Palynological analysis: Stingless bee honeys were qualitatively and quantitatively pollen analyzed, by using the acetolysis
technique (Louveaux, Maurizio, & Vorwohl 1978).
HCL acid in 10% water solution, cold HF 70% for 24 hours, 30% HCL 60[degrees]C, washed with distilled warm water, and 12 minutes acetolysis
at 100[degrees]C washed with glacial acetic acid and distilled water.
Pre-anthesis buds of the three Apiaceae species were collected and transported in glass vials with 70% alcohol to the Palynology Laboratory belongs to the Botanical Department of the National Museum at the Universidade Federal do Rio de Janeiro-UFRJ (Rio de Janeiro, RJ, Brazil), where they were submitted to the acetolysis
method with modified proposals (Erdtman, 1960; Melhem et al., 2003), for the morphological characterization of the pollen grains and reference slide assembly for each botanical species.
The samples were treated in the laboratory with 10% KOH, sieved to remove larger pieces of plant material, undergone acetolysis
and heavy liquid separation in zinc bromide/hydrochloric acid for the samples in which some mineral residue was present especially in some clayey peat or organic soil samples (Wood et al.
After cleaning the samples, pollen grains were further dehydrated in glacial acetic acid and prepared for melissopalynological analysis using the acetolysis
method (Erdtman, 1960).
Briefly, a sample of 10g of crude honey was dissolved in 20ml of distilled water, centrifuged at 2000rpm during 10min, washed once with 10ml of distilled water, centrifuged again and part of the sediment embedded in glycerin jelly without previous acetolysis
. The pollen sediment was transferred to 75 x 25mm slides; after being warmed slightly, the melted jelly with pollen sediment was covered by a 22mm cover glass and the latter sealed with paraffin wax (Nair 1960).
Cellulose undergoes acetolysis
with acetic/nitric reagent forming acetylated cellodextrins which get dissolved and hydrolyzed to form glucose molecules on treatment with 67% [H.sub.2]S[O.sub.4].This glucose molecule is dehydrated to form hydroxymethyl furfural which forms green coloured product with anthrone and the colour intensity is measured at 630 nm using spectrophotometer (Lambda-45 Perkin Elmer, Shelton CT 06484, USA).
Microscope slides of the pollen samples were made with the acetolysis
method (Erdtman 1960; Barth & Luz 1998; Terrab et al.