Microbiology MOOC Flashcards
gram-positive versus gram-negative bacteria
utilize different forms of quorum sensing systems.
bacterium can turn pathogenic when different combinations of the quorum sensing genes are activated
e.g. the virulence of S. aureus increases significantly.
Commensal bacteria can produce a quorum sensing effect on the Vibrio cholerae
that changes its gene expression and restricts its ability to become established in the gut and cause disease
fimbriae
which enable them to stick to surfaces.
containment measures
prevent transmission of pathogens
autoclave, incineration or chemical disinfectants
decontamination
biological safety cabinet
BSC
HEPA filters in the cabinet
remove bacteria and viruses from the air as it exits the ventilated workspace inside, preventing pathogens from being released into the environment.
biosafety levels
depend on:
- infectivity
- potential severity
- transmissibility
- prevention/treatments
when using water
it should be sterile
aseptic techniques:
- close to a Bunsen burner; sterilise air
- flame the neck of the vial
pipettes
do not touch glass vial
use sterile spreader
to cover agar with suspension
colony morphology
unique
Streak plate
- culture of one species
- sterilise innoclation loop in bunsen
- cool the loop to touch the colony
- transfer to agar plate
- repeat then streak
Tryptone Soya Agar
TSA
vortexer
to release microbes
Microscope slide preparation
- pure culture
- sterilise innoculation loop to place sterile water onto slide
- take a single sample from colonies
- spread into water to create a smear
- pass slide through the bunsen burner to heat-fix it
heat-fixing
firmly attaches bacterial cells to the slide so that the stain doesn’t wash them off
Gram staining
- flood the smear with crystal violet for on minute
- tip off excess
- flood with gram iodine and leave for a minute
- tip of excess and keep it tilted, decolurising with ethanol until it runs almost clear
- rinse with tap water
- flood with saphronin to counterstain the smear for 45s
- rinse with water
- blot dry with absorbant paper
Identification of gram positive bacteria
- immersion oil on stain smear
- x100 objective lens
- cells are stained purple and rectangular - rod
Gram positive bacteria appearance explanation
have a wall that has a thick layer of peptidoglycan, which retains the crystal violet-iodine dye after washing with ethanol; appear purple
Gram negative bacteria appearance explanation
- have a much thinner layer of peptidoglycan and an outer membrane that contains porin channels
- when the cells are washed with ethanol the crystal violet-iodine dye is washed out and the cells decolourize.
Further gram negative identification
- A pink dye (safranin or fuchsine) is used to counterstain, so that they are visible under the light microscope.
- Gram positive cells also take up the counterstain but it is not noticeable because the crystal violet stain is much darker
Gram negative bacteria
- outer membrane phospholipid bilayer
- porins
- LPS in outer membrane
- thin peptidoglycan layer between inner and outer membranes
- high abundance of Braun’s lipoprotein
- periplasmic space
- inner cell membrane (cytoplasmic membrane) phospholipid bilayer
lipopolysaccharide (endotoxin)
- composed of a chain of polysaccharides that are anchored to the outer membrane via lipid A.
- chemical composition differs between bacterial strains. - can induce a strong immune response and cause toxic shock.
peptidoglycan
- composed of amino sugars that are cross-linked to form a crystal lattice
- provides structural support to the cell and helps to withstand osmotic pressure
Braun’s lipoprotein
links the outer membrane to the peptidoglycan layer and helps provide structural support to the cell
Periplasmic space
contains periplasm
periplasm
- gel-like matrix that fills the space between the outer membrane and the inner cell membrane
- contains a variety of different proteins that play roles in important cellular processes including nutrient uptake, protein folding and the elimination of waste and toxic compounds.
colony forming units per ml =
number of colonies / (dilution x volume plated)
underestimate inaccuracies:
- did not count all colonies (repeat!)
- laboratory culture conditions
effect of laboratory culture conditions
- e.g. oxygen conditions might exclude anaerobes or microaerophiles
- e.g. temperature may not be at optimum
how many microbes are there in soil?
between 1 and 100 billion (1x10^9 - 1x10^11)
BLAST
basic local alignment search tool; searches a large database to find nucleotide sequences related to or similar to a sequence you input
want a low e value
0.0001 - homologous
expected value - how similar the input to the output
Sequencing
PCR
Antibiotics act by
disrupting a specific cellular component (eg cell wall, cell membrane) or biosynthetic pathway (protein synthesis, nucleic acid synthesis, folate synthesis) within a bacterial cell.
Penicillin
- a Beta-lactam antibiotic
- inhibits penicillin binding protein enzyme that cross-links peptidoglycan layers in the bacterial cell wall.
- specific to bacteria because only they have this polymer in their cell wall
- more effective against Gram positive bacteria because they have a much thicker layer of peptidoglycan in their cell wall
- when a bacterial cell replicates, it makes small cuts in the peptidoglycan layer in order to divide and to incorporate more peptidoglycan into the cell wall as it grows.
- when a bacterial cell replicates in the presence of penicillin the peptidoglycan layer becomes weaker and weaker, and the cell ultimately bursts when it can no longer withstand the internal osmotic pressure.
defence mechanisms against antibiotics
- produce a slightly modified cellular component that is no longer inactivated by the antibiotic (target modification)
- produce an inactivating enzyme that modifies or destroys the antibiotic
- produce an entirely new enzyme that is not sensitive to the antibiotic to bypass the inactivated component
- reducing the ability of the antibiotic to reach the target by either decreasing uptake of the antibiotic or actively removing it from the cell using an efflux pump
Penicillin resistance
- in Gram positive bacteria is predominantly via target modifications that prevent penicillin from binding to the PBP
- in Gram negative bacteria is more commonly achieved by production of inactivating enzymes (𝛽-lactamases)
Campylobacter jejuni is…
… a Gram negative corkscrew-shaped bacterium that lives as a commensal in the intestines of healthy birds, causing no obvious harm or benefit to the host.
