Microbial basics

Question 0

Molecular Koch’s postulates

Answer 0

Falkow - genes -> pathogenic traits

same principles

• identify gene present in sick, not present in healthy
• disruption reduces virulence

Question 1

Koch’s postulates

Answer 1

1. organism in all sick animals, not in any healthy
2. isolate -> pure culture
3. inoculate -> same disease
4. re-isolate -> pure culture

requires infectable animal model…
requires culture growth
difficult with obligate intracellular (no culture growth)

Question 2

Classes of micro-organisms

Answer 2

Bacteria -

• many pathogens, some photosynthetic
• rigid cell wall
• require organic energy source

Fungi

• some pathogens, non-photosynthetic
• rigid cell wall (but diff than bacteria)

Not covered in this course:
Algae - photosynthetic, no pathogens
Protozoa - some pathogens, no rigid wall, unicellular, nonphotosynthetic
(ex amoeba)

Question 3

Characteristics of micro-organisms

Answer 3

Unicellular (but can behave collectively, communicate)

Eukaryotic - fungi, protozoa, algae
Prokaryotic - bacteria
diff chromosomes, organelles, movement, wall

Question 4

Chromosomes (eukaryotic vs pro)

Answer 4

Eukaryotic - vary in number, mitosis

Prokaryotic

• one circular (occassionally multiple)
• segregation but no mitosis

Question 5

Basics of bacterial identification

Answer 5

Morphology of colonies
Morphology and staining of individuals
 - Gram stain
 - cocci, bacilli, etc
Metabolism - ie fermentation of specific nutrient
Antigens (specific assays)

PCR - used clinically for specific detection

Question 6

Organelles (eukaryotic vs pro)

Answer 6

Eukaryotic - membrane-bound organelles
- ex mitochondria with ox-phos enzymes

Prokaryotic - no organelles
- oxidative phosphorylation on cytoplasmic membrane

Question 7

Cellular movement (eukaryotic vs pro)

Answer 7

Eukaryotic

• cytoplasmic streaming
• cilia or flagella (9 doublets + 2 of microtubules, covered by membrane)

Prokaryotic

• no cytoplasmic streaming (too dense)
• flagella - repeating flagellin subunits in hollow tube, no membrane or microtubules

Question 8

Cell wall (eukaryotic vs pro)

Answer 8

Eukaryotic

• plants, algae - cellulose
• fungi - chitin (acetyl glucosamine) + beta 1,3 glucan
• often have sterols in cell membrane

Prokaryotic

• peptidoglycan polymer - muramic acid, D-amino acids, etc
• key for specificity of antibiotics

Question 9

Protein synthesis

Answer 9

Eukaryotic

• RNA splicing and transport before translation
• 80S ribosome

Prokaryotic

• simulataneous transcription and translation (no splicing)
• 70S ribosome

Question 10

Bacteria shapes

Answer 10

Rigid cell wall - don’t change with osmotic pressure
Usu about 1 micron
“Pleomorphic” - changes shape

Cocci - round - can be elongated (Pneumococcus, Gonococcus) or perfect sphere (Staph)
Bacilli - can have variations in shape (ex coccibacilli)
Vibrio - curved (Cholera)
Spirochete - not actually rigid, spiral (ex Treponema pallidum, Borrelia burgdorferi)

Question 11

Bacterial division

Answer 11

Binary fission -> filament if incomplete separation

Long axis -> chain
 - Streptococci, all bacilli
Random -> cluster
Stick together - Corynebacterium -> stacks
 - Staphylococcus

Question 12

Cell membrane

Answer 12

Lipid bilayer
No sterols (vs eukaryotic)

• oxidative enzymes (similar to inner mitochondrial)
• cell wall synthesis
• selective/active transport
• secretion of toxins

Question 13

Cytoplasmic inclusions

Answer 13

Ribosomes (70S, antibiotic target)
Granules - nutrient storage without increased osmolarity
- high molecular weight lipid
- glycogen
- metachromatic = polymerized phosphate (ex Diptheria)

Question 14

Cell wall structure

Answer 14

25-30% of cell weight

Glycosidic bond -> chain
- n-acetyl glucosamine (NAG)
- muramic acid (= NAG + lactic acid)
- glycosidic bond broken by lysozyme
Cross-linking - necessary for stability and rigidity
- via tetrapeptide on lactic acid (lots of variation, weird amino acids)

Question 15

Gram negative structure

Answer 15

Thin cell wall (only peptidoglycan)
Outer membrane
- lipopolysaccharide (LPS) = antigen!
- lipid A embedded = toxin, polysaccharide attached = antigen (repeating oligosaccharide = “O” antigen)
- anchored to cell wall
- pores in outer membrane - non-specific for small (block vanco)
- generally more resistant
Periplasmic space = between cytoplasmic and outer membrane
(lots of enzymes for intake of nutrients, penicillinase)

Question 16

Gram positive structure

Answer 16

No outer membrane
Thicker cell wall - peptidoglycan + carbohydrate polymer
- ex ribitol phosphate (sugar-OH) in Staph = teichoic acid
- polymers are antigens

Question 17

Gram stain

Answer 17

Crystal violet + iodine -> insoluble complex
Decolorizer
- Gram negative washes out (EtOH degrades outer membrane -> thinner cell wall)
- Gram positive retained dt thick cell wall
Counterstain

Gram positive can appear negative if old (wall breakdown)

Question 18

Capsule

Answer 18

Non-essential, polysaccharide coat
Affects pathogenicity (ie resists phagocytosis)
Can be antigen

Not super solid - particles floating free, detect in urine or CSF for dx

Question 19

Flagella

Answer 19

All motility is via flagella!

10-20 nm x 8-12 microns (longer than cell)
Flagellin -> repeating polymer, hollow tube
Requires electron micrograph, special stains
Either polar or peritrichous

Movement - can have chemoreceptors for chemotaxis

• attractant (sugar, aminos) -> counter-clockwise spin -> straight line
• repellant -> clockwise spin -> “tumble”

Question 20

Pili

Answer 20

aka fimbrae (interchangeable)
Adhesion (almost all bacteria have some form)
- recognize and bind to sugars on mammalian cells
- not sole mechanism (ex Gram + glycocalyx, Gram (-) adhesins in outer membrane)
Examples
- F pili - “donor” bacteria during conjugation
- type IV - push and pull across surface -> twitching motility, aggregation
- Gonococcus - adheres to epithelial cells

Question 21

Sporulation

Answer 21

Stimulus (nutrient deprivation) ->
Sporulation (forms within bacillus - characteristic location and shape) ->
Spore can persist for long periods, resistant to boiling, drying, chemicals, staining (low H2O content, protein coat) ->
Germinate to vegetative in favorable conditions

Ex:

• Bacillus anthracis -> lungs
• Clostridium tetani - soil -> wounds