Preventing microbial contamination

Contamination Risk of Mammalian Cell Cultures

Introduction to Contamination Risks

  • Contamination of cells in culture can arise from several sources, including:
    • Other cell lines
    • Reagents and supplies such as pipettes and culture vessels
    • Equipment including tissue culture hoods and incubators
    • Laboratory personnel

Primary Contamination Sources

  • Laboratory Personnel: Humans carry approximately 10,000 microorganisms per cm² of skin.
  • Indoor Air: Contains 30 to 1,000 microorganisms per liter.
  • Chemical Exposure: Fumes from lab chemicals, cleaning agents, and environmental Volatile Organic Compounds (VOCs) can dissolve into culture media or incubator atmosphere, leading to:
    • Cytotoxicity
    • Stress granule formation in cells
    • Impaired cell health

Reducing Contamination Risk

  • Constant potential for contamination necessitates proper precautions:
    • Using reagents of validated quality and sterility
    • Quarantining new cell lines until tested for contamination
    • Performing routine maintenance of all equipment
    • Cleaning all laboratory equipment
    • Proper training for all cell culture personnel

Types of Contaminants

Bacterial Contamination

  • Bacteria Characteristics:
    • Unicellular, typically a few micrometers in diameter
    • Shapes include spheres, rods, and spirals
  • Growth and Appearance:
    • Most common biological contaminants in cell culture
    • Infected cultures appear cloudy with potential surface films
    • Sudden drops in pH are common due to bacterial metabolism
  • Metabolic Activity:
    • Aerobic or anaerobic bacteria metabolize glucose and amino acids rapidly via fermentation or respiration
    • Major byproducts include organic acids (lactic, acetic, formic acid) that lower medium pH
  • Visual Identification:
    • Under low-power microscopy, bacteria appear as tiny granules among cells. E.g., Contaminated 293 cells show shimmering granules under phase-contrast microscopy.

Yeast Contamination

  • Characteristics:
    • Unicellular eukaryotic microorganisms in the kingdom Fungi, size ranges from a few micrometers up to 40 μm
  • Growth and Impact:
    • Cultures become turbid during advanced stages of contamination
    • pH changes occur only in heavy contamination, often resulting in an increase
  • Metabolic Byproducts:
    • Many yeasts metabolize amino acids producing ammonia (NH₃), which elevates pH
  • Microscopy:
    • Yeast appears as individual ovoid or spherical particles, budding smaller particles under phase contrast images.

Mold Contamination

  • Mold Characteristics:
    • Multicellular eukaryotic organisms in the kingdom Fungi, grow as filaments called hyphae
  • Growth Pattern:
    • Mycelium refers to a colony of hyphae with genetically identical nuclei
    • Initial pH stability in contaminated cultures, rapid increase as infection progresses
  • Visual Identification:
    • Microscopic examination reveals thin, wispy filaments or dense clusters of spores. May appear white, yellow, or black in culture.

Mycoplasma Contamination

  • Characteristics:
    • Very small size, no cell walls, capable of passing through standard filters
    • Often goes unnoticed as they do not induce obvious changes in cultures
  • Impact on Cell Culture:
    • Alters metabolism, growth rates, gene expression, and drug responses, impacting experimental validity
    • Most frequent contaminant in mammalian cell cultures
  • Detection Methods:
    • Cell lines are screened using Hoechst DNA stain, which binds to AT-rich regions of mycoplasma DNA for microscopy detection
    • PCR-based testing has become routine in cell culture labs

Viral Contamination

  • Characteristics:
    • Microscopic infectious agents that hijack host cell machinery for reproduction
    • Extremely small size complicates detection and elimination
  • Cell Impact:
    • Typically do not adversely affect cell cultures from species other than their specific host
  • Microscopy:
    • Electron micrographs depict distinct viral particles in infected cells alongside signs of viral morphogenesis
  • Viral Morphogenesis:
    • The process of viral assembly and maturation into infectious particles in host cells, involving self-assembly of viral proteins into capsids.

Microbial Contamination Check

  • Detection of Contaminants:
    • Bacterial contamination usually visible within days due to turbidity, visible particles, and rapid pH decline indicating acidity
    • Fastidious bacteria may grow slowly, complicating detection.
    • Fungal contamination may not show initial pH change, identifiable through filamentous structures.
    • Yeast larger than bacteria, appear as round or ovoid particles.
  • Microbacterial Media: Include blood agar, thioglycollate broth, tryptic soy broth, BHI broth, Sabouraud broth, YM broth, etc.
  • Composition of Media:
    • Typically includes a carbon source (glucose), nitrogen source (peptone/ammonium salts), salts for minerals, growth factors, or vitamins based on the microbe.
  • Antibiotic Use Caution:
    • Antibiotics can mask contamination; their routine use can lead to resistant strains and possible outbreak of low-level contaminations.

Microbial Contamination Treatment

  • Dealing with Contamination:
    • Discarding the culture and starting over is preferred, especially for unique or irreplaceable cells.
    • Identification of contaminants and antibiotic sensitivity testing is recommended if treatment is considered
  • Culture Process:
    • If using antibiotics, culture cells for 1-2 weeks followed by 1-2 months without antibiotics before retesting with a sensitive method
    • Retesting ensures contaminants do not reappear and should be carried out periodically.

Cross-contamination

  • Occurs when cells from one culture unintentionally enter another, often due to poor aseptic techniques, shared reagents, mislabeling, or splashes
  • Regular testing of cultures is critical to avoid misidentified or overgrown cultures
  • If contamination is found, discard and start afresh with new stock.

Biosafety Overview

  • Precautions in Cell Culture:
    • Dependent on source of biological material, procedures, and laboratory conditions
    • Identify risks and implement precautions before starting work

Biosafety Levels (BSL)

  • Biosafety Level 1 (BSL-1):

    • Risk Level: Low, for non-pathogenic organisms.
    • Requires standard lab practices such as wearing lab coats, gloves, and eye protection.

  • Biosafety Level 2 (BSL-2):

    • Risk Level: Moderate; pathogens causing mild disease, such as Staphylococcus aureus, some mammalian viruses.
    • Requires limited access, mandatory PPE, and a BSC for aerosol-generating procedures.

  • Biosafety Level 3 (BSL-3):

    • Risk Level: High; airborne pathogens like Mycobacterium tuberculosis, requiring strict controls and specific facilities.

  • Biosafety Level 4 (BSL-4):

    • Risk Level: Extreme; life-threatening pathogens with no treatments/vaccines, requiring maximal containment.

Cell Culture Waste Disposal

  • BSL-1 waste treated as biohazard with lower risks than BSL-2.

  • Disposal Procedures:

    • Liquid media: 10% bleach solution for 30 minutes before disposal.
    • Flasks and plates: Biohazard bag followed by autoclaving.
    • Sharps in designated containers.

  • Autoclaving:

    • Utilizes pressurized steam (121–134 °C for 30-60 min) to denature proteins and destroy microbes.

  • Bleach (NaOCl):

    • Active antimicrobial agent; reacts with water to form hypochlorous acid (HOCl) that penetrates microbial cell walls to disrupt structure and function.

Personal Protective Equipment (PPE)

  • Common Items:
    • Gloves to protect hands from chemicals/pathogens
    • Gowns/lab coats for skin and clothing protection
    • Masks/respirators for airway protection
    • Face shields/goggles for eye/face protection
    • Shoe covers/boots to protect feet and prevent contamination

Safe Laboratory Practices

  • Always wear PPE and wash hands before leaving the lab and after handling hazardous materials.
  • Avoid consumables in the lab, and minimize aerosol/splash creation.
  • Decontaminate work surfaces pre/post-experiment, handling sharps with care.

Setting Up a Cell Culture Lab

  • Critical success factors include providing an aseptic environment for cell manipulation and optimal growth conditions:
    • Clean Bench/Laminar Flow Hood: Maintains aseptic environment via HEPA-filtered air and UV light for sterilization.
    • Incubators: Maintains controlled environments (37°C, 5% CO₂) with humidity trays.
    • Storage Conditions: Requires liquid nitrogen tanks for long-term storage of cells.

Equipment Essentials:

  • Inverted microsopes, centrifuge, water baths, cell counters, aspiration pumps, refrigerators/freezers.
  • Additional Supplies Needed:
    • Cell culture vessels, pipettes, media, and waste containers.

Cell Culture Hood Types

  • Class I: Provides personnel/environment protection, but not for the cultures.
  • Class II: Most common, designed for handling BSL-1, 2, and 3 materials, ensures aseptic conditions
  • Class III: Gas-tight cabinets for BSL-4 work, maximum protection for personnel/environment.

Airflow Characteristics of Class II BSC

  • Maintains unidirectional flow of HEPA-filtered air, protecting both user and culture.
  • Should comfortably accommodate one user, be easily cleanable, with adequate lighting.

Cell Culture Hood Layout

  • Keep workspace clean and uncluttered with direct sightlines for effective operations
    • Disinfect items with 70% ethanol prior to placement in the hood.

Cleaning Protocols

  • All surfaces in CO₂ incubators especially should be disinfected with 70% ethanol to avoid corrosion typically caused by bleach
  • CO₂ Incubators:
    • Provide ideal environmental conditions for cell growth (temperature, humidity).

Disinfectants Consideration:

  • 70% Ethanol vs Quaternary Ammonium Compounds:
    • Ethanol: Fast-acting but can be harmful to some plastics; effective against a broad range of microbes.
    • Quats: Broader spectrum, with safer residue but limited effectiveness against spores and some viruses.

Aseptic Technique Overview

  • Fundamental to keep cells free from microbial contamination.
  • Aseptic Protocols:
    • Sterile work area, personal hygiene, and use of sterile media/reagents are essential for efficient culture.

Aseptic Technique Best Practices

  • Disinfect surfaces with 70% ethanol before/after use, and employ UV sterilization methods routinely.
  • Always work quickly to minimize contamination and adhere to strict personal hygiene measures to protect cultures.

Aseptic Technique Checklist

  • Confirm setup and cleanliness of the BSC or hood prior to starting experiments.
  • Use sterile pipettes/tools exclusively, minimize exposure time of vessels to the environment.
  • Properly dispose of waste and re-disinfect surfaces.