![]() Widest range of power settings and transfer times Comparison of Western Blotting Transfer Systems Filter papers and membranes are pre-wet and packaged in single-use packages, simplifying assembly of the transfer stack. Transfer conditions must be optimized for proteins of different molecular weights, e.g., to prevent under-transfer (incomplete transfer of proteins out of the gel) or over-transfer (loss of proteins passing completely through the membrane).Ī recent development, these systems utilize specialized apparatus that use proprietary filter papers and specialized buffers to rapidly and efficiently transfer proteins. Both the voltage and the distance between the electrodes then play a major role in governing the rate of elution of the proteins from the gel. The electric field strength (measured in V/cm) that is generated between the electrodes is the driving force for transfer. ![]() Voltage is applied between the electrodes and proteins migrate to the membrane following the current that is generated by the applied voltage across the electrodes. During this process, the membrane and gel are placed together, with filter paper between two electrodes. The most common method of transfer in western blotting is electrophoretic transfer, where an electric field is used to elute proteins from gels and transfer them to membranes. After staining with carbol fuchsin, acid-fast and non-acid-fast bacteria both appear the color fuchsia (a pinky-purple color).Gel and Membrane Setup for Electrophoretic Transfer Acid-fast bacteria and non-acid-fast bacteria begin transparent. In addition, the stain is used to determine the presence of acid-fast bacteria from lung tissue in patients undergoing antibiotic therapy.įigure 1: Diagram shows how cells change color during the steps of the acid-fast stain by comparing acid-fast bacteria and non-acid-fast bacteria. Because of this feature, this stain is extremely helpful in identification in diseases caused by acid-fast bacteria, particularly tuberculosis and leprosy. The decolorized non-acid-fast cells then take up the counterstain, which in our case is methylene blue.Īcid-fastness is an uncommon characteristic shared by the genera Mycobacterium and Nocardia (weakly acid-fast). The smear is then rinsed with a very strong decolorizer (acid-alcohol), which strips the stain from all non-acid-fast cells but does not permeate the cell wall of acid-fast organisms. Steam helps to loosen up the waxy layer and promotes entry of the primary stain inside the cell. This is further assisted by the addition of heat in the form of heat (steam). The primary stain used in acid fast staining, carbol fuchsin, is lipid-soluble and contains phenol, which helps the stain penetrate the cell wall. Non-acid fast bacteria will also take up the carbol fuchsin, but the acid alcohol decolorizer will remove it from wall since the primary dye does not bind strongly to the cell wall. Once in, it will not come out: But the acid-alcohol decolorizer will take it out of the non-acid fast cell walls since the primary dye does not bind strongly to the cell wall. Steam is used to get the carbol fuchsin primary dye to go into the cell wall. They are usually characterized using the acid-fast staining procedure. Acid-fast bacteria stain poorly with the Gram stain procedure, appearing weakly Gram-positive or Gram-variable. The phenol in the carbol fuchsin facilitates the dye going into the waxy wall of the bacterium. The ability of the bacteria to resist decolorization with acid-alcohol confers acid-fastness to the bacterium. Although difficult to stain, once the stain goes into the cell wall, the cell will not de-stain or decolorize easily. Acid-fast bacteria, of which there are very few-the major genus Mycobacterium, have a high concentration of mycolic acid, a lipid, in their cell walls. This type of cell wall is resistant to most compounds, therefore acid-fast microorganisms require a special staining technique. Acid-fast microorganisms are characterized by wax-like, nearly impermeable cell walls they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids. \)Īcid fast stain is a differential stain used to identify acid-fast organisms such as members of the genus Mycobacterium.
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