Light microscopy

uses visible light to see objects that are invisible to the naked eye. It can magnify objects by 1000x, and can easily observe cell size, shape, and motility. However, it can not see objects that are less than its wavelength

magnifies objects by 100,000x to reveal fine details in cell structure due to its smaller than light electron wavelength. Don’t bend through glass and travel poorly through air, so they are shot from a source through a vacuum focused by 2 electromagnetic lenses where the specimen sits between the them.

shot electrons pass through a thin section of the specimen, allowing detailed images of the internal structure. It provides high-resolution views of organelles and other internal components.

• Dark areas on image correspond to dense portions of specimen

• Thin-sectioning can distort cells

most common microscope that is easy to use; light passes through specimen, then a series of magnifying lenses evenly illuminate the entire field of view.

• Compound microscopes: have two types of magnifying lenses: ocular (10x) and objective (4x, 10x, 40x & 100x) -> condenser lens shown above only focuses light, not magnify it.
• Calculating total magnification: objective lens times ocular lens (ex: 10x times 100x = 1000x)
• Resolution: stops it from being able to see less than 0.2 micrometers. (Can see the shape of bacteria and can’t see viruses)
• Refraction oil

•  oil must sit between a slide and a lens because air causes refraction and prevents the light from reaching the objective lens.
• Won't this distort the image? No, we are able to do so because oil and glass have the same refraction index.

1. Commonly used stain for bacteria
2. Differential stain: can distinguish bacteria from each other
3. Steps
1. Sample is heat-fixed: This ensures the bacteria adhere to the slide and are killed for easier observation.
2. Crystal violet (primary stain or dye): The sample is flooded with crystal violet dye, which stains all cells purple.
3. Iodine (mordant): Iodine is added next, which binds with the crystal violet in the cell, forming a complex that makes the dye less soluble.
4. Ethanol wash (decolorization): Ethanol is used to rinse the sample. Here’s the key:
• In Gram-positive bacteria, the thick peptidoglycan layer traps the violet-iodine complex, retaining the purple color.
• In Gram-negative bacteria, the thinner peptidoglycan layer cannot retain the complex, so the violet color is washed away. Gram-negative bacteria have an outer membrane (outside the peptidoglycan layer) that’s disrupted by ethanol, creating gaps. When ethanol is applied, it weakens this thin structure further, allowing the dye complex to wash out.
5. Safranin (counterstain): The sample is then stained with safranin, which gives Gram-negative bacteria a pink color, while Gram-positive bacteria remain purple.

This differential staining allows you to distinguish between Gram-positive (purple) and Gram-negative (pink) bacteria based on the structure of their cell walls.

Capsule stain

1. staining for microbes that are surrounded by a gel-like layer that protects and increases pathogenicity. They stain poorly so a negative stain is often used
1. India ink added to wet mount around bacteria to show contrast.

used to stain organisms that are immune to the gram stain (crystal violet) due to cell wall that contains high concentration of mycolic acid (waxy fatty acid). Same steps are used with a different type of stain. Ex. mycobacteria

1. Acid fast bacteria are susceptible to this staining.

flagella used for prokaryotic motility are too thin to be seen with a light microscope. This stain coats the flagella to thicken it and make it visible.

1. uses fluorescent dyes or natural pigments that emit light when exposed to a specific wavelength (often UV light). The microscope detects this emitted light, allowing certain structures to glow and enhancing contrast.

    

   some bind to compounds (DNA), certain microbes (mycolic acid), and are changed by cellular processes (living or dead cells) respectively.
1. Immunofluorescence

1. Fixation: process of treating (and killing cells) that preserve cell structure using chemicals like formaldehyde that chemically link proteins together or heat.
• Heat fixing/smear: heating a sample on a slide so that bacteria stays in place, staining the sample (usually transparent), and washing it to remove excess for examination with a microscope.
• Dye used is typically positively charged because bacterial surface is negatively charged
• During negative staining, acidic dyes that have a negative charge are used, so it doesn’t stick to the dye (no heat smear).

2. Permeabilization: disrupts the cell membranes to allow entry of the stain into the cell (detergent or organic solvent). Not necessary if stain binds to outside of the cell.
3. Staining: immerse the sample into a dye solution.
4. Mounting: attaching the sample to a slide to examine growth or growing the sample directly on the slide.
5. Mordant: a chemical that combines with the primary stain to make it insoluble so it is less likely to wash away.

1. Technique used to tag specific proteins with a fluorescent compound (fluorophore) by using antibodies to deliver the tag. Tagging a protein unique to a microbe can detect that organism.
1. Requires microscopes that have lamps that can enter fluorophores at one wavelength of light and exit at another wavelength.

How we classify bacteria

1. Shape: does not tell you how related bacteria are to each other
2. Grouping: how cells are arrange after binary fission; still unreliable
3. Cell wall: help distinguish between gram-positive bacteria and gram-negative bacteria, two major types of bacteria

Common shapes

• Spherical: coccus
• Cylindrical: rod (bacillus)

Variety of other shapes: vibrio, spirillum, spirochete

• how cells are arrange after binary fission; still unreliable

• Diplococcus: divide into two cocci on the same plane

• Long chains: cocci is on a straight path side by side

• Cubical packets: 3D clump of cocci perpendicular to one another

• Grapelike clusters: cocci divide in several planes at random.

• Thin, delicate membrane that surrounds the cytoplasm & defines the boundary of the cell
• Critical permeability barrier between cell & external environment
• Structure: phospholipid bilayer embedded with proteins made of a hydrophilic head and hydrophobic tail
• Semipermeable: allow in salts and small polar compounds and block larger molecules from passing through unless they use a transport system (useful for organelles, DNA, proteins) or aquaporins for water passage.

By phagocytosis in eukaryotic bacteria

• Using transport systems (permeases or carrier), most molecules pass through proteins functioning as selective gates.
• They span the entire membrane and are highly specific (carriers transport certain molecule types)

• Facilitated diffusion: passive transport where molecules move down the concentration gradient (high to low) and don't require energy (rarely used in prokaryotes)
• Active transport: Movement against a concentration gradient that require energy like (ATP or proton motive force) by ABC transporters

  • can be im- or exporters of waste, signals to other cell, and large proteins

  • Protein secretion: the active transport of proteins out of the cell (ex: extracellular enzymes, external structures)
    • Proteins tagged for secretion by protein export pump have a signal sequence of amino acids.
    • Prokaryotes use a variety of secretion systems

• strong, rigid structure (keeps shape) that prevents cell lysis (breakdown of the cell caused by damage to the cell membrane)
• can help distinguish between gram-positive bacteria and gram-negative bacteria. Also made of peptidoglycan that can only be found in bacteria.
  • Makes these two bacteria respond differently to antibiotics because our immune system sees them differently.

• Gram negative bacteria in particular causes a lot of major health concerns such as salmonella, e-coli
• Gram positive also cause major health concerns

basic components of the Gram-positive and Gram-negative cell envelope -> refer to image

1. Gram-positive cell envelope: plasma membrane is surrounded by a thick layer of peptidoglycan that interacts with the external environment
2. Gram-negative cell envelope: the plasma membrane is covered by a layer of periplasm sandwiching a layer of thin peptidoglycan, and an outermembrane has contact with the external environment. 

Describe the outer membrane of the Gram-negative bacteria

- semipermeable

- relies on porins -> transport proteins that are open channels

- open channels: only allow simple/small molecules to go through, and thus do not use ATP (go down the concentration gradient)

- Also relies on active transport

membrane + cell wall + capsules

• Protects inside of bacteria from the outside
• What the immune system deals with
• Where pathogenic bacteria put proteins that infect their host
• Antibiotics attack some part of this envelope

1. Peptidoglycan: 3D net that protects bacteria from stress and gives it its shape (cell wall of bacteria)
1. Pepti -> contains peptides (chains of amino acids)
2. Glycan -> contains glycans (sugars or carbs)
3. A cell wall subunit is made of 2 glycans NAG and NAM (wall glycan) and 5 AA's (wall peptides)
   • NAG is found in many species including mammals
   • NAM is a derivative of NAG that has a modification allowing for chemical bonds w/ peptides (unique to bacteria)
   • Wall peptides are made of L- and D-form AA's w/ D-form poking differently

Polymerization of subunit via their sugars leads to the formation of long glycan strand with alternation NAM and NAG sugars

the side-cain AA group of an AA in one wall peptide is linked to the c-terminus of a neighboring wall peptide (after D-alanine is removed).

1. has as many as 30 layers of glycan chains and is permeable to sugars and AA's
1. Teichoic acids: surface polymers of ribitol- or glycerol-phosphate tethered to the plasma membrane (lipoteichoic acid) or attached to peptoglycan (wall teichoic acid). They can be attached with sugars & D-alanine.

1. has 1 or 2 layers of peptidoglycan and don't make teichoic acids. The outer membrane holds on to peptidoglycan using lipoprotein.
1. Outer membrane: lipid bilayer made of one lipopolysaccharide (LPS) layer facing the external environment, where lipo means lipid and polysaccharide means sugars special to bacteria; LPS replaces the phospholipids a regular membrane has with its own hydrophilic head, and the other layer facing the internal environment is made of phospholipids.

2. Medically important because it can cause symptoms of infections by live bacteria (endotoxin -> LPS is a toxin part of the bacteria)
3. Acts as a barrier to antibiotics.
4. LPS differs between different bacteria so it can be used for identification.

Periplasmic space in the gram-negative bacteria

sandwhiches the peptodiglycan layer(s) and sits between the cytoplasmic membrane and outer membrane

• filled with proteins because exported proteins accumulate unless specifically moved across outer membrane—porin protein channels are too small for proteins to pass through

• Lipid A: the part of LPS that remains in the cell's interor; made up of the hydrophilic head / hydrophobic tail

Exists on the cell's exterior

- Core oligosaccharide

- O-Antigen: change to avoid infection by bacteriophages stopping it from binding to the bacteria.

- Both of these vary by bacteria in same species, so it's used to identify variants.

1. Because the sugars and wall teichoic acids are unique to bacteria and are targets for antibodies and the human body's immune system.
1. penicillin interferes with peptidoglycan synthesis by preventing cross-linking of adjacent glycan chains (transpeptidation) and rupture due to osmotic stress. It's usually more effective against gram-positive bacteria than gram-negative (due to thickness of peptidoglycan net.
2. Lysozymes (enzyme found in tears, saliva, etc.) break bonds linking glycan chains. This destroys the structural integrity of peptidoglycan.

1. No, some bacteria lack a cell wall, so their immune to penicillin and lysozyme or enzymes that attack peptidoglycan. Their cytoplasmic membrane contains sterols that increase strength.
2. Ex: mycoplasm (varies in shapes)

1. Found in both gram-positive and -negative bacteria
   • slime layers: gel-like layers outside cell wall that protects or allows attachment/adhere to surface; can also avoid detection from immune system
   • Capsule: made of polysaccharides or AA polymers (D form); made under specific conditions that vary between bacteria
     • Once attached, cells can grow as biofilm: community of bacteria that lives on the surface in a gel matrix (ex: dental plaque)

1. rotate to provide motility (swim); made of flagellum protein that is embedded in the cell membrane(s) in the cell wall

1. Peritrichous: distributed over an entire surface
2. Polar flagellum: singe flagellum at one end of cell; some bacteria have tuft (multiple) at one or both ends

• Filament: composed of flagellin and has a hollow core
• Hook: connects filament surface
• Basal body: anchors to cell wall & cytoplasmic membrane that take energy from proton force to rotate flagella around

• Shorter, thinner, and functionally different than flagella
• Fimbriae: allow surface attachments (using receptor proteins)
• Twitching (extension of pili to grab objects) and gliding motility involve pili (don't rotate)
• Sex pilus used to join bacteria for DNA transfer (grabbing other bacteria)