Gram staining and lectin binding properties of Myxosporea and Sporozoea

The staining method developed by Christian Gram was introduced as a simple and highly selective tool for demonstrating myxosporean and coccidian sporogonic stages. When using standard blood staining procedures for those enigmatic parasites it is sometimes difficult to distinguish them from fish host tissue. They clearly exhibit a partial Gram-positive reaction in histological sections, but staining is variable in air dried fish organ imprints. To visualize the Gram-negative background of different host tissue components in histological sections, the conventional safranin counterstain of the Gram protocol may be modified as follows: after application of 2% crystal violet (basic violet 3) and Lugol's solution, sections are stained with 0.1% nuclear fast red-5% aluminum sulfate and 0.35% aniline blue (acid blue 22) dissolved in saturated aqueous picric acid. Replacement of the Gram-specific dye crystal violet with 2% malachite green gave similar results in organ imprints containing myxospores or coccidia, but only in sections containing myxosporea. Staining for 1 min with an aqueous solution of 0.5% malachite green and followed 1 min washing was sufficient for rapidly demonstrating the parasite spores in organ imprints of both myxosores and oocysts. With regard to the role of acid mucopolysaccharides and other carbohydrates in the Gram reaction of spores, alcian blue 8GX staining was compared to the binding of FITC-labeled WGA, GS I and GS II. Each lectin was applied at 20 μl/ml PBS, HEPES for 1 hr. Whereas WGA yielded a nonspecific pattern like the alcian blue staining, GS II resulted in a pattern similar to the Gram staining results. This binding was weak in untreated specimens, but was significantly enhanced when digested first within trypsin overnight in a humid chamber at 37 °C. The binding of GS II to both myxosporidian and coccidian spores suggests that they are both composed of polymers containing N-acetyl-D-glucosamine residues. Furthermore, the results suggest that this hexosamine plays a key role in the Gram reaction.