BIOL 2460 Chapter 2

Wavelength

length between peaks

Amplitude

height of peaks

Frequency

rate of peaks in time

Reflection

wave bounces off material

Absorbance

wave is captured

Transmission

Wave travels through

Interference

the interaction between waves that meet

Diffraction

bending or scattering of a wave by object opening

Refraction

Change in direction and/or speed

Refractive index

degree of change in transmission speed= speed of light in vaccum/ speed of light through material

If light enters a substance with a ___________ refractive index it _____________ and bends __________ the normal line (_________from the boundary)

HIGHER , SLOWS DOWN, TOWARD , AWAY

Convex lens

A lens that is thicker in the middle than at the edges that bends light rays towards one another. Occurs on a curved boundary to meet a focal point (microscope, glasses, contacts)

Concave lens

a lens that is thicker at the edges than in the middle that bends light rays away from one another. refracts light away from a focal point. (flashlights)

Higher frequency waves have

higher energy (protons move faster)

Ultraviolet

Have the most energy

Infrared

Have the least energy

Magnification

ability of a lens to enlarge the image of an object

Contrast

creation of stark difference in coloration

Resolution

ability to tell that two separate points/objects are separate (clarity)

Shorter wavelengths-

Longer wavelengths

Higher resolution

Lower resolution

Numerical aperture

ability to gather light by lens

Antonie van Leeuwenhoek utilized

1st simple microscope

Galileo Galilei used a

Compound Microscope

Robert Hooke

first to observe "small chambers" in cork and call them cells.

Who invented the mircroscope?

unknown

Brightfield microscope

A microscope that uses visible light for illumination; the specimens are viewed against a white background.

Darkfield microscope

Specimens appear bright against a dark background; no staining is required, live specimens may be observed

Phase-contrast microscope

light microscope that uses refraction and interference to enhance contrast; useful in examining living, unstained cells (endospores and organelles)

Differential interference contrast microscope

This microscope, similar to the phase contrast, is used to observe minute surface irregularities but at a higher resolution. However, the use of polarized light limits the variety of observable specimen containers.

Fluorescence microscopy

uses a fluorescent dye that emits fluorescence when illuminated with ultraviolet radiation. Used to distinguish living from dead cells, and find pathogens and specific molecules

Confocal microscopy

a light microscope that uses fluorescent stains and laser to make two- and three-dimensional images, Useful for examining thick specimens such as biofilms

Two-Photon Microscopy

Cells are stained with fluorochrome dyes. Two photons of long-wavelength (red) light are used to excite the dyes. Good for viewing thicker material (e.g. brain slices, embryos, organs, etc.)

Transmission electron microscope (TEM)

a microscope that passes an electron beam through very thin sections (ultramicrotome). Can only view specimens that are cut into thin, dehydrated slices. Stained with electron-dense heavy metals

Scanning Electron Microscope (SEM)

A microscope that uses an electron beam to scan the surface of a sample, coated with metal atoms. Examines the surface of specimens on a 3-D detailed image. More dehydrated (critical point dying with liquid CO2), sputter-coated with metal.

Scanning Tunneling Microscope (STM)

microscope that measures electrons that leak or "tunnel" from the surface of the specimen. Shows a sample's surface atom by atom with ultra-high resolution, without the use of electron beams or light.

Atomic Force Microscopy (AFM)

uses a metal- and-diamond probe inserted into the specimen.

Produces three-dimensional images.

Wet mount (smear)

a glass slide holding a specimen suspended in a drop of liquid (such as water) for microscopic examination. Good for viewing live specimens

fixed mount (smear)

Good for staining since cells are stuck to the plate

Basic stain

(+) charge on the ion (cationic). Ex: Methylene Blue, crystal violet, Basic fuchsin, Malachite green. stains negatively charged molecules and structures.

Acidic stain

(-) Charge on the ion (anionic). Ex: India Ink, rose Bengal, Eosin, acid fuchsin. Stains negatively charged molecules and structures

Positive stain

Dye/stain is absorbed into cells

Negative stain

stains the background, not the cell

Simple stain

A method of staining microorganisms with a single basic dye emphasizes structures. Helps to highlight the cell

Differential stains

Differentiates organisms based on stain interactions, 2+ stains

Gram stain

A staining method that distinguishes between two different kinds of bacterial cell walls. Gram-positive= thick cell wall, Gram-negative= thin cell wall

Gram stain steps

1. Heat fix smear

2. Primary Stain (crystal violet)

3. Mordant (iodine)

4. Decolorizer (alcohol)

5. Counter Stain (safranin)

positive- purple to blue

negative- pink or red

Acid Fast Stain

A staining procedure for identifying bacteria that have a waxy cell wall. Used for detection of mycolic acid and mycobacterium spp. Ziehl-Neelsen method (w/ heat), Kinyoun method (w/o heat)

Acid Fast stain steps (Ziehl-Neelsen method)

1. Heat fix smear

2. Primary Stain (carbolfuschin)

3. Decolorizer

4. Counter Stain (methylene blue)

Acid-fast bacteria are-red

Non Acid fast bacteria are- blue

Endospore stain

A differential stain used to detect the presence and location of spores in bacterial cells.

Endospore stain steps (Schaeffer-Fulton method)

1. heat fix smear

2. primary stain (malachite green)

3. decolorizer (water)

4. counter stain (safranin)

endospore- green

other structures-pink

Flagella stain

The staining agent adheres to and coats the otherwise thin flagella, making them visible with the light microscope.

Flagella stain steps

1. No heat smear

2. Primary stain (specialized)

3. Decolorizer (water)

4. Counter stain (carbol fuschin)

Capsule stain

Diagnostic tool for detecting protective coatings. Dyes do not penetrate the capsule.

Capsule stain steps

1. no heat smear

2. primary stain (india ink)

Eye piece (ocular lens)

10x magnification

Objective lenses

4-100x

Total Magnification

ocular lens x objective lens

Direct immunofluorescence

fluorophore is conjugated directly to the antibody molecule that recognizes and binds the molecule of interest

indirect immunofluorescence

An immunofluorescent diagnostic technique in which the fluorochrome is not attached to the primary antibody that recognises the target antigen, but to a secondary antibody that binds the primary antibody. Causes a higher intensity fluorescence.