Gram positive bacteria have thick peptidoglycan

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Gram Positive Bacteria have thick Peptidoglycan Layers that the Ethanol cannot fully penetrate. The Crystal Violet-Iodine Complex is retained in Gram Positive Bacteria, so they stain Purple. Gram Negative Bacteria have thin Peptidoglycan Layers and so the Ethanol can easily penetrate, dissolve the Crystal Violet-Iodine Complex, and wash it out. So Gram Negative Bacteria do not have any Crystal Violet-Iodine Complex in their Cell Walls and will not stain Purple. If we stop now, Gram Positive Bacteria will look Purple and Gram Negative Bacteria will not look Purple. That’s not Ideal. So we’ll add a Counter-Stain (Safranin). The Safranin is taken-up by Gram Positive Bacteria, but their Peptidoglycan Layers are already full of the Crystal Violet-Iodine Complex. So Gram Positive Bacteria will still look Purple. The Safranin is taken-up by Gram Negative Bacteria and their Peptidoglycan Layers have no other Stain. So Gram Negative Bacteria look Pink. You will be tested on your Ability to perform a Gram Stain.
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Lab 3 Page 22 Isolations Pseudomonas sp. Most of you probably have Pseudomonas on a Benzoate Plate. If you don’t, “borrow” it from someone who has it. Check your Plates for white, slightly shiny Colonies. Make a Smear from an isolated Colony, confirm that the Bacteria in this Colony are Gram Negative Rods and streak a new Nutrient Agar Plate (not Benzoate Plate) from the same isolated Colony. This will be your first Gram Stain of your Pseudomonas . Don’t be surprised if you think you have Gram Negative Cocci; Pseudomonas tends to be a very, very short Rod (kind of like a Rugby Ball). Bacillus sp. Everyone should have Bacillus by now. Check your Plates for the characteristic Matte Appearance of Bacillus Colonies. Make a Smear from an isolated Colony, confirm that the Bacteria in this Colony are Gram Positive Rods and streak a new Nutrient Agar Plate from this same isolated Colony. Skin Bacteria Everyone should have Micrococcus luteus (Yellow Colonies surrounded by a Zone of Clearing on YGC) or Staphylococcus epidermidis (White Colonies surrounded by a Zone of Clearing on YGC). Make a Smear from an isolated Colony, confirm that the Bacteria in this Colony are Gram Positive Cocci and streak a new YGC Plate from the same isolated Colony. Enteric Bacteria And everyone probably has E. coli (from the Purple Colonies surrounded by that Fly-Butt Green Sheen on your EMB Plate) or Enterobacter aerogenes (from the Hot Pink Colonies on your EMB Plate). Pick an isolated Colony on your Nutrient Agar Plate, make a Smear, and check if the Bacteria in this Colony are Gram Negative Rods. If they are, go ahead and streak a new Nutrient Agar Plate from this same isolated Colony. ____________________________________________ “Powerful you have become, Dooku. The dark side I sense in you.” I have become more powerful than any Jedi. Even you.’ Much to learn, you still have.” © 2002 LucasFilm Ltd.
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