Sterility Assurance & Microbiology

USP Sterility Testing:

Sterility testing has been used to determine the acceptability of products labeled “sterile.” The USP now has a single test using finished products. This test is meant to be the reference procedure for determining sterility of a finished product lot. The test options listed below are described in the USP. These tests include:

Test Method Incubation Time (days)
Product Immersion 14
Membrane Filtration (open filter) 14
Membrane Filtration (closed Steritest) 14

Bacteriostasis/Fungistasis:

All sterility test methods shall be validated using this test method. The USP states that prior to conducting a sterility test, the level of bacteriostatic and/or fungistatis activity shall be determined. Validation is performed by adding selected bacteria and fungi to the challenge test container with product to demonstrate that low numbers (<100 CFUs) of test or challenge organism can be detected. The organism chosen should be based on media types used in the validation study e.g. three organisms use per the ISO standards and six for USP.

Bioburden Testing:

At the time product design and validation studies are conducted, product bioburden testing should be performed to estimate the total viable number of organisms on the product. The number of samples are selected e.g. 10 finished non-sterile units and bioburden testing is performed to determine the total viable population of organism of a product prior to sterilization. The methods offered are listed below

  • Aerobic
  • Aerobic with extended incubation
  • Aerobic plus spores
  • Aerobic plus spore, and fungi
  • Aerobic plus spore, fungi, and anaerobes

Bioburden data may be used to establish process requirements, calculate sterilization dose during validation, and determine initial and ongoing manufacturing process control.

Sterilization Validation:

FDA and regulatory bodies require that all manufactures sterilization process be validated and controlled. The design and validation of a sterilization process will form the foundation of a Sterility Assurance Program. NAMSA offers programs for process design and Installation Qualifications, Operational Qualification, and Biological Performance Qualifications to assure the effectiveness of a validated sterilization process. All phases of a program are closely managed by our experienced NAMSA Consulting Service personnel. These programs are conducted in accordance with the appropriate AAMI/ANSI/ISO?USP Guidelines e.g. ISO 11137 (Radiation) and ISO 11135 (EO). The testing associated with these studies includes:

  • Bioburden Recovery Validation
  • Bioburden Testing
  • Bacteriostasis/Fungistasis Testing
  • Sterility Testing
  • EO Residual Testing
  • LAL Testing
  • Biological Performance Testing
  • Packaging Shelf Life Validation

Biological Indicator Performance Testing:

Studies may be designed to demonstrate biological indicator survival /kill of Inoculated product or spore carriers to determine the resistance value under controlled specified cycle conditions. These tests are performed in resistometers signed to assure controlled sterilization conditions. The vessels performance meets AAMI/ISO standards where applicable. The following performance tests are available:

  • Resistometer Studies
  • Survival Kill Test (EO,Steam,Dry Heat)
  • D-value Studies (EO,Steam,Dry Heat)
  • Validation of Reduced Biological Indicator Incubation Time
  • Population Verification or species: Test is performed in accordance with USP to determine the viable count of suspension and spore carriers. These tests may be performed in conjunction with sterilization validation studies.

Product Inoculation:

This procedure involves inoculating bacillus spores directly to the product. The procedure can be conducted in conjunction with sterilization validation studies and pilot D-value studies. In addition to product inoculation NAMSA can place Biological carriers with spores embedded inside devices.

Reusable Device Studies – Sterilization, Cleaning and Disinfection

Manufacturers of reusable medical devices are responsible for supporting their product claims for reuse. This can be accomplished by developing instruction for reprocessing the device and conducting tests that validate the instructions. These studies are based on the guidelines of ANSI/AAMI/ISO 17665-1:2006, AAMI TIR12:2010, ISO 17664:2004 and AAMI TIR30:2003

Test Code Test Description
M/S Product Inoculation for reusable / reprocessed devices
M/S BI Placement
M­/S Cleaning Validation
M/S Disinfection Validation – High, Intermediate and low level
M/S Functionality of reusable / reprocessed devices
M/S Sterilization Validation
M/S Repeat Sterilization for reprocessed devices

Growth Promotion Testing of Culture Media

Growth Promotion Testing is performed on culture media to ensure that the media will support the growth of microorganisms prior to use in microbiology assays. This test is based on USP guidelines and can be performed on solid or liquid media.

Test Code Test Description
MG044-000 Growth Promotion Testing of Culture Media – Liquid, Qualitative, Per Organism / Per Lot of Media)
MG044-001 Growth Promotion testing of Culture Media – Plated, Quantitative, Per Organism / Per Lot of Media)

Antimicrobial Tests

Manufacturers often incorporate antimicrobial agents into multiple dose solutions, medical devices and general textiles to inhibit or kill microorganisms. Manufacturers of these antimicrobial products test the effectiveness of the antimicrobials to support label claims, for submission to FDA, or for market advantage. The choice of the appropriate test method is often influenced by the antimicrobial agent used in the product and may warrant consultation prior to choosing the most appropriate test method.

AATCC TM 147 is a qualitative method used to determine antibacterial activity on treated textiles. A 25mm x 50 mm sample is laid transversely across five parallel streaks of an 18-24 hours broth culture of each organism Staphylococcus aureus and Klebsiella pneumoniae. Other suitable species can also be used, depending on the intended end-use of the product. After incubation, plates are examined for interruption of growth along the streaks of inoculum and beneath the test sample and for a clear zone of inhibition measured in millimeters beyond the test sample edge.

Test Code Test Description
MG103-000 Antimicrobial Finishes on Fabrics, AATCC Test Method 147 – Parallel Streak
MG103-001 Each Additional Sample
MG103-002 Each Additional Organism – First Sample
MG103-003 Each Additional Organism – Additional Sample

 

AATCC TM 100 is a quantitative method to evaluate the bactericidal activity of the antimicrobial agent. A 48mm disc of the test sample is inoculated with a 1-2 x 105 CFU of Staphylococcus aureus and Klebsiella pneumoniae. Plate counts are performed at zero time of inoculation and after 24 hours of incubation. The percent of bacterial reduction is determined from the counts taken at each time point. Other time points and organisms can also be used depending on the intended end-use of the product.

Test Code Test Description
MG102-000 Antimicrobial Finishes on Fabrics, AATCC Test Method 100
MG102-001 Each Additional Sample
MG102-002 Each Additional Organism – First Sample
MG102-003 Each Additional Organism – Additional Sample
MG102-004 Antimicrobial Finishes on Fabrics, AATCC Test Method 100 – Fungal
MG102-005 Each Additional Sample

 

AATCC TM 174 is a method that evaluates bacteriostatic (Part I), bacteriocidal (Part II and fungistatic (Part III) activity of carpet treated with antimicrobial agents. Part I and Part III are qualitative test methods. Part 1 evaluates antimicrobial activity using Staphylococcus aureus and Klebsiella pneumoniae. Results are reported as inhibition of growth under the sample and/or zone of inhibition around the sample. Part III evaluates antifungal activity using Aspergillus niger. Results are reported as growth or no growth on the test article as well as any growth free zone around the sample. Part II provides results that express the amount of bacteria killed during a 24 hour contact period. A 48mm disc of the test sample is inoculated with a 1-2 x 105 CFU of Staphylococcus aureus and Klebsiella pneumoniae. Plate counts are performed at zero time after inoculation and after 24 hours of incubation. The percent of bacterial reduction is determined from the counts taken at each time point. Other time points and organisms for all three assays can also be used depending on the intended end-use of the product.

Test Code Test Description
Antimicrobial Activity Assessment of Carpets, AATCC Test Method 174
MG104-000 Part I
MG104-001 Additional Sample
MG105-000 Part II
MG105-001 Additional Sample
MG106-000 Part III
MG106-001 Additional Sample

 

ASTM E 2149 is a quantitative test for non-leaching antimicrobial technologies and provides bactericidal results. An example of a test organism used in this method would be Klebsiella pneumoniae. Counts are obtained at zero and 1 hour of exposure and percent reduction is determined. Other time points and organisms for can also be used depending on the intended end-use of the product.

Test Code Test Description
MG120-000 ASTM E2149 – Standard Test Method for Determining the Antimicrobial Activity of
Immobilized Antimicrobial AgentsUnder Dynamic Contact Conditions
MG120-001 Each Additional Time Point

 

Kirby-Bauer Antimicrobial Susceptibility Test is qualitative tested used for leaching antimicrobials or antibiotic sensitivity testing. The typical organisms used in the standard test are Staphylococcus aureus and Escherichia coli. Mueller-Hinton Agar plates are inoculated individually in a lawn pattern with each organism and the test article is placed on the inoculated agar surface. Plates are incubated for 18-24 hours. Results are presented by inhibition in the contact area and zone of inhibition around the test article in millimeters. Other organisms can also be used depending on the intended end-use of the product.

Test Code Test Description
MG109-000 Kirby-Bauer Susceptibility Test
MG109-001 Each Additional Sample
MG109-002 Each Additional Organism – First Sample
MG109-003 Each Additional Organism – Additional Sample

 

AATCC Test Method 30, Part IV is a qualitative test method. The test article is inoculated with 3 different fungi and incubated in humid conditions for 28 days. After incubation the test article is examined for fungal growth.

Test Code Test Description
MG101-000 AATCC Test Method 30, Part IV – 3 Molds
MG101-001 Each Additional Sample

 

AATCC Test Method 30, Part III is a qualitative test method. The test article is placed on an agar surface that has been seeded with Aspergillus niger. Once the test article is placed on the agar surface the sample is also inoculated with Aspergillus niger. Plates are incubated for up to 14 days based on the agar used. Results are reported as no growth, microscopic growth or macroscopic growth. Any growth free zone around the test sample is also reported.

Test Code Test Description
MG100-000 AATCC Test Method 30, Part III – Mildew and Rot Resistance of Textile Material
MG100-001 Each Additional Sample

 

Minimal inhibitory concentration is conducted to determine the lowest concentration required by an antibiotic or antimicrobial that will inhibit more than 99% of the bacterial population.

Test Code Test Description
MAMSF Minimum Inhibitory Concentration

 

New York State 63 Test is a quantitative method that measures bactericidal activity of antimicrobial treated hydrophobic test articles. Two organisms, Staphylococcus aureus and Klebsiella pneumonia are used in the method. Samples are challenged with the test organisms and after 24 hour exposure the percent kill is calculated using surviving population on untreated versus treated samples

Test Code Test Description
MG078-000 New York State 63 Test

 

ASTM G-21 was developed by ASTM to determine whether polymeric material resists fungal attack. A fungal spore suspension using five different fungi at a population of 8.0x 105 to 1.2 x106 CFU/mL is sprayed over the test sample. The samples are then placed in humidified incubation. Growth of fungi is recorded on day 7, 14, 21, and 28 days and rated on a scale of 0 (no growth) to 4 (heavy growth).

Test Code Test Description
MG107-000 ASTM Test G-21 Fungal Resistance
MG107-001 Each Additional Sample

 

USP <51> Preservative Effectiveness Test was designed to verify the efficacy of a preservative system to reduce bacterial populations and prevent the growth of fungi in multiple does injectables and solutions. Three bacteria, one yeast and one mold for used for inoculation. This method is validated using USP <1227> Microbial Recovery from Pharmacopeial Articles.

Test Code Test Description
MG018-000 Preservative Effectiveness Test USP 51- 5 Organisms, 1 Replicate
MG018-001 Duplicates
MG018-002 14 Day Rechallenge
MG117-000 USP <1227> Validation of Microbial Recovery from Pharmacopeial Articles

 

ASTM E2180 is a quantitative test method to evaluate antimicrobial effectiveness of antimicrobials that are incorporated or bound on hydrophobic surfaces. Staphylococcus aureus, and Klebsiella pneumoniae or Pseudomonas aeruginosa are the organisms used in this method. Untreated and treated samples are inoculated with the test organisms and incubated for 24 hours. After incubation the surviving population is counted and percent reduction of organisms is calculated from the untreated control samples. Other organisms can also be used depending on the intended end-use of the product.

Test Code Test Description
MAMSF ASTM E 2180 Determining the Activity of Incorporated Antimicrobial Agent(s) in Polymeric or Hydrophobic Materials

 

ISO 22196 is a quantitative test method to determine antimicrobial effectiveness on plastic or non-absorbent surfaces. Staphylococcus aureus and Escherichia coli are the organisms used in this method. Other organisms can also be used depending on the intended end-use of the product. Log reduction is determined from inoculated treated and untreated test articles after 24 hours of exposure.

Test Code Test Description
MAMSF ISO 22196 Measurement of Antibacterial Activity on Plastic Surfaces

 

ISO 20743 is a quantitative test method to determine the antimicrobial effectiveness of antimicrobial treated products. Staphylococcus aureus and Klebsiella pneumoniae are the organisms used in this method. Other organisms can also be used depending on the intended end-use of the product. Log reduction is determined from inoculated treated and untreated test articles after 24 hours of exposure.

Test Code Test Description
MAMSF ISO 20743 Determination of Antibacterial Activity of Antibacterial Finished Products

Bacterial Endotoxins Test USP <85

Endotoxins are substances, produced by Gram negative bacteria, which can induce a fever response in humans. The Bacterial Endotoxins Test (BET), also known as the Limulus Amebocyte Lysate (LAL) Test detects and quantifies endotoxins present in a test article. The BET is used as a release test for medical devices and parenteral solutions labeled non-pyrogenic, and to evaluate cleaning processes intended to reduce pyrogen potential. Acceptance limits for each test article are based upon its clinical end use.

Validation of the test method is conducted by testing multiple product lots, evaluating test article, negative control and endotoxin- spiked product positive controls (PPC)

Three test techniques are available, each employing a lysate derived from the circulating cells of the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus).

Kinetic chromogenic (quantitative method) – In this method, the endotoxin in a preparation of the test article, reacts with the test reagents to release a chromophore. The rate of color change in the solution is spectrophotometrically evaluated. Results are interpolated against a standard curve.

Kinetic turbidimetric (quantitative method) – In this technique, endotoxin in the test article preparation reacts with test reagents, resulting in turbidity. The concentration of endotoxin in the test article is measured spectrophotometrically, identifying the time required to reach a predetermined level of turbidity. Results are interpolated against a standard curve.

Gel Clot (semi-quantitative method) –In this test, endotoxins are detected based upon the clotting of lysate in the presence of endotoxin. The reaction of the test article preparation is compared to a series of standard preparations.

Associated Test Codes:

Test Code Test Description
V0027_300 USP Limulus Amebocyte Lysate Testing – Kinetic Chromogenic
V0027_302 USP Limulus Amebocyte Lysate Testing – Kinetic Chromogenic, Concurrent Dilution
V0027_500 USP Limulus Amebocyte Lysate Testing – Kinetic Chromogenic, Validation
V0707_000 USP (LAL) Test – Kinetic Turbidimetric Method (Pos, Neg, Inhib)
V0707_001 USP (LAL) Test – Kinetic Turbidimetric Method (Pos, Neg, Inhib) Concurrent Dilution
V0707_002 Validation of USP (LAL) Test – Kinetic Turbidimetric Method as an End Product Endotoxin
V0184_000 USP (LAL) Endotoxin Testing – Gel Clot Method
V0184_200 USP (LAL) Endotoxin Testing – Gel Clot Method – Product Validation (3 Lots)
V0184-201 USP (LAL) Endotoxin Testing – Gel Clot Method – Product Validation (Single Lot)

Microbial Characterization and Identification:

In order to understand the microbial flora associated with a medical device or pharmaceutical, qualitative assessment of isolated organisms is employed. These assessments allow the manufacturer to see shifts in the types of organisms isolated from manufacturing environments, product bioburden, and sterility tests.

Microbial Characterization: The characterization process provides a preliminary microscopic and macroscopic evaluation of microorganism isolates. The organism is characterized by it’s Gram Stain reaction. Additionally, colony characteristics such as size, elevation, pigmentation, texture, margin and form are documented.

Spore Stain: In the spore stain process, rod-shaped bacilli are evaluated for presence or absence of endospores. Organisms with endospores display higher resistance to sterilants and disinfectants.

Bacterial Identification –Automated identification equipment is used to develop a ‘fingerprint” of the organism. The fingerprint is compared against a library of previously identified organisms, to determine the identity (genus and species) of organism.

Fungal IdentificationMacroscopic and microscopic characteristics of a mold culture are used to determine the identify (genus or genus/species) of the organism.

Associated Test Codes:

Test Code Test Description
MG003_000 Gram Stain and Colony Morphology
MG004_000 Spore Stain
MG011_001 Identification of Microorganisms – Aerobic Bacteria/Yeast
MG048_000 ID of Micro-Organisms – Mold

Microbial Limits:

Microbiological Examination of Nonsterile Products (USP)

This testing, formerly known as Microbial Limits Testing, determines determine if a test article meets established specifications for microbiological quality per recommendations outlined in USP <1111> Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use. Acceptance limits are based upon the clinical end use of the test article.

USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests Enumeration is made, under aerobic conditions of mesophilic bacteria and fungi. The results are expressed as the TAMC (total aerobic microbial count) and TYMC (total yeasts and mold count). Testing also includes preliminary methods validation , determining the suitability of the test methods to recover mesophilic aerobic organisms.in the presence of the test article.

USP <62> Microbiological Examinatino of Nonsterile Products: Tests for Specified Microorganisms – In these test procedures, the test article is evaluated for the presence or absence of objectionable organisms such as Bile Tolerant Gram Negative organisms, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Clostridia, Salmonella and Candida albicans. The combination of organisms for which the test article is screened is selected based upon its end use. Prior to routine screening of samples per this method, the microbial recovery method is validated to verify that the target organism(s) can be isolated from the test article preparation. Should suspect objectionable organisms be isolated from the test article, bacterial identification techniques are used to confirm the presence/absence of the organism.

Associated Test Codes:

Test Code Test Description
MG017_000 Microbial Limits USP – Prep
MG017_000 Microbial Limits USP – Screening
MG010_001 Identification of Microorganisms – Aerobic Bacteria/Yeast

Environmental Monitoring (EM) Studies

The microbiological quality of a manufacturing environment affects the resulting bioburden of a product produced in that environment. Microbial monitoring is conducted in medical device and pharmaceutical cleanrooms and controlled environments to verify if required microbial cleanliness levels are being maintained. The number of microorganisms isolated from air or surfaces are enumerated.

Further evaluation of organism isolated from EM studies, e.g. microorganism identification, can be conducted to determine which organisms are present, and at what frequency.

Associated Test Codes:

Test Code Test Description
MG092_001 Environmental Monitoring of Viable Organisms