Chapter 2: Microscopy

How does a Bright-field microscope work?

Light passes through the specimen and then a series of magnifying lenses

What are the 6 main parts of the Bright-field microscope?

Ocular piece

Objective lens

Specimen stage

Condenser lens

Light source

Iris diaphragm lever

Rheostat

What does the ocular lens do?

Magnifies the image 10-fold (10x)

What does the condenser lens do?

Focuses the light

What does the Iris diaphragm lever do?

Controls the amount of light that enters the objective lens

What does the objective lens do?

Provides different magnifications based on the lens chosen.

What does the rheostat do?

Controls the brightness of the light.

Which two lens magnify the specimen?

Objective and ocular

Total magnification

The product of the objective and the ocular lens (10x)

Resolution definition

The minimum distance between two points at which those points can be observed as separate.

What is the maximum resolving power of the light microscope?

0\.2 um

What is immersion oil used for?

To displace air between lens and specimen when using high powered 100 x objective. It prevents the refraction of light and keeps rays from missing opening in objective lens.

What is contrast?

Determines how easily cells can be seen. Bacteria lack contrast but while using stains increase it, it kills the microbes.

Dark-Field microscope

Increases contrast by directing light towards the specimen at an angle.

Only light scattered by the specimen enters the objective lens and therefore those cells appear as bright objects against a dark background.

Phase-Contrast microscope

Increases contrast by amplifying the difference between refractive index of dense material and surrounding medium to make the cells and other dense material appear darker.

Differential Interference Contrast microscope

Depends on differences in refractive index

Separates light into two beams that pass through specimen and recombine

Light waves are out of phase when recombined causing a 3D appearance

What is epifluorescence?

UV light projected onto, not through, the specimen

Confocal microscopy

Detects fluorescence

Uses a laser beam and mirrors to illuminate and image fine slices of a specimen

It is like a CAT scan

Multiphoton microscopy

Detects fluorescence

Like confocal but less energy is used

Doesn’t damage the cells as much and allows for time-lapse images

Light penetrates deeper to give interior views of relatively thick structures

How is electron microscopy similar to light microscopy?

Uses electromagnetic lenses and electrons instead of glass and light rays

Fluorescent screen replace glass lenses, visible light, and eye

Image is captured on electron micrograph rather than film

Since it uses electrons rather than light the wavelength of electrons are \~1,000 times shorter than light meaning greater resolution and higher magnification

Electron microscopes

Magnify images 100,000x

Specimen must be in a vacuum

Large and expensive

Specimen preparation is complex

Transmission Electron Microscope (TEM)

Beam of electrons pass through or scatter (dark areas = dense material)

Thin sectioning used to view internal details

Freeze-fracturing, freeze-etching reveal shape of internal structures

Scanning Electron Microscopy (SEM)

Observes surface details

Surface is coated with thin film of metal

Beam of electrons is scanned over surface

Electrons released from specimen are observed to produce a 3D effect

Wet Mount

Uses a drop of unstained liquid specimen

Views microbial motility

Stained specimen procedure

1. Create a smear of the microbe
2. Dry the smear
3. Heat fix the cells
4. Stain the cells using different dyes depending on your goal

Types of Dyes

Basic dyes: Carry positive charge and are attracted to largely negative cellular components

Acidic dyes: Carry negative charge and are repelled by the negatively charged cell wall

Simple staining

Involves one dye

All cell components will look the same color

Shape and arrangements can be viewed

Differential Stains

Gram stain

Acid-fast staining

Gram Staining

Most common

Identifies two major groups of bacteria according to cell wall structure

Gram positive - purple, thick layer of peptidoglycan

Gram negative - pink, thin layer of peptidoglycan

Gram Stain Steps

1. Primary stain with crystal violet - all cells stain purple
2. Uses iodine as a mordant - cells remain purple
3. Decolorizes cells with alcohol - only gram-negative cells become colorless
4. Counterstain with safranin - gram-positive stay purple an gram-negative cells turn pink

Acid-fast staining

Primary stain uses carbol fuschin (red)

Counterstain uses methylene blue

Acid-fast microbes are red/pink

What does Acid-fast staining test for?

Mycobacterium - Causes tuberculosis and Hansen’s disease (Leprosy)

Cell wall contains high levels of mycolic acid, a waxy fatty acid that prevents uptake of dyes

Can presumptively identify agents in clinical specimens

Capsule stain

Used on microbes surrounded by gel-like layer

a negative stain is used by adding India ink to the wet mount

Endospore stain

Detects members of genera including Bacillus and Clostridium because they form endospore

Resist gram stain and appear clear

Uses heat to facilitate uptake of primary dye malachite green by endospore

Counterstain, usually safranin, is used to visualize other cells

Flagella stain

Flagella are too thin to be seen with a light microscope

Stain coats flagella to thicken and make visible

Presence and distribution can help in identification

Fluorescent Dyes and Tags

Some dyes bind to structures in all cells

Some are changed by cellular processes: distinguish between living and dead cells

Immunofluorescence

Uses fluorescent dye-antibody labels to tag unique microbe protein

Common prokaryotic cell shapes

Coccus - spherical

Bacillus - rod, cylindrical

Fluorescence microscopes

Cells/ materials either naturally fluorescent or tagged with fluorescent dyes

Molecules absorb light at one wavelength and emit light at longer wavelengths

Most are epifluorescent

Other variety of prokaryotic shapes

Vibrio - bent rod

Spirillum - loose spiral

Spirochete - tighter spiral

Pleomorphic - no fixed shape

Prokaryote Groupings

Most prokaryotes divide by binary fission the stick together forming characteristic groupings

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Ex.

Diplococcus - two spherical cells

long chains

cubical packets

grapelike clusters