Microbiology UTA 2460 Chapter 2

Wavelength

length between 2 peaks on a wave

amplitude

the height of a wave's peak

frequency

rate of peaks in a given time

longer wavelengths = _____ frequency (lower/higher)

lower

reflection

wave bounces since it cannot pass through

absorbance

wave captured

transmission

wave passes through object

interference

the interaction of two or more waves that results in a single wave

constructive interference

The interference that occurs when waves combine to make a wave with a larger amplitude resulting in louder noise (e.g. megaphone)

destructive interference

The interference that occurs when waves combine to make a wave with a smaller amplitude (e.g. noise cancelling ear buds)

diffraction

Occurs when an object causes a wave to change direction and bend around it

retraction

change direction and/or speed

refractive index

extent which material slows transmission speed

If light enters a substance with a ____ refractive index (lower/higher) it will _____ (slow down/ speed up)

higher, slow down (and bends toward the normal line away from the boundary)

convex lens

refraction occurs on a curved boundary to meet focal point, used to magnify(microscope)

focal point

the image point when light entering the lens is parallel

focal length

the distance from the center of a lens to the focal point

concave lens

refracts light away from focal point (flash light)

higher frequency waves have

higher energy (photons move faster)

flourescence

refracting non-visible light into visible light

white light

the retina receives visible light of many different frequencies

lowest frequency of visible light (color)

red

highest frequency of visible light (color)

violet

dispersion

colors retracted in different directions

phosphorescence

light emitted following a delay after absorption

magnification

ability of a lens to enlarge an object

resolution

the ability to tell that two separate points or objects are separate

2 factors that affect resolution

1. wavelength 2.numerical aperature

shorter wavelength equals

higher resolution

longer wavelength equals

lower resolution

numerical aperture

the measure of a lens's ability of a lens to gather light (the higher the numerical aperature the better the resolution)

contrast

visible differences between the parts of a specimen

Antonie van Leeuwenhoek (1632-1723)

"father of microbiology" first to utilize simple microscope

Galileo Galilei

used compound microscope that passed light through 2 sets of lenses

Robert hooke

first to observe cells through a cork using a compound microscope

cell is latin for

small room

Who Invented the Microscope?

Unknown. Hans and Zacharias Janssen MAY have invented the simple and compound microscope.

microscope types

1.light microscope 2.electron microscope 3.scanning probe microscope

path of light

CondenserIlluminator (light source)-->Specimen-->Objective lens-->Ocular lens

Types of light microscopes (use light to view images)

Brightfield, Darkfield, Phase-contrast, Fluorescence, Confocal, Two-photon

brightfield microscope

uses 2 or more lenses to produce a dark image on a light background, most common, used at UTA

Monacular

single eyepiece

binocular

two eyepieces

ocular lens

typically magnifies 10x

objective lens

magnifies 4x-100x

total magnification

the product of ocular lens and objective lens magnification (both lenses work together)

stage

platform of the microscope

specimen

item being viewed

course focusing knob

used for large-scale movements with 4⨯ and 10⨯ objective lenses

fine focusing knob

used for small-scale movements, especially with 40⨯ or 100⨯ objective lenses.

illuminator

high intensity bulb below the stage

condenser lens

located below the specimen, focuses light from the light source onto the specimen

rheostat

dimmer switch, controls intensity of illuminator

when images are magnified, they become ____

dimmer

oil immersion lens

a special lens designed to be used with immersion oils.

darkfield microscope

brightfield with modified condensor(Light that is viewed through objective lens is reflected/refracted from specimen), increases contrast without staining, produces light image on a dark background, often creates high contrast

phase contrast microscope

uses refraction and interference to increase contrast w/o stain, good for viewing live specimen, oldest/simplest type of microscope

differential interference contrast "Nomarski" microscope

similar to the phase contrast, two beams of light are created in which the direction of wave movement (polarization) differs, this results in high-contrast images of living organisms with a three-dimensional appearance.

Fluorescent Microscope

Fluorochromes are used to absorb & refract into visible light, direct & indirect

indirect immunofluorescence

causes higher intensity fluorescence

Immunoflourescence

method of tagging antibodies with a luminating dye to detect antigen-antibody complexes

antibodies

Proteins that attach to specific pathogens to kill or inhibit them

Fluorescent microscope uses

used to identify pathogens, find particular species in environment, find location of molecules and structures in a cell, distinguish between living vs dead cells

Confocal microscope

uses a laser to scan multiple z-planes, useful in examining thick specimens, modified fluorescence to avoid "bleaching"

Two-Photon Microscope

Modified confocal scope with near infrared light that minimizes light scattering through tissue by scanning technique & long wavelength light, useful for examining living cells in thicker material

Give 3 examples of specimens examined by two-photon microscopes

brain slices, embryos, organs

electron microscopy

use electrons and have short wavelengths (No use of light), cannot be used on living material, can magnify up to 100,000x

What are the 2 types of electron microscopy?

Transmission electron microscopy (TEM) &

Scanning electron microscopy (SEM)

Transmission Electron Microscope (TEM)

A microscope that uses an electron beam to study the internal structure of thinly sectioned specimens, Forms image based on varying opacity of specimen

Scanning Electron Microscope (SEM)

A microscope that uses an electron beam to study the surface of a cell or other specimen. Electrons are bounced off specimen w/ coating

Scanning Probe Microscopy (can magnify up to 100,000,000x)

Atomic scale microscopy (Viruses and smaller), 2 types: STM & AFM

Scanning Tunneling Microscope (STM)

uses a probe passed horizontally above a specimen to map the structure of surfaces at an atomic level

Atomic Force Microscopy (AFM)

establishes constant current and measures variations in height of probe tip

staining specimens (for light microscopy)

wet mount- good for viewing live specimens, simplest type, used at uta

fixed mount (smear)- good for staining, fixation achieved by either heating or chemically treating

basic dye

the chromophore is a cation (+)

acidic dye

the chromophore is an anion (-)

basic stain

positively charged ions (dye absorbs into cells)

acidic stain

negatively charged ions (dye absorbed into background)

simple stains

single stain, emphasize structures

differential stains

2+ stains, differentiates organisms

gram stain

created by Hans Christian (1884) to distinguish different cell wall components

steps for gram staining

1. Heat fix smear

2. primary stain crystal voilet (stains cells purple or blue)

3. mordant (iodine)

4. decolorizer (alcohol) Gram (+) will appear purple/blue

Gram (-) will appear colorless

5. counter stain (safranin)

Gram (+) will appear purple/blue

Gram (-) will appear pink/red

acid fast stain

Diagnostic tool for detection of mycolic acid and Mycobacterium,

2 types: Ziehl-Neelson method (w/ heat) & Kinyoun method (w/o heat)

steps of acid fast stain

1.Heat fix smear

2.Primary Stain (carbolfuschin)

3.Decolorizer

4.Counter Stain (methylene blue)

capsule stain

Diagnostic tool for detection of cells with capsules, dyes do not penetrate capsule

steps for capsule stain

1. no heat smear

2. primary stain (india ink)

(+) and (-) staining used to visualize capsules (halos)

endospore stain

identification of endospore formers using the Schaeffer Fulton Method

Steps of Schaeffer Fulton Method

1.Heat fix smear

2.Primary stain (malachite green)

3.Decolorizer (water)

4.Counter stain (safranin)

endospores= green

vegatative cells= pink

flagella staining

used to reveal the presence, number, and arrangement of flagella on bacteria that have them

Steps of Flagella Staining

1.No heat smear

2.Primary stain (specialized)

3.Decolorizer (water)

4.Counter stain (carbol fuschin)

Prepping specimens for electron microscopy

TEM and SEM

TEM

Samples analyzed must be have very thin sections (cut by tool called ultramicrotome), Specimen embedded in plastic resin and dehydrated, Stained with electron dense heavy metals

SEM

More dehydrated- critical point drying with liquid CO2

Sputter- coated with metal