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what is up lovely people it's mitochosis
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perfectionist where medicine makes
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perfect sense let's continue our
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microbiology playlist today is video
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number two in the last video we had an
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introduction about microbiology today we
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will talk about the gram stain the
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technique of the gram stain and we will
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compare between gram positive and gram
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negative the purple versus the ping gram
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is a name of a scientist so this should
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be upper class g please watch the
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previous video before this one microbes
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are what bacteria fungi viruses and
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parasites technically viruses are
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particles not microorganisms and
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technically parasites do not belong with
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microbes because many of them are
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macroscopic not microscopic mr graham
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discovered slash invented the gram stain
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and this is very useful to differentiate
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between many gram-positive bacteria
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versus gram-negative bacteria and if you
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have watched my last video you already
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know that gram positives are divided
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into gram pods of i and gram
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positive rods gram negatives are also
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cocci and rods hickory dickory dough
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it's either a caucus or a rod bacteria
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versus humans prokaryotes versus
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eukaryotes both of them have cell
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membrane aka plasma membrane or
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cytoplasmic membrane but only bacteria
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do have a cell wall humans do not hence
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the idea behind the class of antibiotics
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known as cell wall synthesis inhibitors
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which include the beta-lactam
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antibiotics which include penicillin
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here is a schematic diagram to show you
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the basic cell structure of a bacteria
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outer is here as you go here we are
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digging deeper to the inside of the cell
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so what do we have outside cell wall
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followed by cell membrane aka plasma
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membrane or cytoplasmic membrane and
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then what do we have here cytoplasm when
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you dig deeper you find the nucleus
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please don't forget that the bacteria
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are prokaryotes they do not have a
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nuclear membrane like this beauty right
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here this is just a schematic diagram do
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not take it very seriously the cell wall
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is made of what peptidoglycan how about
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the cell membrane it's a lipid bilayer
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very important it's a phospholipid
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bilayer and then what we have proteins
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and then what we also have carbohydrates
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then you go cytoplasm nucleus easy peasy
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if you want to be a pro cell wall has
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two things and cell membrane has two
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things number one the cell wall has
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peptidoglycan
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and tachoic acid the cell membrane of
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the bacteria has all of this lovely
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stuff little bit by layer protein
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carbohydrate and
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lypotechoic acid so teucoic acid is in
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the cell wall but lipotechic acid is in
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the cell membrane the way i remember it
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is that lypotechoic belongs with the
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lipid bilayer this is the simplest form
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of the structure of the bacteria but the
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gram negative bacteria begged to
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disagree she said i'm gonna change this
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i'm gonna have a different structure
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okay i still have a cell wall and the
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cell membrane all right but above that
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cell wall i have an outer membrane
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something that the gram positive do not
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possess okay what's your cell wall
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peptidoglycan just like the good old
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days what's your cell membrane it's my
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lipid bilayer some proteins some carbs
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just like the good old days but now it's
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called the inner membrane because there
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is another outer membrane that is
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slightly similar but not identical and
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where's the cell wall now it's in
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between the outer and the inner
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membranes and we call this space what
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the inter membrane space it's the space
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between the two membranes and here is a
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wonderful comparison between the
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structure of the gram positive versus
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gram negative bacteria here is the point
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of comparison here is my left column for
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gram positive here is my right column
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for the gram negative and here is the
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middle column for what is common between
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the two of them so let's go outer
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membrane gram positive i don't have it
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gram negative i do have it thank you how
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about the inter membrane space or the
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periplasm or the periplasmic space gram
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positive i don't have it you know why
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because i don't have outer membrane by
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definition the inter membrane space is
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between two membranes outer and inner
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since i don't have outer i don't have
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anything in between duh how about you
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gram negative i do have an inter
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membrane space or a periplasmic space
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next how about cell wall both of us have
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it both of us have this lovely
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peptidoglycan
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in the gram positive it is super thick
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but in the gram negative it is thinner
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more layers of peptidoglycan in the gram
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positive but less layers of
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peptidoglycan in the gram negative
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that's why the wall is thicker in the
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gram positive as compared to the gram
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negative all right let's talk about the
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inner membrane which is the cell
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membrane or plasma membrane or
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cytoplasmic membrane both of us have it
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and this is good old phospholipid
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bilayer plus proteins plus carbohydrates
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now let's dig deeper into more details
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the outer membrane is only present in
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the gram negative what's the function
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first of all it has polarines what's
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that if it ends in in it's usually a
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protein a protein pore a hole in the
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wall some lovely people think that this
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is just a hole in the wall not true if
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you look at it under electromicroscopy
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it's a very complex structural protein i
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mean it's really complex it's like
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looking at the architectural design of a
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freaking hospital it is huge and why do
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you need a pore to transport molecules
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molecules coming into the bacteria
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molecules leaving the bacteria what else
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do we have
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lipopolysaccharides love it lipo because
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it has lipid or fat poly many saccharide
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sugar oh i have sugar and fat what's
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that it acts as an endotoxin it is toxic
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to your body and it is part of the
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membrane of the gram-negative bacteria
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that's why it's endo because it's part
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of the membrane itself it's endogenous
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to it it's not secreted by the bacteria
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to the outside world it is part and
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parcel of the gram-negative's outer
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membrane this is very antigenic
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translation an antigen is something that
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will generate an antibody your body will
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make an antibody against this antigen oh
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which part of it the polysaccharide
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oh oh my goodness this is dangerous oh
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my goodness this is going to be a great
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story because here is an example of an
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antigen antibody reaction happening in
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my own body moreover this
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lipopolysaccharide endotoxin is also
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pyrogenic what does pyro mean you know
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the type of glass that you can put in
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the oven without breaking it what do
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they call it pyrex you know why because
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pyro means heat pyro means favor oh so
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pyrogenic means pro fever and
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pro-inflammation absolutely which part
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of the lps is pyrogenic it's the lipid a
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the lipid a is going to activate
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interleukin-1 and tnf alpha
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interleukin-1 is the hero of fever and
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acute inflammation tnf alpha is the hero
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of recruiting white blood cells and
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activating the endothelium all right
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thank you tell me about the
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intermembrane space which is peculiar to
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the gram-negative bacteria it contains
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peptidoglycan really because remember
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the previous slide the peptidoglycan and
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the gram negative was present in the
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inter membrane space so it's the exact
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same peptidoglycan moreover as molecules
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try to leave the gram-negative bacterial
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cell some of them will leave some debris
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behind eg beta lactamase and this is
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going to be super important in the story
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of resistance to antibiotics i give you
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antibiotics why didn't you respond it's
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thanks to the bacterial betalectumase
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hashtag antibiotics resistance next tell
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me about the cell wall it is thicker in
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the gram positive with more
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peptidoglycan layers but it's thinner in
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the gram negative with less
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peptidoglycan layers okay tell me more
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in gram-positive there is techoic acid
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in the cell wall you do not find techoic
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acid in the gram-negative the tochoic
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acid is specie-specific which means that
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the choice acid of the staph aureus
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which is gram-positive is different from
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the tychoic acid of another
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gram-positive bacteria it differs by
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species it's also a polymer okay it's a
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complex molecule made of many monomers
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it contains phosphate it's covalently
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bound to the peptidoglycan
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and it binds fibronectin this might seem
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gibberish to you right now but just wait
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good things happen to those who wait in
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the next videos this is going to be more
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clear tell me about the pepsi glycan why
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call it peptide because it has peptide
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side chains why call it glycan because
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the main part the backbone is made of
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what glyc which is sugar which is carb
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that's why we call it glycogen oh it's
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carbohydrate that's right glycan is
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carbohydrate it is sugar how can we link
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the sugar backbone with the peptide side
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chain the link is called
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transpeptidase cross-linking so what's
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the process cross-linking when you cross
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linking with peptides what's the name of
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the enzyme that you need trans peptidase
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for the peptidoglycan what's the
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function of the peptidoglycan well it
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provides rigid support for the bacteria
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and it protects the bacteria from
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osmotic damage but hermeticosis i as a
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human do not have cell wall how do i
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protect my own cell from osmotic damage
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you have a cell membrane the cell
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membrane can also protect your cell from
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osmotic damage okay i get it tell me
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more about the inner membrane or the
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cell membrane or the cytoplasmic
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membrane phospholipid bilayer plus carbs
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plus proteins what kinds of proteins
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enzymes because all of your enzyme
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macromolecules are what proteins and
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they also include penicillin binding
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protein and this is how penicillin
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destroy many bacteria because penicillin
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the drug is gonna bind penicillin
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binding protein which is a protein in
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the bacteria and it's gonna destroy the
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bacteria how does it destroy the
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bacteria penicillin is a cell wall
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synthesis inhibitor when penicillin
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binds to its penicillin binding protein
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it's gonna interfere with these
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bacterial proteins and therefore you
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will not be able to synthesize the wall
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of the bacteria a bacteria without wall
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will die very soon due to lack of
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support and lack of asthmatic protection
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go to hell bacteria but many bacteria
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are resistant to penicillin thanks to
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the beta-lactamase and other mechanisms
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let's review gram-positive versus gram
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negative gram positive easy very thick
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cell wall peptidoglycan plus tachoic
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acid after this i have cell membrane
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there is no outer or inner there is just
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one membrane it's lipid bilayer protein
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carbohydrates this is where you find the
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enzymes many of them will help me make
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the cell wall and this is where you find
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the penicillin binding protein which is
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the receptor for the drug known as
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penicillin and then cytoplasmic nucleus
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easy peasy how about the gram negative
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well i have outer membrane and inner
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cell membrane outer membrane has what
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lps and polarines the lps is the
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endotoxin the lps is the pro fever pro
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inflammation and i have an inner cell
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membrane just like this doofus right
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here but what is peculiar and unique to
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me is the presence of the periplasmic
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space between the outer and inner
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membranes and this is where you find my
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pepsi glycans do i have to colic acid
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the answer is no do i have lps the
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answer is yes now let's talk about the
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gram stain technique let's look at this
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quote some people live for the day or
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for the weekend other people think long
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term 20 years ahead 30 years ahead 50
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years ahead hashtag neurosurgeons the
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free market is ruled by those who are
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able to see and plan long range the
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better the mind the longer the range i'm
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gonna take this quote and absolutely
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ruin it for the sake of microbiology of
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course the gram stain is ruled by those
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bacteria with a thick peptidoglycan cell
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wall the thicker the wall the purpler
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the stain but if your wall is thin
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you're going to be pink hermeticosis
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you're trying to say that since
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gram-positive bacteria have a thicker
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cell wall they will appear purple and
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since gram-negative bacteria have a
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thinner cell wall they're going to
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appear pink absolutely correct wonderful
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so here is how you do it you start by
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your sample that contains the bacteria
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could be a sputum sample could be a
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urine sample could be a blood sample
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stool sample all kinds of samples and
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you add it to your glass slide okay add
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to this crystal vital stain and you can
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add iodine if you want to strengthen the
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stain what's the function of the crystal
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viral stain it binds peptidoglycan which
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is part of the cell wall of the bacteria
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okay i get it now if it's gram-positive
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it is purple so far if it's
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gram-negative it is also purple so far
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but wait we will add ethanol to wash out
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the crystal vital stain oh
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you will wash out the gram negative
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because it have a thinner cell wall it
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cannot retain the crystal violet but
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look at the gram positive bacteria you
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cannot wash it away why they have a very
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thick
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peptidoglycan cell wall layer you cannot
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wash their absorption to the crystal
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violet imagine that you have a very
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thick jacket and i stained it with some
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horrible sauce and then i have a very
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thin undergarment i also stained it with
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the same sauce which one do you think is
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easier to wash oh of course the thin
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undergarment bingo that's the exact
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point when you add ethanol it washes the
00:14:34
gram negative but it cannot wash the
00:14:36
gram positive next you add a
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counterstain a pink stain such as
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safranin or wait for it because the
00:14:45
correct pronunciation of this is fossen
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fossen what's that this is fuchsia which
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is this color right here i.e the pink so
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this is how the gram stain help you
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differentiate between the gram positive
00:14:58
which will remain purple all long
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because you did not remove the purple in
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the first place but the gram negative
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started purple then the purple was
00:15:06
washed out then they acquired the pink
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of the counter stain counter to what
00:15:12
counter to the original crystal violet
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stain oh i get it and this is how gram
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helped us differentiate between gram
00:15:19
positives and gram negative gram
00:15:22
positive i'm either a caucus or a rod if
00:15:24
you're a caucus we can classify you
00:15:27
based on the presence or absence of
00:15:29
catalase into staphylococcus i do have
00:15:33
catalase or streptococcus i do not have
00:15:36
catalase her minericosis what the flip
00:15:39
is the function of catalase ace enzyme
00:15:41
catalase because it's a catalyst oh yeah
00:15:44
enzymes are callous i know this from
00:15:45
chemistry awesome bacteria has invaded
00:15:48
your body right yeah your body is trying
00:15:50
to defend itself your body is gonna
00:15:53
throw
00:15:54
harmful chemicals hydrogen peroxide in
00:15:57
the face of the bacteria okay but some
00:16:00
bacteria are smart some bacteria have
00:16:03
catalase which is gonna convert your
00:16:06
harmful chemicals into harmless
00:16:08
molecules that will not harm the
00:16:09
bacteria these bacteria are sneaky and
00:16:12
this is one more mechanism of evasion of
00:16:16
bacteria from your immune system in the
00:16:18
next video we will dig deeper into the
00:16:21
gram positive bugs bugs is a straight
00:16:23
term not a technical term because
00:16:26
technically bugs belong to parasites not
00:16:29
bacteria so when you say gram-positive
00:16:32
bugs versus gram-negative bugs we're
00:16:34
talking tongue-in-cheek of course
00:16:36
this microbiology playlist here on
00:16:38
youtube is going to help you with
00:16:40
microbiology stuff if you want to add
00:16:42
pharmacology to it go to my website
00:16:44
medicosisperfectnetis.com
00:16:46
and download my antibiotics course it
00:16:48
comes with 40 videos talking about
00:16:50
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00:16:53
and anti-parasitic medications and for a
00:16:56
limited number of students you can get
00:16:58
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00:17:01
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00:17:05
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00:17:12
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00:17:14
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00:17:17
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