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USP 81 Turbidimetric Testing For Antimicrobial Medical Products

What is microbiology testing?

Microbiology testing identifies the presence and the type of microbes in a manufacturing environment, medical product, or medical device. Specific regulatory testing for microbiology may include assays such as microbial enumeration testing, particulate analysis, yeast analysis, antifungal activity assessment, growth promotion testing, microbial limits testing, zone of inhibition testing, water analysis, bacteriostasis/fungistasis testing, bacteria identification, yeast identification, fungi identification, gram-negative staining, product inoculations, biological indicator tests, and BI incubation time reduction studies. Microbiology tests are governed by USP 61 and USP 62.

Microbiology testing often requires sample collection from a product, a surface, a water source, or the air. Once samples are obtained, various microbiology testing can be performed. Below are links to articles related to multiple microbiology tests and testing methods. Firstly, further details on USP 788 particulate matter testing can be found here. Microbial characterization methods can be found here, whereas the top techniques for microbiology examination are here. Lastly, information on how to record and evaluate microbiology data is here.

What is zone of inhibition testing, and what medical products need zone of inhibition testing?

An antimicrobial agent’s potency can be proven by its inhibitory effect on microorganisms. Antimicrobial growth reduction is often assayed using a microbiological zone of inhibition test, as chemical methods can be unreliable. The exception is liquids, gels, and ointments containing preservatives. These products should be examined using preservative efficacy testing (PET). USP 81 specifies two methods for zone of inhibition testing: the cylinder-plate (or plate) assay and the turbidimetric (or tube) assay. Table 1 (reproduced from USP 81) lists which antibiotics are assessed with cylinder-plate inhibition testing vs. a turbidimetric assay.

Table of antimicrobial Inhibition Assay Preferences By Antibiotic
Table 1. Antimicrobial Inhibition Assay Preferences By Antibiotic

How is a turbidimetric test performed (USP 81 tube method)?

The turbidimetric assay evaluates how well a substance or extract inhibits the growth of a microbe in a microbial growth medium. Antibiotic stock solutions are prepared using USP reference standards as indicated in Table 2 below (reproduced Table 7 of USP 81). When the assay is performed, the antibiotic stock is serially diluted in a ratio of 1:1.25. The median dilution should have the concentration level of S3 in Table 2. For sample solutions, unknown potency is determined by unit weight or volume. A sample stock solution is prepared through extraction in the unit weight (or volume) desired. The sample stock is diluted to the S3 concentration in Table 2.

Table of turbidimetric Antibiotic Stock Solution & Dilution Information
Table 2. Turbidimetric Antibiotic Stock Solution & Dilution Information

Stock solutions of the test microorganism (typically Enterococcus hirae (ATCC 10541) for gramicidin assays or Staphylococcus aureus (ATCC 9144) are grown in a liquid medium (not agar). Sterile saline is used to suspend the microbial stock solutions and plate them on agar plates or the surface of a 250 mL Roux bottle under the conditions specified in Table 3 (reproduced USP 81 Table 8).

Table of turbidimetric Test Microorganism By Antibiotic Type
Table 3. Turbidimetric Test Microorganism By Antibiotic Type

After incubation, test organisms are harvested and quantified for use in the turbidimetric assay as inoculum. Grow harvested microorganisms with S3 concentrations of antibiotic stock solution. If absorbance levels as indicated in Table 4 below are reached, the turbidimetric assay is verified and ready for unknown test samples.

Table of turbidimetric Assay Antibiotic Absorbance
Table 4. Turbidimetric Assay Antibiotic Absorbance

Once the test organism concentration and activity with the antibiotic stock are confirmed, sterile glass or plastic test tubes (at least 3 tubes for each sample solution and the antibiotic dilutions) can be prepared in a single rack. Prepare five test levels (via serial dilution) of the antibiotic standard and a single test amount of the sample solution that correlates to the antibiotic’s median dilution value. Use Table 5 (USP 81 Table 10) below to determine the volume of microbial inoculum and volume of sample or antibiotic to add to each of the prepared test tubes. The test diluent is used as a negative control. Filled tubes are then incubated at the times and temperatures desired for the experiment. After incubation, organism growth is inhibited by adding 0.5 mL of dilute formaldehyde to each tube, unless Tylosin is used. Heat inhibition is used for Tylosin instead.

Table of antibiotic Inoculum & Dilution Volumes
Table 5. Antibiotic Inoculum & Dilution Volumes

How are turbidimetric test results assessed?

Each tube with USP reference stock dilutions, control, or sample solution is assessed with a spectrophotometer set to 580 or 530 nanometers. The measured absorbances are used to create a standard curve and estimate the potency of the antimicrobial sample compared to the USP reference standard. A standard curve is created by plotting the corrected absorbance measurements versus the log of the standard concentration values. Calculate the standard curve line equation by performing a standard unweighted linear regression (either with nature log or with base 10 log). The minimum for an accurate percentage coefficient of determination is not less than 95%. To calculate the potency of the product sample, take the antilog and multiply the result by any applicable dilution factor. Potency can also be expressed as a percentage of the reference standard concentration value.

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Summary

Overall, zone of inhibition testing is a type of microbiology test that assesses the potency of antimicrobial agents. These antimicrobial agents may be assessed alone, combined with a material, or as a product coating. USP 81 turbidimetric zone of inhibition testing is described herein. Turbidimetric testing uses a liquid antibiotic standard and suspended microorganism standard grown together to test the antimicrobial activity of a sample. This test is performed in tubes, and the antimicrobial activity of a sample is assessed based on the absorbance of the antimicrobial solution after exposure to live microbes for a set time. Preservative efficacy testing (PET) may be needed instead of a turbidimetric or cylindrical zone of inhibition testing to verify the potency of antimicrobial preservatives contained within a liquid, gel, or ointment-based cosmetics or medical products. All in all, ensure you choose a contract testing organization that can support you with appropriate microbiology, preservative efficacy, or zone of inhibition testing for your unique cosmetic or medical device needs.

MycoScience is a contract manufacturing organization specializing in sterile syringe and vial filling for parenteral products. MycoScience also offers testing services, including Preservative Efficacy Testing/Suitability Testing, Bioburden Testing, Microbial Aerosol Challenge Testing, Cytotoxicity Testing, Cleaning Validations, Accelerated Aging, Microbiology Testing, EO Residual Testing, Bacterial Endotoxin Testing, Package Integrity Testing, Sterilization Validations & Environmental Monitoring services medical devices and allied industries. MycoScience is an ISO 13485 certified facility.

References

Michael J. Akers. Sterile Drug Products Formulation, Packaging, Manufacture, and Quality. Drugs and the Pharmaceutical Sciences. Informa Healthcare. 2010.

United States Pharmacopeial Convention. <51> Antimicrobial Effectiveness Testing. Rockville, MD, USA. 2021. (USPC <51>).

United States Pharmacopeial Convention. <81> Antibiotics- Microbial Assays. Rockville, MD, USA. 2021. (USP<81>).

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