Add Gram’s iodine for 1 minute- this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall. Rinse sample/slide with acetone or alcohol for ~3 seconds and rinse with a gentle stream of water. The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet.
What reagent is applied to Decolorize the Gram-negative bacteria and thus differentiate it from the gram positive quizlet?
Safranin is the secondary dye used in the Gram stain. This dye will stain the decolorized Gram-negative cells, which will appear in pink.
How do you distinguish between Gram positive and Gram-negative bacteria?
Gram positive bacteria have a thick peptidoglycan layer and no outer lipid membrane whilst Gram negative bacteria have a thin peptidoglycan layer and have an outer lipid membrane.
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 happens to the Gram-negative cell during decolorization?
What’s Going On? The decolorizing mixture dehydrates cell wall, and serves as a solvent to rinse out the dye-iodine complex. In Gram-negative bacteria it also dissolves the outer membrane of the gram-negative cell wall aiding in the release of the dye.
What are the four reagents used in Gram stain quizlet?
List the four reagents used for the Gram stain in the proper order.
- Crystal violet (primary stain)
- Iodine (mordant)
- Alcohol wash (decolorization)
- Safranin (counterstain)
What is the best procedure for decolorization quizlet?
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 is the function of pili and fimbriae?
Pili. Pili or fimbriae are protein structures that extend from the bacterial cell envelope for a distance up to 2 μm (Figure 3). They function to attach the cells to surfaces. E.
What are the functions of the individual stains and reagents used in Gram staining?
The first reagent is called the primary stain. Its function is to impart its color to all cells. The second stain is a mordant used to in- tensify the color of the primary stain. In order to es- tablish a color contrast, the third reagent used is the decolorizing agent.
How do you Decolorize a Gram stain?
Gram Decolorizer Solution: Mix equal volumes of 95 % ethanol and acetone. Gram Safranin Solution: Dissolve 2.5 g of safranin O in 100 ml of 95 % ethanol to make a stock solution. Working solution is obtained by diluting one part of the stock solution with five parts of water.
What is the Decolorizer in the Gram stain quizlet?
The decolorizer is Ethanol. It is added to chemically change the shape of the dye molecule and trap it in the cell wall.
Why is the decolorization important in gram staining?
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 happening during the decolorization step?
The decolorization step is the step that rinses the crystal violet from the Gram-negative cell wall, but not the Gram-positive. This allows the differential staining of the Gram-negative cell wall with safranin.This helps the crystal violet remain in the gram-positive cells during decolorization.
What happens if you over Decolorize in the Gram stain procedure?
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 is counterstain in microbiology?
A counterstain is a stain with colour contrasting to the principal stain, making the stained structure easily visible using a microscope.
What are the 4 reagents used in Gram staining?
Reagents:
- Crystal violet (primary stain)
- Iodine solution/Gram’s Iodine (mordant that fixes crystal violet to cell wall)
- Decolorizer (e.g. ethanol)
- Safranin (secondary stain)
- Water (preferably in a squirt bottle)
What are the four reagents used in a Gram stain?
This method employs the use of four reagents. These reagents consist of crystal violet, Gram’s iodine, a decolorizing agent, and safranin.
What is the proper order of the reagent used in Gram staining?
Explanation: Gram staining is a type of differential staining. In this process the fixed bacterial smear is subjected to the following staining reagents in the order listed: crystal violet, iodine solution, alcohol (decolorizing agent), and safranin.
When doing a Gram stain procedure What color is a Gram-negative cell after Decolorizer has been added?
Decolorizer is added, dissolving the outer layer of the Gram negative cell and causing enough damage in the thin peptidoglycan layer to allow the CV-I complex to escape from the cell when the cell is washed. 5. At the completion of the washing process the Gram negative cell is, in theory, colorless.
What is the purpose of mycolic acid in the mycobacterial cell wall quizlet?
What is the purpose of mycolic acid in the mycobacterial cell wall? It reinforces the cell wall and makes the bacterium resistant to certain chemicals and dyes.
What is the purpose of immediately rinsing the slide after decolorization?
The decolorization of the cells is the most “operator-dependent” step of the process and the one that is most likely to be performed incorrectly. Rinse with water to stop decolorization. Rinse the slide with a counterstain (safranin or carbol fuchsin) which stains all cells red.
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