When cleaning glass beads, it's not sufficient to simply sterilize of cells, but also destroy all DNA, and perhaps even labile organic molecules such as antibiotics, proteins, cofactors, quorum signals — molecules that could interfere with sensitive experiments. Many researchers use ethanol like a panacea, but DNA is insoluble in ethanol (hence it being the main component in DNA purification wash buffers). Ethanol is fine for preventing microbial growth on used beads and drying of residue on them, which otherwise makes cleaning more difficult.

For routine cleaning, you need to destroy organic molecules. Some protocols use an HCl solution, but it's been found that even a strong acid-wash is inferior to simple bleach in destroying DNA¹ ². A good protocol may look as below.

Dilutions of bleach (commonly 10%) ought to be used within a week or two to maintain enough hypochlorite to be effective, so make only squirt-bottle quantity dilutions. Use deionized water in dilutions, as metals in hard water accelerate hypochlorite decomposition, and store in an opaque container or dark cabinet if possible, as hypochlorite is photolabile.

¹ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943653/
² https://www.ncbi.nlm.nih.gov/pubmed/1571142

Procedure

  1. Ensure used beads are collected soaking in water or 20% ethanol to ease cleaning.
  2. Fully immerse beads in a fresh 20%V/V solution of commercial bleach (Chlorox, ≈5% NaOCl) made using diH2O. Let soak for ≥30 min, stirring a several times in the interim. Strain out bleach.
    • Shaking overnight in pure bleach is likely much overkill. Doubling the minimum 10% concentration is a sufficient excess.
  3. Rinse beads in four iterative volumes of water to dilute away residual bleach: thrice with tap water and lastly with deionized water. For each rinse, stir thoroughly and strain out all rinse water.
  4. Dry beads in an oven in the same container.
    • Or use another trusted, immaculate, detergent-free container.
  5. Aliquot into glass culture tubes (or otherwise). Autoclave on the pipette tip cycle (vacuum, not gravity) with a 30 min sterilization time.
    • Some autoclave beads for as long as 2 hr for sensitive PCR applications or resilient spores.
    • If bead aliquots in culture tubes are damp, attempt to dry them in the oven.
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