MicroscopMicroscopyy

Observing Observing Microorganisms Microorganisms

Through a MicroscopeThrough a Microscope

Units of Units of MeasurementMeasurement

Microorganisms are so small that Microorganisms are so small that metric prefixes may be unfamiliarmetric prefixes may be unfamiliar

centi = 1/100 or 10centi = 1/100 or 10-1-1

milli = 1/1000 or 10milli = 1/1000 or 10-2-2

micro = 1/1,000,000 or 10micro = 1/1,000,000 or 10-6-6

nano = 1/1,000,000,000 or 10nano = 1/1,000,000,000 or 10--

99

The InstrumentsThe Instruments

D arkfie ld M icroscopy

P hase C ontrast M icroscopy

D ifferentia l In terference C ontrast M icroscopy

F luorescence M icroscopy

C onfocal M icroscopy

C om pound L ight M icroscope

S canning E lectron M icroscopy

Transm iss ion E lectron M icroscopy

A tom ic F orce E lectron M icroscopy

E lectron M icroscopes

Compound Light Compound Light MicroscopeMicroscope

Uses visible lightUses visible light Has at least 2 sets Has at least 2 sets

of lensesof lenses Can achieve Can achieve

maximum 2000X maximum 2000X magnificationmagnification

Resolution of Resolution of objects as small as objects as small as 0.2 0.2 mm

Light MicroscopyLight Microscopy

In a In a light microscope light microscope visible lightvisible light passes through the specimen and passes through the specimen and then through glass lenses.then through glass lenses. The lenses refract light such that the The lenses refract light such that the

image is magnified into the eye or a image is magnified into the eye or a video screen.video screen.

Microscopes vary in magnification and Microscopes vary in magnification and resolving power.resolving power.

MagnificationMagnification is the ratio of an object’s is the ratio of an object’s image to its real size.image to its real size.

Resolving powerResolving power is a measure of image is a measure of image clarity.clarity. It is the minimum distance two points It is the minimum distance two points

can be separated and still viewed as two can be separated and still viewed as two separate points.separate points.

Resolution is limited by the shortest Resolution is limited by the shortest wavelength of the source, in this case wavelength of the source, in this case light.light.

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Light MicroscopesLight Microscopes

The minimum resolution The minimum resolution of a light microscope is of a light microscope is about 2 microns, the size about 2 microns, the size of a small bacteriumof a small bacterium

Light microscopes can Light microscopes can magnify effectively to magnify effectively to about 1,000 times the about 1,000 times the size of the actual size of the actual specimen.specimen. At higher At higher

magnifications, the magnifications, the image blurs.image blurs.

Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Fig. 7.1

Resolution of Light Resolution of Light MicroscopesMicroscopes

Brightfield IlluminationBrightfield Illumination

Usual operationsUsual operations Specimens must be Specimens must be

stained for viewingstained for viewing Best magnification Best magnification

and resolution with and resolution with the oil immersion the oil immersion objectiveobjective

Oil has same Oil has same refractive index as refractive index as glassglass

Darkfield MicroscopyDarkfield Microscopy

Only light reflected from specimen enters Only light reflected from specimen enters objective lensobjective lens

Organism appears light against a dark Organism appears light against a dark fieldfield

Useful for examining Useful for examining Live organismsLive organisms Microorganisms which cannot be stained by Microorganisms which cannot be stained by

standard methodsstandard methods Treponema pallidum, the causative agent of Treponema pallidum, the causative agent of

syphilis syphilis

Phase-Contrast and Phase-Contrast and Differential Interference Differential Interference

(DIC) Microscopy(DIC) Microscopy

Uses wave nature of Uses wave nature of lightlight

One set of light rays One set of light rays are direct and one set are direct and one set are reflectedare reflected

Makes detailed images Makes detailed images of internal structure of of internal structure of living microorganisms living microorganisms possiblepossible

Image in greyscaleImage in greyscale

Uses differences in Uses differences in refractive indicesrefractive indices

Uses 2 beams of lightUses 2 beams of light Resolution higherResolution higher Brightly colored Brightly colored

imageimage Image appears nearly Image appears nearly

three-dimensionalthree-dimensional

DICPhase-Contrast

Electron micrographsElectron micrographs

Fluorescence MicroscopyFluorescence Microscopy

When illuminated with When illuminated with short short light some light some dyes emit light with dyes emit light with longer longer

Enables viewing of Enables viewing of cells located on an cells located on an opaque surface such opaque surface such as a soil particleas a soil particle

When illuminated with When illuminated with UV or halogen light UV or halogen light source preparations source preparations glowglow

Bovine pulmonary artery endothelial cells. Photometrics, Ltd.

Fluorescent stain of cellFluorescent stain of cell

Confocal Confocal MicroscopyMicroscopy3-D confocal microscopy of Salmonella-infected macrophage (green) with XY-slice showing bacteria (red) inside the cell

•Preparations also stained with fluorochromes

•Exceptionally clear two-dimensional images

•Three-dimensional images obtained by computer construct

Electron MicroscopyElectron Microscopy

Beam of electrons has shorter Beam of electrons has shorter so so gives better resolution than visible gives better resolution than visible lightlight

Electromagnetic lenses rather than Electromagnetic lenses rather than glassglass

Done in a vacuumDone in a vacuum Can resolve to 0.5nm and magnify up Can resolve to 0.5nm and magnify up

to 100,000 times.to 100,000 times. Specimen must be dry….deadSpecimen must be dry….dead

Transmission ElectronTransmission Electron MicroscopyMicroscopy

(TEM)(TEM) Resolves objects as Resolves objects as

close as 2.5nmclose as 2.5nm Magnification Magnification

10,000 to 100,000X10,000 to 100,000X Ultra-thin sections Ultra-thin sections Specimens must be Specimens must be

dehydrateddehydrated Preparation of Preparation of

specimen may specimen may generate artifactsgenerate artifacts

Lambda Bacteriophage DNA

(TEM x153,000)

Scanning Electron Scanning Electron MicroscopyMicroscopy

(SEM)(SEM) Resolves objects as Resolves objects as

close as 20 nmclose as 20 nm Magnification Magnification

between 1,000 and between 1,000 and 10,000X10,000X

Whole specimensWhole specimens 3-dimensional view 3-dimensional view

of specimenof specimen Specimen Specimen

dehydrateddehydratedSlime Mold Fruiting Structure, Lamproderma sp.

(SEM x290)

Scanning Tunneling Scanning Tunneling Atomic ForceAtomic Force

Thin metal probe Thin metal probe scans specimenscans specimen

Resolving power Resolving power much greater than much greater than electron microscopeselectron microscopes

no special specimen no special specimen preparationpreparation

detailed views of detailed views of silicon chips & DNA silicon chips & DNA moleculemolecule

Metal and diamond Metal and diamond probe forced down probe forced down along surface of along surface of specimenspecimen

3-dimensional image3-dimensional image no special no special

preparation of preparation of specimen is requiredspecimen is required

views of detailed views of detailed structure of biological structure of biological moleculesmolecules

Preparation of Microscopy Preparation of Microscopy SpecimensSpecimens

Microorganisms must be spread over Microorganisms must be spread over the surface of a slide (smear)the surface of a slide (smear)

Microorganisms must be attached to Microorganisms must be attached to the slide (fixed)the slide (fixed)

Microorganisms must be colored Microorganisms must be colored (stained)(stained)

Making the Smear and Making the Smear and Fixing ItFixing It

Put a small amount Put a small amount of organism into a of organism into a drop of water on a drop of water on a clean microscope clean microscope slide & spread .slide & spread .

When dry pass When dry pass through the flame through the flame of a Bunsen burner of a Bunsen burner or flood with or flood with methyl alcoholmethyl alcohol

StainsStains

DyesDyes Negative StainNegative Stain

stains the background not the organismstains the background not the organism Simple StainsSimple Stains

everything stained a single coloreverything stained a single color Differential StainsDifferential Stains

distinguish among bacteria based on distinguish among bacteria based on particular characteristics particular characteristics

Characteristics of DyesCharacteristics of Dyes

Basic DyesBasic Dyes Chromophore is in Chromophore is in

the positive ionthe positive ion Used to stain most Used to stain most

bacteriabacteria Used alone as Used alone as

simple stains in simple stains in combination for combination for differential stainsdifferential stains

Acidic DyesAcidic Dyes Chromophore is in Chromophore is in

the negative ionthe negative ion Used in negative Used in negative

staining & for staining & for staining nuclear staining nuclear materialmaterial

Gram StainGram Stain The Gram Stain is the single most The Gram Stain is the single most

important test in microbiology. The important test in microbiology. The principal utility of the Gram Stain rests on principal utility of the Gram Stain rests on its speed and simplicity. Most bacteria may its speed and simplicity. Most bacteria may be divided in two groups by this procedurebe divided in two groups by this procedure

developed by the Danish physician Hans developed by the Danish physician Hans Christian Gram to differentiate Christian Gram to differentiate pneumococci from pneumococci from KlebsiellaKlebsiella pneumoniapneumonia

difference between Gram-positive and difference between Gram-positive and Gram-negative bacteria is in the structure Gram-negative bacteria is in the structure of the cell wallof the cell wall

ProcedureProcedure

ResultsResultsG+ cocci G- rods

Websites with more samples of gram stained bacteria

GRAM STAINED IMAGES OF MEDICALLY IMPORTANT BACTERIALoyola University Medical Center

http://www.meddean.luc.edu/lumen/DeptWebs/microbio/med/gram/slides.htm

GRAM STAIN TUTORIALhttp://www.courses.ahc.umn.edu/pharmacy/5825/GSPage05.html

Acid Fast StainAcid Fast Stain

Acid Fast Staining is used primarily for detection of Acid Fast Staining is used primarily for detection of organisms with a thick outer coat composed of true organisms with a thick outer coat composed of true waxes, mycolic acids and phosphatideswaxes, mycolic acids and phosphatides

Mycobacteria are not decolorized and retain the stain, Mycobacteria are not decolorized and retain the stain, appearing pink under the light microscope (hence are appearing pink under the light microscope (hence are 'fast', like color-fast clothes).'fast', like color-fast clothes).

Acid Fast Stain can also be used to identify several Acid Fast Stain can also be used to identify several protozoa, such as Cryptosporidium and Isospora belli. protozoa, such as Cryptosporidium and Isospora belli. These two coccidia have recently acquired greater These two coccidia have recently acquired greater clinical significance because of their widespread clinical significance because of their widespread occurrence in immuno- compromised patients, such as occurrence in immuno- compromised patients, such as those infected with HIV. those infected with HIV.

Special StainsSpecial Stains

Spore StainSpore Stain

Capsule StainCapsule Stain

Flagella Stain Flagella Stain

Bacillus subtilis

Streptococcus pneumoniae

Pseudomonas aeruginosa