- MCB Incubation at 42–44°C: This temperature range is used to favor the growth of thermophilic organisms, which thrive at higher temperatures, and is critical for specific microbial cultures, such as E. coli in Molecular Cloning Buffer (MCB).
- 0.45 Micron Filter for Water Analysis: A 0.45-micron filter is commonly used in water analysis because it captures bacteria but allows viruses and smaller particles through. 0.2-micron filters, though finer, are typically used for sterilization because they can block most bacteria and some viruses.
- R2A for Water Analysis: R2A (Reasoner's 2A Agar) is used to cultivate bacteria that grow slowly in nutrient-poor environments like water. It's ideal for heterotrophic plate count (HPC) analysis in potable water systems.
- Data Integrity: Ensuring that data is accurate, complete, consistent, and reliable throughout its lifecycle, crucial for compliance in regulated industries like pharmaceuticals and healthcare.
- ALCOA+: Stands for Attributable, Legible, Contemporaneous, Original, and Accurate. The "+" adds Completeness, Consistency, Enduring, and Available to emphasize maintaining high-quality data integrity.
- Salmonella Detection in 10g Sample: Testing 10g rather than 1g ensures greater sample representation and increases the likelihood of detecting contamination in microbiological limits testing (MLT).
- Instruments in Microbiology: Common instruments include autoclaves (for sterilization), incubators (e.g., Thermo Fisher), microscopes, centrifuges, and microbial identification systems like the VITEK 2 system (bioMérieux).
- Autoclave Calibration: Calibration involves using biological indicators (spores) and calibrated sensors to confirm the autoclave is reaching the correct temperature and pressure consistently.
- Autoclave Revalidation: Periodically reassessing the autoclave to confirm that it still meets performance criteria using methods like biological indicators and temperature mapping.
- Water Analysis Method: Common methods include membrane filtration, heterotrophic plate counts (HPC), and biochemical oxygen demand (BOD) testing.
- Water Sampling: Water samples are typically collected using sterile containers, often after flushing the system, and stored at 2–8°C to prevent microbial growth.
- Limits of Environmental Monitoring (EM): The microbial limits depend on the cleanliness classification of the environment, such as ISO Class 5 for aseptic areas, with defined limits for colony-forming units (CFU).
- Purified and Treated Water Limits: Purified water typically must meet limits like ≤100 CFU/mL, while treated water may have higher limits based on its intended use.
- Water USP Chapter: USP Chapter <1231> provides guidance on water quality for pharmaceutical use.
- Environmental Monitoring USP Chapter: USP Chapter <1116> outlines procedures for microbial control in cleanrooms.
- Water System Qualification: Involves design qualification (DQ), installation qualification (IQ), operational qualification (OQ), and performance qualification (PQ) to ensure the system meets specifications.
- Compressed Gas USP Chapter: USP Chapter <1207> focuses on the quality control and testing of compressed gases in pharmaceutical settings.
- Growth Promotion Test (GPT): Ensures that media can support the growth of microorganisms by inoculating with low levels of organisms and verifying adequate recovery.
- MLT (Microbial Limits Test): Tests for the presence of specified microorganisms in pharmaceutical products, ensuring that contamination levels remain within permissible limits.
- Lab Roles & Responsibilities: Includes sample collection, microbiological analysis, media preparation, sterilization validation, and documentation according to regulatory standards.
- F0 Value: A sterilization metric that represents the time required to kill a specific bacterial load at 121°C. It helps in validating sterilization processes.
- Alert and Action Limits: These are predefined microbial thresholds that trigger investigation (alert) or corrective action (action).
- Setting Limits and Formula: Limits are set based on historical data and risk assessment. Formula for microbial control can involve calculating the mean ± 3 standard deviations.
- Trend Charts: Visual representations used in microbiology labs to monitor and track data trends over time, such as microbial counts in environmental monitoring.
- Differential Pressure in Pharmaceuticals: It ensures air flows in the right direction (from clean to less clean areas) and helps maintain contamination control.
- Incubator Temperature and Limits: Incubators typically operate at temperatures like 20–25°C for environmental samples and 30–35°C for microbial cultures, with strict tolerance to ensure accurate results.
- Pathogen Testing in MLT: Pathogens like E. coli or Salmonella are tested by inoculating selective media, followed by biochemical and serological confirmation tests.
- Environmental Monitoring Location Selection: Based on risk assessment, high-traffic and critical control areas are selected for monitoring.
- Nitrogen Gas Monitoring: Involves testing for microbial contamination in nitrogen gas lines, often using membrane filtration.
- Colonies in Compressed Air Sampling: If colonies are observed, an investigation is initiated to identify the source, and corrective actions are implemented to prevent recurrence.
- Gram Staining: A differential staining technique that classifies bacteria as Gram-positive (thick peptidoglycan layer) or Gram-negative (thin layer with outer membrane).
- Five Gram-positive and Gram-negative Bacteria: Examples include Staphylococcus aureus (G+), Bacillus (G+), Clostridium (G+); Escherichia coli (G-), Pseudomonas (G-).
- RVSEB Sterilization at 115°C: Lower temperature sterilization is used for heat-sensitive materials where higher temperatures might degrade the product.
- VITEK Report: A VITEK report includes microbial identification details, antibiotic susceptibility, and test interpretations, often used in clinical microbiology.
- EM Plates Incubation: Plates are incubated at 20–25°C for fungi and yeasts, then at 30–35°C for bacterial recovery.
- R2A Plates Incubation at 30–35°C for 5 Days: R2A agar is incubated at this temperature for up to 5 days to allow slow-growing heterotrophic bacteria to develop. This extended incubation ensures the recovery of organisms that may not grow on other media or in shorter time frames.
- Decontaminating Media: Typically, media decontamination involves autoclaving at 121°C for 15-20 minutes to sterilize, ensuring all microorganisms are killed.
- Fumigation in Microbiology Lab: This involves the use of chemical disinfectants (like formaldehyde or hydrogen peroxide vapor) to disinfect the entire laboratory environment. Fumigation is followed by proper air purging to ensure safety.
- Exposing EM Plates After Fumigation: After fumigation, EM plates are exposed to assess the effectiveness of the fumigation process and to confirm that the environment is free of microbial contamination.
- Fumigation Solution: Common fumigants include formaldehyde gas, hydrogen peroxide vapor, or quaternary ammonium compounds. Specific solutions depend on the type of fumigation being performed.
- Composition of Minncare and Dettol: Minncare is typically a mix of peracetic acid and hydrogen peroxide used for disinfecting water systems. Dettol contains chloroxylenol, a disinfectant that kills bacteria and viruses.
- Plates Incubated in Inverted Position: Incubating plates upside down prevents condensation from dripping onto the media, which could interfere with bacterial colony growth and cause contamination.
- Using Saline for Swabs: Sterile saline is used as it maintains osmotic balance, providing an isotonic solution that supports microorganism survival without altering their physiological state during swabbing.
- Measurement for Swab on Instruments: Typically, a 10 cm² area is swabbed for microbial testing in cleanrooms, using validated swab techniques to ensure consistent sampling.
- Water Sample Storage at 2–8°C: Water samples are stored at low temperatures to inhibit microbial growth, maintaining the integrity of the sample until testing.
- Monthly Calibration of Balance and Limits: The balance calibration process involves using standard weights to ensure accuracy within the specified limits. Typically, balances must be accurate to within ±0.1 mg for analytical balances.
- Oxidation Test: This test evaluates the oxidative stability of substances, determining how well they resist degradation when exposed to oxidizing agents.
- Pathogen Confirmation Tests: These involve biochemical tests (like Triple Sugar Iron, SIM tests) and molecular assays (PCR) to confirm the identity of pathogens like Salmonella, E. coli, etc.
- Selecting EM Location in Manufacturing: Locations are chosen based on a risk assessment of critical zones, typically high-traffic or cleanroom areas where contamination is most likely.
- Revalidation of LAF (Laminar Air Flow): Revalidation of LAF involves airflow velocity testing, filter integrity testing (using DOP/PAO), and microbial monitoring to ensure continued performance.
- Difference Between Laminar Air Flow (LAF) and Biosafety Cabinet: LAF provides unidirectional airflow to protect the product from contamination, while a biosafety cabinet offers personnel, product, and environmental protection through HEPA filtration.
- Temperature Inside Microbiology Lab: Typically maintained between 20–25°C with humidity control to prevent fluctuations that could affect microbial growth or testing conditions.
- Receiving New Media in the Lab: New media undergoes quality checks, including pH testing, sterility testing, and growth promotion testing before being accepted for use.
- Passages Used in Microbiology: Bacterial cultures are often limited to a set number of passages (commonly 5) to prevent genetic drift and ensure strain consistency.
- Why Only 5 Passages?: After several passages, bacteria can undergo mutations, which may alter their characteristics, making them less representative of the original strain.
- Sensors Inside the Autoclave for Revalidation: Typically, 12 sensors are placed at different points inside the autoclave during revalidation to ensure uniform temperature distribution.
- UV Lamp Burning Hours in LAF: UV lamps are typically used for 8-12 hours in biosafety cabinets or LAF hoods to sterilize the surfaces, and they are replaced after a defined usage period, often every 1000–2000 hours.
- Using 70% IPA (Isopropyl Alcohol): 70% IPA is more effective than 100% because the presence of water helps the alcohol penetrate microbial cell walls, leading to better denaturation of proteins and cell death.
- Calibrating Micropipettes: Micropipettes are calibrated by dispensing a known volume of water and weighing it to ensure accuracy and precision, with the acceptable range being within ±1-2%.
- Media Weighing for SCDA and pH: For SCDA (Soybean Casein Digest Agar), approximately 40g/L is weighed, and the pH is adjusted to 7.3 ± 0.2 before sterilization.
- Not Using SCDA for Water Testing: SCDA is not ideal for water testing because it supports a wide range of fast-growing bacteria, potentially masking the detection of slower-growing organisms in water samples.
- Limit of TOC (Total Organic Carbon): For purified water, the limit is typically ≤500 ppb (parts per billion), but this can vary depending on the specific water system and its validation.
- TOC Instrument at Water Plant: The TOC instrument displays organic carbon levels in real-time. The limit typically displayed for purified water is ≤500 ppb, depending on the specification.
- Flushing Water for Sampling: Water systems are flushed for a minimum of 1–2 minutes, or until a steady-state flow is achieved, to ensure a representative sample from the system.
- TOC Sampling: TOC samples are taken in sterile, low-TOC glass containers and analyzed immediately or stored at 2–8°C to avoid organic contamination.
- Exposing EM Plates for 4 Hours: EM plates are exposed for 4 hours to capture airborne contamination over a significant time period, ensuring a representative sample of the microbial load in the air.
- Air Sampling for 10 Minutes/1000 Liters (USP Chapter): According to USP Chapter <1116>, air sampling for a specified time is used to monitor the microbial quality in cleanrooms, typically 10 minutes for 1000 liters of air.
- Water System Validation (Phase 1: 2–4 Weeks): This is a testing phase where the water system's performance is assessed daily for 2–4 weeks to ensure compliance with microbial and chemical specifications.
- Autoclave at 121°C for 15 or 26 Minutes: The sterilization time depends on the load type. 15 minutes is standard for typical loads, while 26 minutes is used for larger or denser items that require more time to achieve sterility.
- Growth Promotion Test for SCDA: SCDA is tested by inoculating known microorganisms and assessing whether the media supports their growth to ensure its effectiveness in promoting microbial recovery.
- Checking PLC Reports Daily: Daily checks involve verifying critical parameters like temperature, pressure, and cycle times to ensure that the autoclave or other sterilization systems are functioning correctly.
- Validation: A documented process of ensuring that a system or equipment consistently produces results that meet predetermined specifications.
- New Instrument Qualification: Involves installation qualification (IQ), operational qualification (OQ), and performance qualification (PQ) to confirm that the instrument meets operational and performance criteria.
- Number of Incubators in the Lab: Typically, microbiology labs have multiple incubators to accommodate different temperature requirements (e.g., 20–25°C and 30–35°C), with the number depending on the size of the lab.
- Mapping of Incubators: Temperature mapping is done to ensure uniform conditions throughout the incubator, often using data loggers placed at various locations inside the incubator.
- Releasing EM Plates: EM plates are released after being checked for media integrity, sterility, and growth promotion to ensure they are ready for use in environmental monitoring.
- MLT 1:100 Dilution: This dilution is used to reduce the concentration of microorganisms, making them easier to count and ensuring that they fall within a quantifiable range.
- Why 10g in MLT: A 10g sample size provides a more representative test of microbial contamination in a product, especially for detecting low levels of contamination.
- MLT Limits for Raw Materials/Finished Products: These limits vary depending on the product type and regulatory guidelines but generally ensure that microbial contamination is within acceptable levels.
- BET (Bacterial Endotoxin Test): This test detects and quantifies endotoxins from Gram-negative bacteria, which are critical contaminants in injectable products and medical devices.
- Sterility: Sterility refers to the complete absence of viable microorganisms. Sterility testing is conducted on pharmaceutical products to ensure they are free from contamination.
- Media Filling: This is the process of filling culture media into sterile containers or plates, often used for sterility testing or microbial growth studies.
- Autoclave Breakdown – Next Steps: If an autoclave breaks down, alternate sterilization methods (if available) are used, and repairs are initiated. Pending loads may need to be retested or rester.
- Ensuring Autoclave Sterilization for Each Cycle: To ensure proper sterilization in every autoclave cycle, biological indicators (BIs) such as Geobacillus stearothermophilus spore strips, chemical indicators, and thermocouples are used. These are placed in the most challenging areas of the load to monitor for proper time, temperature, and pressure conditions.
- If Environmental Monitoring Crosses the Alert Limit: When environmental monitoring (EM) results exceed the alert limit, the situation is investigated. The steps include reviewing environmental conditions, identifying potential sources of contamination, re-sampling, and corrective actions like increased cleaning, disinfecting, or equipment calibration.
- Reporting Excursions in Microbiology Department: Excursions (deviations from expected limits) are documented using a formal incident reporting system. The event is investigated, root cause analysis is performed, and corrective and preventive actions (CAPA) are implemented. This ensures compliance with GMP (Good Manufacturing Practices).
- Concluding There is No Laboratory Error in Microbiology: To rule out lab errors, multiple factors are investigated, such as reviewing sample handling procedures, verifying equipment calibration and functionality, ensuring proper media storage and preparation, and checking for any deviations from SOPs (Standard Operating Procedures). The analyst’s work is scrutinized to confirm adherence to protocols.
- Performing E. coli Test: Testing for Escherichia coli typically involves selective media such as MacConkey or EMB agar, where E. coli produces distinct colonies. Confirmation involves biochemical tests (like IMViC series), and further identification may use molecular techniques like PCR.
- RODAC (Replicate Organism Detection and Counting): RODAC plates are used in surface sampling for environmental monitoring. These plates are pre-filled with a culture medium and are directly pressed against surfaces to capture any microorganisms present, which are then incubated to assess colony-forming units (CFU).
- Size of Petri Plates in Microbiology Lab: Typically, microbiology labs use 90 mm or 100 mm diameter petri plates. Larger plates may be used in certain cases for high-volume sampling or specific applications.
- Hot Air Oven Temperature for Drying Glassware: Hot air ovens used for sterilizing and drying glassware typically operate at temperatures of 160–180°C for durations of 2–3 hours, depending on the size and type of load.
- Coliform Test: This test determines the presence of coliform bacteria, which indicates potential contamination by fecal matter. Water samples are tested using methods like the Most Probable Number (MPN) or membrane filtration, with results indicating whether the water meets safety standards.
- Feller Correction in Air Samplers: The Feller correction accounts for the possibility that some microorganisms may overlap on an air sampler’s sieve plate, leading to an underestimation of microbial counts. Air sampler sieves typically have around 300–400 holes, depending on the design.
- Deviation: A deviation refers to any departure from an approved procedure or standard. In a lab setting, deviations must be documented, and the root cause must be investigated. Corrective actions are implemented to prevent recurrence.
- Water Doll Test in EM: This test is conducted to assess the effectiveness of surface and environmental cleaning in critical areas. The "doll" refers to a swab test or a media plate that collects environmental samples after cleaning, and the samples are then incubated to detect residual contamination.
- Culture Storage and Handling: Microbial cultures are stored in cryogenic vials or slants at ultra-low temperatures (e.g., -80°C) to preserve viability. They are handled aseptically to avoid contamination, and proper labeling ensures traceability.
- Treated Water Sampling with Sodium Thiosulphate: Sodium thiosulphate is added to water samples to neutralize chlorine or other disinfectants that may interfere with microbial testing. The water is then sterilized and tested.
- Hold Time Period of Media: The hold time of prepared culture media refers to the time between preparation and use. For most media, the hold time is up to 2–4 weeks if stored properly at 2–8°C, although some media require immediate use.
- Why Cool Media to 45°C Before Pouring Plates: Cooling media to 45°C before pouring ensures that the media is cool enough not to kill heat-sensitive microorganisms, yet warm enough to remain liquid for easy pouring.
- 21 CFR Part 11: This is a regulation by the FDA (Food and Drug Administration) that outlines the criteria under which electronic records and electronic signatures are considered trustworthy, reliable, and equivalent to paper records. It includes guidelines on audit trails, data security, and validation of computerized systems.
