Staining lipids

Further investigation by Kresling confirmed earlier observations that saponification of the fat gave an alcohol of high molecular weight. He was unable to detect cholesterol in the tubercle lipids. It was found that ethereal hydrogen chloride would remove the acid-fast staining lipid fraction from the bacillary cell. 

Oil red O is used to stain lipids, fatty acids, triglycerides, and cholesterol to make them visible under light microscopy. Like other lysochromes (fat stains), oil red O is more soluble in lipids than in its alcohol solvent which causes it to preferentially stay in lipid droplets (Kiernan, 1981). This stain is often used to demonstrate adipogenic differentiation of stem or progenitor cells. 

Figure 22.6 Microscopic observation of bodipy-stained lipid bodies in selected microalgae. Left panel, bright field right panel, green fluorescence field. Green fluorescence indicates lipid bodies.

The effect is more than just a matter of pH. As shown in Fig. XV-14, phospholipid monolayers can be expanded at low pH values by the presence of phosphotungstate ions [123], which disrupt the stmctival order in the lipid film [124]. Uranyl ions, by contrast, contract the low-pH expanded phase presumably because of a type of counterion condensation [123]. These effects caution against using these ions as stains in electron microscopy. Clearly the nature of the counterion is very important. It is dramatically so with fatty acids that form an insoluble salt with the ion here quite low concentrations (10 M) of divalent ions lead to the formation of the metal salt unless the pH is quite low. Such films are much more condensed than the fatty-acid monolayers themselves [125-127]. 

Some substances, e. g. penicillin and pyrazolinone derivatives, are poorly detected by iodine staining with detection limits of 2-4 pg substance per chromatogram zone [6, 7]. The limits for lipids and for opium alkaloids lie with 50-500 ng [8] in the middle nanogram range [9]. 

The cell walls of mycobacteria contain three structures peptidoglycan, an arabinogalactan polysaccharide and long chain hydroxy fatty acids (mycolic acids) which are all covalently linked. Additional non-covalently attached lipid components found in the wall include glycolipids, various phospholipids and waxes. The lipid-rich nature of the mycobacterial wall is responsible for the characteristic acid-fastness on staining and serves as a penetration barrier to many antibiotics. Isoniazid and ethambutol have long been known as specific antimycobacterial agents but their mechanisms of action have only recently become more clearly understood. 

Several of these morphological factors are illustrated in Figure 1. Figure lA is of the fat portion of bacon and has been stained for connective tissue. It is noted that fat tissue is not all lipid but has an extensive connective tissue component ranging from fairly thick layers to delicate layers defining each adipose cell. Figure IB is from a finely chopped emulsion. Connective tissue pieces are stained dark, the protein matrix is gray and the... 

Isolated hepatocytes incubated with ionic iron rapidly undergo lipid peroxidation. Some studies have not shown a consequent decrease in viability (as indicated by uptake of trypan blue or release of enzymes). This is probably a result of short incubation times, as changes in viability lag behind increases in lipid peroxidation, and may not occur for more than 2 h after lipid peroxidation begins (Bacon and Britton, 1990). Recent studies have shown strong correlations between increased lipid peroxidation [production of thiobarbituric acid (TBA) reactants] and loss of cell viability (trypan blue staining) (Bacon and Britton, 1989). The significance of the lag between lipid peroxidation and decreases in cell viability is as yet uncertain. 

Figure 4. Electron micrographs of the polymerized vesicles of Lipid 1 (stained by 1% uranyl acetate).

Another limitation of 2D gels is that membrane proteins are underrepresented. Because membrane proteins account for approximately 30% of total proteins (Wallin and Von Heijne, 1998), this is a serious problem for characterization of the proteome. The relative lack of membrane proteins resolvable on 2D gels can be attributed to thee main factors (i) they are not abundant, and therefore are difficult to detect by standard staining techniques, (ii) they often possess alkaline pi values, which make them difficult to resolve on the pH gradients most often used for isolelectric focusing, and (iii) the most important reason for under representation may be that membrane proteins are poorly soluble in the aqueous media used for isoelectric focusing (Santoni et al., 2000). Membrane proteins are designed to be soluble in lipid bilayers and are therefore difficult to solubilize in water-based solutions. 

Information concerning myelin structure is also available from electron microscope studies, which visualize myelin as a series of alternating dark and less dark lines (protein layers) separated by unstained zones (the lipid hydrocarbon chains) (Figs 4-4 to 4-7). There is asymmetry in the staining of the protein layers. The less dark, or intraperiod, line represents the closely apposed outer protein... 

Figure 5.29 Negative-stained electron micrograph of DCs,9 PC tubule from 5-mg/ml sample in methanol-water (85 15). Solvent conditions and lipid concentration were adjusted to obtain sample of two-bilayer tubules as shown in enlargement of tubule edge in bottom panel. Bar = 200 nm (top) 50 nm (bottom). Reprinted with permission from Ref. 125. Copyright 1998 by the American Chemical Society.

Figure 5.38 (a) Negative-stained transmission electron micrograph of nanotubules formed from equimolar mixture of DCg PC and DNPC (2 mM total lipid concentration) stored at 4°C just prior to deposition, (b) Freeze-fracture electron micrograph of twisted ribbons at 27°C. Bars = 100 nm. Reprinted with permission from Ref. 153. Copyright 2001 by the American Chemical Society. 

Aldehyde groups stain pink Lipids and fatty acids—Sudan III Unfixed or fixed frozen sections Take sections to 50% aqueous ethanol... 

Lead citrate/uranyl acetate6 Step 1 Float or immerse sections for 10-30 min on filtered 1-2% aqueous uranyl acetate (or in EtOH) wash with ultrapure H20 (three beakers of 50 mL each) by dipping grids held with a forceps dry for 5 min Step 2 Place drops of lead citrate (lead carbonate free) onto a wax surface (parafilm or dental wax) in a Petri dish line edges of dish with pellets of KOH float grid with sections (sections face down) for 4-5 min (if overstained 2-3 min and dilute stain) wash grids with sections in ultrapure H20 Nonselective enhancement of membrane contrast, ribosomes, and nuclear material proteins and lipid droplets... 

Iodine vapour Place the dried plate in a sealed tank containing a few iodine crystals Dark yellow-brown spots appear within a few minutes where lipids have absorbed the iodine. Unsaturated lipids are more intensely stained. Glycolipids do not stain significantly... 

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