Microbiology Midterm #1

Microrganisms

Anything microscopic, except for large bacteria, algae, and microscopic worms. Includes bacteria, archaea, and eukaryotes. (such as fungi, algae, and protozoa)

What are smaller organisms good for?

Energy efficiency and there is a larger surface area to volume ratio

Coccus

Rod

Spirillum

Spirochete

Budding and appendaged bacteria

Filamentous

Light Microscopy

Using a compound microscope, light is the source of protons. You use an objective lens and an ocular lens. The light interacts with the objective to create a contrast. (100x magnification)

Absorption- Light Microscopy

The object blocks part of the light.

Reflection- Light Microscopy

The wavefront of the light bounces off the surface.

Refraction- Light Microscopy

The light bends when it enters a substance that changes its speed.

Scattering- Light Microscopy

A small fraction of the incident light is scattered in all directions. Occurs when the dimensions of the object are close to the wavelength of incident light. When you shine light, a little of it will get dispersed. The shorter the wavelength, the better the resolution.

Brightfield

With brightfield, oftentimes people use dyes which are normally positively charged. Sometimes the dyes are detrimental for bacteria.

Phase Contrast

Accentuates the contrast of bacteria compared to background

Dark field

The environment is black because there is no scattering. Light is caught.

Differential interference contrast

Creates a shadow of sides, looks 3D (like an illusion)

Fluorescence

Have to have flor. Emit light at high fluorescence after activation with a laser. Can use dyes

Gram Stain

Gram negative bacteria stains negative which is pink and gram positive bacteria stains positive which is purple. The difference is in the cell walls.

Electron Microscopy

Electron microscopes that use electrons instead of photons and electromagnets instead of lenses. You need to work under a vacuum because you do not want electron interference with the air.

Transmission Electron Microscopy

Uses stains made of heavy metals like uranium. The highest resolution is 0.2 nm. You need to make thin sections (20-60nm) for visualization of internal structures. It can magnify up to one million. You can only see the surface, see what is inside, and make slices.

Cryo electro tomography

Uses a sample that is flash-frozen, thus in a near-native (hydrated) state. No staining is involved. The imaging occurs under cryogenic conditions to maintain the sample hydrated and reduce damage from the electron beam.

Tomography

Acquisition of projected images from different angles; no thin sectioning required if the sample is thin (< 0.5 μm)

Scanning Electron Microscopy

It uses a thin film of heavy metals like gold. it has magnification from 15X to 100,000X and uses an electron beam to scan back and forth, creating a 3d rendering. It is good for looking at the surface of bacteria.

Scanning Electron Microscopy vs Cryo Electron tomography

SEM uses one image, and Cryo Electron scans with many different images to get the 3D aspect.

Bacterial Cell Envelope

The cell envelope interacts with the environment and adds protection. All Cell envelopes have cytroplasmic membranes, and the bacterial cell envelope also has a cell wall. The cell wall includes all of the external layers outside the cytoplasmic membrane. Peptidoglycan is one of the main components and gram negative bacteria also have an outer membrane. In the TEM image, the black line is the peptidoglycan.

The cytoplasmic membrane- A phospholipid bilayer

The bilayer is very fluid and have the viscosity of light grade oil. It is missinging sterols. It has a highly selective permeability barrier that allows the cell to concentrate specific metabolites and excrete waste materials. Water and dissolved gases (O₂, CO₂) and lipid-soluble molecules readily cross the phospholipid bilayer, unlike ions and charged molecules. The bilayer likes sterols such as cholesterol.

Peptidoglycan provides mechanical support and resistance to the intracellular osmotic pressure in hypotonic environments

When this occurs, lysozyme is produced as an enzyme for the first line of defense. The cell ends up breaking because of too much pressure, and water moves via osmosis. Peptidoglycan is like a tire that keeps the pressure in. With rod shapes, peptidoglycan keeps it in the rod shape, but when the shape is lost, protoplast becomes spherical.

Peptidoglycan dictates cell morphology

Peptidoglycan is a strong eleastic fabric and not a brick wall. Glycan strands connected by peptide bridges forming one giant molecule that surrounds the cytoplasmic membrane. Multiple layers in Gram–positive bacteria; mostly only 1 layer in Gram-negative bacteria.

Peptidoglycan

A meshwork permeable to solutes and small proteins

Basic Unit of Peptidoglycan

There is a N-acetyl-muramic acid, meso-diaminopimelic acid (DAP), and amino acids in the D configuration. It will always have two DAlas next to each other. The 3rd position of peptide stem will be an amino acid with a free amine because the amine is used for cross linking via a peptide bond.

The peptidoglycan biosyntehtic pathway

Peptidoglycan synthesis (Pink is the peptides and purple and blue are the glycan strands)

Transglycosylation occurs, which is the addition of disaccharide-peptides to the growing glycan strand. Transglycosylation is the linking. Then Transpeptidation occurs, when a peptide cross-links adjacent glycan strands (which confers rigidity). Transpeptidation uses a free amine to bond to the fourth amino acids.

Enzymes invovled in Peptidoglycan synthesis

Penicillin-binding proteins (PBPs):

High-molecular-weight PBPs are involved in peptidoglycan synthesis: transglycosylase (a) and transpeptidase (b) activities, or just transpeptidase activity (b). Low-molecular-weight PBPs involved in peptidoglycan processing: carboxypeptidases (e) or endopeptidases (d).

Other processing enzymes: lytic transglycosylases (c); amidases (f), N-acetylglucosaminidase activity (g).

In order to expand, bonds must be broken by enzymes.

The Gram negative outer membrane

The outer membrane is rigid relative to the cytoplasmic membrane and, together with the monolayered peptidoglycan, it provides mechanical resistance to the intracellular osmotic pressure.The outer membrane is a semi-permeable barrier because of the presence of porins. The pink things are porins, and are less selective. Nutirients can go through the porins but lysosyme can’t. Lipopolysaccharide (LPS) interacts with the environment and it is not fluid. LPS does provide resistence to turbo pressure.

Bacteria can form microcompartments with Protein shells to sequester specific metabolic activities

A polyhedral protein shell encapsulates enzymes involved in carbon fixation, including Rubisco, which converts carbon dioxide into energy-rich molecules. Rubisco is a very poor enzyme, so in order to increase its activity, it must be condensed. CO2 gets concentrated, and CO2 goes into the shells and turns into bicarbonate. Bicarbonate goes into carboxysome, and then turns into CO2.

Major cytoskeletal systems- In Humans

Microtubules (tubulin)

Microfilaments (actin)

Intermediate filaments (intermediate filament  proteins)

FtsZ assembles into a ring-like structure at the site of division

FtsZ ring constricts to create new cell poles

FtsZ ring directs peptidoglycan synthesis across the cell width, resulting in cell pole formation. The ring is right at the site of division.

FtsZ is required for cell division

Without FtsZ the cells will continue to grow but won’t divide. Removal of xylose (inducer) from the environment results in repression of ftsZ expression and a block in cell division (cell filamentation).

MreB is an actin homolog found in many rod-shaped bacteria

In live cells, short filaments rotate around the cell circumference. The current hypothesis is that they direct circumferential peptidoglycan synthesis. Glycan moves in one direction and peptides more in anaother. MreB is mainly found in rod shaped bacteria.

MreB maintains the cell width constant during cell elongation

MreB is a rod shape determinant. Spherical species lack MreB.

Crescentin is an intermediate filament-like protein in Caulobacter crescentus

Spontaneous polymerization occurs in the absence of nucleotides or cofactors. Crescentin assembles into a filamentous structure beneath the cytoplasmic membrane on one side (inner curvature) of the cell. Crescentin self-assembles inside the cell and is structurally similar to animal intermediate filament proteins.

Crescentin mediates the cell curvature of Caulobacter crescentus

Major cytoskeletal systems- Analogous Systems in Bacteria

Tubulin-like (FtsZ)

Actin-like (MreB, others)

Intermediate filament-like (crescentin, others)

Bacterial cell replication

Growth of an individual cell continues until it divides into two cells, typically after doubling its genome, ribosomes, macromolecules, metabolites and inorganic ions.

Generation

Interval for the formation of two cells from one during exponential growth

Growth rate

The change in cell number or cell mass per unit of time.

Generation time

The time required for the cell population to double during exponential growth. Also called doubling time.

Growth parameters (for exponential growth)

Microbes can grow into colonies visible to the naked eye

Viable counts using dilution and plating method

You perform a serial dilution then plate it. Then you can count. It tells you the number of fiable cells in the tube and the density of the cells.

Counting chamber for direct cell count using microscopy

The number of cells under the big square is counted (in practice, several squares are counted and the numbers are averaged). Because the volume under the grid is known (e.g., 10^-4 mL), this value can be converted to the number of cells per mL. The chamber holds a specific volume under square grids. The counting chamber provides Instant measurements; it counts live and dead cells. It is not a good method for cell suspensions of low density (<106 cells/mL), and motile cells must be immobilized. If the cells are lysed, you can distinguish live from dead cells. The number of cells per square decreases at low density.

Turbidity Growth Measurements

Using a spectrophotometer you can measure light that didn’t hit anything. The more bacteria there is, the more the light will be scattered. However, you need to use other technidues in addition to this for calibration. When the cell denisity is too much, the light gets rescattered, and you will need to dilute for the correct measurement. With this, you get instant measurements, you can count live and dead cells, and you need a standard curve to convert optical density (OD) measurements into cell number per mL.

Calibration of turbidity measurements

The Optical density (OD) is proportional to cell numbers; so you can use a standard curve for conversion. However, it is less reliable at high OD.

Microbe growth is dependent on many environmental factors

•Nutrients

•Temperature

•Water availability

•pH

•Oxygen

•Saline/osmolarity

•Pressure

•Radiation

•Others

Microbes are not “culturable”

We can’t meet all conditions needed to grow in pure culture (e.g., needs other co-existing organism to grow)

Extremophiles

Microbes that live in environments considered as extreme. e.g. psychrotolerant organisms: grow near 0º C but have optima of 20-40º C, found in temperate climates (psychrophiles are pickier and die outside ideal range)

The benefits are exploitability/applicability in lab experiments.

Temperature classes of microorganisms

The higher the temperature, the faster the enzymatic activity that facilitates growth. at a certain threshold temperature = denaturation. Microbes cannot regulate their temperature.

What happens if you put a thermophile in lower temperature (outside its ideal range)?

It becomes more stiff and rigid, therefore has lower activity

Sterilization (e.g., autoclave)

If boiling a solution, some bacteria are heat-resistant because they have SPORES so the fix is to autoclave (increases pressure to increase temperature)

Pasteurization (doesn’t kill all organisms and is for heat sensitive foods)

Used when we cannot sterilize (e.g., lots of protein denaturation in milk) but want to reduce amount of organisms, which will still grow overtime hence fast expiration)

The longer the time of heat exposure = the greater the reduction in bacteria

Irradiation

Causes irreparable DNA damage (UV, x-rays, 𝜸-rays, electrons)

Deinococcus radiodurans

A radiation-resistant microorganism originally isolated from an irradiated can of meat. They have evolved to this degree of resistance via  desiccation and have a very efficient DNA repair mechanism

Microbial growth control

•Heat

  Sterilization (e.g., autoclave)

  Pasteurization (doesn’t kill all organisms; for heat-sensitive foods)

•Irradiation

  (UV, X-rays, γ-rays, electrons) 

•Filter sterilization

  for heat-sensitive liquids or gases

•Chemical growth control

  antimicrobial agents

Filter sterilization

This removes microorganisms from heat-sensitive liquids or gases using membrane filters, typically with pores, without heat or radiation. Common methods include syringe filters for small volumes, vacuum filters for intermediate, and cartridge/capsule filters for large-scale production. This is meant for heat-sensitive liquids or gases. Most bacteria 0.5-5 μm in size and the typical pore sizes are: 0.3-0.5 μm = small enough to trap most bacteria but large enough to let liquid/gas pass through

Chemical - antimicrobial agents

Substances that kill or inhibit the growth of microorganisms (bacteria, viruses, fungi, parasites), acting through mechanisms like disrupting cell membranes, DNA inhibition, or protein denaturation. Common types include alcohol (70% ethanol/isopropanol), phenolic compounds (triclosan, hexachlorophene), halogen-releasing agents (chlorine bleach, iodine), heavy metals (silver, copper), and soaps/quaternary ammonium compounds. Arrows show when the antimicrobial agent was added during the exponential growth of the bacterial culture.

Bacteriostatic

Arrests bacterial growth (# of viable plateaus, not increases) If the antimicrobial agent is removed, bacterial growth would recover (reversible)

Bacteriocidal

Substances that actively kill bacteria by disrupting cell walls, membranes, or essential enzymes, If the antimicrobial agent is removed, bacteria cannot recover (irreversible), count continues declining

Bacteriolytic

Bacteriocidal but ALSO cells lyse completely = non-visible, unable to count

Antibiotics

Drugs that selectively inhibit bacterial growth or kill bacteria (and not the host). Growth inhibition can be enough for an antibiotic to be effective because the reduced population size is now enough for our immune system to combat.

Bacterial susceptibility vs. host sensitivity (e.g., toxicity, allergic reaction)

An antibiotic might be good at killing bacteria (high BS) but still be unusable if the patient has severe side effects (high HS). A good antibiotic ensures both high BS AND low HS!!

Minimal inhibitory concentration (MIC)

Lowest concentration of an antimicrobial drug (e.g., antifungal, antibiotic) that will inhibit the visible growth of a given microorganism after incubation in a growth medium.

• start at high concentration, then gradually increase dilution to lower concentration

• **does not tell use anything about bacterial killing just inhibition

• example to the right: MIC = 1.0 i.e. the tube with 1.0 μg/mL is the lowest concentration where you see no visible bacterial growth (clear tube) i.e. growth successfully inhibited

Kirby-Bauer disk susceptibility test

Known amounts of an antibiotic are added to filter-paper disks, then they diffuse through the agar to establish a concentration gradient where the lowest point of the gradient is where the bacteria is resistant enough.

• the larger the gray circles = the farther the antibiotic has diffused (greater radius) = the more lethal it is against the bacteria → clinical significance? indicates that this is the antibiotic to be using for patients

• performed under standardized conditions & in standard-sized zones of inhibition for each antibiotic

• results usually reported as sensitive, intermediate, or resistant, based on zone of inhibition’s size:

  • if observed zone of inhibition size >= standard zone size → microorganism deemed susceptible to antibiotic

if observed ZOI size < standard zone size → microorganism deemed resistant

The size of a zone of inhibition in a KB test is inversely related to the minimum inhibitory concentration (MIC), which is the amount of antibiotic required to prevent bacterial growth in an overnight culture. The MIC (in µg/ml) can be calculated from known standard-curve graphs based on the diameter of the observed inhibition zone diameter (in millimeters).

β-lactam antibiotics

Can inhibit penicillin-binding protein (PBP) activity:

• β-lactam antibiotics (penicillins (5-member ring), cephalosporins (6-member ring)) structurally mimic D-Ala-D-Ala — make a covalent bond with PBPs to block transpeptidation. Using the active site serine in PBP attacks the carbonyl carbon of the β-lactam ring → ring opens → forms a stable acyl-enzyme complex (covalent bond) → PBP permanently inactivated

  • (Enz = example of a PBP)

High-molecular-weight PBPs

Involved in peptidoglycan synthesis: transglycosylase (a) and transpeptidase (b) activities, OR just transpeptidase activity (b).

Low-molecular-weight PBPs

Involved in peptidoglycan processing: carboxypeptidases (e) or endopeptidases (d)

Site of antibiotic action

Many bactericidal antibiotics also generate reactive oxygen species (ROS), superoxide (O2•—), hydrogen peroxide (H2O2), & hydroxyl radical (OH•) by affecting metabolism 

• ROS damage DNA, proteins, & lipids

Rapid rise of antibiotic resistance & decline of antibiotic development

Daptomycin affects the function of bacterial cell membranes. Linezolid is a protein synthesis inhibitor

Antibiotic resistance mechanisms:

• prevent access to target

  • target site protection

• pump the antibiotic out of the cell

  • ex. multi-drug efflux pumps in gram– and gram+ bacteria

    • a single pump can often export diff kinds of antibiotics

    • ↑ expression of efflux pumps → multi-drug resistance

    • either chromosomally- or plasmid-encoded

• alter the target

  • ex. mutation

• inactivate the antibiotic (cleavage or modification)

  • ex. hydrolysis

    • β-lactamases

  • ex. steric hindrance