVE:

Units pulled for laboratory testing should represent each phase of the production process. If certain steps have been omitted, these units are not acceptable samples. Appropriate samples have been through each phase of production (including inspections, packaging, labeling, etc.) except for the sterilization phase.

REJECTED SAMPLES:

Rejected products can be used for laboratory testing if they have been through the same processes that normal units have been through. Rejected units which have been handled repeatedly by several persons, thrown onto the floor or picked out of trash cans are not acceptable samples. Appropriate rejected samples are units with discoloration, poor seals and joints, mis-fitting components, etc.

SIMULATED PRODUCT:

Some clients will produce special units for laboratory testing because of the expensive nature of the item. Once again, this practice is only acceptable if the simulated products go through the exact procedures as the regular units. Using specially made units instead of randomly selected ones, should be documented and justified.

Nelson Experts:

• Trent Teeples 801-290-7896, 800-826-2088

Sample Requirements

GENERAL SAMPLING STRATEGIES

SAMPLING:

The method of obtaining samples for either bioburden or sterility tests might influence the test results observed. The preferred method for evaluating products or processes is to obtain random samples. These samples may be selected from routine production which should include products produced at different times during the production of a single lot. The production lot selected to be sampled needs to be representative of typical processing and conditions. Samples may be obtained from in-process rejected materials if those materials have been subjected to the same processing conditions used for the remainder of the lot.

ASEPTIC:

Be as clean as possible when sampling. Samples for sterility testing should be double bagged before they are sent to be dosed. This provides an extra sterile barrier to take into the clean room and can cut down on the number of false positives.

RANDOM:

Random samples are pulled throughout the production lot, not at the beginning or the end of the production day. Random samples are taken from each area of the production room and not from one specific location. Random samples are pulled from various individuals and are not taken from one technician alone.

REPRESENTATIVE:

Units pulled for laboratory testing should represent each phase of the production process. If certain steps have been omitted, these units are not acceptable samples. Appropriate samples have been through each phase of production (including inspections, packaging, labeling, etc.) except for the sterilization phase.

REJECTED SAMPLES:

Rejected products can be used for laboratory testing if they have been through the same processes that normal units have been through. Rejected units which have been handled repeatedly by several persons, thrown onto the floor or picked out of trash cans are not acceptable samples. Appropriate rejected samples are units with discoloration, poor seals and joints, mis-fitting components, etc.

SIMULATED PRODUCT:

Some clients will produce special units for laboratory testing because of the expensive nature of the item. Once again, this practice is only acceptable if the simulated products go through the exact procedures as the regular units. Using specially made units instead of randomly selected ones, should be documented and justified.