Bee incubation medium preparation based on Kaatz (1985) amino acids (AA) (50 ml):

  1. Dissolve L-Cysteine  (5 mg) in 2 ml 1M HCl,
  2. Add L-Tyrosine (10 mg) to 40 ml of double distilled water.
  3. Dissolve the remainder amino acids one after the other: 60mg L-alanine, 51mg L-arginine, 20mg L-aspargine, 10mg L-aspartic acid, 25mg L-glutamic acid, 100mg L-glutamine, 20mg L-glycine, 20mg L-histidine, 10mg L-isoleucine, 55mg L-lysine, 5 mg L-methionine, 20 mg L-phenylalanine, 330mg L-proline, 20mg DL-serine, 15mg L-threonine, 5mg L-tryptophan, 15mg L-valine. .
  4. Add double-distilled water to make up the volume to 50 ml.

Inorganic Salts (IS) (20 ml):

Dissolve 179mg KCl, 40.6mg MgCl2*6H20, 49.3mg MgSO4*7H2O, 69mg NaH2PO4*H2O, 33.6 mg NaHCO3, in 20 ml DDW.

Sugars (15ml):

Dissolve 400mg glucose, 250mg fructose and 6700 mg sucrose in 15ml DDW.

Mixture of all components

  1. Mix the three groups of components (AA, IS and sugars) with piperazine-N,N′-bis(2-ethanesulfonic acid buffer (PIPES) (756 mg).
  2. Titrate with NaOH until PIPES is completely dissolved.
  3. When all components are dissolved, add NaCl (100mg).
  4. Adjust the pH with NaOH to 6.7.
  5. Add 2ml of CaCl2*2H2O (56.45mg/ml in DDW) to the medium.
  6. Bring the medium to a final volume of 100 ml.
  7. Sterilize the medium by filtration via Millipore 0.22 µm.