During the decolorization step, the gram positive bacteria become dehydrated, trapping the crystal violet-iodine complexes within the multi-layered peptoglycan wall. If you skip this step, gram negative bacteria retain the purple dye. If you decolorize properly, the saffrin dye will dye the gram negative bacteria pink.
What would happen if the decolorization step was skipped in a Gram stain?
Do NOT decolorize for a full minute!
If the decolorizer is left on too long, even gram positive cells will lose the crystal violet and will stain red. The staining procedure is here.
What is the purpose of the decolorization step?
11.3.
Decolorization is carried out to remove natural pigment existing in chitin. This is often accomplished by the addition of acetone to chitin residue under reflux condition for a period of time [30,50].
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.
What would be the color of a gram positive bacterium if Decolorizer is not added?
Red b/c the crystal violet molecules wouldn’t form a complex (that remains in thick peptidoglycan layer of Gram-positive bacteria) and would be washed away. What would be the appearance of all cells if the decolorizer were not added? Purple.
What happens to the gram positive cell wall during decolorization quizlet?
What happens to the Gram-positive cell wall during decolorization? The decolorizing agent dehydrates the peptidoglycan….. Removing water from or dehydrating the peptidoglycan allows the decolorizing agent to shrink the spaces through which the crystal violet-iodine complexes might be able to pass.
What happens to 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 would happen if you forget to counterstain at the end of the gram reaction procedure?
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 decolorization in microbiology?
decolorize. (dē-kŏl′ĕ-rīz″) [″ + colorize] To remove dye from a stained microscopic specimen, usually with an acid-alcohol wash.
What counterstain is used why is it necessary could colors other than red be used?
Could colors other than red be used? Saffranin is the counter stain used, it is necessary so gram negative bacteria can be identified.Gram stains tell more than just the morphology of the bacteria, it also tells if an organism is gram-positive or gram-negative.
How does alcohol affect Gram staining?
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. However, caution must be used since applying the decolorizer too long will remove dye complexes from the Gram positive cells as well.
What is the Decolorizer in the Gram stain and why is it important quizlet?
The decolorizer is Ethanol. It is added to chemically change the shape of the dye molecule and trap it in the cell wall.
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 would happen if the decolorizing step is too long too short or omitted?
-If the decolorizing step was too long the color will wash off the cell and the cell possibly explode. -If the decolorizing step is too short/omitted, both will be purple, it’ll hold all the die.
Which bacteria is affected by the Decolorizer ethanol?
The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram-negative Bacteria. The outer membrane of the gram-negative cell is lost from the cell, leaving the peptidoglycan layer exposed.
What happens if you forget the mordant in a Gram stain?
If missed, then the bacteria would remain purple and give a false positive result.
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 was done incorrectly if at the end of the procedure all cells within a mixed culture of S aureus and M smegmatis appear red?
You INCORRECTLY perform an acid-fast stain on a smear from a mixed culture of Mycobacterium smegmatis (acid-fast cells) and Staphylococcus aureus (non-acid-fast cells). Your mistake is that you forget to use acid-alcohol during the procedure.
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 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.
What does an Endospore stain tell you?
Endospore staining is a technique used in bacteriology to identify the presence of endospores in a bacterial sample. Within bacteria, endospores are protective structures used to survive extreme conditions, including high temperatures making them highly resistant to chemicals.
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