Chapter 3 Microscopes

Compound light microscope

uses a series of lenses and visible light

• Most common

• visible light

• 2000x max magnification

• Resolution of 0.2um

Resolution

ability to distinguish between two points

ability of lenses to distinguish fine detail and structure

Magnification

mag of objective lens x mag of ocular lens

When do you use oil?

Immersion oil- on highest magnification

Darkfield microscope

microbes that are invisible in normal light microscope or can’t be stained; darkfield condenser blocks light

• light specimen, dark background

Phase-contrast microscope

details of internal structures in microbes; light wave phases: peaks and valleys

able to see inside of cell better

Fluorescence microscopy

uses UV light, specimen may naturally fluoresce or is stained with fluorescent dyes

Confocal microscopy

constructs 3D images using a computer, scans slices of an image, computer puts together

Electron microscopy

objects smaller than 0.2 um (viruses/internal cell structure)

uses beam of electrons instead of light

Electromagnets instead of glass lenses

Transmission (TEM)- beam passes through specimen

• 10,000-100,000x

• Shows internal structures

• Requires complex prep, kills, microbe, causes some disortion

Scanning (SEM)- gives 3D views

• 1,000-10,000x

• Beam is directed to surface of specimen

Staining

Attaching dye to microbe

Microbe must be fixed- attached to slide

Kills and perserves microbe

Microbe attached to slide = smear

Fixing - keeps microbe from being washed off slide with stain

• Basic dye- color in positive ion

  • used in bacteria

• Acidic dye- color in negative ion

Simple stain

single basic dye

• applied to smear, then washed

Differential stain

distinguish between bacteria

• reacts differently with different bacteria

• Gram stain and acid-fast stain

Gram stain

Distinguishes bacteria into 2 groups

• gram positive and gram negative

  • 1. Smear covered with crystal violet (primary stain = color to all cells)

  • 2. Washed and covered with iodine, intensifies stain (mordant- improve stain binding)

  • 3. Washed with alcohol (removes purple from some bacteria but not others) (decolorizer)

  • 4. Washed and stained with safranin (red) (counterstain)

  • 5. Washed and visualized

Peptidoglycan

cell walls of bacteria

Gram positive

retain purple color, thick peptidoglycan layer

• crystal violet - iodine complex can’t be washed out

More easily killed by penicillins

Gram negative

lose purple and are dyed red, thin peptidoglycan layer and LPS

more resistant, can’t penetrate LPS

Acid-fast stain

binds to bacteria with waxy material in cells

• mycobacterium

1. Stained with red dye carbolfuchsin

2. washed and covered with alcohol (removes red from non acid-fast bacteria)

3. stained with methylene blue

Acid-fast bacteria-red (carbolfuchsin is soluble in lipids of cell wall)

non acid-fast bacteria-blue

Negative staining

• for microbes with capsules

• microbe is mixed with colored particles

• Particles can’t enter through capsule, giving contrast between microbe and colored background

Endospore staining

Malachite green stains endospores withing pink stained cells

Flagella staining

structures of cell movement

Carbolfuchsin