A Modification of the Tannic Acid Phosphomolybdic Acid-Dye Stain for Demonstrating Myoepithelial Cells in Formalin Fixed Tissue

A modified tannic acid-phosphomolybdic acid-dye procedure is used for staining myoepithelial cells in formalin fixed surgical and autopsy material. Paraffin section are brought to water, mordanted for 1 hr in Bouin's fixative previously heated to 56 C, cooled while still in Bouin's, rinsed in tap water until sections are colorless, rinsed in distilled water, treated with 5% aqueous tannic acid 5-20 min, rinsed in distilled water 30 sec or less, treated with 1% aqueous phosphomolybdic acid 10-15 min, rinsed 30 sec in distilled water, rinsed in methanol, stained 1 hr in a saturated solution of amido black or phloxine B in 9:l methanol:acetic acid, rinsed in 9:l methanol:acetic acid, dehydrated, cleared and mounted. Myoepithelial cells of sweat, lacrimal, salivary, bronchial, and mammary glands are blue-green with amido black or pink with phloxine B. Fine processes of myoepithelial cells are well delineated. Background staining is minimal and the procedure is highly reproducible.     

Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl₂. Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.     

Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-β-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI^q carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.The use of fluorescent proteins as probes has proven to be a vital tool in elucidating spatial and temporal patterns of gene expression, protein localization, and protein-protein interactions in vivo. In addition to well-known contributions to cell biology and biochemistry, the use of fluorescent proteins to label bacteria has provided insight into complex host-pathogen interactions and routes of entry (14, 23, 29).The zebrafish has become an attractive model for in vivo infection studies. As an optically clear organism at the embryo and larval developmental stages, zebrafish are amenable to visualization during challenge with fluorescently labeled pathogens. In addition, fertilization in the zebrafish occurs ex utero, thereby simplifying infection by either static immersion or microinjection. The visualization of infection in situ can be conducted on the whole embryo using confocal or wide-field epifluorescence microscopy, an approach not possible in other model systems. To date, several zebrafish infection models have been established employing both fish and mammalian pathogens, including Edwardsiella tarda (21), E. ictaluri (14, 23), Staphylococcus aureus (16), Streptococcus pyogenes and S. iniae (18), and Pseudomonas aeruginosa (4). Illustrating the value of fluorescently labeled bacteria in studies of microbial pathogenesis, Van der Sar and colleagues established the zebrafish as a model for Salmonella enterica serovar Typhimurium infections using DsRed-labeled bacteria (30). Similarly, O''Toole and colleagues made use of a green fluorescent protein (GFP)-labeled strain of Vibrio (Listonella) anguillarum to elucidate the natural route of invasion of this fish pathogen (20). However, obstacles to the construction of fluorescently labeled microbes have prevented many from taking advantage of this technique to permit the real-time in situ analysis of infection. These obstacles include the nontransformable nature of many pathogens and the toxicity of RFP expression from high-copy-number plasmids.Recently, two transgenic lines of zebrafish that express GFP in cells of the myeloid lineage have been developed. Lawson et al. described a transgenic (Tg) zebrafish that utilizes the protooncogene fli1 promoter to drive the expression of enhanced GFP (EGFP) (15). The fli1 gene is preferentially expressed in endothelial and hematopoietic cells. Intended for the study of vascular development, Tg(fli1::EGFP) zebrafish not only have fluorescent endothelial cells but also macrophages (15), making this zebrafish line attractive to those who study innate immunity. It has been shown that zebrafish macrophages possess strong phagocytic properties and migrate to sites of bacterial infection (13). In 2006, Renshaw and his colleagues engineered a novel Tg(mpo::GFP) zebrafish that expresses GFP under the control of the neutrophil-specific myeloperoxidase promoter (22). In this study, they characterized the GFP-labeled neutrophils, noting their capability of chemotaxis at wound sites (22). Given the recent progress in zebrafish genetics with regard to these GFP-expressing transgenic lines, there was a clear need for a simple method for the RFP labeling of Gram-negative bacteria. Such a method would facilitate further studies of microbial pathogenesis in the zebrafish and could further elucidate phagocyte (GFP labeled)-pathogen (RFP labeled) interactions, kinetics of bacterial clearance, and mechanisms of phagocyte migration to sites of bacterial infection.For the purposes of this study, we employed dimeric (d) and monomeric (m) red fluorescent protein variants d-Tomato, td-Tomato, m-Orange, and m-Cherry, which were constructed by Shaner et al. (25). These variants were chosen based on combinations of their high relative brightness and decreased RFP maturation times compared to those of the original RFPs (25). We describe here a simple method for the RFP labeling of Gram-negative bacteria.     

Nucleic Acid Enzyme Studies of Nonfermentative Gram-Negative Bacteria Using Thin-Layer Chromatography

A rapid and easy to perform procedure for determining the nucleic acid enzyme reactions of intact bacterial cells was developed. Overnight organism growth on triple sugar agar was tested for nucleoside phosphotransferase, nucleosidase, and nucleotidase activity. Reaction products were detected by means of thin-layer chromatography and fluorescence. Characteristic patterns were seen with certain strains of nonfermentative gram-negative bacteria, which indicated that these tests could aid in classification and identification of E0-1 group, Pseudomonas multivorans, P. cepacia, P. maltophilia, certain other Pseudomonas species, and Herellea vaginicola.     

An Improved Stain for Bacteria in Soil

A stain for bacteria in soil is proposed different from that originally employed in the introduction of pre-treatment of the soil smears with dilute acid. The procedure found best is: A suspension of soil in a 0.015% solution of gelatin is dried on a slide, treated 1-3 minutes in 40% acetic acid, washed and dried on a boiling water bath; while still on the water bath it is stained about one minute with 1% aqueous rose bengal. It is suggested however that under certain conditions better results may possibly be obtained by substituting hydrochloric for acetic acid and one of the phloxines or one of the erythrosins for rose bengal.     

An Improved Stain for Bacteria in Soil

A stain for bacteria in soil is proposed different from that originally employed in the introduction of pre-treatment of the soil smears with dilute acid. The procedure found best is: A suspension of soil in a 0.015% solution of gelatin is dried on a slide, treated 1-3 minutes in 40% acetic acid, washed and dried on a boiling water bath; while still on the water bath it is stained about one minute with 1% aqueous rose bengal. It is suggested however that under certain conditions better results may possibly be obtained by substituting hydrochloric for acetic acid and one of the phloxines or one of the erythrosins for rose bengal.     

Detection of Arginine Dihydrolase in Nonfermentative Gram-Negative Bacteria by Use of Thin-Layer Chromatography

Thin-layer chromatography was used to detect the presence of the arginine dihydrolase system in nonfermentative gram-negative bacteria. The test was positive for the fluorescent pseudomonads as well as Pseudomonas maltophilia, whereas other Pseudomonas sp., Mima, and Herellea were negative. This procedure can be completed in a few hours and may be useful in the clinical laboratory.     

The Use of Picric Acid with the Gram Stain in Plant Cytology

The technic described involves the use of a saturated solution of picric acid in absolute alcohol in the process of dehydration following the gentian-violet-iodine stain as applied to plant cytological material. The method is suitable for both paraffin sections and smears of pollen mother cells fixed in Navashin's or Flemming's solutions. Differentiation in clove oil is very easy since cytoplasm destains immediately, while chromatic material destains very slowly following picric acid. Chromosomes are stained more distinctly than with the usual Gram stain and do not fade.     

The Growth of Gram-Negative Bacteria in the Hen's Egg

Summary: Bacteriological and chemical methods were used to follow the course of infection in eggs, incubated at 27°, the air cells of which had been inoculated with a suspension of washed bacteria. In the 3–4 days following inoculation, limited bacterial multiplication occurred in the inner membrane of the air cell but very few organisms entered the albumen. These populations then remained static or decreased slightly until renewed multiplication occurred 12–30 days after inoculation. This was induced by contact of the yolk and the shell membranes: it occurred on the 12–20th day in eggs in which the yolk moved towards the site of inoculation, but later when the yolk moved in the opposite direction. At this time there was a general infection of the egg contents and significant changes occurred in the pH and glucose concentration in the albumen. In eggs that had been inoculated with chromogenic and/or proteolytic bacteria, the first macroscopic changes of the contents were seen at this time. The rate and extent of the initial multiplication was influenced by the composition of the fluid used to suspend the washed bacteria and, in all instances, the fastest multiplication occurred when iron was added to the inoculum. Moreover, renewed multiplication occurred when iron was added to the albumen of eggs in which the bacteria were in the stationary phase.     

Extractable Lipids of Gram-Negative Marine Bacteria: Phospholipid Composition

Phospholipid compositions of 20 strains of marine and estuarine bacteria were determined. Results showed that phospholipids of marine bacteria differed very little from those of nonmarine organisms with phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol being the predominant phospholipids in all strains examined. Lyso-phosphatidylethanolamine occurred in significant quantities among a number of the marine bacteria, and two of the isolates contained significant quantities of poly-beta-hydroxybutyrate. Effects of age and growth temperature on the phospholipid composition were also investigated. It is suggested that phylogenetic relationships among bacteria may be correlated with phospholipid composition.     

Tannic Acid in Histology: An Historical Perspective

The development of tannic add as a reagent in histological methods is traced against a background of widespread use in science and technology from times of antiquity. Numerous light microscopic methods involving tannic add, particularly in conjunction with iron and silver, have been described for a variety of tissue components. In most applications, tannic add functions as a mordant. Current use is generally restricted to methods based on its affinity for collagen. The most significant histological use of tannic add in contemporary times is as an adjunct to conventional glutaraldehyde-osmimn-heavy metal fixation and staining for ultrastructural studies of tissue structures not normally clearly demonstrated. Tannic add reacts with various components by mechanisms which are often not fully understood.     

Tannic Acid-Iron Alum Reaction: Stain of Choice for Macroscopic Sections of Brain to be Embedded in Plastic

As a macroscopic stain for gross brain sections to be embedded in plastic, tannic acid-iron alum is superior to the generally recommended LeMasurier's variation of the Berlin blue technique because of its greater permanency in plastic. However, as originally adopted for use with brain tissue by Mulligan, the intense black staining of gray matter is too dark for plastic embedded specimens. A modification of this method designed to overmme this difficulty is described. Staining procedure: Wash formalin-fixed brain slices overnight in running water. Wash in distilled water, 2 changes, 30 minutes each. Place slices individually in Mulligan's solution at a temperature of 60-65 C for 4 minutes. Rinse in ice water for 10 seconds. Mordant in 0.4% tannic acid in distilled water for 1 minute. Wash in running tap water for 1 minute. Develop in 0.08% ferric ammonium sulfate in distilled water until gray matter is light gray, about 10-15 seconds. Wash in lukewarm running water for 1 hour, then gently hand-rub whitish film from myelinated surfaces. Store briefly in 3% formalin or 25% glycerine if necessary depending on plastic embedding procedure to be followed.     

A Polychromatic Stain for Use in Parasitology

In sensory systems, insight into synaptic arrangements on cells of known physiological response properties has helped our understanding of the structural basis for these properties. To carry out these types of studies, however, synaptic types in the region of interest must be defined. Unfortunately, defining synaptic types in the brainstem has proved to be a challenging enterprise. Our study was done to classify synapses in the gustatory part of the nucleus solitarius using objective quantitative criteria and a cluster analysis procedure. Cluster analysis allows classification of a population of objects, such as synaptic terminals, into groups that exhibit similar characteristics. Six terminal types were identified using cluster analysis and subsequent analyses of variance and post hoc tests. Unlike classification schemes used for the cerebral cortex, where synaptic apposition density thickness and shape of vesicles is useful (Gray's Type I and II synapses), the concentration of vesicles in a terminal was a more useful measurement with which to classify terminals in the nucleus solitarius. To validate that vesicle density (vesicles/μm²) is a useful defining characteristic to classify terminals in the nucleus solitarius, terminals of a known type were used. GABAergic terminals were identified using postembedding immunohistochemical techniques, and their vesicle density was determined. GABAergic terminals fall into the range of two of the terminal types defined by the cluster analysis and, based on vesicle density, two types of GABAergic terminals were identified. We conclude that vesicle density is a helpful means to identify synapses in this brainstem nucleus.     

Tannic Acid Supplemented Fixation Improves Ultrastructural Evaluation of Respiratory Epithelium in Children with Recurrent Respiratory Tract Infections

Samples of the respiratory mucosa of children with recurrent respiratory infections suspected of having primary ciliary dyskinesia are routinely fixed with glutaraldehyde before ultrastructural examination. This standard technique, however, may not be optimal for visualizing ciliary components or for preserving several cellular and extracellular structures during dehydration and embedding procedures. In this study, brushes of nasal (28 samples) and/or tracheal (9 samples) mucosa from 32 children with recurrent respiratory tract infections were examined. Twenty-nine samples were fixed with glutaraldehyde supplemented with tannic acid to determine if the ultrastructural analysis of respiratory epithelium and bronchial secretions could be improved. Eight samples were conventionally fixed with glutaraldehyde alone. Lesions of the cellular membrane and damaged cells were easily visualized using tannic acid supplemented fixation. Internal ciliary structures including individual microtubules and dynein arms were also more clearly observed. In addition, the internal structure of microvilli of the respiratory epithelium could be studied and the presence of phospholipid-rich surfactant-like material within nasal and tracheal secretions were visualized after tannic acid supplemented fixation. We suggest that addition of tannic acid during fixation is useful for accurate ultrastructural evaluation of respiratory mucosa in both clinical and experimental situations.