Bacterial component and morphology

• Cell Wall


  Also referred to as the peptidoglycan [main component], or murein layer
• Cell Wall

  • Phospholipid bilayer and protein are the other components
  • Gives the bacterial cell shape and strength to withstand changes in environmental osmotic pressures that would otherwise result in cell lysis
  • Gives structure to the shape of the cell
  • Gives strength; resists any mechanical disruption
  • Acts as an osmotic barrier to the passage of larger substances
  • Location of electron transport chain = energy is generated
  • Protects against mechanical disruption of the cell
  • Offers some barrier to the passage of larger substances
• Gram Staining


  Very important step in the workflow of bacterial identification for the reason that it can define already whether the bacteria is gram positive or negative

  
• Gram Stain Reaction


  Differential staining method (using 2 stains; initial & secondary) → cell wall and target

  
• Gram Staining Components

  • Crystal Violet – Primary Stain
  • Gram's Iodine – Mordant [strengthen the affinity of the stain to the bacterial cell wall]
  • Acetone Alcohol – Decolorizer [acetone in 95% ethanol or ethyl alcohol]
  • Safranin – Secondary Stain/Counterstain [red]

  
• Before the staining process, the bacterial cell wall is colorless

  
• Fixation Methods

  • Physical [heat]
  • Chemical [methyl alcohol]
• Peptidoglycan layer/Murein layer

  • Gram positive – thicker peptidoglycan
  • Gram negative – thin peptidoglycan
• Periplasmic space


  Can only be seen in gram negative
• Gram positive


  Only have 1 layer of membrane
• Gram negative


  Has 2 layers of membrane
• Fixation


  Attach bacterial cell on surface of slide para pag nag undergo ng staining process di siya ma wwash-out

  
• Physical Fixation


  Passed through flame; most common because of availability
• Chemical Fixation


  Drip the slide; ideal
• Mordant


  Strengthen the affinity of a stain to the cell wall of the bacterial cell
• Decolorization is the most crucial step in any staining technique because siya na mag ddiferentiate
• Periplasmic Space


  Gel-like substance that either contains enzymes that are degradative
• Degradative enzymes


  Important for invasion; capable of degrading the host's surface; mabilis makakapag-attach; degrade mucus (1st line of defense)
• Gram Negative organisms have greater virulence
• Periplasmic space assists in the capture of nutrients from the environment
• Gram Positive Cell Wall


  Has a very thick protective peptidoglycan (murein) layer
• Teichoic acid


  Provide rigidity to the cell wall by attracting magnesium and calcium
• Lipoteichoic acid


  Acts as regulator of autolytic wall enzyme, regulate the antigenic properties (has antigen)
• Antigenic properties


  Stimulate the immune response of the host
• Autolytic wall enzyme


  ex: muramiclase (anti-microbial enzyme); kaya nilang i-lyse yung sarili nila on their own
• Gram Negative Cell Wall

  • Thin peptidoglycan layer
  • Periplasmic Space
  • Outer membrane: proteins, phospholipids, and lipopolysaccharide (LPS)
  • Inner membrane: Phospholipid bilayer, periplasmic space, murein layer
• Functions of the Outer Membrane


  • It acts as a barrier to hydrophobic compounds and harmful substances
  • It acts as a sieve, allowing watersoluble molecules to enter through protein-lined channels called porins
  • It provides attachment sites that enhance attachment to host cells
• Gram Variability


  A characteristic exhibited by gram positive bacteria where the bacteria has the capability of demonstrating both reactions
• Natural gram variability

  • Mobiluncus spp., and Gardnerella vaginalis
• Acquired gram variability


  For gram positive bacteria, can occur in old culture [has been in the refrigerator for more than 24 hrs] or fresh culture [in the incubator for less than 24 hrs]
• Acid stain


  Stains basic component
• Alkaline Stain


  Stains acidic component
• Contributing factors to gram variability

  • Use of old culture
  • pH of staining reagents
  • Bacterial autolysis
  • Staining reaction time

  
• Staining reaction time

  • Primary stain – 1 minute
  • Mordant – 1 minute
  • Decolorizer – until colorless [2 seconds]
  • Secondary Stain – 30 seconds-1 minute
• The staining reaction time depends on the manufacturer's instructions
• Guiding rules in the gram stain reaction of medically important bacteria

  • All COCCI are Gram POSITIVE except: Neisseria, Branhamella/Moraxella, Veillonella [NVM] sp.
  • All BACILLI are Gram NEGATIVE except MBCCLLEARK [MyBELLCRACK]
  • Mycoplasma spp., Ureaplasma spp., and Acholeplasma spp. usually have a gram-negative reaction NOT because it has a gram-negative cell wall but because they DO NOT HAVE a CELL WALL
  • Spirals are very difficult to stain using gram staining, however, stainable spirals are usually gram negative
  • Mycobacterium & Nocardia spp. have a gram-positive cell wall structure, however, because 60% of the cell wall is made of hydrophobic lipids mainly mycolic acid, it affects its permeability this makes it difficult to gram stain

  
• Acid Fast Cell Wall


  Contain a waxy layer of glycolipids and fatty acids (mycolic acid)
• Acid Fast Staining


  Specifically designed for a subset of bacteria whose cell walls contain long-chain fatty (mycolic) acids
• Acid Fast Staining


  Differential staining method: will differentiate acid fast [pink/red] from non-acid fast [blue/green] [no mycolic acid – nonacid fast]
• Mycolic acids render the cells resistant to decolorization, even with acid alcohol decolorizers