What would happen to the cells if you use too much alcohol in Gram staining?
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. Add the secondary stain, safranin, to the slide and incubate for 1 minute.
What is the role of alcohol in 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 happens if Decolorizer is not left on long enough?
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. … 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 happens if you Decolorize a Gram stain too much?
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 add too much Decolorizer in a 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.
Why 95 Ethanol is used in Gram staining?
Gram-negative cell walls contain a high concentration of lipids which are soluble in alcohol. The decolorizer dissolves the lipids, increasing cell-wall permeability and allowing the crystal violet-iodine complex to flow out of the cell. The color of the counterstain must contrast with that of the primary stain.
What does alcohol do to Gram negative bacteria?
Alcohol exposure can promote the growth of Gram-negative bacteria in the intestine, which may result in accumulation of endotoxin.
Why do we heat in ZN staining?
The cell wall of Mycobacterium possesses mycolic acid that makes it impervious to staining by aqueous staining solutions. Heating the slide helps to soften the mycolic acid on the bacterial cell wall as in conventional ZN stain.
What happens if you don’t use Decolorizer?
what is the primary stain? … what happens when yo don’t decolorize in a gram stain? all cell’s will appear gram positive due to the peptidoglycan layer of the gram negative not being broken down enough to release the crystal violet and accept the safranin. what is the purpose of a negative stain?
What happens if you forget 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.
What happens if you put too much bacteria on a slide?
Holding the slide with a pair of forceps pass it through the interface of the yellow and blue flame,-this is the hottest region of the flame- 5 times. The slide should be in this region for no more than 1 second. If it gets too hot the bacteria will rupture (or the slide will break).
What color would a gram positive bacterium be if you did too much rinsing?
Gram’s method involves staining the sample cells dark blue, decolorizing those cells with a thin cell wall by rinsing the sample, then counterstaining with a red dye. The cells with a thick cell wall appear blue (gram positive) as crystal violet is retained within the cells, and so the red dye cannot be seen.
Why is iodine used in Gram staining?
The first step in gram staining is the use of crystal violet dye for the slide’s initial staining. The next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine complex to prevent easy removal of dye. … With the dissolution of the lipid layer, gram negatives lose the primary stain.