Aseptic Culture Technique

The culture phase is the most vulnerable period in the cytogenetic workflow. A failure in aseptic technique leads to two distinct but catastrophic outcomes: microbial contamination (which destroys the specimen) or cross-contamination (which destroys the diagnosis by mixing two patients’ cells). The laboratory scientist’s strict adherence to sterile protocols and workflow discipline is the only barrier against these errors

Prevent Microbial Contamination

The rich media used for human cells (containing serum and glucose) is an ideal growth environment for environmental pathogens. Once introduced, bacteria and fungi outcompete human cells, causing culture failure

• Engineering Controls (The Hood)
  • All work is performed in a Class II Biological Safety Cabinet (BSC). The vertical laminar flow of HEPA-filtered air creates a sterile curtain
  • Airflow Discipline: The laboratory scientist must avoid rapid movements or overcrowding the hood, which disrupts the air curtain and draws in non-sterile room air. Air grilles must never be blocked
• Personal Aseptic Technique
  • PPE: Gloves and dedicated lab coats (long sleeves) are mandatory to contain skin shedding. Gloves must be sprayed with 70% ethanol immediately upon hood entry
  • The “No-Pass” Rule: A non-sterile object (like an arm or hand) must never pass over an open sterile container. This prevents skin flakes or dust from falling into the culture
  • Specimen Segregation: “Dirty” specimens (skin, POCs) should be processed last or in a dedicated hood to protect “clean” specimens (bloods/marrows)
• Maintenance & Remediation
  • Incubators: Water pans (humidity source) must be cleaned regularly with antifungal agents to prevent mold
  • Visual Checks: Turbidity (cloudiness) or a Yellow color change (acidic pH) in the media indicates bacterial overgrowth. Contaminated vessels must be isolated immediately to protect other patients

Prevent Cross-Contamination Between Cultures

Cross-contamination occurs when cells from “Patient A” are transferred to the culture of “Patient B.” This can lead to a false diagnosis (e.g., a normal karyotype masking a leukemic clone) or a chimeric result. Unlike microbial contamination, this error is often invisible until analysis

• The Golden Rule: One Patient at a Time
  • Only one patient’s: specimen tubes and culture vessels should be open in the hood at any given moment. The workspace must be cleared and wiped between patients
  • Prohibition: Never have multiple patients’ tubes on the rack simultaneously
• Liquid Handling Discipline
  • Tip Changes: A pipette tip must never be reused between vessels. If a tip touches Patient A’s tube and then enters the common media bottle, the entire bottle becomes contaminated with Patient A’s cells
  • Aliquotting: Media should be poured into secondary disposable containers (e.g., 50 mL tubes) for each patient rather than pipetting directly from the stock bottle
  • Splash Prevention: Avoid vigorous “blowing out” of pipettes, which creates micro-droplets that can land on adjacent surfaces or tubes
• Maternal Cell Contamination (MCC)
  • Specific to prenatal samples. Laboratory scientists must dissect maternal decidua from fetal villi (CVS) and discard the first few mL of amniotic fluid (Amnio) to minimize the risk of culturing maternal cells instead of fetal ones