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Cytogenetics

Operate & Maintain Equipment

The cytogenetics laboratory relies on a controlled environment to mimic the human body (in vivo) conditions for cell growth. The equipment that maintains these parameters - incubators, refrigerators, water baths - requires rigorous daily monitoring and preventative maintenance. A failure in equipment usually means the death of all patient cultures

The \(CO_2\) Incubator (The Artificial Body)

The incubator is the most critical piece of equipment. It maintains the physiological conditions required for cell division

  • Temperature (\(37^{\circ}\text{C}\))
    • Operation: Must be maintained at \(37.0 \pm 0.5^{\circ}\text{C}\)
    • Why?: Cells stop dividing if too cold (\(<35^{\circ}\text{C}\)) and die rapidly if too hot (\(>39^{\circ}\text{C}\))
    • Maintenance
      • Daily: Check digital display against a calibrated NIST-traceable thermometer placed inside. (Digital displays drift!)
      • Monthly: Clean interior surfaces to prevent fungal growth
  • \(CO_2\) Concentration (5%)
    • Operation: Most mammalian cell media (RPMI 1640) use a Sodium Bicarbonate buffer system. This buffer requires an atmosphere of 5% \(CO_2\) to maintain a neutral physiological pH (7.2–7.4)
      • Too Low \(CO_2\): pH rises (becomes basic/purple). Cells stop growing
      • Too High \(CO_2\): pH drops (becomes acidic/yellow). Toxic to cells
    • Maintenance
      • Fyrite Test: The internal sensor (IR or TC) must be cross-checked weekly using a chemical gas analyzer (“Fyrite”) or external gas meter to verify the actual percentage
      • Auto-Zero: Modern sensors auto-calibrate to room air (0.04% \(CO_2\)) periodically
  • Humidity (95–99%)
    • Operation: A water pan at the bottom maintains saturation
    • Why?: Prevents the media in the flasks from evaporating. Evaporation increases the salt concentration (osmolality) of the remaining media, killing the cells
    • Maintenance: Keep the water pan filled with sterile distilled water containing a fungicide/bactericide (e.g., Copper sulfate)

Biological Safety Cabinet (The Sterile Zone)

Used to protect the specimen from contamination and the worker from biohazards

  • Operation
    • Class II Type A2 is standard. Air is HEPA filtered before hitting the workspace (protects product) and before exhausting (protects environment)
    • Sash Height: Must be kept at the indicated level to maintain proper airflow velocity
  • Maintenance
    • Daily: Wipe down with 70% Ethanol before and after use. UV light (if used) should only be on when not in use
    • Annual: Certification by a professional engineer to verify airflow velocity and HEPA filter integrity

Temperature-Controlled Equipment

  • Refrigerators (\(4^{\circ}\text{C}\)) and Freezers (\(-20^{\circ}\text{C}\))
    • Use: Reagent storage
    • Maintenance: Daily temperature logs. Continuous monitoring systems (e.g., Rees) alarm if the temp goes out of range (e.g., door left open)
  • Water Baths (\(37^{\circ}\text{C}\))
    • Use: Warming media before use
    • Maintenance: High risk of contamination (“pseudomonas soup”). Must be drained and cleaned weekly. Use floating distinct “beads” or specialized algicides
  • Slide Drying Oven/Hotplate
    • Use: Baking slides (\(60^{\circ}\text{C}\) or \(90^{\circ}\text{C}\)) to “age” them for banding
    • Maintenance: Verify temperature with a surface thermometer

The Microscope

  • Use: Analysis
  • Maintenance
    • Daily: Clean oil from 100x lens
    • Weekly: Check Kohler illumination
    • Annual: Professional cleaning and alignment

Centrifuges

  • Use: Harvesting (pelleting cells)
  • Operation: Must be balanced
  • Maintenance
    • Tachometer Check: Annually verify that the RPM displayed matches the actual spin speed (critical for G-force calculation)
    • Cleaning: Clean buckets with bleach if tubes break

Environmental Monitoring (Oxygen?)

  • Hypoxic Culture: While standard incubators use ambient oxygen (~20%), some specialized stem cell or embryo cultures use Tri-Gas Incubators to lower \(O_2\) to physiological tissue levels (e.g., 5% \(O_2\)) using Nitrogen gas displacement. This reduces oxidative stress but is less common in routine clinical cytogenetics

Summary of Maintenance Frequency

  • Daily: Record temps (\(37^{\circ}\), \(4^{\circ}\), \(-20^{\circ}\)), check \(CO_2\) display, check humidity pans
  • Weekly: Fyrite (\(CO_2\) verification), change water baths
  • Annually: Certify Biosafety Hoods, calibrate Centrifuges/Pipettes/Thermometers