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Cytogenetics

Detect, Identify, & Control Contamination

Once the culture is established in the incubator, the laboratory scientist shifts from “active manipulation” to “active surveillance.” The monitoring phase is critical because contamination spreads. A single fungal spore in one flask can sporulate and infect an entire incubator of precious patient samples within days. Therefore, daily visual inspection is mandatory to detect, identify, and control biological invaders before they compromise the entire laboratory’s workload

Detection: Visual & Chemical Indicators

The first sign of contamination is usually macroscopic (visible to the naked eye). Scientists inspect cultures daily by holding the vessels up to the light or placing them on an inverted microscope

  • Turbidity (Cloudiness): Healthy culture media is crystal clear. Bacterial contamination causes a diffuse “milky” or “foggy” appearance that swirls when the tube is agitated. This indicates high bacterial load (\(>10^6\) organisms/mL)
  • pH Shift (Color Change): The Phenol Red indicator in the media acts as a biological alarm
    • Rapid Yellowing (Acidic): Bacteria metabolize glucose rapidly and produce acidic byproducts (lactate/acetate). If a tube turns bright yellow overnight while others remain red/pink, it is likely infected
    • Rapid Purpling (Alkaline): While less common for bacteria, some fungi or Pseudomonas species can break down proteins (deamination), releasing ammonia and raising the pH
  • Colony Formation (Fungal): Fungi often appear as discrete floating spheres (“cotton balls”) or fuzzy mats on the cell surface. They may not cause immediate turbidity or pH changes
  • Microscopic Signs
    • Brownian Motion: Under the inverted microscope (10x or 40x), bacteria appear as tiny, shimmering, vibrating dots in the background of the human cells. Unlike cellular debris, they move independently and purposefully
    • Hyphae: Fungi appear as long, branching filaments (mycelia) that grow over the human cell monolayer

Identification: Knowing the Enemy

Identifying the type of contaminant helps determine the source of the breach and the potential for salvage

  • Bacteria (Bacilli/Cocci)
    • Appearance: Tiny rods or spheres. Fast growth (24 hours)
    • Source: Usually skin flora (Staph/Strep) from the laboratory scientist (glove breach, talking in the hood) or water bath splashes
  • Fungi (Mold/Yeast)
    • Appearance: Branching filaments or budding yeast. Slower growth (48–72 hours)
    • Source: The environment. Spores travel in the air (HVAC issues) or reside in incubator humidity pans. Common in “dirty” specimens like POCs or skin
  • Mycoplasma
    • Appearance: Invisible: to the naked eye and light microscope. Does not cause turbidity
    • Detection: Requires specialized stains (DAPI/Hoechst) or PCR testing. It appears as “starry sky” fluorescence on the human cell membrane
    • Danger: It alters cell metabolism and chromosome morphology (fuzziness/breakage), leading to diagnostic errors without killing the culture outright

Control & Remediation: The Response Protocol

When contamination is suspected, immediate action is required to protect the specific patient sample and the surrounding cultures

  • Isolation (Containment): The moment a tube looks suspicious, it must be removed from the incubator. Do not open it to “check.” Opening a sporulating fungal culture in the lab can contaminate the entire room. Tighten the cap and seal it
  • Salvage Attempt (For Irreplaceable Specimens)
    • If the specimen is critical (e.g., bone marrow or amnio) and no backup exists, a “Wash and Rescue” may be attempted if caught early
    • Protocol: Centrifuge the sample \(\rightarrow\) Discard the contaminated supernatant \(\rightarrow\) Wash the pellet 2–3 times with sterile saline \(\rightarrow\) Resuspend in fresh media containing “Shock” Antibiotics (e.g., Penicillin/Streptomycin + Gentamicin + Fungizone/Nystatin)
    • Note: This rarely works for heavy bacterial loads but may control slow-growing fungi enough to get a harvest
  • Disposal: If the specimen is replaceable (e.g., blood) or the culture is dead, the vessel is discarded in biohazard waste. It should be autoclaved to kill the pathogens
  • Decontamination of Equipment
    • The incubator shelf where the contaminated tube sat must be wiped with disinfectant (Quaternary ammonium or 70% Ethanol)
    • If the contamination is fungal, the entire incubator may need to be emptied and decontaminated (copper sulfate treatment) to kill spores

Mycoplasma Control

Since Mycoplasma is invisible, it requires a proactive control strategy

  • Routine Testing: Labs typically test cell lines or random culture batches periodically (e.g., quarterly) using PCR or molecular kits to ensure the lab is Mycoplasma-free
  • Prevention: Use media/serum that is certified Mycoplasma-free. Filter-sterilize reagents through 0.1-micron filters (standard 0.2-micron filters do not stop Mycoplasma)