1. Why is it best if the Gram stain can be performed using cultures less than 24

Question

1. Why is it best if the Gram stain can be performed using cultures less than 24 hours old? What results would you expect to see if you were using a mixed smear of Escherichia coli and Staphylococcus epidermidis and 2. a. left the decolorizer on for 60 seconds? b, forgot to add the decolorizer (95% EtOH)? c. forgot about what you were doing and left the decolorizer on for 60 seconds? d. forgot to add the Gram-s iodine? e. made a smear that was way to thick? f. forgot to use the counterstain? 3. Why do the G+ cells retain the crystal violet while the G-cells do not? 59

Explanation / Answer

1.As the cultures age the organisms may lose their ability to retain the primary stain, especially Gram-positive organisms. Gram-stains made from cultures that are greater than 24 hours old may appear to be Gram-variable, where some cells will appear purple, while others will appear red.

2.Escherichia coli is a gram negative bacterium so it has less thick peptidoglycan which makes it to take up less strain.Staphylococcus epidermidis is a gram positive bacterium that takes up more gram staining due to the presence of thick walled peptidoglycan.

(a) When the smear is left on decoloriser for 60 seconds , E.coli gets decolorised as thin walled cell wall and S.epdermidis retains the stain being thick walled cell wall.

(b)When forgot to add decoloriser both strains will be purple.

(c)When forgot about all and left in decoloriser all strains will be decolorised.

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