Evaluation of intracellular lipids by standardized staining with a Sudan black B fraction.

The selective staining of neutral lipids in Human Amnion cells in tissue culture was achieved using a particular fraction of the lipid dye, Sudan black B and a standardized staining procedure. The fraction, termed SBB-I, was isolated by thin-layer chromatography. The cytophotometric assessment of intracellular neutral lipids, stained with SBB-I, is described and applied to the study of changes in granulocytic neutral lipids in leukemia.     

Decomposition of Sudan Black B by Ultraviolet Light and Gases; its Lipid and Protein Staining Properties

The bluish-black spots of lipid-containing materials stained with a saturated solution of Sudan black B in 55% ethanol were found to fade and change color to brownish-pink shades in 5 min if exposed to ultraviolet light. Spots that were exposed to daylight for 6 hr on a sunny day lost 14% of their original color intensity but the decrease was less on cloudy days. Exposure to H₂S initiated fading and color change in 2 hr. Exposure to HCl vapors restored the original color but not its intensity. Spots kept in darkness and wrapped airtight showed a decline of 2.5% in color intensity after 96 hr and no obvious color change. The speed and extent of change of color and fading of the various fractions of the dye separated by means of paper chromatography were different. Heat coagulated serum proteins were stained blue with commercial Sudan black B solution in 55% ethanol.     

Decomposition of Sudan Black B by Ultraviolet Light and Gases; its Lipid and Protein Staining Properties

The bluish-black spots of lipid-containing materials stained with a saturated solution of Sudan black B in 55% ethanol were found to fade and change color to brownish-pink shades in 5 min if exposed to ultraviolet light. Spots that were exposed to daylight for 6 hr on a sunny day lost 14% of their original color intensity but the decrease was less on cloudy days. Exposure to H₂S initiated fading and color change in 2 hr. Exposure to HCl vapors restored the original color but not its intensity. Spots kept in darkness and wrapped airtight showed a decline of 2.5% in color intensity after 96 hr and no obvious color change. The speed and extent of change of color and fading of the various fractions of the dye separated by means of paper chromatography were different. Heat coagulated serum proteins were stained blue with commercial Sudan black B solution in 55% ethanol.     

Beta-elimination: an unexpected artefact in proteome analysis

Two persistent myths, ingrained in the electrophoretic literature of the last thirty years, namely carbamylation and deamidation, have been recently dispelled (Herbert et al., J. Proteome Res. 2002, in press). We report here, for the first time, a noxious and unexpected artefact in proteome analysis: beta-elimination (or desulfuration), which results on the loss of an H(2)S group (34 Da) from cysteine (Cys) residues for protein focusing in the alkaline pH region. With such an elimination event, a dehydro alanine residue is generated at the Cys site. In turn, the presence of a double bond in this position elicits lysis of the peptide bond, generating a number of peptides of fairly large size from an intact protein. The first process seems to be favored by the electric field, probably due to the continuous harvesting of the SH(-) anion produced. The only remedy found to this noxious degradation pathway is the reduction and alkylation of all Cys residues prior to their exposure to the electric field. Alkylation appears to substantially reduce both beta-elimination and the subsequent amido bond lysis.     

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.     

Extraction of osmium-containing lipids by section staining for TEM

Postfixation with osmium-ferrocyanide or OSO4 renders lipid droplets in rat liver and kidney homogeneously electron dense without additional section staining. In sections of the same block that have been single stained by uranyl acetate or lead citrate, lipid droplets show a more electron translucent center surrounded by a dense rim. In sections double stained with uranyl acetate and lead citrate, lipid droplets frequently appear as empty vacuoles, from which the electron dense content has been completely extracted.     

Evidence against the existence of acetylated Sudan Black B

Summary The nature of acetylated Sudan Black B (aSBB) has been investigated, and it has been found, by thin layer chromatography, that each fraction of aSBB has an R_f which is the same as that of a similar fraction of Sudan Black B (SBB). However, aSBB has been found to have fewer fractions, 9–12 than SBB, 14–16. The two major fractions from aSBB and SBB were examined, and a great similarity was found between the absorption spectra of the respective fractions of aSBB and SBB. The major fraction of aSBB was investigated by mass spectroscopy and found to have a similar molecular weight to that expected of SBB. This demonstrates that aSBB is not in fact acetylated, and that the components of aSBB are chemically no different from the corresponding components of SBB.     

Evidence against the existence of acetylated Sudan Black B

The nature of acetylated Sudan Black B (aSBB) has been investigated, and it has been found, by thin layer chromatography, that each fraction of aSBB has an Rf which is the same as that of a similar fraction of Sudan Black B (SBB). However, aSBB has been found to have fewer fractions, 9-12 than SBB, 14-16. The two major fractions from aSBB and SBB were examined, and a great similarity was found between the absorption spectra of the respective fractions of aSBB and SBB. The major fraction of aSBB was investigated by mass spectroscopy and found to have a similar molecular weight to that expected of SBB. This demonstrates that aSBB is not in fact acetylated, and that the components of aSBB are chemically no different from the corresponding components of SBB.     

Quantification of lipids and protein in thin biofilms by fluorescence staining

The efficiency of removing unwanted biofilm from surfaces in industrial water systems was examined by fluorescence microscopy and image analysis. A quantitative assay for in situ determination of biofilm components was developed and tested on thin biofilms grown in reactors as well as real biofilms sampled from a fish processing factory. Different fluorescent dyes for in situ detection of protein, lipid and total organic matter were tested. It was possible to determine the approximate amounts, concentrations and coverage of the different components by correlating the fluorescent intensity of the biofilm components to standard solutions immobilised as a biofilm. The quantification methods were evaluated as a strategy for determining the efficiency of different disinfection/cleaning procedures, showing that quantification of these biofilm components was fast and reliable for optimisation of cleaning in place procedures. However, the approach also showed that bacterial cells, as investigated by culture-independent procedures, were killed but not removed by most disinfection procedures tested, potentially leading to surfaces which are easily recolonised.     

Quantification of lipids and protein in thin biofilms by fluorescence staining

The efficiency of removing unwanted biofilm from surfaces in industrial water systems was examined by fluorescence microscopy and image analysis. A quantitative assay for in situ determination of biofilm components was developed and tested on thin biofilms grown in reactors as well as real biofilms sampled from a fish processing factory. Different fluorescent dyes for in situ detection of protein, lipid and total organic matter were tested. It was possible to determine the approximate amounts, concentrations and coverage of the different components by correlating the fluorescent intensity of the biofilm components to standard solutions immobilised as a biofilm. The quantification methods were evaluated as a strategy for determining the efficiency of different disinfection/cleaning procedures, showing that quantification of these biofilm components was fast and reliable for optimisation of cleaning in place procedures. However, the approach also showed that bacterial cells, as investigated by culture-independent procedures, were killed but not removed by most disinfection procedures tested, potentially leading to surfaces which are easily recolonised.     

Genetic fingerprints of parasites causing severe malaria in a setting of low transmission in Sudan

In this study we intended to examine the extent of genetic diversity of Plasmodium falciparum parasites causing severe malaria (SM). For this purpose, 100 parasite isolates were obtained from patients with SM and uncomplicated malaria, from an area of low and unstable malaria transmission in Sudan. The diversity of infection (DOI) was estimated by relating the number of the different parasite genotypes that were detected to the total number of parasites that were genotyped (parasite population/subpopulation). We used different molecular markers individually (pfcrt-76, pfmr1-86, GLURP size and MSP2 family and size) and as a group to set a multilocus genetic profile for each parasite isolate. The DOI as estimated by MSP2 and GLURP was 0.553 and 0.435, respectively. However, combination of all four molecular markers (multilocus genetic profile) revealed a fingerprint pattern of genetic diversity with a DOI of 0.936, indicating that in SM infection, diversity is the rule and homogeny is the exception. Furthermore, our clinical data suggest that the virulence markers might also be more diverse than expected. In conclusion, the results are unexpected and overturn the assumption that parasites causing SM are a limited subpopulation of virulent parasites or of a clonal nature. However, it was more likely that there was a genetically unique parasite in each infection.