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Cytogenetics

Prepare, Label, & Store Reagents

Cytogenetics is “wet chemistry” biology. The quality of the chromosomal preparation depends entirely on the quality of the reagents used. From the media that feeds the cells to the hypotonic solution that swells them, every liquid must be prepared, labeled, and stored according to strict Quality Assurance (QA) protocols

Reagent Preparation (SOPs)

All reagents must be prepared following Standard Operating Procedures (SOPs). There is no “cooking by feel” in the clinical lab

  • Accuracy: Use calibrated balances and volumetric glassware (graduated cylinders, volumetric flasks) to ensure concentrations are precise
    • Example: Hypotonic solution (0.075M KCl). If made too strong (hypertonic), cells shrink. If made too weak (very hypotonic), cells burst. Precision is non-negotiable
  • Water Quality: Use Type I (Ultrapure) or Type II (Deionized) water. Tap water contains ions and contaminants that kill cell cultures
  • Sterility: Culture media and mitogens must be prepared inside a Biological Safety Cabinet (Laminar Flow Hood) using aseptic technique. Solutions are often filter-sterilized (0.22 \(\mu\text{m}\) filter) rather than autoclaved, as heat destroys vitamins and proteins

Labeling Reagents (OSHA/CLIA)

A beaker of clear liquid on a bench is a safety hazard and a compliance violation. Every container must be labeled immediately

  • Mandatory Label Elements
    1. Content Name: Full chemical name (e.g., “Methanol/Acetic Acid 3:1”)
    2. Concentration: (e.g., “0.075 M”)
    3. Preparation Date: When was it made?
    4. Expiration Date: When does it go bad?
    5. Initials: Who made it? (Traceability)
    6. Storage Requirements: (e.g., “Store at \(4^{\circ}\text{C}\)”)
    7. Hazard Warnings: (GHS Symbols). e.g., “Flammable” for Methanol, “Corrosive” for Acid

Storage Requirements

Different reagents degrade via different mechanisms (heat, light, oxidation). Proper storage extends shelf life and ensures performance

  • Temperature Control
    • Room Temp (\(20\text{--}25^{\circ}\text{C}\)): Giemsa Stain (precipitates in cold), Acids
    • Refrigerated (\(2\text{--}8^{\circ}\text{C}\)): Culture Media (RPMI 1640), Antibiotics (Pen/Strep), Mitogens (PHA), Hypotonic Solution (prevents bacterial growth)
    • Frozen (\(-20^{\circ}\text{C}\)): Serum (FBS), L-Glutamine (unstable at \(4^{\circ}\text{C}\)), Trypsin aliquots, FISH Probes
  • Light Sensitivity
    • Fluorescent probes (FISH) and some stains (e.g., Hoechst) are Photolabile. They must be stored in amber bottles or wrapped in foil to prevent photobleaching
  • Flammables
    • Fixative components (Methanol, Ethanol) must be stored in a dedicated Flammable Safety Cabinet: (yellow cabinet), not on open shelves

Expiration & QC

  • “Open Date”: When a commercial reagent (e.g., a bottle of Trypsin) is opened, the date must be written on the bottle. The expiration date often changes from the manufacturer’s date to a “shorter” stability date once the seal is broken
  • Stock Rotation: Use “First In, First Out” (FIFO) to prevent old chemicals from accumulating in the back of the fridge
  • Quality Control (QC)
    • New batches of critical reagents (e.g., a new lot of Fetal Bovine Serum) must be “parallel tested” against the old lot before routine use to ensure they support cell growth
    • The pH of media is checked (via color indicator: Phenol Red). Orange/Yellow = Acidic (bacterial contamination?); Purple = Basic (old/CO2 loss)

Safety (SDS)

A Safety Data Sheet (SDS) must be available for every chemical in the lab

  • Carcinogens: Some traditional cytogenetic chemicals (e.g., Ethidium Bromide, some mounting media) are toxic
  • Acids/Bases: Proper PPE (gloves, goggles, lab coat) is required when mixing fixative (Acid + Methanol creates an exothermic reaction)