It was a lovely October afternoon-the sun was shining and Sam was having a great

Question

It was a lovely October afternoon-the sun was shining and Sam was having a great tint at the dog park with his dog. Scout As the day came to a dose. Sam was packing up Scout's toys when another dog tried to steal Scout s favorite chew toy. Without a second thought Sam reached for the toy: unfortunately, the dog snapped at Sam and bit him on the forearm. It was a quick bite-it didn't bleed very much and didn't seem very deep. Sam washed the bite with soap and water when he got home and didnt give n another thought. The next morning. Sam noticed that the bite area was a little red and lender. As the day went on, the soreness and redness increased and Sam noticed that the area around the bite was also hurting. By the next morning, there was a pus-filled abscess present and his entire forearm was sore to the touch. Sam decides that should go to the doctor ASAP. At the doctor's office. the doctor drains the abscess aid takes a sample for culturing. She then performs a gram-stain and the bacteria on the slide turns purple a) What is the gram status of the bacterium? b) Explain in detail what would have happened to her gram stain if the lab technician had left the alcohol on too long. (You should explain in terms of what happens to the cell envelope and to the results of her stain)

Explanation / Answer

a) The Gram status of the bacteria is definitely Gram Positive. Because Gram positive bacteria stain purple or violet due to the presence of thick peptidoglycan layer in their cell wall. This peptidoglycan layer retains the crystal violet-Iodine complex after the decolorization step. Thus Gram positive bacteria appears violet or purple after Gram staining.

b) To answer this, we first need to know the detail steps involved in gram staining which is as follows

i) heat fixing of the bacterial smear on to slide.

ii) Add crystal violet (CV)as primary stain and incubte for 1 min then rinse with water to remove unbound CV.

iii) Add Gram's iodine for 1 minute which binds to crystal violet and traps it in the cell

iv) rinse with alcohol for 3 sec

Alcohol reacts with outer lipopolysaccharide membrane of Gram negative bacteria, and the inner thin peptidoglycan layer is left exposed. The CV–I complexes are washed out from the gram-negative cell along with the outer membrane. But in Gram- positive bacteria alcohol shrinks and tightens the thik peptidoglycan layer.The large crystal violet-iodine complex can't penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria.

Now if the decolorizing agent i.e. alcohol is left on too long the crystal violet stain will be removed from both gram-positive and negative cells. then both will take the secondary stain of safranin. then a wrong result will come.

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