Persistence of Pathogenic Bacteria in UV-Sterilized Equipment Shadows

Problem Statement

This experiment tests whether viable bacteria persist on surfaces that are geometrically shadowed during UV-C sterilization. Only survival on defined surface regions is measured. No clinical, institutional, or policy conclusions are drawn.

Hidden Assumption Being Tested

UV-C sterilization inactivates all surface bacteria on exposed objects, regardless of object geometry or shadowing.

What Might Be True Instead

Bacteria may survive in regions that are physically shadowed from UV-C radiation, even when standard exposure parameters are used.

Test Objects

  • Matte stainless steel or plastic cube (5 cm × 5 cm × 5 cm)
  • Matte stainless steel or plastic cylinder (5 cm diameter × 5 cm height)

UV Parameters

  • UV-C wavelength: 254 nm
  • Intensity: 1.0 mW/cm² (verified at 20 cm)
  • Exposure distance: 20 cm
  • Exposure time: 10 minutes
  • Ambient temperature: 21–25 °C

Sampling Locations

  1. Directly exposed surface (top-facing UV source)
  2. Shadowed object surface (opposite UV source)
  3. Flat surface, unshadowed (no object obstruction)
  4. Flat surface, shadowed (behind object relative to lamp)

Biological Material

  • Non-spore-forming BSL-1 or BSL-2 E. coli
  • Overnight culture adjusted to ~10⁸ CFU/mL

Experimental Procedure

  1. Clean all objects and surfaces with 70% ethanol and air-dry.
  2. Inoculate each sampling area (2 × 2 cm) with 10 μL bacterial suspension (~10⁶ CFU).
  3. Allow all inoculated areas to dry completely (~30 minutes).
  4. Arrange objects to produce reproducible geometric shadows relative to the UV source.
  5. Expose setup to UV-C for 10 minutes at fixed distance and intensity.
  6. After exposure, swab each inoculated area using sterile PBS-moistened swabs.
  7. Elute each swab in 1 mL sterile PBS and vortex briefly.
  8. Plate 100 μL (or appropriate dilution) on nutrient agar.
  9. Incubate plates at 37 °C for 24 hours.
  10. Count colony-forming units (CFU).

Controls

  • Positive control: Inoculated samples not exposed to UV-C
  • Negative control: Uninoculated surfaces exposed to UV-C

Binary Falsification Condition

  • Pass: Zero detectable CFU on all shadowed and unshadowed surfaces after UV exposure.
  • Fail: ≥1 CFU detected on any shadowed surface after UV exposure.

Confounders and Execution Constraints

  • UV intensity must not vary during exposure
  • Objects must have matte (non-reflective) surfaces
  • Shadow geometry must be verified before exposure
  • Triplicate runs required for validity

Safety Boundaries

  • Operate UV-C sources with eye and skin protection
  • Follow institutional biosafety procedures
  • No human or animal testing involved

Scope Limitation

This experiment evaluates surface survival only under the defined geometry, organism, and UV conditions. It does not assess air sterilization, biofilms, or medical device sterility claims.

Edge-of-Practice experiments are designed for direct execution and binary falsification. No extrapolation beyond measured outcomes is permitted.