Microbiology Pt. 1

Question 1

Pasteur

Answer 1

• demonstrated sterilisation

- disproved the idea of spontaneous generation

Question 2

Koch’s Postulates

Answer 2

Postulates proving a specific microorganisms causes a specific disease

1. suspected pathogenic organism should be present in all cases of disease and absent in all healthy organisms
2. suspected organism should be grown in pure culture
3. cells from a pure culture of suspected organism should cause disease in healthy animals
4. organism should be reisolated and shown to be the same as the original

Question 3

Characteristics of Bacteria

Answer 3

• single colonies can be grown on agar
• each colony comes from a single cell
• pure culture = culture derived from a single colony
• all cells in the same single colony are genetically identical

Question 4

Bacteria

Answer 4

• <40 phyla, and each phyla has several genera
• each genus has many species
• rod, coccus, spirilla shapes
• rod = cylindrical
• coccus = spherical/oval
• spirilla = curved rods
• unusual shapes = spirochetes and filamentous
  Many bacteria remain in clusters after division and form characteristic arrangements like sheets and chains

Question 5

Streptococcus Pyogenes

Answer 5

• gram positive bacteria
• cocci chains
• produces many virulence factors like enzymes and toxins
• eg. scarlet fever, rheumatic fever, septic sore throat

Question 6

Staphyloccocus Aureus

Answer 6

• gram positive
• groups of grape clustered cocci
• causes skin infections, respiratory infections, toxic shock, scalded skin syndrome
• serious post-op infections
• MRSA > resistant to antibiotics
• virulence factors produced are species specific

Question 7

Bacillus Anthracis

Answer 7

• gram positive
• rod shaped
• anthrax toxin
• pathogenic due to acquisition of certain genes, including genes for anthrax toxin (plasmid transfer)

Question 8

Neisseria Gonorrhoeae

Answer 8

• gram negative
• cocci (diplococcus)
• causes gonorrhae
• relates to N. meningitis

Question 9

E. Coli

Answer 9

• gram negative
• rod shaped
• model organism
• express certain genes causing virulence
• intimate adherence to gut
• transfer of ‘effector proteins’ from bacterium to host cell

Question 10

Energy Sources of Bacteria

Answer 10

All make ATP
Chemical (chemotropy)
- organic chemicals (chemoorganotrophs)
- inorganic chemicals (chemolithotrophs)
Light (phototrophy)
- phototrophs

Question 11

Cell Division

Answer 11

• generation time = time for population to double

- septum formation between cells

Question 12

Tubulin Homologue Fts2

Answer 12

• initiates division by localising in mid-cell

- homologue of eukaryotic cytoskeletal protein tubulin

Question 13

Cell Culture Calculations

Answer 13

• direct relationship between number of cells initially i na culture and those after a certain time period
  N = No2^n
  g = t/n (generation time)
  k = slope of line (specific growth rate)
• optical density doesn’t take into account dead cells so viable count is preferentially used
• count cell colonies and assume each viable cell makes one colony
• plate count x dilution factor = cfu/ml

Question 14

Growth Curve

Answer 14

Time vs. Log10 organisms and vs. Optical Density

1. Log phase
2. Exponential Phase
3. Stationary Phase
4. Death Phase

Question 15

Log Phase

Answer 15

• occurs during cell adaptation to new environment
• inoculim is usually depleted of certain nutrients
• time needed for resynthesis
• some cells may be non-viable
• time varies greatly

Question 16

Exponential Phase

Answer 16

• rate of increase of cell numbers constantly rises
• cell numbers increase at the same rate as cell constituents
• growth rates of cultures vary/depends on many factors
• still limited by nutrients

Question 17

Stationary Phase

Answer 17

• growth limited by lack of nutrients/build up of toxic waste products to inhibitory levels
• cells are not dead > no net change in cell number
• certain genes necessary for survival in stationary phase
• sporulation commences in certain species
• accumulation of storage products

Question 18

Effect of Temperature

Answer 18

One of the key environmental factors for bacteria
Increase: rates of reactions rise, proteins denature at temperatures past the optimum, membranes destabilise
Decrease: membranes gel, membrane transport becomes limited, enzymes become inflexible
Optimal temperatures differ between organisms
- hyperthermophiles, mesophiles, etc.

Question 19

Gram Staining Protocol

Answer 19

1. flood heat fixed smear with dye for 1 min
2. add iodine (forms insoluble purple complex on cell walls
3. decolorise with alcohol (complex released from gram negative cells)
4. counterstain with sanfranin (gram negative bacteria stained pink/red)

Question 20

Gram Positive Bacteria

Answer 20

• thick peptidoglycan layer holds crystal violet complex
• Peptidoglycan layer contains lipotechoic acid and wall techoic acid
• 90% of cell wall is peptidoglycan
• glycan chains running parallel and tightly linked by peptide interbridges
• rigid and inflexible

Question 21

Gram Negative Bacteria

Answer 21

• alcohol can release the crystal violet complex to reveal pink counterstain
• outer membrane is degraded and the thinner peptidoglycan later cannot retain the complex so color is lost
• thin peptidoglycan layer
• periplasm layer on either side of peptidoglycan
• lipopolysacchride & protein outer layer
• outer membrane is not symmetrical - inner layer is phospholipid and outer layer is lipopolysacchrides
• lipoproteins attach the outer membrane to the peptidoglycan
• outer membrane is not permeable to high molecular weight molecules
• permeability due to porins
• porins are TM proteins, trimeric structure forming water filled channels, permeability can be nonspecific/specific

Question 22

Peptidoglycan layer

Answer 22

• forms a rigid layer outside the cytoplasmic membrane of Gram + and Gram - bacteria
• similar composition between bacteria
• made of 2 sugar derivatives and some amino acids
• sugars are linked by 1,4 glycosidic bonds

Question 23

Structure of Peptidoglycan

Answer 23

• glycan backbone
• Gram -: peptides linked together instead of interbridge
• Gram +: interbridge of gly linked together
  Final sequence of G-M-G at end

Question 24

Glycan chain

Answer 24

• N-acetyl glucosamine and N-acetyl muramic acid are connected to form a repeating structure termed a glycan chain
• interbridge can be direct or use amino acids
• strength of glycan bonds reinforced by peptide bonds
• archaea have a pseudopeptidoglycan structure

Question 25

Teichoic Acid

Answer 25

• polymers containing glycophosphate or ribil phosphate
• teichoic acids covalently bound to membrane lipids are lipotechoic acids
• flexbility and attracts ions

Question 26

Lipopolysacchrides (LPS)

Answer 26

• specific, inner and outer core, lipid A
• varies between species
• can be very long due to repetition
• specifies antibody recognition
• lipid A forms outer leaflet
• responsible for LPS toxicity
• causes sickness