Improved Stability of Methanolic Wright's Stain with Additive Reagents

Additive reagents have been investigated to improve the stability of methanolic Wright's stain. The addition of ammonium halides, monoalkyiamine hydrochlorides, dialkylamine hydrochlorides or trialkylamine hydrochlorides to methanolic Wright's stain was found to enhance the stability of stain components in methanol. No change in performance is observed with these additives present. Random precipitation in the stain solution was still observed with the addition of ammonium halides and monoalkyiamine hydrochlorides. No precipitation was found in stain solutions containing hydrochlorides of most dialkylamines and trialkylamines. Of the compounds evaluated, 0.6% diethylamine hydrochloride added to methanolic stain solutions produced the most desirable overall results. Mechanisms of stabilization and precipitation in these stain solutions are proposed, Essentially, separation of the thiazine-eosinate ion pair through interaction with an appropriate additive increases stain stability. The solubilities of thiazine-eosinate or additive cation-eosinate ion pairs in methanol determine the formation of precipitate in such stain solutions.     

Quantitative Determination of Murine Dermal Elastic Fibers by Color Image Analysis: Comparison of Three Staining Methods

We compared three different staining methods to determine if the dermal elastic fiber content of the HRS/Skh-1 hairless mouse could be accurately measured by color image analysis. Comparisons were made among Klig-man's modification of Luna's mast cell stain for elastin, Unna's orcein stain with or without potassium permanganate preoxidation, and Gomori's aldehyde fuchsin stain with potassium permanganate preoxidation. The color image analysis system could be used to identify and quantify murine dermal elastin fibers in sections stained by all three methods. Gomori's aldehyde fuchsin stain with preoxidation demonstrated twice the content of dermal elastic fibers demonstrated by either Kligman's modification of Luna's mast cell stain or Unna's orcein stain with or without preoxidation. Gomori's aldehyde fuchsin method with preoxidation should be considered the stain of choice for evaluating murine dermal elastic fiber content.     

大肠菌群革兰染色技术条件探讨

介绍影响大肠菌群革兰染色正确性的因素：菌株的菌龄、涂片时菌体浓度、染色时间、媒染时间、脱色程度等,通过试验分析,得出大肠菌群革兰染色的最佳条件,并提出验证革兰染色结果正确性的方法。     

Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions

OBJECTIVE: To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies. STUDY DESIGN: Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN. RESULTS: Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm. CONCLUSION: Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.     

A method for removing stain precipitate and destaining sections

Subjecting electron microscope sections to NaOH treatment removes stain precipitate from the section surface. The alkali treatment also extracts stain from the tissue itself. Following this treatment, sections can be restained to obtain clean images. Alternatively, after being viewed or photographed using one stain and then destained, the same sections can be treated with a different stain to obtain additional histochemical information.     

A New Stain for Pollen Fertility Studies

The juice from the berries of Cocculus hirsutum was extracted and used for pollen fertility studies in various crops. Two stains were prepared: P. H. Ramanjini (PHR) stain and modified PHR stain. The modified PHR stain contains lactic acid and produces the best staining differentiation. The intensity of the staining was dependent on the thickness of the pollen cell walls, hence PHR stain is recommended for thick walled pollen grains and the modified PHR stain for pollen with relatively thin walls. The preparation of both the stains are very simple, quick and inexpensive.     

Newer applications of the histological stain prepared from Pterocarpus santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn. has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.     

A Method for Removing Stain Precipitate and Destaining Sections

Subjecting electron microscope sections to NaOH treatment removes stain precipitate from the section surface. The alkali treatment also extracts stain from the tissue itself. Following this treatment, sections can be restained to obtain clean images. Alternatively, after being viewed or photographed using one stain and then destained, the same sections can be treated with a different stain to obtain additional histochemical information.     

Newer Applications of the Histological Stain Prepared from Pterocarpus Santalinus

A histological stain prepared from the heartwood of Pterocarpus santalinus Linn, has been found to be an excellent nuclear stain for various cells of animal and plant origin. As an elastic tissue stain, the results are comparable to standard elastic tissue stains. The striations of voluntary muscle fibers are well shown. The Nissl granules and fibers of cranial nerves in the pons are visualized. When counterstained with light green, it differentially stains muscle and fibrous tissue. The stain can be used as counterstain with certain histochemical procedures with satisfactory results. The preparation and use of this versatile stain are described.     

Analysis of the Microbiota of Black Stain in the Primary Dentition

Black tooth stain is a characteristic extrinsic discoloration commonly seen on the cervical enamel following the contour of the gingiva. To investigate the relationship between black tooth stain and the oral microbiota, we used 16S rRNA gene sequencing to compare the microbial composition of dental plaque and saliva among caries-free children with and without black stain. Dental plaque and saliva, as well as black stain, were sampled from 10 children with and 15 children without black stain. Data were analyzed using the pipeline tool MOTHUR. Student’s t-test was used to compare alpha diversities and the Mann-Whitney U test to compare the relative abundances of the microbial taxa. A total of 10 phyla, 19 classes, 32 orders, 61 families and 102 genera were detected in these samples. Shannon and Simpson diversity were found to be significantly lower in saliva samples of children with black stain. Microbial diversity was reduced in the black stain compared to the plaque samples. Actinomyces, Cardiobacterium, Haemophilus, Corynebacterium, Tannerella and Treponema were more abundant and Campylobacter less abundant in plaque samples of children with black stain. Principal component analysis demonstrated clustering among the dental plaque samples from the control group, while the plaque samples from the black stain group were not and appeared to cluster into two subgroups. Alterations in oral microbiota may be associated with the formation of black stain.     

Haematoxylin-Phloxine-Alcian Blue-Orange G Differential Staining of Prekeratin, Keratin and Mucin

This is a modification of Kreyberg's stain with Alcian blue 8GS used to stain acid much while phloxine B and orange G stain keratin and prekeratin. Procedure: Dewax formalin-fixed paraffin sections in xylene and hydrate through alcohol. Stain in Mayer's haemalum, 10 min; blue in tap water; wash in distilled water; stain in 1% phloxine, 3 min; wash in running water, 1 min; wash in distilled water; stain in 0.5% aqueous Alcian blue in 0.5 acetic acid, 5 min; wash in distilled water; stain in 0.5% orange G dissolved in 2.0% phosphotungstic acid, 13 min; dehydrate quickly in 2 changes of 95% alcohol and 2 changes of absolute alcohol; clear in several changes of xylene; mount in a synthetic resin. Acid mucopolysaccharides are stained turquois blue; prekeratin and keratin are orange to red orange.     

A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.     

Staining Protozoa with Janus Green B

The use of janus green to stain mitochondria has long been known. While using it to study the mitochondria in Trichomonas buccalis it was found to stain the flagella also. This is an easy and quick method of determining the number of anterior flagella of the trichomonads and so of distinguishing the trichomonads from the pentatrichomonads in intestinal infections. This stain proved so useful as a flagella stain that it was applied to numerous protozoa with interesting results.     

Wrinkle-Free Plastic Sections for Light Microscopy

Plastic sections 0.5 to 2 μm thick are routinely used for light microscopy. Although plastic sections have several advantages over paraffin or celloidin sections, a problem that is often encountered with plastic sections is wrinkling (Fig. 1). Wrinkling occurs during staining when sections dried on glass slides are covered with stain and heated to hasten the penetration of the stain. Mounted sections heated on glass slides, but not stained, ordinarily lack wrinkles, even when examined with phase contrast optics. Similarly, mounted sections covered with stain, but not heated, lack wrinkles; unfortunately, such sections fail to stain adequately. Unmounted sections floated on heated drops of stain also lack wrinkles (Millonig 1980). Thus, it is clear that wrinkling occurs only when mounted sections are covered with stain and heated.     

Histochemical properties of sulfated glycoconjugates in developing enameloid matrix of the fish Polypterus senegalus

I investigated the ultrastructural localization and histochemical properties of sulfated glycoconjugates in developing enameloid matrix of the fish Polypterus senegalus, by use of the high iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion methods. HID-TCH-SP stain deposits were localized in the dental basal lamina and in the whole thickness of developing enameloid matrix, particularly closely associated with enameloid collagen fibrils. Most HID-TCH-SP stain deposits in the enameloid were susceptible to testicular hyaluronidase but some stain deposits survived. HID-TCH-SP stain deposits in the basal lamina resisted the enzymatic digestion, and were regularly localized to the internal and external sites of lamina densa at an early stage of development, subsequently tending to be randomly arranged with the increase in thickness of enameloid matrix layer. Furthermore, enzymatic digestion with heparitinase preferentially removed HID-TCH-SP stain deposits in the region of the basal lamina. Thus, it was confirmed that most HID-TCH-SP stain deposits in developing enameloid matrix are chondroitin 4-sulfate and/or 6-sulfate and that the stain deposits in the basal lamina represent heparan sulfate. The chondroitin sulfates tended to disappear with the advancement of enameloid mineralization.     

The influence of Romanowsky-Giemsa type stains on nuclear and cytoplasmic features of cytological specimens

The aim of the present study was to compare the staining pattern of the standard azure B-eosin Y stain with commercial May-Grünwald-Giemsa (MGG) stains on cytological specimens by means of high resolution image analysis. Several cytological specimens (blood smears, abdominal serous effusions, bronchial scrape material) were air dried, methanol fixed and stained with the standard azure B-eosin Y stain and with commercial May-Grünwald-Giemsa stains. Integrated optical density (IOD) and colour intensities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyser. Commercial MGG stains gave much higher coefficients of variation for all parameters than the standard stain. Reproducibility of cell nuclei segmentation versus cytoplasm was significantly better for the standard stain. Contamination of the standard stain with methylene blue partly copied the staining pattern of commercial stains. The standard azure B-eosin Y stain is recommended for high resolution image analysis (HRIA) of cytological samples.     

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.     

The correlation between bismuth and uranyl staining and phosphorus content of intracellular structures as determined by electron spectroscopic imaging

Four groups of intracellular structures can be recognized according to bismuth and uranyl staining and phosphorus content. (1) Those which contain phosphorus and stain strongly with uranyl acetate but not with bismuth (ribosomes, heterochromatin and mature ribosomal precursor granules), presumably because of their nucleic acid content. (2) Those which contain phosphorus and stain with uranyl acetate and bismuth (interchromatin granules, immature ribosomal precursor granules and mitochondrial granules), presumably because at least some of their phosphate is available to react with bismuth. (3) Those which contain little phosphorus but which stain strongly with bismuth and weakly with uranyl acetate (Golgi complex beads), perhaps because some ligand in addition to phosphate reacts with bismuth, and (4) those which do not contain phosphorus and stain with neither uranyl acetate nor bismuth (portasomes). Uranyl staining correlates strongly with the phosphorus content of nucleic acids, proteins and inorganic deposits. Bismuth will stain some phosphorylated molecules but not all. Thus only some phosphates stain with bismuth.     

Mag fura staining of fungi

Mag fura is a fluorescent stain that can be used to identify those parts of filamentous fungi that have active cell membranes. As the stain is held within the chitin of the cell wall, where it responds to locally high concentrations of divalent ions being transported across the cell membrane. This has now been tested on a broad range of fungi, including four filamentous species and three yeasts. The characteristic response of the stain appeared in areas of the cell wall where chitin might be localised. This supports the hypothesis, as well as extending the range of use of this stain.     

A method for rapid and sensitive negative staining of proteins in SDS‐PAGE using 2′,7′‐dichlorofluorescein

We report here a rapid and sensitive technique for negative visualization of protein in 1D and 2D SDS‐PAGE by using 2′, 7′‐dichlorofluorescein (DCF), which appeared as transparent and colorless bands in an opaque gel matrix background. For DCF stain, down to 0.1–0.2 ng protein could be easily visualized within 7 min by only two steps, and the staining is fourfold more sensitive than that of Eosin Y (EY) negative stain and glutaraldehyde (GA) silver stain, and eightfold more sensitive than that of the commonly used imidazole‐zinc (IZ) negative stain. Furthermore, DCF stain provided good reproducibility, linearity, and MS compatibility compared with those of IZ stain. In addition, the potential staining mechanism was investigated by colorimetric experiment and molecular docking, and the results demonstrated that the interaction between DCF and protein occurs mainly via van der waals force, electrostatic interaction, and hydrogen bonding.