What happens if you Decolorize a Gram stain too long?

What happens if you Decolorize a Gram stain too long?

Do NOT decolorize for a full minute! The decolorizer should stay on the slide for no more than 15 seconds! If the decolorizer is left on too long, even gram positive cells will lose the crystal violet and will stain red.

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 are two mistakes you could make that would make a Gram stain less reliable?

Choose two mistakes that you could make that would make a Gram stain less reliable. Putting too much dye and leaving it on too long. What is correct about Simple stain? Always done with basic dye.

How long will you drench the bacterial smear in iodine?

1 minute
Saturate the smear with iodine for 1 minute. 5. Rinse the slide gently with water. 6.

What happens if you forget to use alcohol in a Gram stain?

The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet. However, if the alcohol remains on the sample for too long, it may also decolorize Gram positive cells.

What happens if you don’t Decolorize 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.

Why is my Gram stain not working?

Several factors may affect the results of Gram staining: If the smear is too thick, proper decolorizing will not be possible. If the smear is overheated during heat fixing, the cell walls will rupture. Concentration and freshness of reagents may affect the quality of the stain.

Can iodine be added before crystal violet?

Can iodine be added before the primary stain in a Gram stain? No because the iodine allows the crystal violet stain to bind to the peptidoglycan in cell walls.

How long does Gram staining take?

Add the primary stain (crystal violet) to the sample/slide and incubate for 1 minute. Rinse slide with a gentle stream of water for a maximum of 5 seconds to remove unbound crystal violet. Add Gram’s iodine for 1 minute- this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall.

What happens if iodine is not used in Gram staining?

In the absence of iodine, crystal violet would initially bind to equally well to both Gram positive and Gram negative cells. Thus, both Gram positive and Gram negative cells would appear as pink (due to the secondary stain, safranin). The same result would occur if you destain for too long with the acetone alcohol. Click to see full answer

What happens if you miss a step in Gram staining?

Similarly, what happens if you miss a step in Gram stain? Steps for gram staining: 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 is crystal violet used in Gram staining?

The purpose of Gram’s iodine in the Gram stain is to act as a mordant. That is, a chemical or process that allows the primary stain (crystal violet in this case) to bind more avidly to some cells. In the absence of iodine, crystal violet would initially bind to equally well to both Gram positive and Gram negative cells.

What does crystal violet do in the absence of iodine?

In the absence of iodine, crystal violet would initially bind to equally well to both Gram positive and Gram negative cells. However, the destaining reagent (acetone alcohol) would remove the purple stain from both kinds of cells when applied.