The Histochemical Detection of iron in Tissues

The fixation and staining of iron in tissues is discussed. Procedures for demonstrating iron in hemoglobin and nuclei are also briefly considered.

Lillie's formalin buffered at neutrality gave the optimal fixation for iron. The Prussian blue method was preferred to the Turnbull blue. Lison's procedure of the former, slightly modified, gave the most satisfactory results. When a procedure is required that employs non-iron-containing reagents, Macallum's or Mallory's hema-toxylin and Quincke's ammonium sulfide method are useful. The former, though not entirely specific, is preferable under controlled conditions when the quantities of iron are small. Hemoglobin iron in paraffin sections can be demonstrated by the usual procedures for iron after previous exposure of the section to peroxide, as recommended by Brown. The property of nuclei to adsorb iron from inorganic sources and from hemoglobin can readily be shown; caution is required in interpreting the iron detected in nuclei after Macallum's sulfuric acid hydrolysis.     

Observations on the Staining of Corynebacterium Diphtheriae

Albert's method, of staining diphtheria cultures consists of staining a fixed smear for one minute (some laboratories stain for five minutes) with a solution containing toluidine blue and malachite (or methyl) green, washing with water, and applying Albert's iodine for one minute. This procedure is discussed and criticized, and in addition the mechanism of the stain is elucidated. Also, the procedure which involves staining a fixed smear for one minute with Loeffler's alkaline methylene blue solution is discussed and criticized.

To overcome the objections to the above staining methods, a different method is proposed. This consists of staining a fixed smear with an acid solution of toluidine blue, washing with water, applying Albert's iodine for one minute, washing with water, and finally applying a safranin solution for 15-20 seconds. The theoretical basis for this method is presented.     

The Oxidation Products of Methylene Blue

The mechanism of the oxidation of methylene blue varies with the conditions. The formation of trimethyl thionin (azure B) and of asymmetrical dimethyl thionolin (azure A) is followed under alkaline conditions by that of dimethyl thionin (methylene violet) and under acid conditions by that of monomethyl thionin (named by authors azure C).

Simple and practical methods are given for the preparation of azure A and azure C. The latter product, which has not been obtained from methylene blue hitherto, has valuable staining properties as a nuclear and bacterial stain in tissue and may also be employed satisfactorily as a substitute for azure A in the MacNeal tetrachrome formula as a blood stain or substitute for the Giemsa stain.

Azure B has no particular merit in staining.

Azure C proves to be a very valuable stain. A procedure is given for its use with eosin Y and orange II as counterstains, by which it is possible to demonstrate bacteria in tissue and at the same time the cytological elements of the tissue.     

Combined in situ zymography, immunofluorescence, and staining of iron oxide particles in paraffin-embedded, zinc-fixed tissue sections

Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.     

Combined In Situ Zymography, Immunofluorescence, And Staining Of Iron Oxide Particles In Paraffin-embedded, Zinc-fixed Tissue Sections

AbstractSuperparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.     

Purification of outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae

A system of protein purification, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, that results in purified outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae is described. The proteins, which ranged in apparent molecular mass from approximately 31,000 to approximately 92,000 Da, were located by naphthol blue black staining, eluted from nitrocellulose membranes using 88% formic acid, and precipitated by the addition of concentrated ammonium hydroxide. Up to 65% of the original protein present was recovered by this procedure. The resultant purified protein could then be resuspended in aqueous buffer by brief sonication, making it available for further structural and in vivo immunological analyses. Proteins purified in this manner retain their original antigenicity when probed with polyclonal and monoclonal antibodies, and are structurally unaltered by the purification process. This procedure makes it possible to acquire easily usable quantities of highly insoluble outer membrane proteins of gram-negative bacteria.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.     

A Combination Verhoeffs Elastic and Masson's Trichrome Stain for Routine Histology

A method for the combined staining of elastic, muscle and connective tissue for routine use in histopathology is described. The elastica, stained black by Verhoeffs technique, is contrasted with the muscle and connective tissue stained red and green or blue respectively by a modification of Masson's trichrome. Cell nuclei stain blue-black with Weigert's iron hematoxylin. The procedure takes approximately two hours and is most suitable for the study of vascular pathology in surgical and autopsy sections.     

A combination Verhoeff's elastic and Masson's trichrome stain for routine histology

A method for the combined staining of elastic, muscle and connective tissue for routine use in histopathology is described. The elastica, stained black by Verhoeff's technique, is contrasted with the muscle and connective tissue stained red and green or blue respectively by a modification of Masson's trichrome. Cell nuclei stain blue-black with Weigert's iron hematoxylin. The procedure takes approximately two hours and is most suitable for the study of vascular pathology in surgical and autopsy sections.     

A Staining Procedure for Use in the Brucella Opsonocytophagic Test

The following staining procedure is recommended for use in the Brucella opsonocytophagic test in order to avoid confusing results obtained with stains of the Hasting or Wright type: Fix spreads for 5 minutes or longer in absolute methyl alcohol. Stain for 10 to 30 minutes in a solution of the following: 0.5 g. NaCl, 0.5 g. phenol, 0.5 g. methylene blue, 0.02 g. Na₂HPO₄+12H₂O, 50 cc. distilled water, 50 cc. methyl alcohol. Wash slides gently in water. Air dry. By this procedure, the bacteria and the nuclei of the leucocytes appear deep blue. The cytoplasm of the leucocytes appears faintly green with the cell outline distinctly visible. Cytoplasmic granules do not stain.     

Contrasty Staining And Quantitative Determination Of Mucopolysaccharides On Filter Paper By Means Of Colloidal Iron

The contrast between the Prussian blue color of the mucopolysaccharide spots and paper background was increased by differentiation of the paper strips (dyed in acid colloidal iron solution made up in 60% ethanol) with thioglycolic acid. Thus the trivalent iron bound to the paper background was reduced but that adsorbed by the mucopolysaccharides was precipitated as ferric ammonium thioglycolate. This procedure was found to stain equally well acid (including sulfated) and neutral mucopolysaccharides, even though these varieties exhibited different staining properties by the periodic acid-Schiff and toluidine blue dyeing procedures. Staining of different depolymerization products of hyaluronate was little influenced by their chain lengths. Quantitative determination of the mucopolysaccharide content of the spots was performed by elution of ferrocyanide with hot sodium hydroxide and measuring of the Prussian blue color of the extracts, developed on acidification and addition of FeCl₃.     

Nuclear stains with soluble metachrome metal mordant dye lakes

Summary Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe²⁺ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO₄ sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control.Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe²⁺ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results.Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes.The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes¹. Benzil at pH 13, which prevents the -naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.Assisted by Contract Nol-CB-43912 National Cancer Institute     

A Note on the Detection of Phytopathogenic Bacteria within the Leaf by a Differential Staining Procedure

A simple differential staining procedure for demonstrating infection within the leaf tissue by Pseudomonas tomato, Ps. lachrymans and Xanthomonas vesicatoria has been developed. It is based on (1) clearing of plant tissue with a mixture of glycerol, lactic acid, phenol and water; (2) treating the leaf tissue with boiling KOH; and (3) staining with aniline blue-chloralhydrate. When observed under a light microscope, the bacteria appear dark blue, whereas the leaf tissue appears transparent and colourless.     

Comparison of ferric iron generation by different species of acidophilic bacteria immobilized in packed-bed reactors

Flooded packed-bed bioreactors, prepared by immobilizing four different species of acidophilic iron-oxidizing bacteria on porous glass beads, were compared for their ferric iron-generating capacities when operated in batch and continuous flow modes over a period of up to 9 months, using a ferrous iron-rich synthetic liquor and acid mine drainage (AMD) water. The bacteria used were strains of Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, a Ferrimicrobium-like isolate (TSTR) and a novel Betaproteobacterium (isolate PSTR), which were all isolated from relatively low-temperature mine waters. Three of the bacteria used were chemoautotrophs, while the Ferrimicrobium isolate was an obligate heterotroph. Greater biomass yields achievable with the Ferrimicrobium isolate resulted in greater iron oxidation efficiency in the newly commissioned bioreactor containing this bacterium, though long-term batch testing with organic carbon-free solution resulted in similar maximum iron oxidation rates in all four bioreactors. Two of the bioreactors (those containing immobilized L. ferrooxidans and Ferrimicrobium TSTR) were able to generate significantly lower concentrations of ferrous iron than the others when operated in batch mode. In contrast, when operated as continuous flow systems, the bioreactor containing immobilized PSTR was superior to the other three when challenged with either synthetic or actual AMD at high flow rates. The least effective bacterium overall was At. ferrooxidans, which has previously been the only iron-oxidizer used in the majority of reports describing ferric iron-generating bioreactors. The results of these experiments showed that different species of iron-oxidizing acidophiles have varying capacities to oxidize ferrous iron when immobilized in packed-bed bioreactors, and that novel isolates may be superior to well-known species.     

Observations on the Staining of Corynebacterium Diphtheriae

Albert's method, of staining diphtheria cultures consists of staining a fixed smear for one minute (some laboratories stain for five minutes) with a solution containing toluidine blue and malachite (or methyl) green, washing with water, and applying Albert's iodine for one minute. This procedure is discussed and criticized, and in addition the mechanism of the stain is elucidated. Also, the procedure which involves staining a fixed smear for one minute with Loeffler's alkaline methylene blue solution is discussed and criticized.

To overcome the objections to the above staining methods, a different method is proposed. This consists of staining a fixed smear with an acid solution of toluidine blue, washing with water, applying Albert's iodine for one minute, washing with water, and finally applying a safranin solution for 15-20 seconds. The theoretical basis for this method is presented.     

Standardization of Staining in Giycosaminoglycan Histochemistry: Alcian Blue, its Analogues, and Diamine Methods

Glycosaminoglycans are identified in tissue sections by various histochemical techniques including staining with alcian blue and its analogues, such as cuprolinic blue and cupromeronic blue, or with high and low iron diamine methods. The variation in staining results is particularly confusing in the case of alcian blue, where not only are several different brands of alcian blue available but also several different staining protocols are used. If the results obtained by these techniques are compared, they often do not match. We have developed a dot blot technique for quality control of glycosaminoglycan histochemistry to standardize the staining protocols. This staining technique enables his-tochemists to test particular batches of alcian blue or its analogues for selective glycosaminoglycan staining, thus improving control of histochemical results. The results obtained using the dot blot assay indicate that it is necessary to test each batch of dye individually to obtain valid results in glycosaminoglycan histochemistry.     

Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry

Three metallic ions, NiCl2, CoCl2, and CuSO4, were found to modify the color of the normally brown diaminobenzidine (DAB) reaction. The colors ranged from purplish blue (NiCl2), dark blue/bluish black (CoCl2), to greyish blue (CuSO4). We have found that the CoCl2 + DAB is the ion of choice because: 1) it yields a distinct dark blue color that is easily distinguishable from brown DAB; 2) the blue reaction product is very stable throughout the entire staining procedure; and 3) background staining is minimal. These findings can be applied to the double staining technique of two different antigens in the same section. Among three staining procedures discussed, the avidin-biotin peroxidase complex (Co-DAB)-peroxidase-antiperoxidase (DAB) technique produced the best results because: 1) no antibody elution was needed following the avidin-biotin-peroxidase complex procedure when the CoCl2-DAB modification was used; and 2) no background staining occurred.     

A Method for Staining Certain Bacteria and Antherozoids

A modification of Loeffler's method for staining the flagella of bacteria was employed in staining large forms of bacteria and antherozoids. The bacteria or the antherozoids are killed and fixed in a drop of water on a slide and set aside to dry, before the next step is undertaken. The slide is treated for a period of time, varying from about ten minutes to several hours, in a practically saturated solution of tannic acid. After the slide is thoroly rinsed in water, it is stained with either a single stain or a combination of stains. The slide is then dehydrated with absolute alcohol, cleared, with clove oil, and completed in the usual manner.

The body of the bacterium and that of the antherozoid are well differentiated and the cilia are distinctly brought out by means of the method herein described.

The technic is of especial value in staining the antherozoids of mosses, liverworts, and ferns.     

趋磁细菌的磁小体

趋磁细菌是一类对磁场有趋向性反应的细菌,其菌体能吸收外界环境中铁元素并在体内合成包裹有膜的纳米磁性颗粒Fe₃O₄或Fe₃O_{3S4晶体即磁小体。综述了趋磁细菌的磁小体生物矿化的条件,以及趋磁细菌的铁离子吸收、磁小体囊泡的形成、铁离子的转运到磁小体囊泡及囊泡中受控的Fe3O4生物矿化的分子生物学和生物化学等方面的研究进展,重点介绍了趋磁细菌磁小体合成机制的研究进展及未来研究磁小体的发展方向。}     

Sulfur chemistry,biofilm, and the (in)direct attack mechanism — a critical evaluation of bacterial leaching

It has been shown (a) that bacterial leaching of metal sulfides apparently requires the attachment of leach bacteria to metal sulfides, (b) that exopolymerbound iron compounds are responsible for or at least considerably increase the rate of the biological attack over the chemical rate, (c) that the primary attacking agent in leaching environments is the ferric iron hexahydrate ion, (c) that thiosulfate is the first intermediate sulfur compound, giving rise to a variety of other compounds including polythionate-containing periplasmic granula, and (d) that we have no idea about the actual concentrations of protons, ferrous/ferric and/or other cations, and sulfur compounds in the reaction space between the bacterium and the sulfide surface.