Chapter 4A:Microscopy &

Microbial Classification

1. Types of Microscopy

2. Staining

3. Classification & Nomenclature

1. Types of MicroscopyChapter Reading – pp. 96-107

Visible light

10-8m

700 nm400 nm

Increasing resolving power

Increasing wavelength

Trough

100m10-4m10-12m

Crest One wavelength

103m

UVlight

Infra-red

Micro-wave

Xrays

Radio waves andTelevisionGamma rays

The Electromagnetic Spectrum

Diameterof DNA Ribosomes

Mitochondrion

Typical bacteriaand archaea

VirusesProteins

Aminoacids

Atoms

Largeprotozoan (Euglena)

Human redblood cell

Chloroplasts

Flea

Chickenegg

Pig

Unaided human eye200 µm–

Transmission electron microscope (TEM)10 nm–100µm

Scanningtunneling

microscope(STM)

0.5 nm–10 nm

1 nm 10 nm 100 nm0.1 nm 1 µm 10 µm 100 µm

Scanning electron microscope (SEM)10 nm–1 mm

1 mm 10 mm 100 mm 1 m 10 m

Atomicforce

microscope(AFM)

1 nm–10 nm

Compound light microscope (LM)200 nm–10 mm

Scale of Magnification

Light Microscopy

a typical “bright field” microscope such as used in lab

Microscopeobjective

Refractedlight rays

lost to lens

Light sourceSpecimen

Slide

Glass cover slip

LensesMicroscope

objective

Light sourceSpecimen

Slide

Glass cover slip

Without immersion oil With immersion oil

Immersion oil

Unrefractedlight rays enter lens

Oil Immersion & Light Refraction

Different media (air, water, glass, oil…) bend lightto different degrees. • i.e., have different refractive indexes• immersion oil has refraction index similar to glass,allows more light to enter the lens

Bright vs Dark Field Microscopy

Light refractedby specimen

ObjectiveLight unrefracted

by specimen

Specimen

Condenser

Dark-field stop

Dark-field stop

only light refractedby the specimen

will enter theobjective lens

(“normal” light microscopy)

Nucleus

Phase contrast

Rays in phase

Ray deviated byspecimen is 1/4 wavelength out

of phase.

Phase plate

Rays out of phase

Bacterium Deviated rayis now 1/2

wavelength out of phase.

Phase Contrast Microscopy

• reveals internal detailwithout staining

• useful for live specimens

Enhances misalignment of light waves to create contrast

Bacteria

Nomarski

Differential Interference Contrast (DIC) Microscopy

A variation on phase-contrast microscopy involving a more complex combination of filters and prisms.

• also referred to as “Normarski Microscopy”

• creates an image with evengreater detail and contrast

• image has a 3-dimensional appearance as if it was illuminated from the side

Fluorescent Microscopy Fluorescent dyes or antibodies with a fluorescent tag stick to specific targets.

Under UV light, dye fluoresces, onlylabeled cells or structures are seen.

confocalstandard

Confocal Fluorescence Microscopy

Only light from a given depth or plane is transmitted, “out of focus” light is excluded

Electron Microscopy Electromagnetic lenses focus electron beam onto metal-stained specimen.

• electron beams have veryshort wavelengths• allows far greater resolution

than with light microscopy

Transmission EM (TEM)• thin sections of specimen,

highest resolution

Scanning EM (SEM)• reveals surface features

Other types of Microscopy Scanning-Tunneling & Atomic Force microscopy use special fine-tipped probes to produce highest resolution.

SCANNING-TUNNELING• distance between probe and specimen determined by electron flow between them

scanning-tunneling (STM)

atomic force (AFM)

• deflection of laser aimed at probe tip produces image

ATOMIC FORCE

DNAdoublehelix

PlasmidDNA

2. StainingChapter Reading – pp. 108-111

Why the Need for Stains?Because, no matter how high the magnification or resolution, you need contrast to be able to see anything.

If contrast is not sufficient in the sample orthe microscopic method used, staining canprovide the necessary contrast:

• stains used for viewing bacteria via light microscopy aretypically positively charged chromophores (basic dyes)

• chromophore = “color-bearing” ion of a salt

• bacteria have a net negative charge (i.e., bind positive ions)

General Types of StainsSimple stain• dye that non-specifically stains all organisms, features

Differential stain• dye that binds various structures or organisms differently

Negative stain• dye that stains background, not specimen

Special stain• dye that specifically stains certain subcellular structures

**a mordant is any chemical added to enhance a stain**

Counter stain• a 2nd dye added that is a different color than original dye

Spread culture inthin film over slide

Air dry

Pass slide throughflame to fix it

Fixing the Specimen on a SlideSpecimens must first be “fixed” to the slidesurface before they can be stained.

• generally done by smearing a sample on the slide, airdrying, and passing briefly through flame to “heat fix”• specimens can also be fixed to the slide surface bychemical means

Gram Staining A very common stain to distinguish 2 bacterial types:

Process:1) 1o stain

(crystal violet)

2) mordant (iodine)

3) decolorize* (alcohol)

4) counter stain (safranin)

• retains primarystain (due to thickpeptidoglycan layerand teichoic acids)

Gram positive

• does NOT retainprimary stain, onlycounter stain

Gram negative

1 2 3* 4

* key step

Acid-Fast Staining Most bacteria are not “acid-fast” (i.e., don’t retain

primary stain afteracid wash).

• “acid fast” bacteriadetected by this stain include those in the genera:

• “non-acid-fast”cells revealed by counter stain

MycobacteriumNocardia

acid fast non-acid fast

Special Stains Flagella Stain

Capsule Stain Endospore Stain

• reveal specific subcellularstructures

3. Classification & NomenclatureChapter Reading – pp. 113-115

Species Genus Family Order Class Phylum Kingdom Domain

Ursus americanus(American black bear)

Ursus

Ursidae

Carnivora

Mammalia

Chordata

Animalia

Eukarya

Taxonomic Hierarchy

species can also be subdivided

different strains

Scientific Nomenclature To avoid confusion, every type of organism must be referred to in a consistent way.The current system of nomenclature (naming) has been in use since the 18th century:• every type of organism is referred by its genus name

followed by its specific epithet (i.e., species name)

Homo sapiens (H. sapiens) Escherischia coli (E. coli)

• names are Latin (or “Latinized” Greek) with the genusbeing a noun and the specific epithet an adjective

• name should be in italics and only the genus is capitalizedwhich can also be abbreviated

**strain info can be listed after the specific epithet (e.g., E. coli O157:H7)**

Key Terms for Chapter 4A

• microscopy: bright & dark field, phase contrast, DIC, fluorescent, confocal, transmission & scanning EM,scanneling-tunneling, atomic force microscopy

• resolution, refraction & oil immersion

• simple, differential, counter, negative stains• mordant, chromophore

Relevant Chapter Questions MC: 1-10 FB: 1-5 Labeling SA: 1, 3-6