Mim Lecture 16

Front 1

16.1

immersion oil

Back 1

- used to increase the objective diameter

Front 2

16.2

stains

Back 2

- used to increase contrast

Front 3

16.3

Gram Stain

Back 3

- Gram + and Gram -
- differentiated by physical and chemical properties of the cell wall
- Gram + retains a complex that gram - bacteria cannot
- grame + = purple
- grame - = pink

Front 4

16.4

gram stain procedure

Back 4

- thin smear of bacteria is applied to a glass slide
- sample passed thru a flame to heat fix the bacteria
#1 crystal violet added for about a minute, staining purple
- water wash
#2 iodine added to act as a mordant to form a complex with crystal violet
- excess iodine is washed away with water
#3 alcohol added, washed off in 10 sec, as a decolorizing agent.
- alcohol remove crystal violet-iodine complex
- gram + = purple
- grame - = colorless
#4 counterstaining with safarin: give gram - = pink

Front 5

16.5

gram negative:

Back 5

- alcohol dissolves the lipid outer membrane and the complex is released
- decolorization causes gram - rods to appear colorless while grame + = purple

Front 6

16.6

acid fast stain

Back 6

- used if Mycobacterium is the suspected agent
- boiling is not used anymore
- counterstain is methylene blue
- bacteria of interest will stain red while everything else is blue

Front 7

16.7

mycobacterium cell wall:

Back 7

- very waxy and hydrophobic (lipid-rich)
- resist decolorization with the acid-alcohol and retain primary red stain from the carbolfuchsin

Front 8

16.8

staphylococcus

Back 8

- G+ cocci
- grape-like clusters

Front 9

16.9

streptococcus

Back 9

- G+ cocci in chains

Front 10

16.20

streptococcus pneumoniae

Back 10

G+ diplococci

Front 11

16.11

Corynebacterium (diphtheriae)

Back 11

- G+
- rods in shapes resembling 'chinese characters'

Front 12

16.12

clostridium

Back 12

- G+
- boxcar/brickshape rods

Front 13

16.13

gonorrhea in the male

Back 13

- G- biplococci within a PMC cell
- penile discharge

Front 14

16.14

prokaryotic cytoplasmic membrane:

Back 14

- does not contain sterols (i.e. cholesterol)
- has cell wall (peptidoglycan)

Front 15

16.15

adherence

Back 15

phili
fimbriae
capsule

Front 16

16.16

anti-complementary

Back 16

capsule, protease

Front 17

16.17

anti-phagocytic

Back 17

capsule, leukotoxins

Front 18

16.18

subvert humoral immunity

Back 18

Fc receptors, Ig proteases, endotoxin, cell wall components

Front 19

16.19

subvert cellular immunity

Back 19

superantigens

Front 20

16.20

vaccine antigens and antigens inducing hypersensitivity

Back 20

wall and secreted proteins
lipoproteins
lipids
carbohydrates

Front 21

16.21

cell and tissue damage

Back 21

exotoxins e.g. Cytotoxins, neurotoxins, etc

Front 22

16.22

Capsule (K antigen)

Back 22

- the external to cell wall structure.
- present in both Gram + and Gram -
- usually carbohydrate, but they can be protein

Front 23

16.23

structures of capsules

Back 23

- discrete: as seen from negative-stained slides, where there is a distinct halo around each bacterium that did not take the dye
- some have a slime/glycocalyx structure

Front 24

16.24

capsule of bacillus anthracis

Back 24

contains polyglutamic acid

Front 25

16.25

function of capsules

Back 25

- antigenically diverse
- anti-phagocytic
- anti-complementary
- contribute to adhesion
- true virulence factor - if bacterium lacks its capsule, it loses its virulence

Front 26

16.26

flagella

Back 26

- composed of flagellin helically arranged and are driven by a proton motive force.

Front 27

16.27

fimbriae and pili

Back 27

- fimbriae = Type 1 pili
- hair like protrusions composed of fimbrillin subunits
- act as adhesins by way of lectin (protein that binds a sugar) or protein-protein interactions
- at the tip of each fimbria is a receptor for a complimentary ligand on a host cell
- antigenically variable
- may choose to express or not express fimbriae and/or pili to change antigen types

Front 28

16.28

phase variation

Back 28

- a mode of gene regulation which produces populations of bacteria containing a mixture of cells expressing fimbriae (phase ON) and cells not-expressing fimbriae (phase OFF)

Front 29

16.29

Type II pili

Back 29

- sex pili
- act in conjugation between gram negative bacteria
- pilus will act as a 'lasso' to catch and 'reel in' a bacterium, and their walls fuse, genetic material is exchanged

Front 30

16.30

prokaryotes that lack cell wall:

Back 30

- mycoplasma and ureaplasma
- their cytoplasmic membranes contain sterols, which help to strength the cytoplasmic membrane

Front 31

16.31

Gram + cell wall:

Back 31

- same cytoplasmic membrane as Gram -
- simple
- 1 thick layer of peptidoglycan
- contain teichoic acid

Front 32

16.32

Gram - cell wall:

Back 32

- peptidoglycan layer is reduced to a thin monolayer between a lipid bilayer.
- lipid content of the outer membrane of the bilayer is permeabilized by alcohol inthe gram stain
- consists of an outer membrane and the periplasmic space containing the peptidoglycan.
- lipoproteins link outer membrane to peptidoglycan
- transmembrane protein (OmpA) stabilizes the outer membrane
- amphilphilic (amphipathic)
= porins: ring like structures that penetrate through the outer membrane, allowing protein transport.

Front 33

16.33

peptidoglycan

Back 33

- a glycan chain backbone of the disaccharide N-acetylmuramic acid N-acetylglucosamine
- chains are linked by tetrapeptides attached to N-acetylmuramic acid, forming a 3-D lattice.
- tetrapeptides consist of alternating L and D amino acids (D is non-biological -> very difficult to digest these cell walls)
- 1st and 4th amino acids are alanine
- 3rd amino acid is always a di-amino acid e.g., lysine, diaminopimelic acid, etc
- adjuvant: smallest fragment is muramyldipepti de (MDP)
- thick and undifferentiated.
- 50% of dry weight of bacterium
- amino acids of both the cross-linking and the stem polypeptides can be different: the taxonomy of these microorganisms

Front 34

16.34

peptide bond crosslink:

Back 34

- found only in gram + cell walls
- gives 3D structure of the wall
- in gram - wall D-alanine binds directly to the di-amino amio acid

Front 35

16.35

lipoteichoic acids

Back 35

glycerophosphate polymers terminating in glycolipid anchored in the cytoplasmic membrane
- a lipopolysaccharide cf. LPS of Grame - bacteria
- immunogenic, extend from the cytoplasmic membrane through the wall to the external environment
- ancor wall to cytoplasmic membrane

Front 36

16.36

teichoic acid

Back 36

- ribitol phosphate polymers covalently linked to peptidoglycan
- modified with sugars and/or amino acids
- anchored in the wall but extend to the exterior
- immunogenic
- capture captions
- stablilze wall

Front 37

16.37

Periplasmic space
(Gram -)

Back 37

- lies between the cytoplasmic membrane and outer membrane
- contains a single sheet of peptidoglycan; relatively few peptide bridges and no interpeptide bridges
- embedded in a polysaccharide gel
- contains hydrolytic enzymes (some of which contribute to virulence), sugar trasnport system, nutrient-binding proteins

Front 38

16.38

outer membrane
(gram -)

Back 38

- asymmetric lipid bilayer
- outer leaflet composed of:
proteins: 60%
lipopolysaccharide (LPS) 40%
effective barrier
- molecular sieve
- attachment site for conjugation
0 contains determinants of pathogenesis
- protects the peptidoglycan and periplasm

Front 39

16.39

outer membrane proteins Omp)

Back 39

- porins - transmembrane pores
- lipoprotein - anchors O< tp peptidoglycan
- transmembrane protein (OmpA) - stabilizes OM

Front 40

16.40

lipopolysaccharide

Back 40

- most potent immunostimulatory molecule that is known
- B cell mitogen (non-specifically activate all B cells)
- activate the complement cascade and inudce cytokine release from cells of IR
- causes fever, shock and disseminated intravascular coagulation (DIC) (systemic blood coagulation)

Front 41

16.41

LPS can be divided into three parts:

Back 41

lips A: toxic component of LPS. Lipid A and the Core are important for the integrity of the OM. Thi sis necessary for the viability of the cell

Core- most of the core is essential for cell viability

O antigen - it is attached to the core and extends perpendicularly away from bacterium surface. Not involved in virulence. Some gram - bacteria lack the O antigen

Front 42

16.42

cytoplasmic membrane

Back 42

- most lack sterols (except Mycoplasma and Ureaplasma)
- take care of many of the functions of eukaryotic cytoplasmic organelles, such as electron trasnport and energy production, nutrient uptake and waste excretion.
- ion pumps maintain the membrane potential and the CM is responsible for biosynthesis
- site of attachment for flagella, pili and fimbriae.
- antibiotics that act on the CM tend to act by disrupting the membrane and increasing permeability (e.g.. Bacitracin, Polymixcins)

Front 43

16.43

Nucleoid

Back 43

- occupied by a circular, single molecule of helical dsDNA that is supercoiled.
- chromosome is around 1000um long and contains as many as 3500 genes
- e.coli = 2-3um in length, has a chromosome 1400 um

Front 44

16.44

Extrachromosomal DNA plasmids

Back 44

- independent, circular, double-stranded self-replicating DNA molecules that carry only a few genes
- plasmid genes are non-essential but confer a selective advantage, eg. encode antibiotic resistance, toxin production, unique substrate metabolism
- regularly transmissible between bacteria of the same and different species and genera. (Enterococci are vancomycin resistant. Vancomycin used to be used as a last resort for staph infections, b/c staph is resistant to a lot of antibiotics. However, due to transmission of the antibiotic resistance gene, staph is now also resistant to vancomycin.

Front 45

16.45

binary fission

Back 45

- how bacterium replicates
- occur every 20 mintues under facorable conditions
- does not last long

Front 46

16.46

antibiotics acting on nucleic acid:

Back 46

- antibiotics that disrupt DNA synthesis: metronidazole,
- inhibition of DNA gyrases or topoisomerases: quinolones, nalidixic acid
- inhibition of DNA dependent RNA polymerase: rifampin, rifabutin

Front 47

16.47

ribosomes

Back 47

30S + 50S subunits = 70S ribosome
- proteins and RNA in the prokaryotic ribosome are different to eukaryotic ribosomes

Front 48

16.48

antibiotics acting on protein synthesis

Back 48

- irreversible or reversible binding to 30S ribosomal proteins: Aminoglycosides, tetracyclines
- reversible binding to the 50S subunit: chloramphenicol, macrolides, lindamycin, streptogramins

Front 49

16.49

inclusion granules

Back 49

- polyphosphate metachromatic (volutin: food stores in the cytoplasm)
- glycogen/starch
- lipid
- marker in identifying bacteria (responds to iodine stain).

Front 50

16.50

k. spores

Back 50

- gram + rods only
- in bad environment, bacteria produce spores. cells start out as vegetative cells, environmental trigger => binary fission.
- one daughter nucleoid will be enclosed in a double cell membrane and double cell wall containing only essential biochemical proteins and DNA.
- the cell disrupt and the spore will be released while the other daughter cell dies.

Front 51

16.51

biothreat agents

Back 51

- bacteria than can sporulate are considered biothreat agents
- there are readily aerosolized and are able to survive for a long time wherever they land.
- weaponsized spores have outer charges removed -> airborne longer
- spores are resistant to radiation and other chemical and physical means of disinfection.

Front 52

16.52

clostridium tetani

Back 52

- biothreat agents
- microscopic morphology unique in that the spores are located atht epole of the bacterium cell, giving it a 'tennis racket' appearance
- obligate anaerobe
- toxins released target the CNS and periphearl nerve ends and bind irreversibly
- leads to unopposed muscle contraction

Front 53

16.53

Clostridium botulinum

Back 53

- a muscle relaxant (why botox is used)

Front 54

16.54

bacillus anthracis

Back 54

- spores of B. anthracis are in the middle of the cell and do not bulge out

Front 55

16.55

opportunistic pathogens

Back 55

- sporulating bacteria are usually opportunistic pathogens
- saprophytic, or soil-living, and survive as spores when there isn't enough nutrients
- when the environment is favorable again, they resturn to vegetative cells.
- transformation from a vegetative cell to a spore and vice versa will release toxins in the process, making the environment dangerous for us