Transcription
What is up, lovely people? It's Mitochosis Perfectionist, where medicine makes perfect sense. Let's continue our microbiology playlist. Today is video number two. In the last video, we had an introduction about microbiology. Today, we will talk about the Gram stain, the technique of the Gram stain, and we will compare between Gram-positive and Gram-negative, the purple versus the pink.
Gram is the name of a scientist, so this should be upper-class G. Please watch the previous video before this one. Microbes are what? Bacteria, fungi, viruses, and parasites. Technically, viruses are particles, not microorganisms. And technically, parasites do not belong with microbes because many of them are macroscopic, not microscopic. Mr. Gram discovered/invented the Gram stain, and this is very useful to differentiate between many Gram-positive bacteria versus Gram-negative bacteria. And if you have watched my last video, you already know that Gram-positives are divided into Gram-positive cocci and Gram-positive rods. Gram-negatives are also cocci and rods. Hickory dickory dock, it's either a coccus or a rod.
Bacteria versus humans: prokaryotes versus eukaryotes. Both of them have a cell membrane, aka plasma membrane or cytoplasmic membrane. But only bacteria do have a cell wall. Humans do not. Hence, the idea behind the class of antibiotics known as cell wall synthesis inhibitors, which include the beta-lactam antibiotics, which include penicillin.
Here is a schematic diagram to show you the basic cell structure of a bacteria. Outer is here. As you go here, we are digging deeper to the inside of the cell. So, what do we have? Outside, cell wall, followed by cell membrane, aka plasma membrane or cytoplasmic membrane. And then, what do we have here? Cytoplasm. When you dig deeper, you find the nucleus. Please don't forget that the bacteria are prokaryotes. They do not have a nuclear membrane, like this beauty right here. This is just a schematic diagram, do not take it very seriously.
The cell wall is made of what? Peptidoglycan. How about the cell membrane? It's a lipid bilayer. Very important. It's a phospholipid bilayer. And then, what we have? Proteins. And then, what we also have? Carbohydrates. Then you go cytoplasm, nucleus. Easy peasy. If you want to be a pro. Cell wall has two things, and cell membrane has two things. Number one, the cell wall has peptidoglycan and teichoic acid. The cell membrane of the bacteria has all of this lovely stuff: lipid bilayer, protein, carbohydrate, and lipoteichoic acid. So, teichoic acid is in the cell wall, but lipoteichoic acid is in the cell membrane. The way I remember it is that lipoteichoic belongs with the lipid bilayer.
This is the simplest form of the structure of the bacteria. But the Gram-negative bacteria begged to disagree. She said, "I'm gonna change this. I'm gonna have a different structure." Okay, I still have a cell wall and the cell membrane. All right. But above that cell wall, I have an outer membrane, something that the Gram-positive do not possess. Okay, what's your cell wall? Peptidoglycan, just like the good old days. What's your cell membrane? It's my lipid bilayer, some proteins, some carbs, just like the good old days. But now it's called the inner membrane because there is another outer membrane that is slightly similar but not identical. And where's the cell wall now? It's in between the outer and the inner membranes. And we call this space what? The intermembrane space. It's the space between the two membranes.
And here is a wonderful comparison between the structure of the Gram-positive versus Gram-negative bacteria. Here is the point of comparison. Here is my left column for Gram-positive. Here is my right column for the Gram-negative. And here is the middle column for what is common between the two of them. So, let's go. Outer membrane: Gram-positive, I don't have it. Gram-negative, I do have it. Thank you. How about the intermembrane space or the periplasm or the periplasmic space? Gram-positive, I don't have it. You know why? Because I don't have an outer membrane. By definition, the intermembrane space is between two membranes, outer and inner. Since I don't have outer, I don't have anything in between. Duh. How about you, Gram-negative? I do have an intermembrane space or a periplasmic space. Next. How about cell wall? Both of us have it. Both of us have this lovely peptidoglycan. In the Gram-positive, it is super thick. But in the Gram-negative, it is thinner. More layers of peptidoglycan in the Gram-positive, but less layers of peptidoglycan in the Gram-negative. That's why the wall is thicker in the Gram-positive as compared to the Gram-negative. All right.
Let's talk about the inner membrane, which is the cell membrane or plasma membrane or cytoplasmic membrane. Both of us have it. And this is good old phospholipid bilayer plus proteins plus carbohydrates. Now, let's dig deeper into more details. The outer membrane is only present in the Gram-negative. What's the function? First of all, it has porins. What's that? If it ends in "in," it's usually a protein. A protein pore, a hole in the wall. Some lovely people think that this is just a hole in the wall. Not true. If you look at it under electromicroscopy, it's a very complex structural protein. I mean, it's really complex. It's like looking at the architectural design of a freaking hospital. It is huge. And why do you need a pore? To transport molecules. Molecules coming into the bacteria, molecules leaving the bacteria. What else do we have? Lipopolysaccharides. Love it. Lipo because it has lipid or fat. Poly many. Saccharide sugar. Oh, I have sugar and fat. What's that? It acts as an endotoxin. It is toxic to your body. And it is part of the membrane of the Gram-negative bacteria. That's why it's "endo" because it's part of the membrane itself. It's endogenous to it. It's not secreted by the bacteria to the outside world. It is part and parcel of the Gram-negative's outer membrane. This is very antigenic. Translation: an antigen is something that will generate an antibody. Your body will make an antibody against this antigen. Oh, which part of it? The polysaccharide. Oh, oh my goodness, this is dangerous. Oh, my goodness, this is going to be a great story because here is an example of an antigen-antibody reaction happening in my own body. Moreover, this lipopolysaccharide endotoxin is also pyrogenic. What does "pyro" mean? You know the type of glass that you can put in the oven without breaking it? What do they call it? Pyrex. You know why? Because "pyro" means heat. "Pyro" means favor. Oh, so pyrogenic means pro-fever and pro-inflammation. Absolutely. Which part of the LPS is pyrogenic? It's the lipid A. The lipid A is going to activate interleukin-1 and TNF alpha. Interleukin-1 is the hero of fever and acute inflammation. TNF alpha is the hero of recruiting white blood cells and activating the endothelium.
All right, thank you. Tell me about the intermembrane space, which is peculiar to the Gram-negative bacteria. It contains peptidoglycan. Really? Because remember the previous slide, the peptidoglycan in the Gram-negative was present in the intermembrane space. So, it's the exact same peptidoglycan. Moreover, as molecules try to leave the Gram-negative bacterial cell, some of them will leave some debris behind, e.g., beta-lactamase. And this is going to be super important in the story of resistance to antibiotics. I give you antibiotics, why didn't you respond? It's thanks to the bacterial beta-lactamase. #AntibioticResistance. Next. Tell me about the cell wall. It is thicker in the Gram-positive with more peptidoglycan layers, but it's thinner in the Gram-negative with less peptidoglycan layers. Okay. Tell me more. In Gram-positive, there is teichoic acid in the cell wall. You do not find teichoic acid in the Gram-negative. The teichoic acid is species-specific, which means that the teichoic acid of the Staphylococcus aureus, which is Gram-positive, is different from the teichoic acid of another Gram-positive bacteria. It differs by species. It's also a polymer. Okay, it's a complex molecule made of many monomers. It contains phosphate. It's covalently bound to the peptidoglycan. And it binds fibronectin. This might seem gibberish to you right now, but just wait. Good things happen to those who wait. In the next videos, this is going to be more clear.
Tell me about the peptidoglycan. Why call it peptide? Because it has peptide side chains. Why call it glycan? Because the main part, the backbone, is made of what? Gly, which is sugar, which is carb. That's why we call it glycogen. Oh, it's carbohydrate. That's right. Glycan is carbohydrate. It is sugar. How can we link the sugar backbone with the peptide side chain? The link is called transpeptidase cross-linking. So, what's the process? Cross-linking. When you cross-link with peptides, what's the name of the enzyme that you need? Transpeptidase. For the peptidoglycan, what's the function of the peptidoglycan? Well, it provides rigid support for the bacteria and it protects the bacteria from osmotic damage. But Mitochosis, I as a human do not have a cell wall. How do I protect my own cell from osmotic damage? You have a cell membrane. The cell membrane can also protect your cell from osmotic damage. Okay, I get it.
Tell me more about the inner membrane or the cell membrane or the cytoplasmic membrane. Phospholipid bilayer plus carbs plus proteins. What kinds of proteins? Enzymes, because all of your enzyme macromolecules are what? Proteins. And they also include penicillin-binding protein. And this is how penicillin destroys many bacteria because penicillin, the drug, is gonna bind penicillin-binding protein, which is a protein in the bacteria, and it's gonna destroy the bacteria. How does it destroy the bacteria? Penicillin is a cell wall synthesis inhibitor. When penicillin binds to its penicillin-binding protein, it's gonna interfere with these bacterial proteins, and therefore, you will not be able to synthesize the wall of the bacteria. A bacteria without a wall will die very soon due to lack of support and lack of osmotic protection. Go to hell, bacteria. But many bacteria are resistant to penicillin, thanks to the beta-lactamase and other mechanisms.
Let's review Gram-positive versus Gram-negative. Gram-positive: easy, very thick cell wall, peptidoglycan plus teichoic acid. After this, I have cell membrane. There is no outer or inner, there is just one membrane. It's lipid bilayer, protein, carbohydrates. This is where you find the enzymes. Many of them will help me make the cell wall. And this is where you find the penicillin-binding protein, which is the receptor for the drug known as penicillin. And then cytoplasmic nucleus. Easy peasy. How about the Gram-negative? Well, I have outer membrane and inner cell membrane. Outer membrane has what? LPS and porins. The LPS is the endotoxin. The LPS is the pro-fever, pro-inflammation. And I have an inner cell membrane, just like this doofus right here. But what is peculiar and unique to me is the presence of the periplasmic space between the outer and inner membranes. And this is where you find my peptidoglycan. Do I have teichoic acid? The answer is no. Do I have LPS? The answer is yes.
Now, let's talk about the Gram stain technique. Let's look at this quote. Some people live for the day or for the weekend. Other people think long-term, 20 years ahead, 30 years ahead, 50 years ahead. #Neurosurgeons. The free market is ruled by those who are able to see and plan long-range. The better the mind, the longer the range. I'm gonna take this quote and absolutely ruin it for the sake of microbiology, of course. The Gram stain is ruled by those bacteria with a thick peptidoglycan cell wall. The thicker the wall, the purpler the stain. But if your wall is thin, you're going to be pink. Mitochosis, you're trying to say that since Gram-positive bacteria have a thicker cell wall, they will appear purple, and since Gram-negative bacteria have a thinner cell wall, they're going to appear pink? Absolutely correct. Wonderful.
So, here is how you do it. You start with your sample that contains the bacteria. Could be a sputum sample, could be a urine sample, could be a blood sample, stool sample, all kinds of samples. And you add it to your glass slide, okay? Add to this crystal violet stain. And you can add iodine if you want to strengthen the stain. What's the function of the crystal violet stain? It binds peptidoglycan, which is part of the cell wall of the bacteria. Okay, I get it. Now, if it's Gram-positive, it is purple so far. If it's Gram-negative, it is also purple so far. But wait. We will add ethanol to wash out the crystal violet stain. Oh, you will wash out the Gram-negative because it has a thinner cell wall. It cannot retain the crystal violet. But look at the Gram-positive bacteria. You cannot wash it away. Why? They have a very thick peptidoglycan cell wall layer. You cannot wash their absorption to the crystal violet. Imagine that you have a very thick jacket, and I stained it with some horrible sauce. And then I have a very thin undergarment, I also stained it with the same sauce. Which one do you think is easier to wash? Oh, of course, the thin undergarment. Bingo. That's the exact point. When you add ethanol, it washes the Gram-negative, but it cannot wash the Gram-positive. Next. You add a counterstain, a pink stain, such as safranin. Or wait for it, because the correct pronunciation of this is fuchsia. Fuchsia. What's that? This is fuchsia, which is this color right here, i.e., the pink. So, this is how the Gram stain helps you differentiate between the Gram-positive, which will remain purple all along because you did not remove the purple in the first place. But the Gram-negative started purple, then the purple was washed out, then they acquired the pink of the counterstain. Counter to what? Counter to the original crystal violet stain. Oh, I get it. And this is how Gram helped us differentiate between Gram-positives and Gram-negative.
Gram-positive: I'm either a coccus or a rod. If you're a coccus, we can classify you based on the presence or absence of catalase into Staphylococcus (I do have catalase) or Streptococcus (I do not have catalase). Mitochosis, what the flip is the function of catalase? Ace enzyme. Catalase because it's a catalyst. Oh, yeah, enzymes are catalysts. I know this from chemistry. Awesome. Bacteria has invaded your body, right? Yeah. Your body is trying to defend itself. Your body is gonna throw harmful chemicals, hydrogen peroxide, in the face of the bacteria. Okay? But some bacteria are smart. Some bacteria have catalase, which is gonna convert your harmful chemicals into harmless molecules that will not harm the bacteria. These bacteria are sneaky, and this is one more mechanism of evasion of bacteria from your immune system.
In the next video, we will dig deeper into the Gram-positive bugs. Bugs is a straight term, not a technical term, because technically bugs belong to parasites, not bacteria. So, when you say Gram-positive bugs versus Gram-negative bugs, we're talking tongue-in-cheek, of course. This microbiology playlist here on YouTube is going to help you with microbiology stuff. If you want to add pharmacology to it, go to my website, medicosisperfectionist.com, and download my antibiotics course. It comes with 40 videos talking about antibacterials, antifungals, antivirals, and antiparasitic medications. And for a limited number of students, you can get 25% discount towards any course on my website. Just use promo code SAFE25. Thank you for watching. Please subscribe, hit the bell, and click on the join button. You can support me here or here. Go to my website, download my courses. Go to Picmonic for animated medical mnemonics. Be safe, stay happy, study hard. This is Medicosis Perfectionist, where medicine makes perfect sense. Peace.