Radial Diffusion Assay for Tannins

Results of a radial diffusion test for tannins in buried acorns of red oak. The three weUs in each Petri dish contain extract from acorns from the same collection, but buried for different lengths of time 0 days (kept in cold storage) at top in Petri dish 91 days in the grotmd at lower left in Petri dish 141 days in ground at lower right of Petri dish, (a) Extracts of base of acorns (b) extracts of tips (containing embryo) of acorns (c) extracts of whole acoms. Diameter of rings parallels tannin levels 

MuUer-Schwarze, Hands-On Chemical Ecology Simple Field and Laboratory Exercises DOl 10.1007/ 978-l-4419-0378-5 14, Springer Sdence+Business Media, LLC 2009 

In this assay, the interaction of tannins with protein in an agar gel is quantified. The insolnble precipitates form rings around an origin. The diameter of the rings is proportional to the tannin amonnts present. It needs to be added that not all tannins bind to proteins, and not all precipitates are insoluble. The method of this exercise follows the Tannin Assay as described by Hagerman (1987). 

The material analyzed in this exercise can be known amounts of tannins such as tannic acid (a mixtnre of tannins), or plant extracts with unknown amounts of tannin. This exercise is written for tests with plant materials whose tannin level we wish to determine. These can be bnd scales of trees such as aspen, or different parts of acorns (tip vs. base). 

For five plates, dissolve 1 g of agarose in 100 ml water, resulting in a 1% (w/v) solution. To buffer, add 50 mM acetic acid, 60 pM ascorbic acid, adjusted to pH 5.0 with sodium hydroxide. Bring slowly to boil (on hot plate stirrer) to completely dissolve the agarose. Cool to 45°C. (Warning Failure to let the mixture completely cool will denature the protein). Add 100 mg bovine serum albumin (BSA) [to 0.1% 

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We will try to test ourselves whether free-ranging (or captive) squirrels discriminate between acorns that have been buried for several weeks and those that have not. In a later laboratory experiment we will analyze tannin levels in the apical and basal poles of acorns, and also compare buried acorns with untreated controls in this regard. We will use the Radial Diffusion Assay for Tannins (Chap. 14). 

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