Microbial Observation: Microscopy & Culturing

• The chapter focuses on observing microbial cells, specifically with microscopes, and covers culturing/identifying microorganisms.

Key Microscope Characteristics

• Magnification is the ability to enlarge objects.
• Resolving power is the ability to show detail.

Magnification

• Most microscopes magnify through the interaction of visible light waves and a lens' curvature.
• Magnification refers to the extent of enlargement.

Parts of a Microscope

• Ocular (eyepiece)
• Body
• Interpupillary adjustment
• Nosepiece
• Arm
• Objective lens
• Mechanical stage
• Substage condenser
• Aperture diagram control
• Basewith light source
• Field diaphragm lever
• Light Intensity control
• Coarse adjustment knob
• Fine focus adjustment knob
• Stage adjustment knobs.

Magnification in Two Phases

• The objective lens creates a magnified real image.
• The real image is projected to the ocular lens, where it is magnified again to form the virtual image.

Total Magnification

• Total magnification is the product of the separate magnifying powers of the objective and ocular lenses.
• Calculated as objective power multiplied by ocular power.

Resolution

• Resolution is defined as the capacity to distinguish or separate two adjacent objects.
• Resolution depends on the wavelength of light used to form the image and the characteristics of the objectives.

Quantifying Resolution

• Resolving Power (RP) = Wavelength of light in nm / (2 x Numerical aperture of objective lens)
• Visible light wavelength ranges from 400 nm to 750 nm.
• Numerical aperture of lens ranges from 0.1 to 1.25.
• Better resolution comes from shorter wavelengths and larger numerical apertures.
• Oil immersion objectives have a resolution of 0.2 µm.

The purpose of oil

• Oil is used to fill the gap between the microscope objective lens and the microscope.
• It improves the light gathering ability of the microscope, allowing for sharper images and more accurate measurements.

Variations on the Optical Microscope

• Bright-field microscopy is widely used and shows specimens darker than the surrounding field; use for live and preserved stained specimens.
• Dark-field microscopy shows brightly illuminated specimens surrounded by a dark field; used for live and unstained specimens.
• Phase-contrast microscopy transforms subtle changes in light waves passing through the specimen into differences in light intensity; best for observing intracellular structures.

Fluorescence Microscope

• These microscopes are modified with an ultraviolet radiation source and filter.
• They use dyes which emit visible light when bombarded with shorter UV rays (fluorescence).
• Useful for diagnosing infections.

Scanning Confocal Microscope

• Uses a laser beam of light to scan the specimen.
• Integrates images to allow focus on multiple depths or planes.

Electron Microscopy

• Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds.
• Electron waves are 100,000 times shorter than the waves of visible light.
• Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength.
• Magnification is between 5,000X and 1,000,000X.

2 Types of Electron Microscopes

• Transmission electron microscopes (TEM) transmit electrons through the specimen.
• Darker areas represent thicker, denser parts, while lighter areas indicate more transparent, less dense parts. Scanning electron microscopes (SEM) provide detailed three-dimensional view.
• SEM bombards the surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.

Specimen Preparation for Optical Microscopes

• Wet mounts and hanging drop mounts allow examination of characteristics of live cells: size, motility, shape, and arrangement.
• Fixed mounts are made by drying and heating a film of specimen.
• The resulting smear is stained using dyes to permit visualization of cells or cell parts.

Staining

• Dyes are used to create contrast by imparting color.
• Basic dyes feature a cationic, positively charged chromophore.
• Positive staining is where surfaces of microbes are negatively charged and attract basic dyes.
• Acidic dyes are anionic, negatively charged chromophore.
• Negative staining is where microbe repels dye, and the dye stains the background.
• Simple stains use one dye to reveal shape, size, and arrangement (e.g., Methylene blue stain).
• Differential stains use a primary stain and a counterstain to distinguish cell types or parts (examples: Gram stain, acid-fast stain, and endospore stain).
• Structural stains reveal certain cell parts not revealed by conventional methods: capsule and flagellar stains.

The 6 I's of Culturing Microbes

• Inoculation: Introduction of a sample into a container of media to produce a culture of observable growth.
• Isolation: Separating one species from another.
• Incubation: Maintaining under conditions that allow growth.
• Inspection: Observing cultures for macroscopic and microscopic details.
• Information Gathering: Conducting additional tests for microbial function and characteristics.
• Identification: Attaching a name or identity to the microbe.

Isolation

• If an individual bacterial cell is separated from other cells and has space on a nutrient surface, it wills from a colony, or a mound of cells.
• A colony consists of one species.

Isolation Techniques

• Streak plate technique
• Pour plate technique
• Spread plate technique

Inspection

• If a single species is growing in the container, that is a pure culture.
• If there are multiple species than there is a mixed culture.
• Contaminants are unknown or unwanted microbes in the culture.

Ways to Identify a Microbe

• By cell and colony morphology or staining characteristics.
• DNA sequence
• Biochemical tests to determine an organism's chemical and metabolic characteristics
• Immunological tests

Media: Providing Nutrients in the Laboratory

• Media can be classified according to 3 properies:
• physical state (liquid, semisolid, and solid)
• chemical composition (synthetic or complex)
• functional type (general purpose, enriched, selective, differential, anaerobic, transport, assay, enumeration)

Media: Physical states

• Liquid media is called broth and does not solidify
• Semisolid media contains solidifying agent
• Solid media is firm surface for colony formation. Contains solidifying agent and can be liquefied and nonliquefiable

Agar

• The most commonly used solidifying agent.
• Solid at room temperature, liquefies at boiling (100°C), and does not re-solidify until it cools to 42°C.
• Agar provides a framework to hold moisture and nutrients.
• Not digestible for most microbes

Most Commonly Used Media

• Nutrient broth is a liquid medium containing beef extract and peptone.
• Nutrient agar is a solid media containing beef extract, peptone, and agar.

Chemical Content of Media

• Synthetic media contains pure organic and inorganic compounds in an exact chemical formula.
• Complex/nonsynthetic media contains at least one ingredient that is not chemically definable.
• General purpose media grows a broad range of microbes, usually nonsynthetic.
• Enriched media contains complex organic substances such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes

Selective and Differential Media

• Selective media inhibits the growth of some microbes, while encouraging the growth of desired microbes.
• Differential media allows the growth of several types of microbes and displays visible differences among those microbes.
• Some media can be both selective and differential.

Miscellaneous Media

• Reducing medium contains a substance that absorbs oxygen or slows penetration of oxygen into the medium and are used for growing anaerobic bateria.
• Carbohydrate fermentation medium contains sugars that can be fermented, converted to acids, and a pH indicator show this reaction.

Learn about observing microbial cells using microscopes, including key characteristics like magnification and resolving power. Explore microscope parts, magnification phases, and total magnification techniques. Also covers culturing and identifying microorganisms.