1-fore yo- make sure 2. What result is expected of a mixed smear of E. coli and
ID: 145508 • Letter: 1
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1-fore yo- make sure 2. What result is expected of a mixed smear of E. coli and S. epidermidis if a. the decolorizer is left on the slide for 60 seconds? b. the decolorizer is never added or rinsed off immediately? c. the decolorizer is not rinsed off well? d. the counter-stain is forgotten? e. the mordant was not used? f. your smear was too thick? g. crystal violet was replaced with methylene blue? h. cultures that are 1 week old are used? 3. Why don't the Gram positive cells stain red with safranin?Explanation / Answer
E. coli is Gram Negative bacteria (takes pink color due to counterstain safranin), whereas S. epidermidis is Gram-positive bacteria (takes purple color due to crystal violet).
a) If Decolorizer is left for too long the gram-positive cells will lose the crystal violet stain and will take pink stain, so will look like Gram-negative. The decolorizer should not stay on the slide for more than 15 seconds.
b) All the cells (both Gram positive and Gram negative) will appear purple since the crystal violet will stain both the Gram-positive and Gram-negative bacterial cells, and the presence of safranin is not likely to be detected over the darker crystal violet stain.
c) If decolorizer is not rinsed off well, decolorization of the gram-positive cells will occur, and eventually, the purpose of gram staining will be defeated.
d) If the counterstain is forgotten, it would not be possible to distinguish between Gram positive and Gram negative cells.
e) If mordent is not used then the crystal violet stain will not be retained in the cell, as crystal violet stain would not form a complex and will be washed away. So, the cells will appear red.
f) If your smear is too thick, the cells in the very thicker area can appear Gram Positive, so it will be tough to distinguish the gram positive and gram negative cells.
g) Crystal violet stain can be replaced with methylene blue, no problem in it, as both the stains, are equally effective.
h) Older cultures sometimes stain erratically as the older cultures sometimes form more numbers of endospores due to environmental resistance in their growth as the time passes.
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