1. domain
2. kingdom
3. phylum
4. class
5. order
6. family
7. genus
8. species

• morphology
• biochemistry and physiology
• genetic characteristics

• gram stain or fast stain characteristics
• cell shape
• cell arrangement

1. heat fix cells to slide
2. flood slide with crystal violet for 10 seconds
3. wash with running tap water
4. flood with gram iodine for 10 seconds (wash with water)
5. carefully decolorize with 95% ethanol
6. wash with water
7. flood with safranin (counterstain that is pink) for 10 seconds
8. wash with water and dry air or blot with absorbetant paper
9. examine under oil immersion

• gram pos.- alcohol wash dehydrates thick cell wall and trap complex (blue color)
• gran neg.- cell wall remains permeable, so complexes wash out and have counterstain visible (pink color)

• type of metabolism
• which nutrients can be used
• which products produced
• temperature requirments
• pH optimums
• salt tolerance

• GC content
• DNA fingerprinting (RFLP)
• PCR
• rDNA analysis

• cytoplasmic membrane
• periplasmic space
• cell wall
• outer membrane
• cell appendages
• other structures

• proteins (60-70%)
• phospholipids (20-30%)
• carbs (trace amounts)

• osmotic barrier
• attachment pt of chromosome (inner surface)
• energy generation
• cell wall syn.
• selective permeability
• carrier mediated transport

• transport proteins that simultaneously transport two substances across membrane in opposite direction
  • one against conc. gradient
  • one with conc. gradient
• use proton motive force for energy

• two substances go same direction
  • one against gradient
  • one with grandient
• proton motive force used

• sugar transport across membrane
• substrate phosphorylated by carrier and released into cytoplasm
• accumulate sugars "against" gradient
• most require energy

• space btw cytoplasmic membrane and outer membrane in gram negative bacteria
• contains enzymes that break down large molecules into constituent parts for transport into cells
• some strains concentrate antibiotic hydrolases in periplasm

• function- surround cell, provide rigidity, prevent osmotic lysis
• composition- mainly peptidoglycan

• overlapping latice of two sugars crosslinked by AA bridge
• sugars are NAG and NAM
• found ONLY in bacterial cell wall

• single layer of peptidogylcan
• Braun's LP anchors outer membrane to cel wall
• NAM side chain with unique AA"s
  • mesoDAP always pen-ultimate AA in gran negative cell walls

• multiple peptidogylcan layers
• highly cross linked between layers
• peptidogylcan can contain several alternatives at position three
  • allow for more possible cross links than gram negative

teichoic acid

lipoteichoic acid

• attached to lipids in cytoplasmic membrane
• transverse cell wall and extend into environment
• major antigenic determinents

• wall teichoic acids attach to NAM in cell wall
• role in adhesion (needed for virulence)
• cause gran pos. sepsis
  • inflam. response to fragments of the cell wall/membrane
• PAMPs (antigenic determinents) recognized by innate immune cells

Effect of bacitracin on cell wall synthesis

• only in gram neg
• same comp as cytoplasmic membrane
• proteins float in lipid bilayer include porins and channels
• anchored to wall by Braun's LP
• contains LPS aka endotoxin

O antigen

lipid A

core (outer and inner)

• repeating polysaccharide
• characteristic type for each gram negative bacteria
• can express more than one type
• function
  • imparts resistance to phagocytosis
  • phage receptor
  • adherence receptor

• outer
  • structure
    • variable
    • sugars and amino sugars, including heptose
  • function- connect with O antigen
• inner core
  • structure- mostly KDO

CONNECT O antigen to LIPID A

• glycophospholipid
• floats in outside layer of outer membrane
• connects to inner core

• lipid A is a B cell mitogen
• activates alternative complement pathway
• also pyrogenic
• responsible for gram negative sepsis
• disseminated intravascular coagulation (DIC) and shock caused by it

• porins
• porin like proteins
• TonB-dependent receptors

• linked to energy dep. protein TonB
• highly specific for scarce molecules (ex: Vit B12)

• trimeric proteins that form water filled channels through the membrane
• function- allow passage of ions and small hydrophilic molecules
• ex: OmpF, C, PhoE

• ex: lamB
• function- maltose transport, phage receptor

• selective barrier
• confers negative charge to cell
• phage receptors
• pathogenic properties
• stabilizes mating cells
• keeps enzymes in periplasm

• function- used for motility
• composition- flagellin
  • arranged differently in different species
  • anchored to membrane by complex molecular machinery
• mechanism of action- rotates like propellor and driven by proteins moving across the cytoplasmic membrane

• chemorepellent- flgella tangles, and tumbles aimlessly
• chemoattractant- exhibits net movement towards

common (somatic) pili

sex pili

• function
  • adhension- help colonize mucosal surfaces
  • help prevent phagocytosis
• large numbers within cell

• function- facilitates transfer of F factor to "nonfertile" cell
• structure
  • encoded on extracellular DNA molecules called F factors

ONLY FEW PER "MALE" CELL

• structure-
  • mucopolysaccharide layer surrounding cell
  • slime layer is loose capsule
• FUnction
  • resist phagocytosis
  • adherance
  • prevent dessication

NEED SPECIAL STAIN TO SEE

• structure
  • difficult to kill (must autoclave at 121 C for 10-15 minutes)
  • dormant, highly resistent cells cells formed by some gram positive species

CAN PERSIST over 300 yrs

Structure of endospores

• core- cytoplasm with chromosome
• celll membrane
• thin cell wall
• cortex
  • concentric layers of petidoglycan with unusual crosslinks
  • DPA- calcium chealator causes dehydration of cortex, making it resistant to boiling
• spore coats
  • outer and inner
  • structure- highly disulfide bonds
  • function- imparts resistance to chemicals
• exosporium (outermost layer)

• starvation
• high cell density
• stationary phase growth

90 minute process

1. activation
   • heat or certain chemicals activates L alanine receptor (protease)
2. initiation
   • exposure of protease
   • protease activates cortex lytic enzyme
   • leads to degradation of cortex and rehdyration
3. outgrowth- resume vegetative growth