Evaluation of histochemical staining techniques for visualization of bacterial biofilm-associated extracellular matrix in skin

Biofilms play a significant role in histopathology and are complex structures consisting of bacterial cells embedded in an extracellular matrix that contains polysaccharides, proteins, and deoxyribonucleic acid (DNA). The biofilm matrix limits the effectiveness of topical antibiotic treatment in infected wounds and impedes wound healing and immune responses. The purpose of this study was to visualize the biofilm-associated extracellular matrix using standard histological techniques. The commercially available MatTek epidermal full-thickness skin tissue model (EFT-400) was injured and infected for 24 h with biofilm-forming Staphylococcus aureus. Tissue for paraffin sections was fixed in formalin, microwave-processed, and embedded in paraffin. Serial sections cut to 5 microns were stained with Periodic acid-Schiff reagent, Calcofluor, modified Congo Red/Ziehl carbol fuchsin stain and Feulgen reaction. Stained tissues were evaluated using light and fluorescent microscopy. A detailed analysis of the application of the different staining techniques in demonstration of biofilm-associated extracellular matrix revealed that both carbohydrates and DNA were present. Discussion of the value of each staining technique is presented.     

Sequential histochemical staining for resident and recruited macrophages

A new histochemical technique is described that permits differentiation of resident from recruited macrophages by staining of paraffin sections of tissues from rats and mice. Resident macrophages are identified by their ability to phagocytose and retain intravenously injected colloidal Prussian blue. New macrophages that emigrate into tissue are identified by phagocytosis of a second colloid, iron dextran. Paraffin sections of formalin-fixed tissues are sequentially stained for the presence of the two colloids with different chromogens, the endogenous pseudo-peroxidase activity of colloidal Prussian blue used to catalyze the polymerization of diaminobenzidine and after conversion of iron dextran to Prussian blue, the second colloid used to catalyze the polymerization of tetramethylbenzidine. The staining results in resident macrophages staining brown while newly recruited macrophages stain blue. The studies have shown that colloidal Prussian blue is stable in vivo and neither loses its catalytic activity nor undergoes extensive redistribution. They also show that the technique can be used to measure Kupffer cell recruitment stimulated by complete Freund's adjuvant in rats and tumor-associated macrophage recruitment in subcutaneous and spontaneous liver metastases in mice.     

Toluidine blue staining of cartilage proteoglycan subunits.

Selective binding of toluidine blue to basophilic proteoglycans is the first stage of a staining method which proceeds to the formation of a heavy metal salt of the dyestuff. By means of this procedure matrix complexes of hyalin cartilage were stained such as to display granular subunits about 25 to 35 A in diameter. The findings are interpreted as the demonstration of proteoglycan constituents of chondromucin aggregates.     

Multiple epiphyseal dysplasia. 2. Morphological and histochemical investigation of cartilage matrix, particularly in the pre-calcification stage.

The cartilage of epiphysis and cuboid bones from three Beagle puppies bred from parents with a constant heredity of multiple epiphyseal dysplasia, was examined macroscopically, histomorphologically and histochemically. The puppies were euthanized at the age of one week, three weeks and five weeks, respectively. The investigation showed the foci of the disease develop in a broad subarticular zone where normally strong three-dimensional growth of the cartilage takes place. The first signs of the disease appeared about the time of birth and new foci reached a climax when the puppies were about three weeks old. Histomorphological and histochemical examination of the cartilage showed that the disease developed in a sequence of confluent stages. In the initial stage, there was an accumulation of abnormal cartilage matrix in the chondrocyte and/or chondrocyte lacunae. The abnormal substance consisted apparently of concentrated or free chondroitin sulphates not bound to protein. Then followed a second stage of liquefaction and coalescence of the abnormal cartilage matrix which formed cysts of different sizes, some of them visible macroscopically. In the third stage of calcification of the cyst content and surrounding abnormal cartilage, it was found that the calcium was deposited in grains bound to chondroitin sulphates. The results of the present study of the pre-calcification stage of the disease in Beagle dogs seem to be comparable with or equivalent to the results of the study of the post-natal stage of the disease in children and may explain something of the prenatal pathogenesis in children.     

Glycosaminoglycans in the cartilage of the porcine heart as studied by light microscopic histochemical methods

Glycosaminoglycans involved in the cartilage of the porcine heart have been studied by means of light microscopic histochemical methods. The methods employed consisted of a series of staining procedures for the demonstration of acidic groupings of glycosaminoglycans. In addition, some of these procedures were employed in combination with chemical modification and enzyme digestion techniques. According to the results obtained, the cartilage of the porcine heart contained hyaluronic acid, chondroitin, chondroitin sulfate A and/or C and keratan sulfate. The effects of digestion with enzymes upon the histochemical reactions of the cartilage tissues indicated that hyaluronic acid exists as a glycoprotein link factor of proteoglycan subunits, forming proteoglycan aggregates in tissues.