Biochemistry and Microscopy Review Notes

Biochemistry Review

Macromolecules

  • Macromolecules constitute the majority of organic compounds in a cell.
  • They are large, complex polymer chains made of smaller subunits called monomers.
  • Dehydration synthesis: monomers join to form polymers, releasing water and using energy.
  • Hydrolysis: polymers break down into monomer subunits, adding water and releasing energy.

Dehydration Reaction

  • Monomers link to form a short polymer.
  • Dehydration removes a water molecule, forming a new bond.
  • Results in a longer polymer.

Hydrolysis Reaction

  • Polymers break down into monomer subunits.
  • Hydrolysis adds a water molecule, breaking a bond.

Macromolecules: Carbohydrates

  • Monosaccharides: simple sugars, one sugar molecule.

  • Basic formula: \text{CH}2[O (e.g., \text{C}6\text{H}{12}\text{O}6).

  • Disaccharides: two sugar monomers (e.g., sucrose = glucose + fructose).

  • Polysaccharides: polymers of sugar monomers.

    • Starch: sugar storage in plants.
    • Glycogen: sugar storage in animals.
    • Dextran: a polysaccharide of glucose secreted by some bacteria; a major component of bacterial capsules, forming a protective sugary slime layer.

  • Saccharides and other carbohydrates in both prokaryotic and eukaryotic cells:

    • Directly embedded into a cell’s plasma membrane.
    • Conjugated to proteins (glycoproteins) or lipids (glycolipids).

  • Used to create energy through cellular metabolic processes.

Macromolecules: Lipids

  • Lipids are diverse and generally non-polar.
  • They do not dissolve in polar solvents like water but dissolve in non-polar solvents like chloroform and ether.
  • Triacylglycerol: simplest form of a lipid.
    • Glycerol backbone.
    • Three fatty acids attached (R group).
      • Vary in the number of carbon atoms in the chain and the number of bonds between the carbons (saturated vs. unsaturated).
  • Note: Lipids contain 2x the amount of energy per gram compared to polysaccharides.

Macromolecules: Lipid Structure

  • Ester linkage connects the glycerol backbone to the fatty acids.
  • Examples of fatty acids:
    • Palmitic acid (\text{C}{15}\text{H}{31}\text{COOH}) - Saturated + \text{H}_2\text{O}
    • Stearic acid (\text{C}{17}\text{H}{35}\text{COOH}) - Saturated + \text{H}_2\text{O}
    • Oleic acid (\text{C}{17}\text{H}{33}\text{COOH}) - Unsaturated + \text{H}_2\text{O}
  • Molecule of fat (triglyceride) is formed through these ester linkages.

Macromolecules: Lipid Formation

  • Glycerol interacts with fatty acids to form lipids.
  • Illustrative example: Palmitic acid (\text{C}{15}\text{H}{31}\text{COOH})
    • Carboxyl group and hydrocarbon chain.

Macromolecules: Complex Lipids

  • Complex lipids may contain phosphorus, nitrogen, and sulfur.
  • Phospholipids: make up biological membranes.
    • Amphipathic molecules: contain a polar (hydrophilic) and nonpolar (hydrophobic) portion.
  • Forms a micelle.

Macromolecules: Complex Lipids in Cell Membranes

  • In a cell membrane, phospholipids form two leaflets.
  • Each leaflet houses proteins, sugars, and other lipids and molecules essential for cell communication, motility, structure, and function.

Macromolecules: Proteins

  • Organic molecules that contain carbon, hydrogen, oxygen, nitrogen, and sometimes sulfur.
  • Proteins make up about 50% of a cell’s dry weight.
  • Examples: enzymes, transport proteins, bacteriocidins (kill bacteria), toxins, cell structure, etc.
  • Monomers of proteins are amino acids.
  • Basic structure: alpha carbon (\text{C}α) attached to a hydrogen, a side group (R group), a carboxyl group, and an amino group.
  • Example: tyrosine.

Amino Acids

  • Table 2.4 lists the 20 amino acids found in proteins.
  • Examples include:
    • Glycine (Gly)
    • Alanine (Ala)
    • Valine (Val)
    • Leucine (Leu)
    • Isoleucine (Ile)
    • Serine (Ser)
    • Threonine (Thr)
    • Cysteine (Cys)
    • Methionine (Met)
    • Glutamic acid (Glu)
    • Aspartic acid (Asp)
    • Lysine (Lys)
    • Asparagine (Asn)
    • Glutamine (Gln)
    • Arginine (Arg)
    • Phenylalanine (Phe)
    • Tyrosine (Tyr)
    • Histidine (His)
    • Tryptophan (Trp)
    • Proline (Pro)

Macromolecules: Protein Synthesis

  • Peptide bonds link amino acids together.
  • A water molecule (\text{H}_2\text{O}) is released during the formation of a peptide bond.
  • Amino end (N-terminus) and carboxyl end (C-terminus) define the protein backbone.

Macromolecules: Proteins Functions

  • Proteins serve many important functions in a cell.
  • Enzymes are a cell’s set of catalytic machinery, performing many essential reactions to assemble or break down large molecules.
  • Cell signaling receptors aid in cellular communication.
  • Membrane channels facilitate the transfer of nutrients and waste products across biological membranes.

Macromolecules: DNA

  • DNA is genetic material, double-stranded to form a double helix.
  • Nucleotides bind to form pairs in a specific manner:
    • Adenine binds to thymine (A-T).
    • Cytosine binds to guanine (C-G).

Macromolecules: RNA

  • RNA is single-stranded.
  • Note: some viruses can have double-stranded RNA.
  • RNA does not have thymine (T), but instead has uracil (U).
    • Adenine binds to uracil (A-U).
    • Guanine binds to cytosine (G-C).
  • There are many RNA types, including:
    • mRNA (messenger RNA).
    • tRNA (transfer RNA).
    • rRNA (ribosomal RNA) - very important for phylogenetics!
    • snRNA (small nuclear RNA).
    • siRNA (short interfering RNA).

Microscopy

  • Light bending: Refraction occurs when light moves from one medium to another.

    • Changes the velocity and direction of the light wave.
    • Predicted by a medium’s refractive index.
    • When light travels from air to glass, it bends towards the normal (a line perpendicular to the glass surface).

  • Lenses: refract light towards a single point for viewing.

    • F is the focal point.
    • f is the focal length.
    • Longer focal length = larger magnification.
    • Light microscopes have up to 4 lenses, each with different focal lengths.
    • Achieved by differing the curvature in the lenses.

Microscopy: Types of Light Microscopes

  • Types of microscopes with lenses:
    • Bright field.
    • Dark-field.
    • Phase-contrast.
    • Fluorescent.
  • These are all examples of compound microscopes (2 sets of lenses).
    • The first gathers light from the sample.
    • The second focuses light to eye or camera.
  • Samples are visualized due to differences in contrast (density) between themselves and the medium they’re in.
    • The denser the specimen, the more light is scattered, and the darker things appear.

Microscopy: Compound Microscopes

  • Components include: ocular (eyepiece), body, nosepiece, objective lens, mechanical stage, substage condenser, aperture diaphragm control, base with light source, field diaphragm lever, light intensity control, arm, coarse/fine focus adjustment knobs, and stage adjustment knobs.
  • Magnification levels: 100x, 400x, 1000x.
  • Light path: Visualized image -> Eye -> Eyepiece (ocular) lens (10x) -> Intermediate image -> Objective lens (10x, 40x, or 100x) -> Specimen -> Condenser lens -> Field diaphragm

Microscopy: Compound Microscopes Details

  • Objective lenses are the most important part of a microscope.
    • Magnifies the specimen (usually 3-4 different lenses at different magnifications).
    • Total magnification = ocular magnification x objective magnification.
  • If these become damaged, you will not get an image.
  • Clarity = resolution: the ability to distinguish two points that are close together.

Microscopy: Pigmented Cells

  • Many organisms do not create much contrast between themselves and the background, so a stain is required to make them more visible.
  • Cells that are already pigmented do not need to be stained since they are easily visualized under a microscope.

Microscopy: Types of Stains

  • Simple stains: Dye stains the cell using basic dyes (+).
  • Negative stains: Dye stains the background but not the cell using acidic dyes (-).
  • Structural stains: Used to stain specific cell structures, i.e., flagella, capsule, DNA (nucleoid region), or endospores.
  • Differential stains: Used to differentiate organisms into two different categories (e.g., Gram stain); they don’t stain all cells the same color.

Microscopy: Structural Stains Examples

  • India ink capsule stain of Cryptococcus neoformans.
  • Flagellar stain of Proteus vulgaris (a basic stain was used to build up the flagella).
  • Endospore stain, showing endospores (red) and vegetative cells (blue).

Microscopy: Gram Stain

  • A differential stain developed by Christian Gram in 1884.
  • Used to classify bacteria into two different categories: Gram-positive or Gram-negative.
  • Useful because Gram-positive and Gram-negative species respond differently to antibiotics.
    • Gram-positive: crystal violet complex is too large to be washed from the cell, and cells are purple (crystal violet).
    • Gram-negative: crystal violet complex can be washed from the cell, and cells are pink because of the counterstain (safranin).

Microscopy: Gram Stain Steps

  • Step 1: Crystal violet (primary stain) - Cells stain purple.
  • Step 2: Iodine (mordant) - Cells remain purple.
  • Step 3: Alcohol (decolorizer) - Gram-positive cells remain purple; Gram-negative cells become colorless.
  • Step 4: Safranin (counterstain) - Gram-positive cells remain purple; Gram-negative cells appear red.

Microscopy: Specimen Preparation for Staining

  • Step 1: Fixation
    • Adheres the cell to the slide and kills the sample.
      • Heat: A film of cells on a slide is passed through a flame. Preserves morphology but not cellular structures.
      • Chemical: Fixatives penetrate the cell, binding to cellular components and holding them in place. Used to protect the fine structures within a cell.

Microscopy: Specimen Preparation for Staining 2

  • Step 2: Staining
    • Uses a dye compound to enhance visibility of a specimen under a microscope.
    • Dyes have chromophore groups, which produce a color.
    • Bind to a specimen through ionic, covalent, or hydrophobic bonding.
      • Acidic dyes have a negative charge and are repelled by negatively charged cytoplasm.
      • Basic dyes have a positive charge and are attracted to and retained by a cell’s negatively charged cytoplasm.

Microscopy: Bright-Field

  • The sample is dark, usually fixed (heat-killed), and has been stained/dyed.
  • The background is light, and the sample is dark.
  • The most common type of microscopy used to look at bacteria.
  • Most are parfocal: once a sample is in focus at one magnification, it will remain in focus (mostly) at all higher magnifications.

Microscopy: Dark-Field

  • Living, unstained organisms (usually eukaryotic).
  • A dark field stop forces light towards the specimen from the sides only.
  • Only light reflected or refracted by the specimen enters the objective, making them appear light on a dark background.

Microscopy: Phase-Contrast

  • Living, unstained specimens.
  • It is very difficult to see unstained cells.
  • Phase plates change the amount of refraction of a specimen, which increases contrast between cells and the background.
  • Differentiation of specific bacterial structures.

Differences in Imaging…

  • Different microscopy methods can produce different images of the same cells:
    • bright-field
    • phase contrast
    • dark-field

Microscopy: Fluorescence

  • Some compounds emit light (fluoresce) continuously (e.g., photosynthetic compounds).
  • Others become excited by a secondary light source.
  • Epifluorescent microscopy.
  • Diagnostics and research uses (e.g., vitality stain).

Microscopy: Fluorescence - Fluorochromes

  • Commonly Used Fluorochromes:
    • Acridine orange: Stains DNA and fluoresces orange.
    • Diamidino-2-phenyl indole (DAPI): Stains DNA and fluoresces green.
    • Fluorescein isothiocyanate (FITC): Often attached to antibodies that bind specific cellular components or to DNA probes; fluoresces green.
    • Tetramethyl rhodamine isothiocyanate (TRITC or rhodamine): Often attached to antibodies that bind specific cellular components; fluoresces red.

3D Microscopy: DIC

  • Differential Interference Contrast Microscopy.
  • Living, unstained organisms.
  • Uses polarized light at right angles to make unstained organisms look stained, creating 3D imaging.
  • This method reveals organelle structures, including the nucleus, endospores, vacuoles, and granules, that are not visible with other forms of microscopy.

3D Microscopy: Atomic Force Microscopy

  • Atomic Force Microscopy (AFM).
  • A tiny stylus is placed in close proximity to a specimen, engaging in weak atomic forces with it.
  • The stylus scans the vertical and horizontal aspects of the specimen, feeding digital information to a computer, which creates an image.
  • No fixatives or coatings are required for this technique; specimens are living and hydrated.

3D Microscopy: Confocal Scanning Laser Microscopy

  • Confocal scanning laser microscopy (CSLM) is a computerized microscope that couples a laser to a computer.
  • Laser beams are bounced off of a mirror that forces the beam towards a detector, shone through a pinhole.
  • This imaging process shows one plane of focus, i.e., one slice through an organism.
  • An image is created through assembling all planes collected from this method.

The Limitations of Light Microscopy

  • A type of radiation cannot be used to probe molecules smaller than its own wavelength.
  • Visible light ranges from approximately 0.4 \mu m to 0.7 \mu m.
  • Bacteria and mitochondria (\approx 0.5 \mu m) are the smallest objects that can be seen clearly with a light microscope.

Microscopy: Light vs. Electrons

  • Electron microscopy (EM).
    • Uses a stream of electrons to view a specimen.
    • Much smaller wavelength than light.
    • Has a much higher resolution.
    • Can focus on much smaller objects.

Microscopy: Light vs. Electrons Advantages/Disadvantages

  • Advantages of electron microscopy:
    • Electrons have a shorter wavelength than visible light, and objects can be viewed at a higher resolution.
    • Maximum magnification is significantly higher than a light microscope.
  • Disadvantages of electron microscopy:
    • Specimens must be killed and fixed, so structures of a living specimen can never be observed.
    • Preparation of specimens may damage or distort their true structures.
  • There are two types of electron microscopes:
    • Scanning Electron Microscopes (SEM).
      • Detects electrons that reflect off of a specimen’s surface.
    • Transmission Electron Microscopes (TEM).
      • Detects electrons passing through a thin specimen.

Microscopy: SEM

  • Scanning electron microscopes use beams of electrons that scan over the surface of a specimen.
  • Creates a three-dimensional, black and white image that is magnified up to 30,000 times.

Microscopy: TEM

  • Transmission electron microscopes use electron beams that pass through an ultra-thin specimen to form an image.
  • Creates a two-dimensional, black and white image that is magnified up to 800,000 times.

Microscopy: SEM vs. TEM

  • SEM provides surface details, while TEM provides internal details.

Fold Microscopy

  • A research group wanted to bring accessibility for microscopic diagnosis of disease to third world countries and created something called a fold scope.
  • Many people in third world countries have no access to diagnostic tools, or the equipment available is extremely outdated or damaged.
  • All of the microscope components are present on a piece of paper that can be folded and used on site. The microscope only costs 50 cents to manufacture.