Deep-well plates, "blocks"

  • Generally, shaking cultures of ⅓ well volume can be regarded as the maximum that prevents cross-well contamination, at least at 800 rpm.
  • Culturing 2 mL allows later centrifugation/minipreps in 2 mL tubes, compatible with the minimum shaking well volume of 24- and 48-well blocks.
  • Square wells with pyramidal "V-bottoms" are best for agitation/aeration than round wells and U-bottoms.

  • 24-well block
    • almost 10 mL well volume to the top.
    • 3 mL max shaking culture volume, to prevent cross-well contamination.
    • Other types/lids
  • 48-well block

    • 7.5 mL well volume

    • 2.5 mL max shaking culture volume, to prevent cross-well contamination.

  • 96-well block
    • almost 2 mL well volume to the top.
    • ⅔ mL max shaking culture volume, to prevent cross-well contamination.
    • Other types

Seals

Ideally, seals minimize cross-contamination and evaporation and allow uniform O2/CO2 exchange for all wells, unlike plate lids which favor exchange for wells near the plate edges.
Use of brayer ("plate roller") assures a secure uniform seal around all wells.

  • AeraSeal™ - Excel Scientific
    • Maximal aeration/gas exchange; at the expense of still considerable evaporation over longer culture time. Best used with ≥0.5 mL cultures. Ideal for non-experimental growth (DNA/strain construction).
    • Material: 140 µm thick hydrophobic porous rayon with medical-grade adhesive
    • Sterile, tip-pierceable, -20°C–80°C. Similar or identical to Breathe-Easier membranes.
    • Directions: partially peel off one side of the main bottom paper, and holding via the protected tabs, align and adhere the exposed adhesive to the plate; then adhere the remainder as you fully peel off the paper across its length, ensuring it adheres flat with no bubbles or creases. Complete the seal by rolling a brayer firmly on top. Removing the bottom paper for the tabs is not necessary; the tabs allow the film to be easily peeled on/off later.
    • For normal cloning of plasmids, it is effective to use as many wells as needed, and then save blocks with the AeraSeal first used still affixed, and later use any remaining unused wells with the same seal. This is best done by never fully removing the AeraSeal at one end, thus preserving its original alignment with the wells. The adhesive is strong enough for several rounds of peeling and reaffixing with a brayer.
  • Breathe-Easy™
    • Best for evaporation protection, but slower bacterial growth because of partly impaired gas exchange.
      • David Zong found ~½ fluorescent protein expression with Breathe-Easy compared to open wells, in 96-well assay plate. Wells under a crease in the film grew ~2-fold more than properly sealed wells.
    • Material: polyurethane with acrylic adhesive.
    • "Permeable to oxygen, carbon dioxide, and water vapor"; sterile, tip-pierceable, -80°C–100°C
    • Gas permeability: 553.5 cm3 O2 / 100 in2 · 24 h; 3781 cm3 CO2 / 100 in2 · 24 h
    • Directions: partially peel off the the left side edge of the bottom paper, and holding via the left protected tab, align and adhere the edge of the exposed adhesive to the plate. 
      Place a flat, straight item on the adhered edge and use it to uniformly push down the remainder of the seal's adhesive bottom while you slowly fully peel off the back paper across the seal's length, ensuring it adheres flat with no bubbles or creases. It is essential that the seal is completely flat, or else wells with bubbles/creases will have greater oxygenation and non-uniform growth relative to other wells. Complete the seal by rolling a brayer firmly on top, and only then remove the top paper to expose the membrane.
  • Silicone and santoprene lids can be purchased

Cleaning Procedure

  1. Add bleach to plates to a rough final 10% to wells containing culture, and add 10% bleach to empty, contaminated wells, wetting the well walls. Let sit 20–30 min before draining and rinsing in tap water, scrubbing out any adhering material. Plate should be spotless when held up to light.
  2. Lastly, fill the plate close to its brim with tap water and leave on your bench or in the plate holding tray next to the tip box bench for at least a day, allowing the water and its metals to solubilize and decompose residual hypochlorite. Eventually, drain these and return to the bin.
  3. Sterilization — When plates become limiting, plates in the holding bin should be filled with tap water to the brim, be placed in monolayers in autoclave bins, and be autoclaved 45 min on the liquid cycle. As soon as the cycle is over, while the contents is still hot, the water should be drained and residual water flung out.
  4. Storage — Once dry, plates can stacked inverted on the cabinet shelf wiped dust-free with ethanol and lined with piece of foil. Blocks may be taken one at a time, always stored inverted to protect their insides from dust. Culture with antibiotics and fast-growing E. coli makes more stringent sterility not useful.

 

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