18.104.22.168 Bee incubation medium preparation based on Kaatz (1985) amino acids (AA) (50 ml):
- Dissolve L-Cysteine (5 mg) in 2 ml 1M HCl,
- Add L-Tyrosine (10 mg) to 40 ml of double distilled water.
- 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. .
- Add double-distilled water to make
up the volume to 50 ml.
Inorganic Salts (IS) (20 ml):
KCl, 40.6mg MgCl2*6H20, 49.3mg MgSO4*7H2O,
69mg NaH2PO4*H2O, 33.6 mg NaHCO3,
in 20 ml DDW.
Dissolve 400mg glucose,
250mg fructose and 6700 mg sucrose in 15ml DDW.
Mixture of all components
- Mix the three groups of components (AA, IS and sugars) with piperazine-N,N′-bis(2-ethanesulfonic acid buffer (PIPES) (756 mg).
- Titrate with NaOH until PIPES is completely dissolved.
- When all components are dissolved, add NaCl (100mg).
- Adjust the pH with NaOH to 6.7.
- Add 2ml of CaCl2*2H2O (56.45mg/ml in DDW) to the medium.
- Bring the medium to a final volume of 100 ml.
- Sterilize the medium by filtration via Millipore 0.22 µm.