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Bacterial characteristics - Gram staining

Discover the fascinating world of bacteria! Learn how scientists differentiate bacteria by their shape - cocci, bacilli, and spirilla - and color using the Gram stain method. Learn about similarities and differences in the structure of bacterial cell walls and understand why Gram positive bacteria retain the purple stain while Gram negative bacteria turn pink.

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  • orange juice squid orange style avatar for user gcpuente
    around , why is peptidoglycan included in the periplasmic space of gram negative bacteria but it is NOT included in the periplasmic space of gram positive bacteria?
    (8 votes)
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    • leaf red style avatar for user Shabab Rahman
      I think it's just to do with the definition of the periplasmic space. In gram (+) bacteria, periplasmic means between the plasma membrane and peptidoglycan, and in gram (-) bacteria, periplasmic means between the two plasma membranes (which includes the peptidoglycan layer). Hope that helps!
      (9 votes)
  • leaf green style avatar for user George Grkovic
    Excellent Video! Very nicely done!
    (7 votes)
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  • blobby green style avatar for user Olivia Claire
    Can spirilla be both Gram + and -? Not clear in the diagram.
    (6 votes)
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    • leafers ultimate style avatar for user Mr. Olson
      According to Wikipedia:
      "The two types of spiral cells are spirillum and spirochete, with spirillum being rigid with external flagella, and spirochetes being flexible with internal flagella."

      "A spirillum (plural spirilla) is a rigid spiral bacterium that is Gram-negative"

      "Spirochetes are difficult to Gram-stain but may be visualized using dark field microscopy or Warthin–Starry stain."
      (3 votes)
  • spunky sam orange style avatar for user PratikshyaRoutray557
    what kind of stain is used while gram staining which colors the gram-ve pink?
    (1 vote)
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    • leafers seedling style avatar for user Sara A Logan
      In gram staining the purple color in gram-positive bacteria comes from a crystal violet dye. The pink color from gram-negative bacteria comes from a counter stain of safranin. The steps of gram staining are: 1. Bacteria are fixed to the slide using heat. 2. Crystal violet dye is added for one minute to stain the cell wall. 3. An iodine solution is added for one minute, to trap the purple stain by forming crystal violet-iodine complexes in gram-positive bacteria. The crystal violet-iodine complexes do form in gram-negative bacteria but do not adhere to the cell wall. 4. The slide is washed with ethanol for 20 seconds. This washes away the stain on the gram-negative bacteria but not gram-positive bacteria because it is trapped in the cell wall. 5. A counter stain of safranin is added, this gives gram-negative bacteria a pink color.
      (5 votes)
  • blobby green style avatar for user Inas Abdel Gayoum
    What one thing do you think of when a patient has a diagnosis of infection with a bacterial cell that stains pink in the gram stain?
    (0 votes)
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  • leaf green style avatar for user christie2taylor
    Exactly which layers surrounding the bacteria comprise the cell wall? Is the capsule (or slime layer), outer membrane, and lipopolysaccharide layer consider part of the cell wall?
    (3 votes)
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  • blobby green style avatar for user donejo2017
    After doing a gram stain do you have a specific order of lab tests to identify the bacteria?
    (2 votes)
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    • starky ultimate style avatar for user Mikey Renteria
      Yes! There are a numerous tests to identify bacteria simply by utilizing potential virulence factors that the bacteria may posses. For example, a catalase test may be conducted to check for the enzyme catalase (which is prevalent in bacteria that can digest reactive oxygen molecules), a coagulase test, growth on diverse media with specific environmental pressures, etc. All of these are simple lab test procedures done to identify bacteria. On the contrary, these tests tend to have limitations and can be time consuming when conducting them in practical clinical settings. Thus, molecular diagnostics such as using PCR or FISH to identify the bacteria using their nucleic acid composition is much faster, cost effective, and just simpler (gotta love modern technology).
      (2 votes)
  • blobby green style avatar for user Parveen Sameul
    What are the component of the capsule?
    (2 votes)
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  • piceratops tree style avatar for user diajayadas
    Kind of not related, but Neisseria _meningitidis_ (meningitis) is a gram negative bacteria, but penicillin (an antibiotic that works best on gram positive bacteria) is used to treat meningitis. Why do we use an antibiotic that works best on gram positive bacteria to treat a gram negative bacteria?
    (1 vote)
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  • leaf green style avatar for user studentn
    So what happened with the spirilla bacteria?
    (1 vote)
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Video transcript

- [Voiceover] Let's talk about how we can actually tell bacteria apart. We're going to pretend that we're the scientists or pathologists looking underneath a microscope to see these bacteria. So here's something that they might be looking at, and you can see that there are five examples. So let's just write down what we see. So vertically, in the top two at least, it looks purple, and on the right I can see that they're pink. And there's this thing I can barely see down here, so I'm just not going to write a color for now, but vertically, purple and pink. So now let's look at the rows, and I can see that in the first row that they kind of look like little circles, kind of maybe spherical. So I'm just going to draw a circle here, and in the second row I can see that they're more stretched out and kind of like a rod. So I'm just going to draw this kind of stretched out oval-looking shape or a rod. And this bottom area, I can barely see. So since I've been using a light microscope for the top view, I'm going to use the opposite. It's called dark field microscopy, but don't worry too much about the name. Just basically know that because these don't stain the same way, they have to be viewed differently, and the best thing I can call this is a squiggle, and I hope that makes sense. Let's go over that quickly one more time, before we get into the official names for these things. So first row, I can see that they're round, and that's a sphere, and in the second row, it looks more like a stretched out rod-looking shape, and in the third row it kind of looks like a squiggle. So the single version of this is coccus, or a bunch of them are called cocci. And a single rod is called a bacillus, whereas a bunch of them are called bacilli. And a single squiggle is called a spirochete, and the plural version is spirilla. So now we know about the three main shapes that bacteria can take, but we haven't talked too much about the color yet, and this color comes from a special stain called the Gram stain, and it basically stains the outside of the bacteria. And if it stains well, it stains purple, and if it gets washed off and stained with something else, it turns pink. And if it stains purple, we call that Gram positive, whereas if it stains pink, the bacteria is called Gram negative. So here I've done that with a positive sign and a negative sign. So we're going to take a closer look at this color aspect. So this might look familiar to you, but this is just the general bacterial structure that we've gone over before. And again, because the stain is only on the external layers of the bacteria, pay attention to just the external layer. So even though right here it says capsule, cell wall, and plasma membrane, you'll see in a minute that these layers are really different between Gram positive or Gram negative bacteria, which is shown here actually. So think of this as a zoomed in version of this peeled off bit. It does have the plasma membrane layer, a really thick cell wall layer, and a capsule layer, but for Gram negative bacteria they actually have the plasma membrane again, but then they have a cell wall that is much thinner compared to Gram positive bacteria, and it's followed by another plasma membrane and then a special layer called a lipopolysaccharide layer and then a capsule. And don't worry that you didn't quite catch all that right now, because we're going to now draw them really zoomed in so you can see it. So here is that plasma membrane, and remember that it's just a lipid bilayer with some integral membrane proteins which are drawn here in little blocks. And the unique bit is this really thick peptidoglycan layer, and I'm just going to write this out so you can see it. So, peptidoglycan. So glycan just basically means sugar, like glucose, and these rods that you see here are made of these long chains of sugars. So that's the glycan part. And then there's peptido, which just means peptides, so your proteins, and those are just the connections between these rods that you see here. Oh and I just realized this is a little bit of an optical illusion, but there you go. So it's almost like a brick wall with these kinds of connections. So that's where the word peptidolgycan layer comes from. It's just long sugar chains connected by proteins. And then the final layer is the capsule or the slime layer. So that's Gram positive bacteria. So now let's take a look at the other type of structure. You have this inner membrane, and then you have this very thin peptidoglycan layer. And again, this is made of sugars all along these rod areas, so I'm not going to write that out again, because it's a super-long word, and it's going to take up space. Then you have an outer membrane, so here. And then next is this brown layer, also called the lipopolysaccharide layer, which most people abbreviate as LPS because lipopolysaccharide, LPS, and it's also exactly what it sounds like. There is a lipo part, which is these lipid areas at the bottom, and polysaccharides, which are these sugar chains at the top, polysaccharides, and then this layer is covered by a capsule. And then there's a little bit of space next to the plasma membrane on both. So above this plasma membrane here and between these two plasma membranes over on this side, and this is just called the periplasmic space. So, I'm just going to write it over here, because it's easier. And again, think of it peri, next to, and plasmic, plasma membrane, so next to the plasma membrane space. Between these two layers and above this layer, before the peptidoglycan layer here. So now we can think about why this would show up purple and this one pink. So the Gram stain, when you first apply it to a bacteria, it just stains the whole thing purple, and then you'll wash it off. And because the Gram negative bacteria has this very thin peptidoglycan layer in their cell wall, it washes right off, and later they'll restain it with something called Safranin, which isn't important, but they come in and restain this to make sure that you can see it and it's pink. So, that's Gram negative. But because this Gram positive bacteria has this super-thick peptidoglycan layer, it will keep its purple stain, and it will stay purple. So this should explain why these particular bacteria stain purple or pink. And now you should have a good understanding of one of the main ways to tell bacteria apart, both by their shape and color, and you should also understand where that color comes from, because of their cell wall structure.