Culturing Bean Beetle Microbiota and Phenotypic Analysis

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The method below is based on “Effect of Diet on Bean Beetle Microbial Communities” by Cole et al. to be published in Tested Studies for Laboratory Teaching, Proceedings of the Association for Biology Laboratory Education, Volume 39.

Beetles are sacrificed by freezing them at -800C for 5 minutes. Before collecting and plating the internal microbiota of the beetles, the outside of the beetles is sterilized to avoid growth of external or contaminating microbes.

  • Using sterile tweezers to grasp the beetle, submerge it in the following solutions:
    • 10% Bleach for 3 seconds
    • Sterile water for 10 seconds
    • 70% ethanol for 5 seconds
    • Sterile water for 10 seconds
  • Place the beetle in a sterile microtube and add 450µL 0.9% sterile saline solution.
  • Using the sterile pestle, crush the beetle to release its interior microbiota.
  • Pellet the large debris by spinning in a microfuge for 5 seconds.
  • Plate 100µL of the microbiota solution on each of the following plate types
    • Nutrient agar (NA) – (Difco, #211665)
    • Eosin Methylene Blue (EMB) – selective for gram-negative bacteria – (Fluka, #62087)
    • Blood agar (BA) – selective for fastidious bacteria – (Teknova, #T0134)
    • Phenylethyl alcohol agar (PEA) – selective for gram-positive bacteria – (Criterion, #C6611)
  • The solution will be absorbed by the PEA plate quickly. So, spread this plate immediately after pipetting.
  • Dilute 20µL of the remaining microbiota solution by mixing with 180µL of 0.9% sterile saline and plate 100µL of this onto an NA plate.
  • Plate 100µL of your sterile saline solution on an NA, EMB, BA, and PEA plates to serve as a negative controls.


Describe the unique microbial phenotypes that can observed on the plates and record the number and proportion of bacteria with each phenotype for each plate.

Use characteristics below to describe the phenotypes. For description or images of morphology types see

Color: W (white), O (off-white), R (red), O (orange), Y (yellow), B (brown)

Gloss: S (shiny), M (matte)

Form: C (circular), I (irregular), F (filamentous), R (rhizoid)

Elevation: R (raised), C (convex), F (flat), U (umbonate), Cr (crateriform)

Margin: E (entire), U (undulate), F (filiform), C (curled), L (lobate)