Microbiology W1-2

Light Microscopy and the Discovery of Microorganisms

• Robert Hooke, in 1665, was the first to describe microbes using a microscope.
• Hooke's book, Micrographia, illustrated the fruiting structures of molds.
• Antoni van Leeuwenhoek was the first to describe bacteria using a microscope.

Improving Contrast in Light Microscopy

• Magnification is the ability to make an object appear larger.
• Resolution is the ability to distinguish two adjacent objects as distinct and separate.
• The limit of resolution for a light microscope is approximately 0.2 micrometers, which is half the wavelength of visible light.
• Several types of light microscopy exist:
  • Bright-field
  • Phase-contrast
  • Differential interference contrast
  • Dark-field
  • Fluorescence
• Compound microscopes use two sets of lenses to form an image: an objective lens (magnifies 10-100x) and an ocular lens (magnifies 10-30x).
• The total magnification of a compound microscope is the product of the objective lens magnification and the ocular lens magnification.
• A magnification of 1,000x is needed to reach the 0.2-micrometer resolution limit of most light microscopes.
• Bright-field microscopy uses normal light and visualization depends on differences in contrast or density between the specimen and its surroundings.

Microscopy & Staining

• Staining can improve contrast by using dyes that bind to specific cellular materials.
• Basic dyes, like methylene blue, crystal violet, and safranin, are positively charged and bind to negatively charged cell components, such as nucleic acids, acidic polysaccharides, and cell surfaces.
• Simple staining uses dried cells to highlight the overall shape, size, and arrangement of cells.
• Differential stains differentiate between cell types by staining them different colors.
• Gram staining is a differential stain widely used for identifying bacteria, which differentiates them into two main groups: gram-positive and gram-negative.
  • Gram-positive bacteria stain purple-violet due to a thick peptidoglycan layer in their cell walls.
  • Gram-negative bacteria stain pink because they have a thinner peptidoglycan layer and an outer membrane.

Phase-Contrast Microscopy

• Phase-contrast microscopy enhances image contrast of unstained, live cells.
• A phase ring amplifies differences in refractive index between the cell and its surroundings.
• An image with dark cells on a light background is produced.

Dark-Field Microscopy

• Light reaches the specimen from the sides.
• Only scattered light reaches the lens, resulting in an image that appears light on a dark background.
• Better resolution than bright-field microscopy.
• Excellent for observing motility, such as flagella.

Fluorescence Microscopy

• Used to visualize specimens that fluoresce, meaning they emit light after being illuminated at a different wavelength.
• Cells appear to glow on a black background due to filters.
• Some cells fluoresce naturally (autofluorescence), while others can be stained with fluorescent dyes like DAPI.
• Widely used in microbial ecology to enumerate bacteria in natural samples.

Differential Interference Contrast (DIC) Microscopy

• Uses a polarizer to generate two polarized light beams
• Provides a three-dimensional appearance to structures like nuclei, endospores, vacuoles, and inclusions

Confocal Scanning Laser Microscopy (CSLM)

• Uses a computerized microscope with a laser source to create a 3D image
• Computer focuses the laser on individual layers of the specimen
• Different layers can be compiled for a complete 3D image

Electron Microscopy

• Uses electrons instead of visible light (photons)
• Electromagnets act as lenses
• Operates in a vacuum
• Images are captured using a camera, resulting in an electron micrograph
• Two primary types: Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM)

Transmission Electron Microscopy (TEM)

• Offers significantly higher resolving power (0.2 nm) than light microscopes (200 nm)
• Enables visualization of structures at the molecular level
• Requires thin specimens (20–60 nm) stained with high atomic weight substances for contrast

Scanning Electron Microscopy (SEM)

• Provides detailed, three-dimensional images of surfaces

Cryo-Electron Microscopy (Cryo-EM)

• Used to dissect protein structure
• Involves rapidly freezing a specimen in liquid ethane
• Cryo-samples are then visualized at -170 °C
• 3D structures are determined by analyzing thousands of individual protein particle images
• Applicable for very large protein complexes

Enriched Media

• Contain complex media plus highly nutritious materials, for example serum or blood
• Used to culture fastidious microbes, which are nutritionally demanding

Selective Media

• Contain compounds selectively inhibiting the growth of some microbes but not others

Differential Media

• Contain an indicator, typically a dye, that detects particular metabolic reactions during growth

Considerations for Growth Media

• For successful microbial cultivation, it is crucial to know the nutritional requirements and supply them in the proper form and proportions in a culture medium.
• Cells can be cultivated in liquid or solid culture media.
• Solid media are prepared by adding the gelling agent agar to liquid media.
• When grown on solid media, cells form isolated masses called colonies.

Properties of Bacteria Utilized for Differential Media

• Example: Lactose utilization and anaerobic fermentation in E. coli
• Involves the conversion of lactose to glucose by β-galactosidase
• Under anaerobic fermentative conditions, a mixture of succinate, formate, acetate, lactate, and ethanol is produced
• The products, including e.g., acetate and lactate, are acidic

MacConkey Agar

• A selective and differential medium
• Selective: Bile salts inhibit most Gram-positive bacteria, and crystal violet inhibits certain Gram-positive bacteria.
• Differential: Detects lactose fermentation
• Lactose-fermenting bacteria: Consumes lactose or other six-carbon sugars and metabolizes them.
• Acid production: Reduces the pH, turning the neutral red dye red.
• Composition: Peptone, proteose peptone, lactose, bile salts, sodium chloride, neutral red, crystal violet, agar.

CHROMagar™

• Enzyme-based chromogenic detection system
• Invented by Dr. Alain Rambach in 1979
• Pioneered chromogenic differentiation culture media for detecting E.coli
• Patented a chromogenic system for detecting Salmonella in 1989, known as Rambach Agar
• Established CHROMagar Microbiology in 1993

Typical Appearance of Microorganisms on CHROMagar™

• VRE (Vancomycin-Resistant Enterococci) faecalis/faecium: Pink to mauve
• E. gallinarum/E. casseliflavus: Blue or inhibited
• Other Bacteria: Inhibited

CHROMagar™ E. coli and CHROMagar™ VRE

• CHROMagar™ E. coli: β-galactosidase-based
• Most chromogenic mixes are proprietary

Growth Media and Laboratory Culture

• Microbes are ubiquitous.
• Sterilization of media is crucial.
• Aseptic technique should be followed to prevent contamination.

Viable Plate Counts

• Measures living, reproducing microbial populations
• Two main methods:
  • Spread-plate method
  • Pour-plate method
• Serial dilution is key for accurate counts, which are typically between 30 and 300 colonies

Sources of Error

• Inoculum size, viability, culture medium, and incubation conditions affect accuracy
• Mixed cultures grow at different rates, leading to inaccuracies
• Colony-forming units (CFUs) are reported instead of viable cell numbers to address clumping

Applications of Plate Counts

• Quick and easy
• Used in food, dairy, medical, and aquatic microbiology, as well as water analyses
• High sensitivity
• Can target specific species in mixed samples

The Great Plate Count Anomaly

• Direct microscopic counts often reveal far more organisms than plate counts
• Dead cells are counted microscopically, but not by viable methods
• Different organisms have vastly different growth requirements, biasing culture methods

Turbidimetric Measures of Microbial Cell Numbers

• Cell suspensions are turbid (cloudy) due to cells scattering light.
• Turbidity is measured with a spectrophotometer, and the measurement is called optical density (OD)
• OD is proportional to cell number for unicellular organisms within a certain range.
• A standard curve must be established to relate OD to cell count.

Bacterial Growth Phases

• E. coli can grow quickly, doubling every 30 minutes under optimal conditions.
• Growth typically goes through three phases:
  • Lag phase
  • Exponential phase
  • Stationary phase

E. Coli Culture and Turbidity

• E. coli is typically cultured at 37°C with shaking for aeration.
• Absorbance at 600 nm is commonly used to measure bacterial density, particularly for E. coli.
• Absorbance is correlated to bacterial cell concentration.

Mechanism of a Spectrophotometer

• A beam of light is passed through the sample, and the intensity of the transmitted light (I) is measured, compared to the initial light intensity (Io).
• Transmittance (T) = I/Io
• Optical density (OD) or Absorbance (A) = -log10T

How to Calculate Concentration

• The Beer-Lambert Law relates absorbance (A) to concentration, path length (ℓ) and absorptivity (ɛ):
• A = ɛℓC*
• Absorbance is linearly correlated with concentration within a certain range, typically absorbance < 1.0

Advantages of Turbidimetric Methods

• Quick and easy to perform
• Does not require sample destruction or significant disturbance
• Allows repeated measurements on the same sample

Disadvantages of Turbidimetric Methods

• Can be problematic for microbes that form clumps or biofilms in liquid medium.

Explore the fundamentals of light microscopy and its role in the discovery of microorganisms, illustrated famously by Robert Hooke and Antoni van Leeuwenhoek. This quiz covers different types of microscopy, magnification, and resolution. Test your knowledge on the essential concepts that paved the way for modern microbiology.