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.
What is the purpose of Decolorizer in Gram staining?
A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria.
Why safranin is used in Gram staining?
The safranin is also used as a counter-stain in Gram’s staining. In Gram’s staining, the safranin directly stains the bacteria that has been decolorized. With safranin staining, gram-negative bacteria can be easily distinguished from gram-positive bacteria.
What is the mordant in Gram staining and why is it important?
A mordant is a substance that increases the affinity of the cell wall for a stain by binding to the primary stain, thus forming an insoluble complex, which gets trapped in the cell wall. In the Gram stain reaction, the CV and iodine form an insoluble complex (CV-I), which serves to turn the smear a dark purple color.
Why is decolorization important in Gram staining quizlet?
Decolorization is important because it will rinse the color out of the gram negative cells so they can be dyed with a secondary dye so you can see the difference between the Gram positive and Gram negative cells.
What is the purpose of ethanol in Gram staining?
Ethanol acts as a solvent which causes the crystal violet-iodine complex to dissolve away and rinse out of the cell wall if the cells are gram negative. Recall that gram negative cells have a very thin layer, about 20% of their cell wall, made up of peptidoglycan.
Why is ethanol used in Gram staining?
After staining the sample with crystal violet, ethyl alcohol is used to decolorize the sample. It achieves its purpose by dehydrating the peptidoglycan layer by tightening and shrinking it.
What is the purpose of using safranin?
Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endospore staining. It can also be used for the detection of cartilage, mucin and mast cell granules.
What would happen if you use safranin as the primary stain?
If you reverse the staining procedure that is using safranin first, this will cause all the bacteria to remain red and crystal violet applied later on may cause the gram-negative bacteria to become violet in color and the gram -positive bacteria will remain red.
How does safranin affect Gram-positive cells?
How does safranin affect Gram-positive cells? Safranin penetrates the cell wall, but not enough of it is retained to cause a color change…… In the Gram-positive cell walls, most of the spaces between the molecules that make up peptidoglycan are already occupied by crystal violet/iodine complexes.
What is the purpose of a mordant in a staining procedure?
A mordant or dye fixative is a substance used to set (i.e. bind) dyes on fabrics by forming a coordination complex with the dye, which then attaches to the fabric (or tissue).
What is the importance of Gram staining in microbiology?
A Gram stain is most often used to find out if you have a bacterial infection. If you do, the test will show if your infection is Gram-positive or Gram-negative. A Gram stain may also be used to diagnose fungal infections.
What is the purpose of the mordant iodine during Gram staining?
The mordant is a substance used in conjunction with a dye to increase its staining ability. For example, in Gram stain, Gram’s iodine is used to form a complex with crystal violet that makes the dye molecules larger and better able to adhere to the sample.
Is the counterstain used in the Gram stain is a basic dye?
The counterstain used in the Gram stain is a basic dye. In a completed Gram stain, gram-negative bacteria are colorless. In a completed Gram stain, gram-positive bacteria are purple. If acid-fast bacteria are stained with the Gram stain, they will stain gram-negative.
What was the purpose of the counterstain quizlet?
The counterstain, a dye of different color from the primary stain, is used to give color to the cells that became colorless for not retaining the primary stain after applying the decolorizing agent.
How might excessive decolorization impact the results of the Gram stain?
Over-decolorizing will lead to an erroneous result where gram-positive cells may stain pink to red indicating a gram-negative result, and under-decolorizing will lead to an erroneous result where gram-negative cells may appear blue to purple indicating a gram-positive result.
What happens if you don’t use alcohol in Gram stain?
So if you leave out the alcohol/acetone. The gram negative bacteria will retain their outer cell membrane, and stay purple when your staining is done. Thus both gram positive and negative bacteria will stain purple without the alcohol/acetone step.
What is the purpose of the alcohol used in a Gram stain quizlet?
What is the purpose of the alcohol solution in the Gram stain? The purpose of the alcohol solution is to remove the dye-iodine complex from Gram-negative but not Gram-positive bacteria. It dissolves the peptidoglycan sugar cases of the gram-negative bacteria, allowing them to take on the color of the second stain.
What is the purpose of the safranin in a Gram stain quizlet?
What is the function of Safranin? Used to stain the Gram-negative cell walls since they lost the primary dye during decolorization.
What happens if you don’t use safranin in Gram stain?
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.
What is the function of safranin how did you use it in the experiment?
The safranin is used in the agreement because the safranin is used to peel the stomata and the glycerine which is used in that experiment which helps to keep the stomata wet so that the stomata can be easily visible and the particles of the stomata can be very much visible for the experiment.
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