Microbiology

Microbial Limit Testing (MLT) is essential for assuring patient and consumer safety in the manufacturing of pharmaceutical, healthcare, and cosmetic products. It determines the quantity of viable aerobic microorganisms, the absence of pathogens, and the presence of specific microbial species in a product. MLT can be evaluated at multiple phases of production, from raw materials to finished goods. The ultimate testing frequency depends on the type and intended use of the product. MLT should be performed by qualified personnel with experience in microbiology and related analytical methodologies for optimal results. The MLT technique consists of three phases: suitability testing, microbial enumeration (total aerobic microorganism count and total combined yeast and mold count), and screening for specified microorganisms. Suitability testing ensures that the tests can detect microorganisms in the product and do not contain antimicrobial properties that could lead to false­ negative results. Microbial enumeration assays determine the total number of aerobic organisms, yeast, and mold in a product, whereas the Screening Test for Specified Microorganisms requires an initial suitability analysis and, if necessary, the addition of a neutralizing agent.

Tests:

1. Examination of Non-Sterile Products: Microbial Enumeration Test (MET)
   • USP<61>
   • EP Chapter 2.6.12
   • BP Chapter 82
   • Japanese Pharmacopoeia XV 1st Supplement
      
2. Examination of Non-Sterile Products: Tests for Specified Microorganisms
• USP<62>
• EP Chapter 2.6.13
• BP Chapter 81
• Japanese Pharmacopoeia XV 1st Supplement

Antimicrobial Effectiveness Tests (AETs) are necessary for determining the efficacy of antimicrobial preservatives in pharmaceutical and cosmetic products in preventing contamination over time. These tests are conducted at various time intervals over the course of 28 days and subject products to microorganisms determined by specific guidance criteria or product relevance. These criteria are addressed in guidance documents such as U.S. Pharmacopeia, European Pharmacopeia 5.1.3, ISO 11930, and PCPC M-3 and M-4. Additionally, manufacturers may design custom assays to resolve particular contamination risks. Typically, neutralization validation is conducted as part of the assay, but manufacturers may occasionally perform the assay without it. Injections, multiple-dose containers, topical and oral dosage forms, and other dosage forms containing antimicrobial preservatives must be tested. The testing performed by Iskon Labs satisfies the requirements of USP General Chapter 51 and the European Pharmacopoeia, providing scientifically sound data through triplicate plating and numerous replicate testing.

Tests:

• USP 51: Antimicrobial Effectiveness Testing
• USP-EP

Water activity (aw) is a measurement of the energy status of water in a substance, signifying its structural or chemical bonding. Moisture content alone is a poor predictor of microbial growth, perishability, chemical reactivity, color, odor, flavor, texture, and shelf-life stability. Meters utilizing the dew-point chilled mirror method or the capacitance technique for volatile materials are utilized to measure water activity. Reducing water activity is essential for preventing microbial growth, enhancing the antimicrobial efficacy of preservative systems, and preventing chemical hydrolysis and crystallization in pharmaceutical active pharmaceutical ingredients (APIs). It also assists in determining the frequency of microbial testing in non-aqueous liquids, such as ointments, which may not support microbial growth due to their low water activity. USP (United States Pharmacopoeia) contains two essential chapters on water activity: USP 1112> Application of Water Activity Determination to Nonsterile Pharmaceutical Products and Chapter 922>. Water Recreation.

Tests:
There are two significant USP (United States Pharmacopoeia) chapters that examine water activity.

1. USP <1112> Application of Water Activity Determination to Nonsterile Pharmaceutical Products
2. Chapter <922> Water Activity

In order to identify and quantify microorganisms in water samples, microbiological water testing is essential. We offer specialized testing options to surmount obstacles in the identification of pathogenic species. The accurate and expeditious diagnosis of resident microorganisms in water and water sources is accomplished in the laboratory using protocols designed to produce consistent results. Methods for testing for microorganisms include Coliform and indicator organisms. Coliform is a reliable technique for identifying and quantifying pathogenic microorganisms in water. Coliform in potable water may indicate the presence of pathogenic bacteria or viruses, such as Klebsiella, Escherichia coli, and Enterobacter. The Environmental Protection Agency (EPA) determines the microbial limit for drinking water, as well as the legal limits for more than 90 contaminants in drinking water. States may establish and enforce their own standards for potable water if they are as stringent as the EPA's national standards. The Iskon Laboratories routinely test water samples from a variety of sources, including drinking water, culinary products, personal care products, environmental sources, and recreational sources.

Burkholderia cepacia is a group of Gram-negative, rod-shaped bacteria that pose a significant threat to the health of immunocompromised, mechanically ventilated, and underlying disease patients. These organisms can quickly colonize water systems, surfaces of manufacturing equipment, and non-sterile water-based products. In 2017, the FDA issued a public warning to drug manufacturers regarding the health risk posed by tainted products. Due to BCC's resistance to certain preservatives and antimicrobial agents and the absence of an official diagnostic protocol for detecting BCC, the risk is especially high. The FDA expects manufacturers of non-sterile products to establish procedures to prevent objectionable microorganism contamination, such as adequate quality of materials, sanitary design, maintenance, and cleansing of processing equipment, in order to prevent objectionable microorganism contamination. In addition, they need validated methods for testing finished products, testing in-process materials, investigating malfunctions, and reporting adverse events or quality issues.

The FDA now expects pharmaceutical companies to monitor their completed products for Burkholderia cepacia contamination. There is now an approved USP method for testing Burkholderia cepacia: USP<60> Microbiological Examination of Nonsterile Products-Tests for Burkholderia cepacia Complex.

Environmental monitoring is an essential microbiology practice for assessing and regulating microbial contamination in diverse environments. Product safety and quality are crucial in industries such as pharmaceuticals, food and beverage production, healthcare facilities, and cosmetics. Sampling and testing, contamination control, compliance with regulations, validation of cleansing procedures, root cause analysis, risk assessment and mitigation, and quality assurance are essential components of environmental monitoring. Environmental monitoring employs surface swabbing, air sampling, setting plates, biological indicators, and real-time monitoring systems, among other methods and techniques. By systematically monitoring the environment for microbial contamination, potential hazards can be identified and managed, resulting in enhanced product safety and consumer confidence.

The identification and classification of microorganisms to the species level is a crucial step in microbiology involving microbial identification. It is necessary in clinical and non-clinical settings and involves a variety of examinations and procedures. Among these are sample collection and isolation, macroscopic and microscopic examination, biochemical and physiological tests, serological tests, molecular techniques such as PCR, DNA sequencing, and ribosomal RNA analysis, and mass spectrometry techniques such as MALDI-TOF. Identification of microorganisms is indispensable for disease diagnosis, treatment, public health surveillance, food safety and quality control, environmental monitoring, pharmaceutical and biotechnology industries, and scientific research and studies. Accurate identification facilitates the selection of appropriate antibiotics, antifungals, or antivirals, enhances patient outcomes, and guarantees the safety and quality of food products.

Tests:

• USP 71
• ISO 11737
• USP 1113
• USP 61/62

Bioburden testing is an essential quality control procedure in numerous industries for determining the presence of viable microorganisms in samples and products. It is beneficial for product safety, quality control, process validation, and environmental monitoring. Membrane filtering, flow plate, spread plate, and Most Probable Number (MPN) methods are typical techniques. By detecting microbial concentrations within acceptable limits, these procedures help maintain product safety, quality, and regulatory compliance. Regular bioburden testing is required to prevent contamination-related problems and guarantee the efficacy of manufacturing processes. The choice of technique is determined by the nature of the sample and industry standards.

Tests:

• ANSI/AAMI/ISO 11737
• ANSI/AAMI/ISO 11137
• ISO 11135
• EN 13795

Products that purport to be sterile or free of viable microorganisms are sterile. The majority of these items are pharmaceuticals and medical devices. To confirm a claim of sterility and detect the presence of viable microorganisms that can cause infection or other health problems, sterility testing is required. Several compendia! Sterility Test protocols exist, such as USP <71>, EP 2.6.1, JP, and ANSI/AAMI/ISO 11737-2.

Typically, pharmaceutical products are examined via membrane filtration, whereas medical. devices are examined via direct immersion. Medical devices, such as titanium fasteners, IV tubing, needles, and other solid products, are frequently immersed directly. The samples are deposited directly into the required volume of growth medium, and the test is observed daily or on a consistent basis. Method for Testing Fertility Suitability (Bacteriostasis/Fungistasis) is required prior to routine sterility testing of products. The suitability procedure requires the addition of <100 CFU of organisms specified in the compendia to the sample/plus media and control media without sample. Routine Sterility Testing is performed based on suitability and is not modified unless the method or product formulation is altered.

Tests:

• USP <71>
• EP 2.6.1, JP
• ANSI/AAMI/ISO 11737-2

Endotoxins are a common pyrogen found in medical devices, and if undetected, they pose a significant risk to patients. Endotoxins can infiltrate the bloodstream at high concentrations, causing adverse reactions such as hemorrhagic shock, diarrhea, meningitis, fever, altered resistance to bacterial infection, and a rapid drop in blood pressure. The Bacterial Endotoxin Test, also known as the Lumulus Amebocyte Lysate (LAL) Test, measures the concentration of endotoxins in the cell walls of gram-negative bacteria. It evaluates devices that meet cerebrospinal fluid or the cardiovascular system. Devices in direct or indirect contact with intravascular, intratympanic, intrathecal, or intraocular systems must undergo Bacterial Endotoxin testing to ensure endotoxin levels are below the device specification. In kinetic chromogenic assays, a sample is combined with a lysate, resulting in the release of chromophore from a chromogenic peptide. Gel clot tests involve mixing an equal quantity of test sample and lysate, with the gel remaining at the bottom of the tube if endotoxin is present. In kinetic turbidimetric tests, a sample is combined with lysate, turbidity is increased in the presence of endotoxins, and the onset time required to attain a predetermined absorbance is recorded.

Tests:

• ANSI/AAMI ST72
• USP/NF <161> and <85>
• Ph Eur 2.6 14. Method A, C, and D

The Antibiotic Potency Test is a biological assay that measures the concentration of antibiotic-active pharmaceutical ingredients (APIs) and their metabolites. For distribution within the United States, the FDA mandates that these pharmaceuticals' labeled potency be met. Chapter 81 of the USP General provides validated methodologies for determining the potency of multiple antibiotic-active ingredients based on their efficacy against particular microorganisms. Antibiotic potency can be determined using either the Cylinder-Plate Assay or the Turbidimetric Assay. The Cylinder-Plate Assay extracts the active ingredient(s) from the product, followed by dilution to the median dose and incubation in a Petri dish. The Cylinder-Plate Assay is based on the antibiotic's and its corresponding reference standard's ability to form a clearing zone, thereby eradicating any microorganisms present in the seed media. The Turbidimetric Assay relies on the antibiotic's ability to eradicate a USP-specified microorganism in a solution, thereby decreasing turbidity. Using a formula that compares the size of the zone derived from known quantities of antibiotics contained in the Reference Standard to the size of the sample, the results are calculated. Method Verification is only conducted once for each sample type (formulation), unless the USP method or sample formulation changes.

Stability testing programs in microbiology assess the viability, consistency, and shelf life of microbial products, ensuring their safety and effectiveness. These programs consider environmental factors like temperature, humidity, light, and storage duration, determine shelf life, expiration date, and storage conditions, and ensure regulatory compliance. Sample selection and testing conditions include accelerated and long-term storage. Methods like CFU counts, microscopic analysis, and molecular assays assess microorganism viability, while physicochemical properties like pH, osmolality, appearance, and active constituent potency are evaluated. Data analysis and reporting are conducted to analyze performance and stability profiles. Shelf life is determined based on stability testing results, with an appropriate expiration date assigned. Overall, stability testing programs are essential quality control measures for ensuring the safety, efficacy, and dependability of microbiological products.

Disinfectant and antiseptic efficacy testing evaluates the ability of chemical agents to destroy or inhibit the growth of microorganisms, ensuring their reliability in controlling and preventing the spread of infectious agents. Suspension tests, carrier tests, time-kill kinetics, and in-use testing are typical procedures. These evaluations evaluate the efficacy of disinfectants and antiseptics in real-world settings, such as hospitals and laboratories. The results contribute to the establishment of recommended concentrations, contact times, and application methods for disinfectants and antiseptics, ensuring their safe and effective use against microbial contamination.

Antimicrobial studies evaluate the effectiveness of substances or agents in inhibiting or killing microorganisms. They help clients develop and launch safe, quality products by conducting various tests. These tests include in-vitro time-kill, zone of inhibition, minimum inhibitory concentration (MIC), topical antimicrobial testing, antiplaque and antigingivitis studies, acne and maskne studies, and custom research projects. These tests help verify claims and safety while adhering to regulatory guidelines. Custom research projects and protocols are also offered to assist clients in their evolving needs.

The Ames Mutagenicity Test is utilized to determine the potential mutagenic activity of a medical device/material extract. As part of the genotoxicity suite of tests, the Bacterial Reverse Mutation Assay (Ames test) is performed to determine if leachable from a medical device/material are mutagenic.

This test adheres to the AAMI/ANSI/ISO/10993-3, AAMI/ANSI/ISO/10993-1, AAMI/ANSI/ISO/10993-12, and OECD 471 guidelines as one of the three levels of in vitro assays for genotoxicity.

Cosmetic testing, also known as import testing, is a crucial procedure that assesses the safety, quality, and compliance of cosmetic products prior to their importation and sale in a country or region. It ensures that products comply with regulatory requirements and standards, safeguarding the health and safety of consumers. Safety evaluation, regulatory compliance, quality control, product claim validation, and consumer safety are all components of import testing. It helps prevent the sale of detrimental or non-compliant products and ensures that consumers have access only to safe and compliant products. To reduce the use of animals in cosmetic testing, in-vitro tests and computer simulations are being implemented. Import testing is essential to the cosmetics industry's supply chain, ensuring that consumers have access to only safe and compliant products.

Custom research projects and client-specific protocols in microbiology offer tailored testing services to meet clients' specific needs and requirements. Common tests include microbial identification, antimicrobial susceptibility testing, pathogen detection, environmental microbiology testing, food and water testing, microbial growth and enumeration, biofilm studies, viral load testing, microbiome analysis, and assay development. These services are essential for diagnosing infectious diseases, monitoring outbreaks, ensuring public health and safety, and ensuring compliance with food safety regulations. Iskon Labs' expertise and state-of-the-art technologies enable us to provide accurate and reliable results, supporting scientific investigations and contributing to advancements in microbiological research.

Testing hand sanitizers is essential for ensuring their safety, effectiveness, and quality. It entails analyzing active ingredients, evaluating effectiveness against microorganisms, evaluating contact time, evaluating residual effects, evaluating skin compatibility, evaluating stability and shelf life, detecting microbial contamination, ensuring regulatory compliance, and validating labeling information. Manufacturers and consumers can rely on the quality and efficacy of hand sanitizers, which are essential for maintaining proper hand hygiene and preventing infectious diseases because they adhere to rigorous testing protocols and industry standards. By adhering to stringent testing protocols and industry standards, manufacturers and consumers can have confidence in the quality and efficacy of hand sanitizers, thereby promoting proper hand hygiene and reducing the spread of infection.

Tests:

• (MIC) Minimum Inhibitory Concentration Test (Standard In Vitro Test for Microbial Inhibition)
• ASTM E2315 (Standard In Vitro Time-Kill Procedure)

Microbial Content Testing (USP 61 and USP 62) is utilized to evaluate raw materials, non-sterile pharmaceuticals, over-the-counter (OTC) finished product5, personal care/cosmetic products, and dietary supplements. USP 61 entails quantitative testing for total bacteria, yeast, or mold, whereas USP 62 involves screening for undesirable microorganisms. Both techniques are in accordance with EP 2.6.12 and 2.6.13 of the European Pharmacopoeia. In accordance with USP 61, Iskon Labs also executes suitability and screening for Burkolderia cepacia complex. USP 62 includes assays for Bacillus subtilis, Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Clostridia, and Candida albicans, among others. USP 61 requires method suitability to ensure that the product or material does not inhibit organism recovery, which could lead to false-negative test results. USP 1111 provides recommendations for microbiological specifications based on the nature of the product and its route of administration.

The USP <2021> and <2022> tests are utilized to evaluate nutritional supplements from their basic materials to their finished products. USP <2022> screens for the presence/absence of specified objectionable microorganisms, whereas USP <2021> involves quantitative testing for enumeration of total bacteria, yeast, or mold. Iskon Labs can also perform USP <61> and <62>-based suitability and screening. USP <2021> is a quantitative test for determining the number of bacteria and fungi in non-sterile supplements, including Total Aerobic Microbial Count (TAMC) and Total Yeast and Mold Count (TYMC). USP <2022> focuses on qualitative tests for the presence or absence of specified objectionable or potentially pathogenic microorganisms in non-sterile supplements. To prevent false negative test results, USP requires method suitability or preparatory testing before performing MLT testing on specific products or materials. The chapter on general information includes recommendations for microbiological specifications for botanicals, ingredients, and dietary supplements.

International Organization for Standardization (ISO) Microbial Content techniques are used to determine the levels of bacteria, yeast, and mold in cosmetic items during testing. In addition, any unwanted organisms present in the cosmetic product can be found through qualitative enrichment testing of the samples in a non-selective growth medium followed by subculture to selective/differential media.

Tests:

• ISO 21149 Cosmetics: Microbiology - Enumeration and Detection of Aerobic Mesophilic Bacteria
• ISO 16212 Cosmetics: Microbiology - Enumeration of Yeast and Mold
• ISO 21150 Cosmetics: Microbiology - Detection of Escherichia coli
• ISO 22717 Cosmetics: Microbiology - Detection of Pseudomonas aeruginosa
• ISO 22718 Cosmetics: Microbiology - Detection of Staphylococcus aureus
• ISO 18416 Cosmetics: Microbiology - Detection of Candida albicans