Influence of staining on fast automated cell segmentation, feature extraction and cell image analysis

FAZYTAN, a system for fast automated cell segmentation, cell image analysis and extraction of nuclear features, was used to analyze cervical cell images variously stained by the conventional Papanicolaou stain, the new Papanicolaou stain and hematoxylin and thionin only; the last two dyes are used as the nuclear stains in the two versions of the Papanicolaou stain. Other dyes were also tried in cell classification experiments. All cell images in the variously stained samples could be described by the same nuclear features as had been adapted for the discrimination of conventional-Papanicolaou-stained cells. Variances were lower for thionin-stained cells as compared with hematoxylin-stained cells. By application of spectrophotometry, it was confirmed that the spectra of the cytoplasmic counterstains are superimposed on those of the nuclear stains. It appears that a variety of dyes are suitable as cytologic stains for cell classification by the FAZYTAN system, provided that they achieve sufficiently strong nuclear-cytoplasmic contrast by precisely delineating the chromatin texture.     

Preservation agents influence UV-coloration of plumage in museum bird skins

Plumage colour has always been a major criterion when describing and distinguishing bird taxa. Today, the use of reflection spectrophotometry is the most commonly used technique to study plumage coloration. A major advantage of this method is the opportunity of observing reflection beyond the human colour vision range—including the UV-waveband. Traditional taxonomic and phylogenetic research is often based on bird skins held in collections in natural history museums worldwide. Different agents for preservation have been used to prevent skins from being damaged by arthropod pests. Sometimes, parts of the plumage have been contaminated with stains from preservation agents. When dried, they are almost invisible to the human eye under normal sunlight conditions and cause no obvious change to feather coloration. However, some preservation agents contain fluorescent components which show up brightly when illuminated with UV-light. Furthermore, undetectable to the human eye, stains from these agents annihilate UV-reflection, preventing accurate data collection based on the UV-reflection of bird feathers. Measuring plumage parts which have been accidentally stained will lead to a relative underestimate of UV-reflection. In studying 20,000 samples, we found fluorescent stains in some 300 bird skins of varying ages (1913–2004) in museum collections throughout Europe and the USA. Different preservation agents have been evaluated for their fluorescence properties.     

New Stains for Blood and Bone Marrow Cells

Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an assymetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells.     

New stains for blood and bone marrow cells.

Traditionally, blood and bone marrow cells have been identified based on their characteristic shapes and colors when stained with one of several panoptic stains including Wright's or Giemsa's. As questions arose regarding the origin of normal and leukemic cells, cytochemical stains were developed. These stains help identify cells on the basis of a distinctive metabolite or enzyme. As part of an ongoing tradition in which textile dyes are used for biological staining, several new stains have been applied to hematologic staining. These include C.I. basic blue 41, basic blue 141, basic blue 93, and an asymmetrical polymethine dye. As additional cell-selective stains are developed, we can anticipate further improvements in our ability to identify normal and malignant hematopoietic cells.     

Analysis and Purification of Biological Stains by Gel Filtration

Some sixty biological stains of widely varying type have been subjected to gel filtration chromatography in columns of Sephadex LH-20 (Pharmacia, Uppsala) resin swollen by dimethylformamide saturated with NaCl. Most dyes contained more than one coloured constituent. Measures of their molecular weights were obtained. The use of the method for analysis was as effective as other common chromatographic or electrophoretic procedures but was technically simpler. Preparative use of the method merely involved using a larger column of gel, though occasionally use of ethanol as solvent gave better resolutions and did provide easier recovery of separated dyes.     

A widely applicable analytical system for biological stains: reverse-phase thin layer chromatography

The suitability of reverse-phase thin layer chromatography using a commercial adsorbant and aqueous methanol as an analytical tool for biological stains was investigated. The wide range of applicability of this technique is shown by the fact that of 120 dyes used as biological stains, 84 of diverse chemical character were successfully chromatographed by varying only the water content of the eluent. Unsuccessful chromatography was due either to immobility or streaking. Dyes exhibiting this behavior can be identified prior to chromatography by inspection of their structural formulae. Rf values were found to be significantly correlated with the calculated partition coefficients. This relationship provides information for the identification of dye components revealed by chromatography and a discussion of its use in the chemical characterization of various dyestuffs is presented.     

A Spectrophotometry Analysis of Tissue Staining

By comparing spectral absorption curves of representative staining solutions and of substances stained with these solutions it is shown that information may be obtained regarding chemical changes associated with the staining process. The stains used in these determinations were acid fuchsin, anilin blue, azo-carmine G, basic fuchsin, eosin Y, orange G, picric acid and Sudan IV. The substrates stained were gelatin, tendon, blood plasma, thymus gland and fat.

Aqueous basic fuchsin and fuchsin-sulfurous reagent to which formalin was added (Setoff reaction) are different stains. The spectral absorption curves for staining solutions and substances stained with the solutions were comparable. Within the limitations of the spectrophotometry methods and stains employed, there was no evidence of significant chemical alteration in the chromophore radicals of the stains associated with the process of tissue staining.     

A Widely Applicable Analytical System for Biological Stains: Reverse-Phase Thin Layer Chromatography

The suitability of reverse-phase thin layer chromatography using a commercial adsorbant and aqueous methanol as an analytical tool for biological stains was investigated. The wide range of applicability of this technique is shown by the fact that of 120 dyes used as biological stains, 84 of diverse chemical character were successfully chromatographed by varying only the water content of the eluent. Unsuccessful chromatography was due either to immobility or streaking. Dyes exhibiting this behavior can be identified prior to chromatography by inspection of their structural formulae. R_f values were found to be significantly correlated with the calculated partition coefficients. This relationship provides information for the identification of dye components revealed by chromatography and a discussion of its use in the chemical characterization of various dyestuffs is presented.     

Absorption Ratios of Biological Stains

Absorption ratios are supplied for most of the dyes used as biological stains.

The use of these ratios will enable the analyst to identify the stains conveniently, and will also frequently afford information of considerable value respecting their purity.     

Enzyme‐enabled responsive surfaces for anti‐contamination materials

Many real‐life stains have origins from biological matters including proteins, lipids, and carbohydrates that act as gluing agents binding along with other particulates or microbes to exposed surfaces of automobiles, furniture, and fabrics. Mimicking naturally occurring self‐defensive processes, we demonstrate in this work that a solid surface carrying partially exposed enzyme granules protected the surface in situ from contamination by biological stains and fingerprints. Attributed to the activities of enzymes which can be made compatible with a wide range of materials, such anti‐contamination and self‐cleaning functionalities are highly selective and efficient toward sticky chemicals. This observation promises a new mechanism in developing smart materials with desired anti‐microbial, self‐reporting, self‐cleaning, or self‐healing functions. Biotechnol. Bioeng. 2013; 110: 1805–1810. © 2013 Wiley Periodicals, Inc.     

The Fluorane Derivatives: Methods for the Standardization of Biological Stains: Part II

In this paper the methods are given which are used in determining whether to approve the sale of certain dyes of the fluorane group as certified biological stains. The methods have been worked out by the Commission on Standardization of Biological Stains in cooperation with the Color and Farm Waste Division, Bureau of Chemistry and Soils, U. S. Dept. of Agriculture. The dyes for which the methods are given in the present paper are: Fluorescein, eosin yellowish, ethyl eosin, eosin bluish, erythrosin, phloxine B, and rose bengal. For each of these dyes methods are given under the following headings: (1) identification or qualitative examination; (2) quantitative analysis; and (3) biological tests.     

Metabolism of 25-hydroxycholecalciferol in human promyelocytic leukemia cells (HL-60). Isolation and identification of (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol

Human promyelocytic leukemia cells incubated with 25-hydroxy[26,27-methyl-3H] cholecalciferol (1 microCi) or non-radioactive 25-hydroxycholecalciferol (550 micrograms) produced significant quantities of two vitamin D3 metabolites. The two metabolites were isolated and purified by methanol chloroform extraction and a series of chromatographic procedures. The metabolite purification and elution positions on these columns were followed by radioactivity and their ultraviolet absorption at 310 nm. The two metabolites have been unequivocally identified as (5Z)- and (5E)-19-nor-10-oxo-25-hydroxycholecalciferol by ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry and co-chromatography with synthetic compounds on a high-performance liquid chromatograph. (5E)- but not (5Z)-19-nor-10-oxo-25-hydroxycholecalciferol was able to induce HL-60 cell phenotypic and functional differentiation. However, these two metabolites of 25-hydroxycholecalciferol did not bind specifically to the chick intestinal 3.7 S. receptor protein for 1 alpha,25-dihydroxycholecalciferol. The precise biological role of these metabolites is as yet unclear.     

Improving Biological Dyes and Stains: Quality Testing Versus Standardization

This paper discusses the impact of both standardization and quality testing of dyes and stains in biology and medicine. After a brief review of why standardized dyes and stains are not presently available commercially, two types of testing and ways of improving dye quality are described. National or international organizations could be established to define standardization of dyes and stains. Standardization would be specifically defined as a list of physico-chemical parameters such as elaborated in this paper. Commercial batches of comparable quality may be labeled by the supplier as “standard dye.” a procedure currently performed by the European Council for Clinical and Laboratory Standardization (ECCLS). Also recommended to improve dye quality is commercial dye testing by independent laboratories with subsequent certification for use. This sort of quality control is currently carried out in the United States by the Biological Stain Commission (BSC). The advantages and disadvantages of both techniques and the use of image analysis for the definition of standards are discussed. A combination of both the BSC testing protocols and the ECCLS standards should be established for extended quality control of biological dyes and stains.     

Report from the President

Although the original Commission on Standardization of Biological Stains was first organized in 1921, it was not until 1944 that this was incorporated as an independent, nonprofit organization known as the Biological Stain Commission (see Clark 1974). The certification of dyes, as indicated by special labels purchased by manufacturers or vendors for attachment to the dye containers, originated with the parent organization and has continued to this day. The objectives of the Biological Stain Commission (BSC) are 1) to identify and standardize the content and performance of dyes and dye preparations used in staining biological tissues and products, 2) to issue labels of certification to companies that buy these to inform consumers that their certified dyes meet the specifications of the BSC, 3) to carry out and to support investigations on dyes and their performance, 4) to publish scientific data concerning biological stains and their use, and 5) to maintain, through scientific meetings and correspondence, an active “dialogue” among scientific and industrial personnel concerned with biological stains. The present report summarizes Commission activity and some of the changes that have occurred during the past five years.     

A Brief History of the Biological Stain Commission: its Founders, its Mission and the First 75 Years

The need for batch-to-batch consistency in available dyes and stains used for biological purposes posed a considerable problem for United States scientists following World War I. Prior to that time, most of the acceptable stains in this country were of German origin. In an attempt to standardize the performance of biological stains and dyes, the Society of American Bacteriologists in 1922 appointed Dr. Harold Conn to form the Committee on the Standardization of Biological Stains. To assist him, Dr. Conn recruited scientists from several major professional scientific societies. Mr. Holland Will, a Rochester, NY, vendor of stains, was also instrumental in the Committee's success. This article traces the origin, mission and accomplishments of the product of that Committee, the Biological Stain Commission, through the past 75 years, and focuses on some of the major events that influenced and shaped its development.     

A Brief History of the Biological Stain Commission: its Founders,its Mission and the First 75 Years

The need for batch-to-batch consistency in available dyes and stains used for biological purposes posed a considerable problem for United States scientists following World War I. Prior to that time, most of the acceptable stains in this country were of German origin. In an attempt to standardize the performance of biological stains and dyes, the Society of American Bacteriologists in 1922 appointed Dr. Harold Conn to form the Committee on the Standardization of Biological Stains. To assist him, Dr. Conn recruited scientists from several major professional scientific societies. Mr. Holland Will, a Rochester, NY, vendor of stains, was also instrumental in the Committee's success. This article traces the origin, mission and accomplishments of the product of that Committee, the Biological Stain Commission, through the past 75 years, and focuses on some of the major events that influenced and shaped its development.     

Impact of Aging on the Regenerative Properties of Bone Marrow-, Muscle-, and Adipose-Derived Mesenchymal Stem/Stromal Cells

Mesenchymal stem/stromal cells (MSCs) are promising cell sources for regenerative therapies due to their multipotency and ready availability, but their application can be complicated by patient-specific factors like age or illness. MSCs have been investigated for the treatment of many musculoskeletal disorders, including osteoarthritis and osteoporosis. Due to the prevalence of these diseases in older populations, researchers have studied how aging affects MSC properties and have found that proliferation and differentiation potential are impaired. However, these effects have never been compared among MSCs isolated from multiple tissue sources in the same, healthy donor. Revealing differences in how MSCs are affected by age could help identify an optimal cell source for musculoskeletal therapies targeting older patients. MSCs were isolated from young and old rabbit bone marrow, muscle, and adipose tissue. Cell yield and viability were quantified after isolation procedures, and expansion properties were assessed using assays for proliferation, senescence, and colony formation. Multipotency was also examined using lineage-specific stains and spectrophotometry of metabolites. Results were compared between age groups and among MSC sources. Results showed that MSCs are differentially influenced by aging, with bone marrow-derived stem cells having impaired proliferation, senescence, and chondrogenic response, whereas muscle-derived stem cells and adipose-derived stem cells exhibited no negative effects. While age reduced overall cell yield and adipogenic potential of all MSC populations, osteogenesis and clonogenicity remained unchanged. These findings indicate the importance of age as a factor when designing cell-based therapies for older patients.     

Identification of a unique sertoli cell steroid as 3α-hydroxy-4-pregnen-20-one (3α-dihyroprogesterone: 3α-DHP)

An allylic steroid produced from progesterone by rat Sertoli cells and which does not appear to have been described previously as a product of gonadal or adrenal tissues has been isolated and identified as 3α-hydroxy-4-pregnen-20-one (3α-dihydroprogesterone; 3α-DHP). 3α-DHP appears to be a reactive molecule which is easily oxidized or dehydrated and its identification was possible by a combination of microchemical procedures, derivative formation, specific enzyme reaction, TLC, GC, HPLC, spectrophotometry and mass spectrometry. The biological functions of this Sertoli cell steroid are not known, but it is suggested that 3α-DHP is more than a metabolic waste product because (a) its production rate varies with age and is highest at the onset of meiosis, and (b) there appear to be specific receptors for it in the testis.     

Effect of some negative stains on the calcium transport of sarcoplasmic reticulum

Summary Effect of some negative stains used in electron microscopy on calcium-transporting membranes of the fragmented sarcoplasmic reticulum (FSR) and interaction between stains and calcium have been studied.Calcium transport as well as ATP-ase activity of FSR are affected by stains investigated at concentrations lower than those used in electron microscopy. Degree of alteration varies from stain to stain. Phosphotungstic acid is the most effective inhibitor.Calcium loaded vesicles are depleted during resuspension in various staining solutions, in concentration routinely used for electron microscopy, according to the calcium affinity of the stain. Also in this respect phosphotungstic acid is the most effective.     

A real-time view of life within 100 nm of the plasma membrane

The plasma membrane is a two-dimensional compartment that relays most biological signals sent or received by a cell. Signalling involves membrane receptors and their associated enzyme cascades as well as organelles such as exocytic and endocytic vesicles. Advances in light microscope design, new organelle-specific vital stains and fluorescent proteins have renewed the interest in evanescent field fluorescence microscopy, a method uniquely suited to image the plasma membrane with its associated organelles and macromolecules in living cells. The method shows even the smallest vesicles made by cells, and can image the dynamics of single protein molecules.