An Overview of MicroscopyBy BugLady

Unless otherwise stated, all micrographs and diagrams are the author’s own work.

Apr 9, 2023 1

Microscopy

Resolution and magnification

Different types of light microscopy: principle and use

Electron microscope: principle

Stains

Recommended activity:oMicroscope tutorial

Apr 9, 2023 2

Principles of Light Microscopy

Light passes through specimen and through a series of magnifying lenses

Important factors in light microscopy includeMagnificationResolutionContrast

Apr 9, 2023 3

Principles of Light Microscopy: Magnification

Compound Microscope : microscope has two magnifying lenses

Lenses include ocular lens and objective lens

Lenses combine to enlarge objects Magnification is equal to the product of the ocular lens x

the objective lens: 10x X 100x = 1,000x

Apr 9, 2023 4

Principles of Light Microscopy: Resolution

Resolving power is defined as the minimum distance existing between two points where they still appear as separate

Resolving power determines how much detail can be seen

Naked eye ≈ 0.1mm

Light microscope ≈ 0.2μm

Electron microscope ≈ 2.5nm

Apr 9, 2023 5Courtesy of the CDC

Electron micrograph of Bacillus anthracis: vegetative cell (A) and endospore (B)

Light micrograph of Bacillus anthracis

Principles of Light Microscopy: ResolutionResolutiono Resolution depends on the quality of lenses

and wavelength of illuminating light How much light is released from the lens

oMaximum resolving power of most brightfield microscopes is 0.2 μm (1x10-6) This is sufficient to see most bacterial structures Too low to see viruses

Apr 9, 2023 6

Principles of Light Microscopy Contrast

Reflects the number of visible shades in a specimen

Higher contrast achieved for microscopy through specimen staining

Apr 9, 2023 7

Microscopy: The InstrumentsTwo lenses in the compound microscopeo Ocular lens (x10)

o Objective lens (x4, x10, x40, x100)

Resolution and contrast are controlled by the condenser lens and iris diaphragmo Close iris increases contrast (low mag)

o Open iris increases resolution (high mag)

Bacteria are transparent and must be stained for bright-field microscopy.

Apr 9, 2023 8

Label the MicroscopeArm

Base

Clamp

Condenser

Coarse adjustment

Diaphragm

Fine adjustment

Light control

Apr 9, 2023 9

Light source

Nose piece

Objective lens

Ocular lens

Power switch

Slide positioning

Stage

Microscopy: The InstrumentsRefractive index is the light-bending ability of a medium.

The light may bend in air so much that it misses the small high-magnification lens.

The refractive indexes of oil and glass are similar.

Immersion oil is used to keep light from bending.

Apr 9, 2023 10

Principles of Light Microscopy

Dark-Field Microscopeo Reverse image

Specimen appears bright on a dark background

Like a photographic negative

o Achieves image through a modified condenser

Apr 9, 2023 11

Dark field microscopy of Treponema pallidum, agent of syphilisCourtesy of CDC

Electron Microscopy

Resolution is a function of wavelength: the shorter the wavelength the higher the resolution.

Uses electrons instead of visible light.

The shorter wavelength of electron beam gives greater resolution.

Apr 9, 2023 12

Principles of Electron Microscopy

Uses electromagnetic lenses, electrons and fluorescent screen to produce imageResolution increased 1,000 fold over brightfield microscopeo To about 0.3 nm (1x10-9)

Magnification increased to 100,000xTwo types of electron microscopeso Transmissiono Scanning

Apr 9, 2023 13

Disadvantages of Electron Microscope

No true color

Artifacts

Large depth of field

Destroys sample

Apr 9, 2023 14

Staining

Live or unstained cells have little contrast with the surrounding medium.

Cells are stained with dyes to make them visible.

Unstained specimens are used to observe cell behavior: motility.

Apr 9, 2023 15

Preparation of Specimens for Light Microscopy

A smear is a thin film of a microbial liquid suspension on a slide.

A smear is usually fixed by passing the slide over a flame to attach the sample to the slide.

Most organisms are killed by heat fixing.

Slides must be treated as potential biohazards.

Apr 9, 2023 16

Preparing Smears for Staining

Stains consist of a positive ion and negative ion.

In a basic dye, the chromophore is a cation (Chr+).

In an acidic dye, the chromophore is an anion (Chr -).

Staining the background instead of the cell is called negative staining.

Apr 9, 2023 17

Simple StainsBacteria cell surfaces are slightly negatively charged and basic dyes are used as stains.

Use of a single basic dye is called a simple stain.

Common basic dyes includeo Methylene blue

o Crystal violet

o Safranin

o Malachite green

Apr 9, 2023 18

Differential Stains: Gram StainThe Gram stain classifies bacteria into Gram-positive and Gram-negative bacteria.

The Gram stain is the most frequently used procedure to stain bacteria.

There are as many variations on the Gram stain procedure as there are labs.

Mostly the timing of each step and the decolorizer composition differ.

Apr 9, 2023 19

Gram positive cocci and Gram negative rods, x1,000

Differential Stains: Gram Stain

Apr 9, 2023 20

Color of Gram + cells

Color ofGram – cells

Primary stain: Crystal violet Purple Purple

Mordant: Iodine Purple Purple

Decolorizing agent:Alcohol-acetone

Purple Colorless

Counterstain:Safranin/Carboxyl fuchsin

Purple Pink or Red

Gram Stain Procedure Animation

This animation shows all the steps involved in the Gram stain.http://www.medschool.lsuhsc.edu/microbiology/Flash/gstainN.htm

Apr 9, 2023 21

Differential Stain: Acid-fast Stain

Used to stain organisms that resist conventional staining

Used to stain Mycobacterium tuberculosiso High lipid concentration in cell

wall prevents uptake of dye

o Once stained difficult to decolorize

Apr 9, 2023 22

Mycobacterium smegmatis

Special StainsStain specific structures in the bacterial cell

Endospore staino Staining enhances endospore

o Uses heat to facilitate staining

Capsule staino Allows capsule to stand out around organism

Flagella staino Staining increases diameter of flagella to make

it visible

Apr 9, 2023 23

Klebsiella pneumoniae, x1,000

Bacillus subtilis, x1,000

Morphology of Prokaryotic Cells

Prokaryotes exhibit a variety of shapeso Most common

CoccusSpherical

BacillusRod or cylinder shapedCell shape not to be

confused with Bacillus genus

Apr 9, 2023 24

Neisseria sicca

Bacillus megaterium

Morphology of Prokaryotic Cells

Prokaryotes exhibit a variety of shapeso Coccobacillus: Short round rod

o Vibrio: Curved rod

o Spirillum: Spiral shaped

o Spirochete: Helical shape

o Pleomorphic: Various shapes Clubs, Chinese letters, palisade

Apr 9, 2023 25

Treponema pallidum, a spirocheteCourtesy of CDC

Corynebacterium diphtheriaeCourtesy of the CDC

Morphology of Prokaryotic CellsDivision along a single plane may result in pairs or chains of cells

Pairs = diplococcio Example: Neisseria gonorrhoeae

Chains = streptococcio Example: species of Streptococcus

Apr 9, 2023 26Streptococcus salivarius

Morphology of Prokaryotic Cells

Division along two or three perpendicular planes form cubical packets

Example: Sarcina genus

Division along several random planes form clusters Example: species of Staphylococcus

Apr 9, 2023 27

Micrococcus luteus

Staphylococcus epidermidis