Optical Microscopy Fundamentals

Optical Microscopy

• Utilizes bright light to illuminate specimens, also known as bright field optical microscopy.
• Magnification power indicates the degree to which the specimen's image is enlarged, with common magnifications including 4X, 10X, 40X, and 100X.
• Numerical aperture is a function of sin θ, where 2θ is the angle at which the objective lens views the specimen.
• Focal length is the distance between the lens and the focal point, and equals the length of the microscope tube divided by magnification.
• Working distance is the space between the objective lens and the specimen.
• Parfocal describes lenses that remain in focus when the focal length is changed.
• The ocular (eye piece) is a simple lens system through which the specimen image is viewed.
• Additional prismatic lenses in modern microscopes alter the optical path for improved eyepiece comfort.
• Total magnification power is the overall magnifying capability of a microscope.
• Total magnification is determined with the formula: (objective) * (ocular 10X) * (prismatic lenses 1X).
• Resolution power is the minimum distance at which two points can be distinguished, and is calculated as R = 0.61λ / NA, where λ is the median wavelength.

Types of Optical Microscopes

• Bright field microscopy: The specimen is positioned between the light source and the illuminated objective lens.
• Dark field microscopy: The specimen is illuminated at a low angle, causing it to appear as bright spots against a dark background since transmitted light is not gathered by the objective.
• Phase-contrast microscopy: Employs polarized light to illuminate the specimen through an objective lens that excludes the same polarized light to yield enhanced resolution of unstained samples.
• Interference microscopy: A method akin to phase-contrast, leverages variations in refractive indices within the structure for imaging.
• Fluorescence microscopy: The specimen, once stained with a fluorescent compound, is illuminated at a specific wavelength, typically UV.
• Confocal Scanning Microscopy: Employs a laser to scan specimens at a single focal depth and is the latest technique.

Other Microscopy Techniques

• Electron Microscope: Uses an electron beam to illuminate the specimen.
  • Transmission electron microscopy (TEM): Electron beams pass through ultra-thin specimen sections, creating a shadow image.
  • Viruses begin multiplying by inducing host cells to form more viruses.
  • Scanning electron microscopy (SEM): Scans the specimen's surface with an electron beam to generate a 3D image.
  • Tunnelling electron microscopy: Electrons penetrate the substance's surface through tunnelling.
• Acoustic microscopy: Employs very high frequency sound (3000 MHz) with a low wavelength.

Drawing Magnification

• Drawing magnification uses the formula: 𝑎𝑐𝑡𝑢𝑎𝑙 𝑠𝑖𝑧𝑒 𝑜𝑓 𝑑𝑟𝑎𝑤𝑖𝑛𝑔 / 𝑎𝑐𝑡𝑢𝑎𝑙 𝑠𝑖𝑧𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑝𝑒𝑐𝑖𝑚𝑒𝑛.

Staining Techniques

• Physical Mechanism: Dye gets trapped in a specific structure, forming a large complex that will not wash out.
• Charge attraction: Charged dye molecules adhere to molecules of the opposite charge.
• Chemical Reactions: Dye reacts with a specific molecule inside the structure.
• Enzymatic reaction (histochemistry): Dye is a substrate for a particular enzyme within cells, converting it into a colored molecule.
• Steric Affinity: Dye binds tightly to cell protein due to shape and chemical makeup.
• Antibodies (Immunostaining): Antibodies that target specific cellular molecules are tagged with a visible marker.
• Negative staining: A technique where the background is stained, making the structure of interest appear transparent.

Basic Staining Procedures

• FIXATION: Cells are attached to the microscope slide by heating or treating with chemicals.
• STAINING: The slide is covered with dye and incubated for penetration.
• WASHING: Excess dye is removed using a proper solvent.
• Optional steps:
  • MORDANT: A chemical enhances dye binding to a specific target.
  • DEVELOPER: Chemical modifies the dye bound to its target.
  • COUNTER STAINING: A second stain provides contrast against the first dye.

Simple Staining Examples

• Loeffler's alkaline methylene blue uses methylene blue and water.
• Diehl's carbon fusion uses basic fuchsin, ethyl alcohol, phenol, and water.

Differential Staining

• Gram Staining Method: Uses crystal violet followed by iodine mordant; gram-negative bacteria are counterstained with safranin.
  • Gram-positive bacteria appear violet.
  • Gram-negative bacteria appear pink.
• Gram Staining Procedure:
  • Drop water on slide.
  • Mix colony into water on plate.
  • Fix smear by sliding through a flame.
  • Flood slide with crystal violet for 1 min (rinse).
  • Add iodine for 1 min (rinse).
  • Wash with ethanol for 5-10 seconds.
  • Stain with safranin for 1 min (rinse).
  • Blot slide dry.
• Acid-fast staining (Ziehl-Neelsen Method): Uses Ziehl carbol fuchsin (basic fuchsin, ethyl alcohol, phenol, and water).
  • Fix smear by heating slide.
  • Place on heating rack, flood with Ziehl carbol fuchsin (heat 5 min) then rinse.
  • Wash with 20% sulfuric acid, blot dry.
• Endospore staining (Conklin’s method):
  • Requires heating and saturation with malachite green.
  • Endospores appear bright green.
• Flagella staining (Leifson’s stain method):
  • Increases flagella diameter by adding coating.
• Capsule staining method: Capsules are unstained and appear with a dark background as a negative stain.

Bacterial Motility

• Flagella are protein structures made of flagellin subunits arranged in a left-handed helix, approximately 2-15µm.
• Movement types:
  • Linear Run: Body rotates counterclockwise, propelling forward.
  • Random walk/tumbling: Body rotates clockwise, causing flagella to become undone.
  • Biased Random walk: Combination of random tumbles/runs, lasting longer towards a nutrient gradient.
  • Swarming: Seen on agar where colonies swarm towards nutrients.
  • Gliding: Occurs on solid surfaces in bacteria without flagella.

Metabolic Tests

• Blood agar plate: Contains hemoglobin. Those colonies which secrete haemolysins rupture red blood cells to access iron and amino acids.
• MacConkey Agar: Differentiates Enterobacteriaceae from gram-negative bacteria, usually inhibiting the growth of gram-positive bacteria.
• TSI (triple-sugar-iron) Test: Performed on a slant-butt culture containing glucose, lactose, and sucrose to detect metabolic activity.
  • Detects both acidification of the medium via color change from red to yellow, and H2S production, detected by blackening of the medium.
  • Sugar fermentation results can show acid butt and alkaline slant that indicate glucose fermentation without sucrose/lactose use.
  • The blackened bottom detects H2S production.
  • Bubble presence near the butt signals gas production.

Diagnostic Tests

• Oxidation/Fermentation Dextrose Basal Medium: Two inoculated tubes gauge fermentative or oxidative properties where one has an oil overlay.
  • Positive fermentation shows acidification, with both tubes turning yellow.
  • Positive oxidation is shown where only the aerobic tube turns yellow.
  • Gas production induces bubbles.
• Voges-Proskauer test - methyl red test: Measures acetoin production through methyl red reaction. Positive indicates red, negative is yellow.
• Citrate utilization: determines if citrate is a viable carbon source, with positive tests showing blue discoloration.
• Starch Hydrolysis: Tests if bacteria uses amylase to hydrolyze starch for fermentation with results in Scratch that will be (blue/dark purple).
• TTC Motility Test: Contains a dye indicating bacteria movement and results are motile (red), non-motile (colorless).
• Catalase Test: Tests for catalase enzyme by adding hydrogen peroxide.
  • Bubbles indicate the production of catalase.
  • Oxidase Test results are negative (no color reaction) or Positive (red).
• Urea Decomposition: Detects urease with results in Pink indicates urease.
• Liquefaction of Gelatin: Tests for gelatinase where If liquid indicates gelatinase product, otherwise gelatinase is not produced.
• Degradation of Casein: Some bacteria hydrolyze casein for amino acid use.
  • A cleared surface shows hydrolyzed casein, otherwise results in ground glass appearance surface.
• Indole Production: Detects tryptophan production with results that are Aerobic (red) and Anaerobic (green).

Pathogens

• Acellular Pathogens do not produce spores or other plant penetrating structures with common examples in tobacco mosaic.
• Unicellular Pathogens can resists removal via pili, cell wall adhesin protein and biofilm capsules with examples in Streptococcus mutans.
• Protozoan Pathogens parasitic microbes, reproduce quickly, and evolve mechanisms to reinfect hosts which survive.
• Fungal Pathogens are aided in development via capsules, which makes them are resistant to phagocytic engulfment.
• Helminth Pathogens are parasitic parasitic to humans. includes -Helminths includes flatworms.