Chapter 3 - Oberving Microorganisms through a Microscope

Vocabulary flashcards covering microscopy types, measurements, staining techniques, and key terms from the lecture notes.

Microscope

An instrument used to magnify small objects.

Resolution (resolving power)

The ability of lenses to distinguish two points; higher resolution means finer detail.

Total magnification

Product of the magnification of the objective lens and the ocular (eyepiece) lens.

Oil immersion objective

An objective designed to be used with immersion oil to reduce refraction and improve resolution.

Light microscope (LM)

A microscope that uses visible light to observe specimens.

Brightfield illumination

• The microscope shines light from below the specimen. (This makes the background look bright/white.)

• Dark objects are visible against the bright background (the specimen absorbs or blocks some of the light, so the specimen (or parts of it) looks darker than the background).

• Light from the specimen does not enter the objective (if a part of the specimen blocks the light, that light never reaches your eye - through the objective lens).

Darkfield microscopy

• Makes specimens glow against a dark background.

• An opaque disk blocks direct light, so only scattered/reflected light enters the lens.

Phase-contrast microscopy

• A modification of light microscopy allows examination of living organisms and internal cell structures

• Resembles a darkfield microscope, but internal structures are clearly seen.

Fluorescence microscopy

• It is a modification of compound light microscope

• Uses UV light and fluorescent dyes/antibodies to make specially stained parts of a cell glow in bright colors, so scientists can see them more clearly.

Confocal microscopy

A fluorescence technique that uses laser and a pinhole to produce sharp optical sections.

Transmission Electron Microscope (TEM)

Electron microscope that transmits electrons through a thin specimen; internal structures viewed; magnification 10,000–10,000,000x; ~10 pm resolution.

Scanning Electron Microscope (SEM)

Electron microscope that scans the surface of a specimen; 1,000–500,000x magnification; ~10 nm resolution.

Atomic Force Microscope (AFM)

Microscope that maps surface topography using a probe; ~0.1–10 nm resolution.

Bare light wavelength and resolution

Resolution increases as wavelength decreases (shorter wavelengths allow finer detail).

Ocular lens (eyepiece)

• The lens you look through.

• Enlarges the image formed by the objective lens.

Objective lenses

Primary lenses that magnify the specimen before the ocular lens.

Condenser

Focusing lens system that concentrates light through the specimen.

Diaphragm

Controls the amount of light entering the condenser.

Immersion oil

Oil used with oil immersion objective to minimize light refraction.

Refractive index

• A measure of how much a medium bends light; a higher index increases bending.

• Light may refract after passing through a specimen to an extent that it does not pass through the objective lens

Simple stain

Staining with a single basic dye to highlight the entire microorganism and visualize shape/size.

Chromophore

• The color-bearing portion of a dye.

• Cationic in basic dyes

• Anionic in acidic dyes.

Basic dye

A dye with a positively charged chromophore that colors negatively charged cells.

Acidic dye

A dye with a negatively charged chromophore; often used for negative staining.

Negative staining

Staining the background rather than the cells; often uses acidic dyes; this makes the cell look clear and bright while the background is dark.

Mordant

Substance (e.g., iodine) that helps fix the dye to the cell in differential staining.

Gram stain

Differential stain classifying bacteria as Gram-positive or Gram-negative.

Gram-positive

Bacteria with thick peptidoglycan walls that retain crystal violet-iodine complex.

Gram-negative

Bacteria with thin peptidoglycan layer and outer membrane; decolorized by alcohol and counterstained by safranin.

Decolorization (alcohol wash)

Step in Gram staining that decolorizes Gram-negative cells but not Gram-positive cells.

Counterstain (safranin)

• Second dye in Gram staining that colors decolorized cells (Gram-negative).

• Second dye in a differential stain (e.g., safranin in Gram staining).

Gram stain components

1. Crystal violet (primary),

2. Iodine (mordant)

3. Alcohol (decolorizer)

4. Safranin (counterstain).

Endospore stain

• Stain to visualize endospores.

• Primary stain malachite green.

• Spores appear green

• counterstain with safranin.

Capsule stain

• A capsule stain is a special stain that makes the protective outer layer (capsule) of some bacteria visible by staining the background and/or the cell but not the capsule itself.

Capsule

Gelatinous outer layer surrounding some bacteria; not stained by most dyes.

Endospore

Dormant, resistant structures produced by some bacteria; highly resistant to staining and decolorization.

Flagella stain

Stain technique to visualize flagella by thickening them with mordant and carbolfuchsin.

Mordant

Substance (e.g., iodine) used to fix the primary dye to the cell.

Primary stain

First dye in a differential stain (e.g., crystal violet in Gram staining).

Compound microscope

• An optical microscope that uses two or more lenses to magnify a specimen, allowing for improved resolution and detail.

• The image goes from the objective lens to the ocular lens

• Total magnification = objective lens x ocular lens

Oil immersion

A technique in microscopy that involves using a special oil with a refractive index that matches glass to increase the resolution of the image at high magnifications.

Nanometers (nm)

• A unit of measurement equal to one billionth of a meter.

• Commonly used to measure wavelengths of light and microscopic structures.

Micrometers (μm)

• A unit of measurement equal to one millionth of a meter.

• Used to measure the dimensions of cells and other microscopic objects.

Electron Microscopy

• Uses electrons instead of light to make an image.

• It can show tiny things like viruses that light microscopes cannot see.

Staining

• A technique used in microscopy to enhance contrast in specimens.

• Makes it easier to see cellular structures by applying dyes or stains.

Smear

A laboratory technique for preparing a specimen by spreading it thinly on a slide in order to allow for better visualization of cells under a microscope.

Bacterial smears

Are prepared by spreading a thin layer of bacterial culture onto a microscope slide, making it easier to observe the cellular morphology and arrangement of bacteria.

Inoculating Loop

A tool used in microbiology to transfer and inoculate microorganisms onto culture media. It is typically made of a thin metal or wire loop, allowing for precise handling of samples.

Simple Stains

Are basic staining techniques that involve the use of a single dye, allowing for the visualization of cell shapes, sizes, and arrangements under a microscope.

Acid-fast stain

A special staining technique used to identify bacteria (like Mycobacterium and Nocardia) that have waxy cell walls and do not easily take up normal stains.

Gram-positive

Gram-positive bacteria are bacteria that turn purple after Gram staining because they have a thick cell wall made of peptidoglycan.

Gram-negative

Gram-negative bacteria are bacteria that turn pink/red after Gram staining because they have a thin cell wall and an outer membrane.

Mordant

A chemical used in staining that helps the dye stick better to the cells or makes the stain more visible.

Capsules

• A capsule is a slimy, protective outer covering around some bacteria that helps them avoid drying out and escape the immune system.

• Are a gelatinous covering that is not stained by most dyes

Endospores

A resistant structure formed by some bacteria to survive extreme environmental conditions, endospores can withstand heat, desiccation, and chemical damage.

DIpicolinic acid

A chemical compound found in the core of bacterial endospores that stabilizes the proteins and DNA, contributing to their heat resistance and toughness.

Flagella

• Structures of locomotion

• Long, whip-like tails on some bacteria (and other cells) that help them move around.