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.     

A high-affinity reversible protein stain for Western blots

We describe a reversible staining technique, using MemCode, a reversible protein stain by which proteins can be visualized on nitrocellulose and polyvinylidine fluoride (PVDF) membranes without being permanently fixed to the membrane itself. This allows subsequent immunoblot analysis of the proteins to be performed. The procedure is applicable only to protein blots on nitrocellulose and PVDF membranes. MemCode is a reversible protein stain composed of copper as a part of an organic complex that interacts noncovalently with proteins. MemCode shows rapid protein staining, taking 30s to 1 min for completion. The method is simple and utilizes convenient application conditions that are compatible with the matrix materials and the protein. The stain is more sensitive than any previously described dye-based universal protein staining system. The turquoise-blue-stained protein bands do not fade with time and are easy to photograph compared to those stained with Ponceau S. Absorbance in the blue region of the spectrum offers good properties for photo documentation and avoids interference from common biological chromophores. The stain on the protein is easily reversible in 2 min for nitrocellulose membrane and in 10 min for PVDF membrane with MemCode stain eraser. The stain is compatible with general Western blot detection systems, and membrane treatment with MemCode stain does not interfere with conventional chemiluminescent or chromogenic detection using horseradish peroxide and alkaline phosphatase substrates. The stain is also compatible with N-terminal sequence analysis of proteins.     

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.     

喷粉洁牙和外漂白法在外源性染色牙面上的应用

本实验借用Sharif的方法,用我国常见的、汤色较重的祁门红茶作色素原,与洗必泰液交替浸泡人类离体上切牙,建立牙齿外源性染色模型。选用气流喷砂法和过氧化氢凝胶外漂白法去色后拍摄数码照片,通过计算机对外源性染色牙处理前、后白色度值变化的分析,比较它们的去色效果;并用扫描电镜观察两种方法对牙面的损伤情况。     

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.     

Lead asparate, an en bloc contrast stain particularly useful for ultrastructural enzymology

Lead aspartate is a new en bloc stain for electron microscopy. Its predictable staining depends on chelation that results from the interaction of the two stain components, lead nitrate and aspartic acid, which must be present in a specific ratio. Lead aspartate stain is 0.02 M in lead nitrate and 0.03 M in aspartic acid, adjusted to pH 5.5. Cells or tissues are stained at 60 degrees C for 30 to 60 min. Cells stained en bloc with lead aspartate closely resemble cells stained on grids by lead citrate, except that the former seldom have contamination. En bloc staining with lead aspartate bypasses the grid-staining step so that samples can be viewed and photographed immediately after they are thin-sectioned. The lower pH of the lead aspartate solution allows counterstaining of enzyme reaction products that dissolve in the highly alkaline lead citrate stain. Lead aspartate en bloc staining to enhance contrast should especially benefit studies of ultrastructure requiring a clean and predictably lead stain.     

Evaluation of stain removal and inhibition properties of eight denture cleansers: an in vitro study

Gerodontology 2012; doi: 10.1111/j.1741‐2358.2011.00522.x
Evaluation of stain removal and inhibition properties of eight denture cleansers: an in vitro study Objectives: To determine the ability of eight denture cleansers to remove and inhibit tea‐stain build‐up on acrylic resin. Materials and methods: In the stain removal study, Perspex^® (cast heat polymerised resin) specimens previously soaked in saliva were stained using multiple exposures of chlorhexidine and tea solutions. Specimens were exposed for 1 min to one of the eight denture cleansers for five cycles, washed and dried and their optical density read on a uv/vis spectrophotometer at 295 nm. In the stain inhibition study, clear specimens were exposed to saliva followed by cleansers then tea solution, for five cycles. The build‐up of stain at each cycle was measured, and differences in optical densities from baseline were calculated. Results: All denture cleansers were significantly more effective than water in removing stain (p < 0.05). There were significant differences in cleaning ability between cleansers (p < 0.001), Dentural^® and Kleenite^® were particularly effective. The stain inhibition experiment showed that most cleansers were significantly more effective than water in inhibiting stain (p < 0.05). There were significant differences in inhibition ability between cleansers (p < 0.01). Kleenite^® and Equate were particularly effective. Conclusions: All denture cleansers had a capacity to remove stain and most had an inhibitory effect on staining. Kleenite^® was particularly effective in controlling stain formation.     

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.     

Use of the Gram stain in microbiology

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be 'Gram-positive,' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be 'Gram-negative.' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.     

Regionalization and lateral diffusion of membrane proteins in unfertilized and fertilized mouse eggs

The unfertilized mouse egg has a round and highly villated main body and a "nipple" that is unvillated and buds off on fertilization to form the second polar body. Fluorescent markers stain the body more intensely than the nipple, which has been assumed to result from surface amplification due to microvilli. Using fluorescence recovery after photobleaching and microfluorescence photometry, we have measured the membrane protein diffusion and concentration on the main body and nipple region of unfertilized and on fertilized CD-1 mouse eggs. Two general membrane protein labels were used: rhodamine-labeled succinylated concanavalin A and trinitrobenzene sulfonate visualized with a rhodamine Fab fragment of a sheep anti-trinitrophenyl. We found that while the diffusion coefficient was the same on the nipple and main body, considerably higher recovery was observed on the nipple for both probes. The ratio of intensity of fluorescence on the nipple to main body was significantly lower for the concanavalin A stain than for the trinitrophenyl stain, indicating that true concentration gradients exist beyond those that result from surface amplification. The effect of fertilization was not general. No effect was observed for the concanavalin A stain for either diffusion coefficient or percent recovery. For the trinitrophenyl stain, percent recovery decreased approximately twofold while diffusion coefficient increased approximately threefold.     

The histology of the excretory kidney of the paddlefish, Polyodon spathula

Kidney tissue from the freshwater chondrostean fish, Polyodon spathula , was examined by light microscopy. P. spathula has a glomerular excretory kidney. The tubule leading from the glomerulus, based on a variety of stain reactions, is composed of two major cell types. Also based on stain reactions, it appears that ion reabsorption, hence conservation, occurs in distal portions of the tubules. Also observed were stain reactions of arteriole walls, similar to renin producing cells in other vertebrates.     

Technical aspects of the Haematoxylin-Basic Fuchsin-picric acid (HBFP) stain applied to skeletal muscle

Synopsis The Haematoxylin-Basic Fuchsin-picric acid (HBFP) stain, a new non-enzymatic histochemical technique described previously to detect early myocardial ischemia, was applied to skeletal muscle. Several factors were found which have an important effect on HBFP positivity including ageing in room air of unstained tissue sections, and the precise timing of the differentiation step of this stain. Using carefully standardized techniques, repeatable staining was obtained and a high level of inter-observer consistency in the interpretation of staining results was achieved. Although the technical requirements of this new stain are rigorous, it offers promise and deserves further evaluation in the study of skeletal as well as cardiac muscle disorders. The histological advantages include vivid contrasts and the ability to use the stain on formalin-fixed paraffin-embedded muscle tissue.     

Coomassie brilliant blue staining of lipids on thin-layer plates

Coomassie brilliant blue staining of lipids on silica gel thin-layer chromatography plates is described. This stain proved to be useful for the wide-range detection of simple and complex lipids on thin-layer plates. It can stain several classes of lipids, including cholesterol, cholesterol esters, glycerides, phospholipids, ceramides, and neutral and acidic glycosphingolipids. It stains the spots evenly without a corrosive reagent, and is simple to use and suitable for storage. The visual detection limits of this stain for lipids were 0.05 to 0.5 microgram.     

The Modified Steiner Stain: a New Use for an Old Stain? Staining Cytomegalovirus-infected Cells in Gastrointestinal Biopsies

The modified Steiner stain is a non-specific silver stain for identifying bacteria in formalin-fixed, paraffin-embedded tissues. The principle behind its use is that bacteria are first sensitized using uranyl nitrate solution, making them able to precipitate silver from a silver nitrate solution. It is used routinely for staining gastric biopsies to identify Helicobacter pylori. Upon staining a gastric biopsy from a patient with acquired immunodeficiency syndrome (AIDS) and cytomegalovirus gastritis, we recognized that this technique also stains the viral inclusions of cytomegalovirus-infected cells. We then proceeded to stain 43 consecutive cytomegalovirus-positive gastrointestinal biopsies from 33 immunocompromised patients based on positive cytomegalovirus immunohistochemistry (DAKO-cytomegalovirus monoclonal antibody, clones DDG9 and CCH2). We also stained eight cytomegalovirus-infected, non-gastrointestinal tissue s, including lung, adrenal gland, ovary, skin and neural tissue, to ensure that the stain was staining the cytomegalovirus-infected cells and not argyrophilic or argentaffin neuroendocrine cells of the gastrointestinal tract. In 40 of the 43 cytomegalovirus-infected gastrointestinal biopsies, we saw positive staining with the modified Steiner stain (93% sensitivity). The cytomegalovirus-infected, non-gastrointestinal tissues all stained positively with the modified Steiner stain. Because the modified Steiner stain is frequently used to identify Helicobacter pylori in gastric biopsies, we propose that it be studied further for possible use either as a screen or as a confirmatory tool, or both, for cytomegalovirus inclusions in gastrointestinal biopsies.     

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。     

Differentiation of bone and soft tissues in formalin-fixed, paraffin-embedded tissue by using methylene blue/acid fuchsin stain

OBJECTIVE: To find a staining method for formalin-fixed, paraffin-embedded tissue that would distinguish bone from surrounding soft tissues, including muscle, periosteal tissue and bone marrow. STUDY DESIGN: A variety of stains were tested and compared with hematoxylin-eosin. The potential value of any given stain was evaluated based on its ability to stain bone and soft tissues different colors or shades that could be readily identified in photomicrographs. Stains were evaluated using both endochondral (tibia) and intramembranous bone (calvaria) samples. RESULTS: In contrast to standard hematoxylin-eosin stain, which stains both bone and soft tissues pink, the methylene blue/acid fuchsin stain demonstrates remarkable contrast between bone and other tissues. Methylene blue/acid fuchsin stained bone bright pink and the surrounding soft tissues blue-purple. CONCLUSION: In addition to the superior staining properties of methylene blue/acid fuchsin, other benefits of this stain include its stability, ease of use and low cost. This stain has many potential applications in the study of erosive bone disease in humans and also in animal models for research.     

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.     

Cytologic detection of Wuchereria bancrofti microfilariae in urine collected during a routine workup for hematuria

A 23-year-old Guyanese man experienced intermittent, total, painless, gross hematuria for a month for which he sought medical attention at the Kings County Hospital Center in Brooklyn. Hematuria was accompanied by weakness but not by frequency of urination or burning on urination. Catheterized urine at the time of cystoscopy and each of two subsequent voided specimens examined cytologically contained sheathed microfilariae. Distinguishing features of the microfilariae were well demonstrated with the Papanicolaou stain. The well-stained nuclei, which did not extend into the clear zone, and the distinct, pale-stained sheath led to the positive identification of the microfilariae as Wuchereria bancrofti. The Papanicolaou stain may well be the stain of choice for the identification of microfilariae in the blood. The excellent detail obtained with this routine cytologic stain is as good as that with Giemsa, which does not stain the sheath.     

The compactness of populations of neutrophilic leucocytes as revealed by the new azure B-eosine stain, Pappenheim's stain and the peroxidase reaction

The new azure B-eosine stain shows a greater inhomogeneity of the granule population of human neutrophil leucocytes than Pappenheim's stain. This difference seems to be dependent on the number of azure granules within the single neutrophil.