1. Baker's acid haematein test for phospholipines is specific provided that only a definite positively result is considered. Very pale blues and greys may be caused by other lipoids, which if present in very large masses may possibly show medium to dark blue granules but will not be coloured all through.
2. The mechanism of the test appears to be as follows:
(a) Phospholipine is not fixed by formal-calcium but is restrained from passing into solution by the calcium ions, which play no other part.
(b) Phospholipine combines readily with chromium from the mordanting fluid, and is then rendered insoluble and mordanted. Other substances, acidic and usually containing phosphorus, are mordanted as well.
(c) On staining, blue and brown colorations are formed; in both cases the dye attaches itself to the chromium in the various substrates.
(d) On differentiation, some browns and most blues, particularly those with phosphoric substrates, remain nearly fast, but most browns and the weak blues of certain lipoids (not phospholipines) are greatly reduced or removed entirely. The period of differentiation must not be shortened.
(e) Blue-staining lipoids (phospholipines) are distinguished from other blue-staining substances by an extraction with the lipoid solvent pyridine, after special fixation. The other substances, and any bound lipoid not removable with pyridine, remain.
3. Since the specificity of the test depends on the relatively greater affinity of phospholipines among lipoids for the mordant, the period of chroming must not be lengthened.
4. One reason why some substances are coloured after pyridine extraction but not after acid haematein is that in the former case they are precipitated and so concentrated; in the latter they are not. This is not a general explanation for the whole class of such substances.