Standardization of biological dyes and stains: pitfalls and possibilities

Summary The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review.This paper is dedicated to my academic teacher, Prof. Dr. D.H. Wittekind, on the occasion of his 70th birthday     

Standardization of biological dyes and stains: pitfalls and possibilities.

The present paper gives a review of the actual state of standardization of biological dyes and stains. In a first part general information is given on practical problems encountered by the routine user of dyes with special emphasis on dye contamination. Some theoretical aspects of standardization are discussed. The second part of the paper gives more detailed information on commercial batches of hematoxylin-eosin-, Giemsa- and Papanicolaou-stains and on their standardization. Special problems arising with the application of image analysis techniques are briefly mentioned. User-oriented specifications for the standardization of dyes, stains and staining procedures are given. Fluorescent dyes and dyes used in chromogenic reagents such as the Feulgen-Schiff reaction are not included in this review.     

Quantitative analysis of dyes for automated studies of cell preparations

Spectral studies were made of the dyes in solutions and also of the cytology preparations stained by these dyes. 14 dyes of the blue-violet spectral region were examined. Absorbtion and transmission coefficients of the cell nucleus and cytoplasm were measured. The data obtained allowed to elaborate a quantitative estimation of dye selection. According to this method cresyl-violet was selected as the dye for Videocon LI-421.     

Standardization of reagents and methods used in cytological and histological practice with emphasis on dyes, stains and chromogenic reagents

Summary The need for the standardization of reagents and methods used in the histology laboratory is demonstrated. After definitions of dyes, stains, and chromogenic reagents, existing standards and standards organizations are discussed. This is followed by practical instructions on how to standardize dyes and stains through the preparation of reference materials and the development of chromatographic methods. An overview is presented of the problems concerned with standardization of the Romanowsky-Giemsa stain for cytological and histological application. Finally, the problem of how to convince routine dye and stain users of the need for standardization in their histology laboratories is discussed.     

A model for pattern recognition by cell networks

A study is made of certain properties of a model cell network based on lateral inhibition. It is demonstrated that specific patterns of input activity, termed eigenpatterns, will pass through the network without distortion. Under certain conditions the network will show selectivity for one particular eigenpattern, and the degree of this selectivity is under the control of non-specific parameters of the network.     

Classification and naming of dyes,stains and fluorochromes

A classification of dyes and other colorants is proposed, based on the chemical features responsible for their visibility and generally consonant with the writings of modern color chemists. The scheme differs in several respects from that of the Colour Index (CI), but it retains some traditional small groups of dyes that include biological stains. Natural dyes, recognized as a group in the CI, are placed with or near synthetic dyes with identical or similar chromophores. The new scheme also provides categories for dyes and fluorochromes that do not have places in the CI classification. Some CI categories, including lactones, aminoketones and hydroxyketones, are not recognized in this new scheme, which is adopted in the forthcoming 10th edition of Conn's Biological Stains: a Handbook of Dyes and Fluorochromes for Use in Biology and Medicine. Some rules are also set out for the spelling of trivial names, which has long been inconsistent in scientific literature. The ending '-ine' is used for compounds derived from organic bases (e.g., fuchsine and thionine, not fuchsin or thionin), and names ending in '-in' are for compounds that are not bases or their derivatives (e.g., eosin and phloxin, not eosine or phloxine). Initial capital letters are used only for words that are names of people or places (e.g., Nile blue or Congo red) and for the 'generic' components of CI application names (as in Acid yellow 36). Other words, including trade names that have fallen into common usage are not capitalized (e.g., alcian blue, biebrich scarlet, coomassie blue). The recommended spellings of some dyes differ from those commonly seen in vendors' catalogs and in biological publications, but they are generally consistent with English and American dictionaries, with recent writings in English by color chemists, and with the trivial names of other organic compounds.     

Classification and naming of dyes, stains and fluorochromes.

A classification of dyes and other colorants is proposed, based on the chemical features responsible for their visibility and generally consonant with the writings of modern color chemists. The scheme differs in several respects from that of the Colour Index (CI), but it retains some traditional small groups of dyes that include biological stains. Natural dyes, recognized as a group in the CI, are placed with or near synthetic dyes with identical or similar chromophores. The new scheme also provides categories for dyes and fluorochromes that do not have places in the CI classification. Some CI categories, including lactones, aminoketones and hydroxyketones, are not recognized in this new scheme, which is adopted in the forthcoming 10th edition of Conn's Biological Stains: a Handbook of Dyes and Fluorochromes for Use in Biology and Medicine. Some rules are also set out for the spelling of trivial names, which has long been inconsistent in scientific literature. The ending '-ine' is used for compounds derived from organic bases (e.g., fuchsine and thionine, not fuchsin or thionin), and names ending in '-in' are for compounds that are not bases or their derivatives (e.g., eosin and phloxin, not eosine or phloxine). Initial capital letters are used only for words that are names of people or places (e.g., Nile blue or Congo red) and for the 'generic' components of CI application names (as in Acid yellow 36). Other words, including trade names that have fallen into common usage are not capitalized (e.g., alcian blue, biebrich scarlet, coomassie blue). The recommended spellings of some dyes differ from those commonly seen in vendors' catalogs and in biological publications, but they are generally consistent with English and American dictionaries, with recent writings in English by color chemists, and with the trivial names of other organic compounds.     

Characterization of six textile dyes as fluorescent stains for flow cytometry.

Few fluorescent stains specific for cell constituents other than DNA are available. To assess their potential use as fluorescent stains for flow cytometry, the cell staining specificity of 55 compounds, originally synthesized for use as textile dyes and fluorescent brighteners, was explored and their excitation and emission wavebands determined. From these, six dyes were chosen for more detailed analysis. All six are vital stains, with excitation wavelengths allowing their use with an argon ion laser, and specific for a range of cell structures including mitochondria, Golgi bodies, lipid droplets, nuclear membrane, and endoplasmic reticulum. Concentrations as low as 0.01-0.25 microM were found to be adequate for most purposes, and high background fluorescence was not a problem. Their specificity allows differentiation between non-cycling and cycling cells. The properties of two of the stains allows their combination with propidium iodide or ethidium bromide for simultaneous determination of DNA content profiles. Being vital stains, usable at very low concentrations, and specific for a range of cell organelles, these six stains may be of considerable utility in flow cytofluorometry. We suggest that other textile dyes may be of use in flow cytofluorometry, or that their structures may form a starting point for the synthesis of further fluorescent stains of enhanced specificity.     

Tools for the automated assignment of high-resolution three-dimensional protein NMR spectra based on pattern recognition techniques

One of the major bottlenecks in the determination of proteinstructures by NMR is in the evaluation of the data produced by theexperiments. An important step in this process is assignment, where thepeaks in the spectra are assigned to specific spins within specificresidues. In this paper, we discuss a spin system assignment tool based onpattern recognition techniques. This tool employs user-specified templatesto search for patterns of peaks in the original spectra; these patterns maycorrespond to side-chain or backbone fragments. Multiple spectra willnormally be searched simultaneously to reduce the impact of noise. Thesearch generates a preliminary list of putative assignments, which arefiltered by a set of heuristic algorithms to produce the final results list.Each result contains a set of chemical shift values plus information aboutthe peaks found. The results may be used as input for combinatorialroutines, such as sequential assignment procedures, in place of peak lists.Two examples are presented, in which (i) HCCH-COSY and -TOCSY spectra arescanned for side-chain spin systems; and (ii) backbone spin systems aredetected in a set of spectra comprising HNCA, HN(CO)CA, HNCO, HN(CA)CO,CBCANH and CBCA(CO)NH.     

Establishment of HEp-2 cell preparation for automated analysis of ANA fluorescence pattern.

BACKGROUND: Identification of antinuclear antibodies (ANAs) has large clinical importance for the assessment of autoimmune diseases. HEp-2 cell preparations on microscopic slides are commonly used as antigenic substrate. Methods used for cell preparation are important for ANA pattern analysis; however, these methods differ widely and are mostly not specified. METHODS: HEp-2 cells were fixed using acetic acid-ethanol, methanol-acetone, acetone, formaldehyde, paraformaldehyde, or glutaraldehyde. Morphological analysis was done after haematoxylin-eosin staining and DAPI-staining of cell nuclei. RESULTS: The results demonstrate a high variability of cell and nuclear morphology depending on the used fixatives. Aldehyde fixatives conserved the cell structures best, acetone fixatives revealed remarkable changes. CONCLUSIONS: After selecting appropriate fixation procedures to preserve nuclear structures further experiments are necessary to find out which fixation procedure preserves the disease-linked antigens the best way and are, therefore, suitable to be used in ANA-testing of AABs.     

Standardization of automated cell-based protocols for toxicity testing of biomaterials

Advances in high-throughput screening (HTS) instrumentation have led to enormous reduction of costs (e.g., of pipetting stations) and to the development of smaller instruments for automation of day-to-day routines in small research laboratories. In the biomaterials community, there has been an increasing interest for standardized screening protocols to identify cell type-specific cytocompatible biomaterials suitable for tissue engineering (TE) applications. In this study, the authors established a multiplexed assay protocol for toxicity screening of biomaterials using a low- to medium-throughput robotic liquid handling station (LHS). The protocol contains analysis of viability, cytotoxicity, and apoptosis combined in one assay. This study includes performance results of a side-by-side comparison of the EpMotion 5070 LHS and conventional pipetting/dispensing systems. Critical parameters were optimized each for a given platform. Higher accuracy and reproducibility were achieved for LHS compared to manually treated samples. The practicability and accuracy of the method in a typical small laboratory setting were tested by running daily routine tasks by trained and untrained laboratory staff. In addition, advantages and disadvantages as well as the step-by-step application protocol are reported. The approach described provides a potential utility in screening biomaterials toxicity, allowing researchers to meet the needs of low- and medium-throughput laboratories.     

Three-dimensional pattern recognition: an approach to automated interpretation of electron density maps of proteins

A procedure is outlined for reducing the high resolution electron density map of a protein to a set of connected thin lines which follow the density. The side chain representations are removed from this skeleton leaving primarily main chain, disulfide bridges and very strong hydrogen bonds. Crystallographic and local operators are used to separate one protein molecule from the neighboring chains in the crystal. Provisional α-carbon positions along the skeletal main chain are derived by application of the “4 Å rule”.The application of these methods to the 2.0 Å electron density map of ribonuclease S (Wyckoff et al., 1970) is described. The skeleton of the isolated molecule that is produced in this fashion provides a good over-all view of the three-dimensional folding of the protein. The results suggest that the skeleton representation can be a valuable supplement to the present methods of map interpretation and a significant step towards complete automation of the interpretation process.The three-dimensional pattern recognition procedures described may have much broader applications than the protein structure problem for which they have been developed.     

The INs and OUTs of pattern recognition receptors at the cell surface

Pattern recognition receptors (PRRs) enable plants to sense non-self molecules displayed by microbes to mount proper defense responses or establish symbiosis. In recent years the importance of PRR subcellular trafficking to plant immunity has become apparent. PRRs traffic through the endoplasmatic reticulum (ER) and the Golgi apparatus to the plasma membrane, where they recognize their cognate ligands. At the plasma membrane, PRRs can be recycled or internalized via endocytic pathways. By using genetic and biochemical tools in combination with bioimaging, the trafficking pathways and their role in PRR perception of microbial molecules are now being revealed.     

Transformation and relational-structure schemes for visual pattern recognition

Two models for visual pattern recognition are described; the one based on application of internal compensatory transformations to pattern representations, the other based on encoding of patterns in terms of local features and spatial relations between these local features. These transformation and relational-structure models are each endowed with the same experimentally observed invariance properties, which include independence to pattern translation and pattern jitter, and, depending on the particular versions of the models, independence to pattern reflection and inversion (180° rotation). Each model is tested by comparing the predicted recognition performance with experimentally determined recognition performance using as stimuli random-dot patterns that were variously rotated in the plane. The level of visual recognition of such patterns is known to depend strongly on rotation angle. It is shown that the relational-structure model equipped with an invariance to pattern inversion gives responses which are in close agreement with the experimental data over all pattern rotation angles. In contrast, the transformation model equipped with the same invariances gives poor agreement to the experimental data. Some implications of these results are considered.     

The analysis of some commercial dyes and Romamowsky stains by high-performance liquid chromatography.

High-performance liquid chromatography has been used to quantitate batch variations in commericial samples of thiazine dyes, thiazine eosinates, and Romanowsky-type blood stains. It has been observed that all the dyes and eosinates examined, only methylene blue chloride and thionin were reasonably free of their methylated, demethylated, or oxidized homologs. Large variations in composition were observed between most of the samples of each type examined. In several instances the labeled compound was a minority species. In one instance a dye was apparently mislabeled. Large compositional variation was found between various batches of Wright and Giemsa stains, whereas significant differences between the thiazine composition of these two stain types were minor. Very little compositional variation was observed between the lots of LARC stain examined. The thiazine composition of Ames stain was similar for the three lots examined. Ames stain, however, was found to contain several components of unknown composition which have been linked to degradation products formed when stains are aged in methanolic solution.