Microbio Lecture 3

Two Types of Microscopes

Light and Electrons

Light - bright field, Dark-field, phase-contrast, fluorescence

Electron - SEM, TEM

Human Retina vs Microscope

150 um

0.2 um

Resolution

Is the smallest distance by which two objects can be separated and still be distinguished

The resolution of the human retina is about 150 um

Detection

Ability to determine the presence of an object

Individual cells are resolved through magnification

Certain shapes of bacteria

Prokaryote cell structures are genrally simpler than those of eukaryotes

Bacilli - rod

Cocci - spheres

Spiral forms

Spirochetes; flexible cell wall

Spirilla; rigid cell wall

Resolution depend on certain conditions

1. Contrast between object and its medium

2. Wavelength smaller than the object

3. Magnification

Visible wavelength 400-750 nm

Refraction

Is the bending of light as it enters a substance that slows its speed

Refraction magnifies an image

Resolution

Limits the ability of how well we can see with a microscope

Refractive Index

The measure of bending of a light ray when passing from one medium to another

n = c/v

c = the speed of light in vacuum

v = The phase velocity of light in the medium

What can u see in light microscopy vs electron

Size, shape, color, gram (±), arrangement

Internal structures (TEM), external strcutures (TEM/SEM)

Compound Microscope

Is a system of multiple lenses used to magnify small objects

• Ocular lens (10x)

• Objective lens (4,10,20,100)

Total Magnification / Parfocal

Magnification of ocular multiplied by that of the objective lens

Parfocal refers to objectives that can be changed with minimal or no refocusing

To increase resolution

Use shorter wavelength light - 400 UV is the safest

Increase contrast - add color

Gather as much light

• Use imersion oil

Wet Mount Advan/Disadvan

Advantages: Observation of cells in natural state

Disadvantages: Little contrast between cell and background sample may dry out quickly

Fixation/Staining

Cells are made to adhere to a slide in a fixed position using chemical or heat fixation

Cells are given a distinct color

• Most stains have conjugated double bonds or aromatic rings, and one or more positive charges

Simple stain

Adds dark color specifically to cells, but not to the external medium or surrounding tissue

For example, methylene blue stain

Differential stain

Stains one kind of cell one color and other cell different color or no color

For example. gram stain and Acid-fast stain

Numerical Aperature (NA)

Refractive index (n) multiplies by the sin of the angle of the light cone 0

NA = n sin 0

nm/sin of the angle (usually)

The ability of lens to gather light - higher NA allows greater resolution

Narrow cone of light gives you smaller sin and therefore lower numerical aperture

Dark-Field Microscopy

Microbes will be visualized as halos of bright light against darkness

Opposite to Bright-Field Microscopy

Light shines at oblique angle

Only light scattered by the object reaches objective - resolves very thin objects

Eg. Flagella, thin organisms like spirochetes

Phase Contrast Microscopy

Refracted and transmitted light shifted out of phase

• Reveals differences in refractive index

• Can be used to view live cells and cellular organelles

Fluorescence Microscopy

In fluorescence microscopy, the specimen absorbs shorter wavelength light, and then emits longer wavelength light

• Light is absorbed by a fluorophore, a fluorescent chemical

• Emits or fluoresce different color

Excitation and Emission

The specimen

• Absorbs light of a specific wavelength (excited)

• Emits light at a longer wavelength (the emission wavelength)

Excitation wavelength < Emission wavelength

Immunofluorescence

Fluorophore is combined with antibodies against a specific type of bacterium

Test for syphilis (VDRL), Treponema pallidum shows up as green cells against a dark background

Electron Microscopy

Electrons behave like light waves

• Very high frequency

• Allow very great resolution up to few nanometer

Sample must absorb electrons

• Coated with heavy metal

• Electron beam and sample are in vacuum

• Lenses are magnetic fields

Two Major types of Electron Microscopy (TEM)

Transmission Electron Microscopy (TEM)

• Electrons pass through the specimen

• Resolve objects as close together as 10 pm (picometer)

• Magnify up to 100,000x

Two Major Types of Electron Microscopy: SEM

Scanning electron microscopy (SEM)

• Electrons scan the specimens surface

• Resolve 10 nm and magnify up to 10,000x

The SEM is different from TEM

Electron beam scan across a specimen coated in gold

Second electrons will be liberated from the gold surface

Detector will capture the ejected electrons

• Generate 3D images