In the large elongated, hexagonal cells of the seed membrane there are two other structures to be seen with the aid of high power (250 fold), one appearing as large crystalline druses, while the other consists of extremely fine needles united in bundles.
Fig. 4.
Cross section of the cotyledon, showing “Mitscherlich particles” (Moeller).
By addition of petroleum spirit the former, consisting of fat acid crystals, are dissolved, the latter, remaining unaltered, are considered by Mitscherlich to be theobromine crystals, since their crystalline form closely resembles that of theobromine. A more scientific explanation has not been forthcoming.
The cotyledons are seen under the microscope to consist of a tissue of thin walled cells, without cavities, lying close together, and here and there distributed through the tissue, cells with brownish yellow, reddish brown, or violet coloured contents. These latter are the pigment cells which contain the substance known as cacao-red and analogous to tannin; it, together with theobromine, gives rise to the delicate taste and aroma of cacao. The other cells of the tissue are filled with extremely small starch granules the size of which rarely exceeds 0.005 mm.; with them are associated fat, in the form of spear-shaped crystals, and albuminoid substances.
In order to discriminate between these substances they must be stained by various reagents. According to Molisch[17], theobromine may be recognised, in sections of the seed, by adding a drop of hydrochloric acid and after some time an equal drop of auric chloride solution (3 %) After some of the liquid has evaporated, bunches of long yellow crystals of theobromine aurochloride make their appearance. On addition of osmic acid the fat is coloured greyish brown. On addition to the microscopic section a drop of iodine solution, or better iodozine chloride, the starch becomes blue, while albuminous substances are coloured yellow. Cacao starch granules are very small and cannot well be mistaken for other kinds, except the starch of some spices such as pimento or that of Guarana, prepared from the seeds of Paulinna sorbilis. According to Möller the blue iodine colouration of cacao starch takes place very slowly and it is probably retarded by the large amount of fat present; but the point has been contested by Zipperer and later investigators.
In order to make the starch granules of cacao and the cells containing cacao-red distinctly visible under the microscope, it is advisable to immerse the section in a drop of almond oil, because the addition of water renders the object indistinct in consequence of the large amount of fat present. Another excellent medium for the microscopic observation of cacao is the solution of 8 parts of chloral hydrate in 5 parts of water, as recommended by Schimper.[18]
By these means it may easily be seen that the pigment or cacao red in different sorts of cacao varies more or less in colour.
To complete the account of the microscopic characters of the cacao cotyledon, mention must be made of the small vascular bundles, generally spiral, that are distributed throughout the tissues of the cotyledons and are readily made visible by adding a drop of oil or a drop of chloral hydrate solution.