Why: To identify a specific organelle or another cellular structure and to mark individual cells, it is necessary to counterstain them in immunocytochemistry/immunofluorescence (ICC/IF) assays. How: Counterstaining is often performed with dyes or antibodies specific to the organelle or cellular structure of interest.
What is the purpose of the counterstain?
What is the purpose of the counterstain (safranin)? The counterstain stains the decolorized Gram – bacteria deep pink, so that they can be seen against the purple Gram + bacteria.
Why is counterstain necessary quizlet?
Why is it necessary? Could colors other than red be used? Saffranin is the counter stain used, it is necessary so gram negative bacteria can be identified.
Is counterstain necessary?
A good nuclear counterstain is essential to correct interpretation of immunohistochemical stain responses. The counterstain distinguishes reactive, neoplastic, and necrotic cells as well as neuroanatomic relationships. Necrotic cells have nuclear pyknosis, karyorrhexis, and karyolysis.
Why is the use of a counterstain important in the gram stain?
In Gram staining, crystal violet stains only Gram-positive bacteria, and safranin counterstain is applied which stains all cells, allowing the identification of Gram-negative bacteria as well.Counterstains are sometimes used to separate animals from organic detritus in microbiology studies.
Why is the counterstain of safranin necessary?
Cells are stained with crystal violet dye.A counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells.
Why is decolorization important in a differential stain?
The length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria. The final step in gram staining is to use basic fuchsin stain to give decolorized gram-negative bacteria pink color for easier identification.
How would your results be affected if you omitted the counterstain?
If it were omitted, how would staining results be affected? It it is ommitted then, when flushed with water and the alcohol decolorized, the bacteria will come off. What is the purpose of the alcohol solution in the Gram stain?
What is the purpose of the primary stain mordant decolorizing agent and counterstain?
The decolorizing agent removes the lipid and the crystal violet-iodine complex from the cell wall so that the cell is unstained. The counterstain stains the Gram-negative cell.
What happens if you don’t add iodine to a Gram stain?
If the iodine is not applied during the Gram stain, then gram positive cells will likely stain pink.
What happens if mordant step is skipped?
Mordant step skipped: Gram-positive bacteria will appear Gram-negative. 4. Slide not decolorized: Gram-negative bacteria will appear Gram-positive.
What is the most commonly used counterstain?
DAPI is traditionally the most popular fluorescent nuclear counterstain. Special stains are used to stain certain cell types, microorganisms, and specific proteins, carbohydrates and metabolites found in the tissue matrix and within cells.
What is the best procedure for decolorization?
What is the best procedure for decolorization? Add decolorizing agent until run-off is clear. This method allows the decolorizing agent to dissolve the outer membrane of Gram-negative cells and rinse out the crystal violet from the thin layers of peptidoglycan. This causes the run-off to be purple.
What happens if you don’t counterstain with safranin?
A safranin counterstain is used to stain these Gram-negative cells pink. However, if the safranin counterstain were forgotten, the Gram-negative bacteria would remain unstained, as the original crystal violet stain would have been removed during the ethanol wash, and no additional stain would have been applied.
Why is a counterstain safranin added to the Gram staining procedure?
The gram iodine helps the bacteria retain the dye, and forms a complex with the crystal violet. Why is a counterstain (safranin) added to the Gram staining procedure? Because gram-negative bacteria are colorless after the ethanol is used, it has to have a counterstain to contrast against the slide to make them visible.
Why is safranin used as a counterstain in Endospore staining?
After the initial washing, a counter stain (safranin) is used. The purpose of the counter stain is to stain the vegetative cells that lost the primary stain.
What happens if Decolorizer is left on too long?
It is possible to leave the decolorizer on too long and strip the blue stain out of all the bacteria, even the Gram positive ones. b. If the decolorizer is not left on long enough, the blue color will remain in the Gram negatives and they will appear Gram positive (purple) Page 4 c.
What is the Decolorizer in the Gram stain?
The decolorizer, ethyl alcohol, is the most crtitical step. Ethyl alcohol is a nonpolar solvent, and thus penetrates the cell walls of Gram negative cells more readily and removes the crystal violet-iodine complex.
What step can you omit in Gram staining?
Which step can you omit without affecting determination of the gram reaction? You can omit the counterstain (safranin) and still be able to differentiate between gram positive and gram negative cells.
What happens if you forget crystal violet?
95% ethanol, because it removes the primary stain (crystal violet) from Gram-negative cells. If missed, then the bacteria would remain purple and give a false positive result.
How long will you drench the bacterial smear in safranin?
Apply safranin to the bacterial smear for 45 seconds. Wash with tap water. Apply 95% alcohol drop by drop to the bacterial smear until it runs almost clear. Wash with tap water.
Contents