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.     

Detection of lipids in the plant meristematic cell with the aid of Sudan black staining

As lipids can be a source of artefacts during intracellular localization of enzymes by cytochemical methodsin situ it was the aim of the present work to obtain orientation data on the distribution of lipids in the meristematic plant cells. The different fixation and object embedding methods examined revealed that it is best to fix the material by some formol fixative and without chroming, to embed it in polyethyleneglycol media. An alcoholic solution of Sudan black was found to be most reliable. In the meristematio cells the cytoplasm is usually stained more intensely than the nucleus. The ground cytoplasm is stained weakly while cytoplasmic particles are stained intensely. In some cases an intense black staining of nuclei, particularly in the prolongation zone, can be achieved. The staining intensity of cell components does not decrease on extracting lipids with pyridine. After extracting the dye from stained cell components a browninsh residual coloration remains. Chromatography of Sudan black revealed in all the samples tested slowly moving spots (blue and violet) and rapidly moving ones (red II, yellow, red I, colourless).     

Decomposition of Sudan Black B Causing an Artefact in the Staining of Lipids

Sudan black B was found to be decomposed in acid solutions. This decomposition was rapid below pH 4. Above this pH the decomposition was markedly slower. After mild acid hydrolysis, the product was studied chromatographically and found to contain a basic dye with a pK of about 4, a brown color above, and a red color below pH 4. The nature of the product of hydrolysis indicates that it can stain nucleoproteins and mucopolysaccharides, hence a brownish stain is more indicative of artefact than the staining of lipids.     

Effect of phosphomolybdic acid on the binding of Sudan black B

Paraffin sections of tissues fixed in absolute alcohol or Carnoy's fluid were mordanted in a 1% aqueous solution of phosphomolybdic acid, stained in saturated solutions of Sudan black B, acetylated Sudan black, various solvent and basic dyes in 70% ethyl alcohol for 5 min at room temperature, dehydrated in alcohol and covered in Permount. Sudan black B and other dyes with basic groups stained basement membranes, reticulum and collagen fibers intensely. Acetylated Sudan black, Sudan IV and oil red 0 did not color any tissue structures. Control sections, without pretreatment, did not bind Sudan black B. These findings indicate interaction between basic groups of the dye and free acid groups of phosphomolybdic acid.     

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.