Lecture 2 - Chapter 4 - Microscopy, Staining And Classification

Front 1

Microscopy

Back 1

•View objects that are not visible to the naked eye

Types Of microscopes •Light

•Electron

•Probe

Front 2

Magnification

Back 2

•Increase In the size of a projected image

•Caused By refraction of light through lens

•Refraction Changes the focal point

-change in magnification

Front 3

Resolution

Back 3

•Distinguish Points of an object close to each other

•Distance is the minimum distance between distinguishable points

•Directly correlated to the wavelength of the electromagnetic radiation

Front 4

Contrast

Back 4

•Difference In luminance or color that allow objects to be distinguishable

Examples

•Staining •Changing Light phases - increase the differences in amplitude of light

Front 5

Light (Optical) Microscopy

Back 5

•Electromagnetic Radiation (light, UV) to create a magnified image of the object

•Brightfield microscope

•Darkfield microscope

•Phase Microscope

•Fluorescence Microscope

•Confocal Microscope

Front 6

Bright Field Microscopy

Back 6

•Contrast is formed when different areas of the specimen w/various density absorb different amounts of light

•Illumination from below the specimen

•Objective Lens projects an image between the objective & ocular lens

•Ocular Lens further magnify that image

Front 7

Bright Field Microscopy: Simple Microscopes?

Back 7

Single Line for increasing magnification & resolution

Front 8

Bright Field Microscopy: Compound Microscopes?

Back 8

Uses Multiple lens to increase magnification & resolution

Front 9

Dark Field Microscope

Back 9

•Suitable For live or unstained specimens

•Illumination From below the specimen

•Condenser Lens focus light onto the specimen at oblique angle

•light scattered by the specimen will be transmitted to the objective lens

•light that did not hit the specimen will miss the lens and not collected

Front 10

Phase Contrast Microscopy

Back 10

•Takes advantage of the wave properties of light

•Light Scattered by the specimen will have different amplitude & phase compare to light absorbed by the specimen

•Special Equipment required to detect these phase changes

Front 11

Fluorescence Microscopy

Back 11

•Substances absorb electromagnetic radiation outside of the visible spectrum & releases the energy back as visible light

•Utilizes this property to increase contrast

•Uses fluorescent dye or antibodies & UV light to make specimens fluoresce

Front 12

Confocal Microscope

Back 12

•Uses fluorescence to increase contrast

•Point Illumination of 1 plane of the specimen

•Pinhole Aperture limits light collected by the lens to those that are in focus with the plane being studied

Front 13

Electron Microscopy

Back 13

•Beam Of electron instead of electromagnetic radiation to illuminate the specimen

-TransmissionElectron Microscopes -ScanningElectron Microscopes

Front 14

Transmission Electron Microscope

Back 14

•Transmit high energy electron beam through the specimen

•Electrons From the beam are either scattered, refracted or transmitted through unaffected

•Specimens Must be very thin (100 nm)

Front 15

Scanning Electron Microscope

Back 15

•Scans The specimen w/a beam of high energy electrons

•Electrons from the beam hits specimen, lose energy through a variety of ways

•Loss of energy can be detected & used to create an image

Front 16

Probe Microscopy

Back 16

•Physical probe scan & generate an image

•A tip of several nanometer in size

Front 17

Staining

Back 17

•Allow microorganisms to become visible

•Increases Contrast

Front 18

Preparation For Staining

Back 18

•Smear And Fix

•Spreads the organisms onto a thin film

•Fixing Maintains structure of the organism & causes to attach to slide

Front 19

Simple Stains

Back 19

Uses 1 stain

Front 20

Differential Stains

Back 20

More than 1 stain are needed to stain the microorganisms

Front 21

Most common Differential stains?

Back 21

Gram stain & acid fast

Front 22

GramStain process?

Back 22

•Primary stain (e.g. Crystal Violet)

•Treated/mordant (e.g. Iodine) to bind the primary stain (makes the dye less soluble)

•Ethanol Or Acetone used to dissolve the thin cell wall of the gram negative cells

•Counterstain(e.g. Safranin)

Front 23

Acid-Fast Stain process?

Back 23

•Primary stain (carbolfuchsin) while heating the slide

•Cool & Treat w/HCl to decolourize the smear

•Counterstain with methylene blue

•Prevents the uptake of Gram stains

•Cover Smear with blotting paper

Front 24

Most common Acid-Fast Stain?

Back 24

Mycobacterium because of their waxy lipids in their cell wall

Front 25

Endospore Staining

Back 25

•Resistant To uptake of most stains

•Use Heat to facilitate the uptake of malachite green

•Counterstain/safranin

Front 26

Negative Stains

Back 26

•Does not stain the capsule well

•Stains the interior of the bacteria and the surrounding regions

•Leaves A clear halo around the bacteria

Front 27

Taxonomy

Back 27

•Domain - (Bacteria,Archaea Eukaryota)

•Kingdom

•Phylum

•Class

•Order

•Family

•Genus - proteins

•Species - Tiger, lion not same species

Front 28

What is the modern Taxonomic classifications?

Back 28

Based Mainly on comparing organisms at a molecular level