You will need to adjust the procedure to match your new decolorizing mix.įalse Gram-negative stains can cause a plethora of issues for patients, so if these fixes aren’t working for you, please call the technical help team at 85 or email at If you switch suppliers or make your own decolorizer, the new decolorizer can impact performance. A 50/50 acetone decolorizer will decolorize much quicker than the 25/75 mix. Examine acetone in your procedure – The main decolorizing factor in your protocol is the acetone.Read the QC slides – If the Quality Control slide is being over-decolorized, then most likely the specimen is also matching.25% acetone is the slowest, and 75% acetone is the fastest. Bacteria with a predominantly peptidoglycan cell wall stain blue/purple with this technique and since they retain the primary stain are called Gram positive. The acetone percentage determines the rate of decolorizing. Change your decolorization length of time – Reduce the time in the solvent and rinse quickly, or use a decolorizing solution that is easier to control for the decolorizing step.
Reduce the time the specimen is exposed to the counterstain to under 30 seconds. If the specimen is overexposed to the counterstain, however, it can replace the crystal violet-iodine complex in gram-positive cells. Reduce time in counterstain – By rinsing the slide with a counterstain, safranin, all cells are stained red.Open bottles lose more than 90% of available iodine in 30 days, while closed bottles lose about 50% in the same time frame. Keep iodine bottles closed when not in use. Increase iodine exposure – Insufficient iodine exposure due to lower concentration of iodine can affect decolorization.The counterstain stains both gram-negative and gram-positive. To avoid this issue, don’t use more than a five-second water rinse at any stage of the procedure. Rinse the slide with a counterstain (safranin or carbol fuchsin) which stains all cells red. Crystal violet is susceptible to washout with water before binding to iodine, creating a false negative test. Reduce water rinse time – Excessive washing can cause excessive decolorization.In order to reduce the risk of speedy decolorization, reduce the heat during fixation. This can make cells more easily decolorized during the subsequent protocol steps. Excessive heat during the fixation process alters cell morphology and breaks down cell walls. Reduce heat during fixation – Gram staining works by first staining bacterial properties within the cell wall, then removing the excess stain using a decolorizer.
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