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