3.3.1. Fixing honey bee gut samples from fresh bees

  1. Dissect the gut from the bee in PBS (0.8% NaCl [w/v], 0.02% KCl [w/v], 0.115% Na2HPO4 [w/v], 0.02% KH2PO4 [w/v], pH 7.5) or Insect Ringer’s solution.
  2. Freshly prepare formaldehyde fixative mixing 9 vol. of PBS and 1 vol. of 40% formaldehyde solution (i.e. 4% formaldehyde in PBS). It would be better to use more than 10 volumes of the fixative to that of a specimen.
  3. Fix the tissue by soaking the gut samples in the freshly prepared formaldehyde fixative, overnight at 4˚C.
  4. After fixation, wash the tissue in PBS at least three times for 10 min at room temperature.
  5. Follow with at least three washes in 75% ethanol for 30 min at room temperature.
  6. Transfer the gut samples to absolute ethanol for storage until used. You can keep the samples in absolute ethanol at least a week, and perhaps longer.