Skip to main content

Cytogenetics

Prevent Microbial Contamination

In clinical cytogenetics, the culture vessel is a rich, warm incubator filled with glucose, amino acids, and serum - a perfect breeding ground for bacteria, fungi, and mycoplasma. If microorganisms enter the system, they inevitably outcompete the human cells, rapidly acidifying the media and releasing toxins that destroy the specimen. Unlike established cell lines, patient specimens are often irreplaceable. Therefore, the prevention of microbial contamination is not merely a technical preference; it is the primary determinant of culture success and diagnostic yield

The Sterile Environment: Engineering Controls

The first line of defense is the physical workspace. All specimen manipulation occurs within a Class II Biological Safety Cabinet (BSC), also known as a Laminar Flow Hood

  • Laminar Flow: The BSC maintains a curtain of HEPA-filtered air that flows vertically from the ceiling of the cabinet down to the work surface. This creates a sterile zone and prevents room air (laden with dust and spores) from entering the culture vessel
  • Airflow Discipline: The laminar curtain is easily disrupted. Rapid arm movements, overcrowding the hood with equipment, or blocking the front/rear grilles creates turbulence that can suck contaminants into the sterile zone. Scientists must work deliberately and keep the airflow path clear
  • UV Sterilization: Germicidal UV lamps are often used when the hood is not in use (e.g., overnight) to degrade DNA and kill surface microbes. However, UV is only effective on surfaces it directly hits; it does not penetrate clutter

Personal Aseptic Technique

The laboratory scientist is the single biggest source of contamination (skin flakes, respiratory droplets, clothing fibers). Strict behavioral protocols minimize this risk

  • Personal Protective Equipment (PPE)
    • Gloves: Must be worn at all times. They should be pulled over the cuff of the lab coat to seal the wrist gap. Gloves must be sprayed with 70% Ethanol immediately upon entering the hood and whenever touching a non-sterile object
    • Lab Coats: Must be clean, buttoned, and designated for the culture room only (not worn in the breakroom or general lab). Long sleeves cover the skin of the arms, which sheds bacteria constantly
  • The “No-Pass” Rule: A critical motor skill. The laboratory scientist must never pass a non-sterile object (like a hand/arm) over an open sterile container
    • Correct: Hold the bottle in the left hand, unscrew the cap with the right, and pipette from the side
    • Incorrect: Reaching over an open flask to grab a pipette
  • Minimizing Aerosols: Avoiding the vigorous “blowing out” of pipettes or splashing media. Micro-droplets can carry bacteria from the rim of a tube into the air or onto the hood surface

Reagent & Specimen Handling

Contamination can be introduced via the fluids used in the culture

  • Aliquotting: Media bottles (500 mL) should be aliquotted into smaller working volumes (e.g., 50 mL tubes). If a 50 mL tube gets contaminated, only that aliquot is lost. If the main 500 mL bottle is contaminated, every patient culture fed with that bottle is destroyed
  • Specimen Segregation
    • “Dirty” vs. “Clean”: Specimens with a high risk of contamination (e.g., Products of Conception passed vaginally, Skin Biopsies) should be processed last, after all sterile bloods and marrows are finished. The hood must be fully decontaminated after “dirty” cases
    • One Patient at a Time: Only one patient’s tubes should be open in the hood at any given moment. This prevents cross-contamination (mixing up patients) and limits exposure time

Equipment Maintenance

The incubators and water baths are potential reservoirs for contaminants

  • Water Baths: The \(37^\circ\text{C}\) water bath used to warm media is a prime fungal breeding ground. It must be cleaned weekly with antifungal agents and filled with distilled water containing bacteriostatic additives
  • Incubators
    • Humidity Pans: The water tray at the bottom of the incubator maintains humidity but is prone to fungal growth (often appearing as pink slime or floating “cotton balls”). It requires regular changing and Copper Sulfate or commercial biocide additives
    • Shelves: Spills inside the incubator must be wiped immediately with 70% ethanol. Monthly “deep cleans” involve removing all racks and autoclaving them

Detection & Remediation

Despite best efforts, contamination occurs. Early detection salvages the lab

  • Visual Indicators
    • Turbidity: Bacterial growth makes the clear media look cloudy or milky
    • Color Change: Bacteria produce acid rapidly, turning the Phenol Red indicator Yellow. Fungi may not change the pH immediately but appear as floating white/fuzzy colonies
  • Remediation
    • Isolation: The contaminated vessel must be removed from the incubator immediately to prevent spores from spreading to other patients’ cultures
    • Salvage (Antibiotics): If the specimen is irreplaceable (e.g., marrow), the laboratory scientist may try to wash the cells and re-culture them in media with “heavy” antibiotics (Gentamicin, Kanamycin, Nystatin). However, success is limited once the culture is overtaken