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 sporo gonic stages. When using standard blood staining procedures for those enigm atic parasites it is sometimes difficult to distinguish them from fish host tissue. They clearly exhibit a partial Gram-positive reaction in histologi cal sections, but staining is variable in air dried fish organ imprints. To visualize the Gramnegative background of different host tissue components in histological sections, the conventional safranin counterstain of the Gra m protocol may be modified as follows: after application of 2% crystal viol et (basic violet 3) and Lugol's solution, sections are stained with 0.1% nu clear fast red-5% aluminum sulfate and 0.35% aniline blue (acid blue 22) di ssolved in saturated aqueous picric acid. Replacement of the Gram-specific dye crystal violet with 2% malachite green gave similar results in organ im prints containing myxospores or coccidia, but only in sections containing m yxosporea. Staining for I min with an aqueous solution of 0.5% malachite gr een and followed 1 min washing was sufficient for rapidly demonstrating the parasite spores in organ imprints of both myxosores and oocysts. With rega rd to the role of acid mucopolysaccharides and other carbohydrates in the G ram reaction of spores, alcian blue 8GX staining was compared to the bindin g of FITC-labeled WGA, GS I and GS II. Each lectin was applied at 20 mul/ml PBS, HEPES for 1 hr. Whereas WGA yielded a nonspecific pattern like the al cian blue staining, GS II resulted in a pattern similar to the Gram stainin g results. This binding was weak in untreated specimens, but was significan tly enhanced when digested first within trypsin overnight in a humid chambe r at 37 degreesC. 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 hexosam ine plays a key role in the Gram reaction.