Use of eriochrome cyanine R in routine histology and histopathology: is it time to say goodbye to hematoxylin?

Abstract

Eriochrome cyanine R (ECR) is a synthetic anionic dye that forms complexes with cations such as iron. We found that an iron-ECR (Fe-ECR) mixture provided either nuclear or myelin staining depending on the differentiator used. Selective nuclear staining was obtained by differentiation in an aqueous HCl solution, pH 0.95, followed by a wash in slightly alkaline tap water; the pH difference facilitated control of differentiation. When used with an eosin B counterstain, results were nearly indistinguishable from standard hematoxylin and eosin (H & E) staining. Nuclear staining with Fe-ECR provides tinctorial features similar to regressive aluminum-hemateins as well as resistance to acidic solutions such as those of iron hemateins. Fe-ECR also stained selectively intestinal cells of the diffuse neuroendocrine system (DNES). In addition to its use as an H & E substitute, acid differentiated Fe-ECR produced acid-resistant and selective nuclear counterstaining in combination with Alcian blue, and in the Papanicolaou and van Gieson techniques. With alkali differentiation, Fe-ECR produced selective myelin staining, which was compatible with neutral red counterstaining. Myelin sheaths were stained aqua blue. Fe-ECR could be used for both cytological and histological samples, and was suitable for use in automated tissue stainers. ECR also is less expensive than hematoxylin. Hematoxylin still may be preferred as a nuclear counterstain for some immunostaining methods for which Fe-ECR mixtures probably are too acidic.     

Isolating Mortierella alpina strains of high yield of arachidonic acid

AIMS: To develop a fast isolation method for arachidonic acid-producing fungi of high yield. METHODS AND RESULTS: Relation between the staining degree of mycelia of Mortierella alpina stained with triphenyltetrazolium chloride (TTC) and arachidonic acid content in the fungal lipids was investigated. Results showed that staining degree of mycelia stained with TTC increased when arachidonic acid content in mycelia lipids increased. This finding was used to isolate strains of high arachidonic acid yield. Arachidonic acid producing fungi were selectively isolated from soil at a low temperature of 4 degrees C and the mycelia of these isolates were stained with TTC. CONCLUSIONS: The strain M. alpina M6 that had the highest staining degree had the highest arachidonic acid content (72.3%). The yield of arachidonic acid in this strain reached 4.82 g l(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: A fast and effective method to isolate strains of high arachidonic acid yield was established according to the finding that staining degree of mycelia of M. alpina was positively correlated with arachidonic acid content in mycelia lipid.     

Growth and viability of mycorrhizal extraradical mycelia associated with three temperate orchid species

Growth and enzymatic activities of extraradical mycelia (ERM) of native mycorrhizal symbionts associated with three orchid species, Dactylorhiza fuchsii, D. majalis and Platanthera bifolia, were studied. ERM extracted from the mycorrhizosphere of these species showed features typical for fungi that form orchid mycorrhiza. In the first pot experiment, three different treatments were applied on tubers of D. fuchsii transplanted from a natural site: control (no specific treatment), reinoculated (surface-sterilized tubers reinoculated with mycorrhizal fungi-colonised roots), and benomyl (nonsterilized tubers treated with fungicide). However, no significant differences in ERM growth and intensity of root mycorrhizal colonisation at harvest were observed among these treatments. ERM associated with reinoculated D. fuchsii plants showed significantly higher alkaline phosphatase (ALP) enzymatic activity at week 36 than at week 24, but no differences were observed for NADH diaphorase activity. Benomyl application significantly reduced ALP activity in comparison with reinoculated plants at week 36. In the second experiment, plants of all three species were either untreated (control), or repeatedly treated with benomyl. Similarly to the results of the first experiment, benomyl application did not reduce the ERM growth of mycorrhizal symbionts associated with D. majalis and D. fuchsii. The low ERM growth associated with benomyl-treated P. bifolia was probably caused by poor root system development in this treatment. Significantly higher mycorrhizal colonisation was found for D. fuchsii compared to P. bifolia in control treatments at the end of cultivation. The ERM of native symbionts of the three orchid species studied seemed to have a different growth pattern over time and responded differently to fungicide application.     

Phloxine B, a versatile bacterial stain

The data presented suggest that Phloxine B, a color additive for food, drugs, and cosmetics has a potential use as a nontoxic, faster (<2 min), inexpensive (350 tests for <1 cent material) and simpler to use alternative to Gram staining. Using Phloxine B staining it was possible to differentiate among gram-negative and gram-positive bacteria by visual determination under normal room lighting, light microscopy, fluorescence microscopy and confocal microscopy. This work demonstrated that Phloxine B can be used as a differential versatile bacterial stain and establishes a correlation between the staining properties of the dye and its bactericidal effect.     

An alternative Alcian Blue dye variant for the evaluation of fetal cartilage

BACKGROUND: The most comprehensive evaluation of vertebrate skeletal development involves the use of Alizarin Red S dye to stain ossified bone and various other dyes to stain cartilage. The dye used most widely to stain fetal cartilage in rodents and rabbits is Alcian Blue 8GX. However, the global supply of this specific dye has been exhausted. Several forms of the dye marketed as Alcian Blue 8GX are now available, although they are not synthesized via the original 8GX manufacturing process. METHODS: One new Alcian Blue 8GX form and two Alcian Blue dye variants were evaluated in rats and rabbits using standard staining procedures. The staining quality of these dyes were evaluated relative to the original form of Alcian Blue 8GX based on cartilage uptake of the dye, clarity of the cartilaginous components, staining intensity of the dye, and overall readability of the specimens under stereomicroscopic evaluation. RESULTS: Staining with the newer form of Alcian Blue 8GX resulted in poor staining quality. The Alcian Blue-Pyridine variant performed well, although staining intensity was less than optimal. The Alcian Blue-Tetrakis variant provided staining characteristics that were most similar to the original form of Alcian Blue 8GX. CONCLUSIONS: Alcian Blue-Tetrakis was markedly better in its ability to stain fetal cartilage than the newer form of Alcian Blue 8GX.     

A Primary Study of the Subgroups of T Lymphocytes in MHV-3 Induced Chronic Viral Hepatitis

To study the contribution of T cell subsets in the pathogenesis of Murine hepatitis virus Type 3(MHV-3) induced chronic viral hepatitis in C3H/Hej mice,ninety C3H/Hej mice were chosen to individually receive 10 plaque forming units(PFU)of MHV-3 intraperitoneally.The changes of virus titer and pathology in liver tissue were examined by standard plaque assay and by the hematoxylin/eosin(HE) staining method from 2 days post MHV-3 infection.The ratios of T cell subsets including CD3 CD4 CD8-,CD3 CD4-CD8 ,CD3 CD4-CD8-,CD3 CD4 CD25 ,CD3 CD4 CD25-and CD3 CD4-CD25 T lymphocyte of total T lymphocytes in blood,spleen and liver were examined at 0,2,4,6,8,10,12,15,20,25,30,40 days post MHV-3 infection by flow cytosorting.We observed that the virus titer raised and showed persistent virus duplications and inflammatory changes in the livers of C3H/Hej mice from 2 days post MHV-3 infection.The double negative T cell(DN Treg cell) and CD4 CD25 T cell ratios increased significantly from 2 days post MHV-3 infection in C3H/Hej mice,and CD3 CD4 CD8-,CD3 CD4-CD8 ,CD3 CD4 CD25-and CD3 CD4-CD25 T cell ratios decreased accordingly.In conclusion,the changes of virus titer and pathology in the livers of C3H/Hej mice post MHV-3 suggest their contribution to viral persistence.Further characterizations of DN Treg cells are that infection indicates that MHV-3 could induce the chronic inflammation in livers of C3H/Hej mice.The increase of the DN Treg cell and CD4 CD25 T cell ratios in C3H/Hej mice post MHV-3 infection suggests that DN Treg cells and CD4 CD25 T cells may both have important suppressive immunomodulation functions in the development of chronic viral hepatitis and have important roles in the virus persistent infection.Further characterizations of DNT cell and CD4 CD25 T cell are under investigation.     

Specific detection and quantification of culturable and non-culturable mycobacteria in metalworking fluids by fluorescence-based methods

Aims: To optimize and evaluate fluorescence microscopy assays for specific assessment of mycobacteria and co‐contaminants, including culturable and non‐culturable sub‐populations, in metalworking fluids (MWF). Methods and Results: Auramine‐O‐rhodamine (AR) staining and LIVE/DEAD BacLight? Bacterial Viability staining (L/D staining) were adapted and evaluated for detection/quantification and differentiation (viable vs non‐viable) of the MWF‐associated mycobacteria and the background bacterial flora, respectively. The AR staining method was found to be specific to MWF mycobacteria with a minimum detection limit of 10 cells ml^?1 and was comparable to the QPCR in quantification efficiency in MWF matrix. The L/D staining‐based microscopy allowed differential quantification of viable vs non‐viable cells. In general, a 3‐log difference was observed between the L/D microscopy count and culture count accounting for the presence of non‐culturable fraction in the bacterial population in in‐use MWF. Conclusions: The optimized AR staining‐ and the L/D staining‐based microscopy methods have the potential for rapid, specific and differential assessment (viable vs non‐viable) of MWF‐associated mycobacteria and co‐contaminants in field MWF. Significance and Impact of the study: Early detection of MWF mycobacteria by rapid, low‐cost, less‐skill intensive and culture‐independent fluorescence‐based microscopy methods will facilitate timely intervention to protect the machine workers from occupational hazards.     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEF

Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20-50 ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS.