Microscope

THE STUDY OF MICROBIAL STRUCTURE: MICROSCOPY AND SPECIMEN PREPARATION*

Scale*

Discovery of MicroorganismsAntony van Leeuwenhoek (1632-1723)first person to observe and describe micro-organisms accurately*Figure 1.1b

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Lenses and the Bending of Lightlight is refracted (bent) when passing from one medium to anotherrefractive indexa measure of how greatly a substance slows the velocity of lightdirection and magnitude of bending is determined by the refractive indexes of the two media forming the interface*

Lensesfocus light rays at a specific place called the focal pointdistance between center of lens and focal point is the focal lengthstrength of lens related to focal lengthshort focal length more magnification*

*Figure 2.2

The Light Microscopemany typesbright-field microscopedark-field microscopephase-contrast microscopefluorescence microscopesare compound microscopesimage formed by action of 2 lenses*

The Bright-Field Microscopeproduces a dark image against a brighter backgroundhas several objective lensesparfocal microscopes remain in focus when objectives are changedtotal magnification product of the magnifications of the ocular lens and the objective lens*

*Figure 2.3

*Figure 2.4

Microscope Resolutionability of a lens to separate or distinguish small objects that are close togetherwavelength of light used is major factor in resolutionshorter wavelength greater resolution*

*working distance distance between the front surface of lens and surface of cover glass or specimen

*Figure 2.5

*Figure 2.6

The Dark-Field Microscopeproduces a bright image of the object against a dark backgroundused to observe living, unstained preparations*

*Figure 2.7b

The Phase-Contrast Microscopeenhances the contrast between intracellular structures having slight differences in refractive indexexcellent way to observe living cells*

*Figure 2.9

*Figure 2.10

The Differential Interference Contrast Microscopecreates image by detecting differences in refractive indices and thickness of different parts of specimenexcellent way to observe living cells*

The Fluorescence Microscopeexposes specimen to ultraviolet, violet, or blue lightspecimens usually stained with fluorochromesshows a bright image of the object resulting from the fluorescent light emitted by the specimen*

*Figure 2.12

*Figure 2.13c and d

Preparation and Staining of Specimensincreases visibility of specimenaccentuates specific morphological featurespreserves specimens*

Fixation process by which internal and external structures are preserved and fixed in positionprocess by which organism is killed and firmly attached to microscope slideheat fixingpreserves overall morphology but not internal structureschemical fixingprotects fine cellular substructure and morphology of larger, more delicate organisms*

Dyes and Simple Stainingdyesmake internal and external structures of cell more visible by increasing contrast with backgroundhave two common featureschromophore groupschemical groups with conjugated double bonds give dye its colorability to bind cells*

Dyes and Simple Stainingsimple staininga single staining agent is usedbasic dyes are frequently useddyes with positive chargese.g., crystal violet*

Differential Stainingdivides microorganisms into groups based on their staining propertiese.g., Gram staine.g., acid-fast stain*

Gram stainingmost widely used differential staining proceduredivides Bacteria into two groups based on differences in cell wall structure

*

*Figure 2.14primary stainmordantcounterstaindecolorizationpositivenegative

*Figure 2.15cEscherichia coli a gram-negative rod

Acid-fast stainingparticularly useful for staining members of the genus Mycobacteriume.g., Mycobacterium tuberculosis causes tuberculosise.g., Mycobacterium leprae causes leprosyhigh lipid content in cell walls is responsible for their staining characteristics*

Staining Specific StructuresNegative stainingoften used to visualize capsules surrounding bacteriacapsules are colorless against a stained background*

Staining Specific StructuresSpore stainingdouble staining techniquebacterial endospore is one color and vegetative cell is a different colorFlagella stainingmordant applied to increase thickness of flagella*

Electron Microscopybeams of electrons are used to produce images wavelength of electron beam is much shorter than light, resulting in much higher resolution*Figure 2.20


The Transmission Electron Microscopeelectrons scatter when they pass through thin sections of a specimentransmitted electrons (those that do not scatter) are used to produce imagedenser regions in specimen, scatter more electrons and appear darker*

*EMFigure 2.23

Specimen Preparationanalogous to procedures used for light microscopyfor transmission electron microscopy, specimens must be cut very thinspecimens are chemically fixed and stained with electron dense material*

Other preparation methodsshadowingcoating specimen with a thin film of a heavy metalfreeze-etchingfreeze specimen then fracture along lines of greatest weakness (e.g., membranes)*

*Figure 2.25

*Ebola

*Fly head

The Scanning Electron Microscopeuses electrons reflected from the surface of a specimen to create imageproduces a 3-dimensional image of specimens surface features*

*Figure 2.27

Newer Techniques in Microscopyconfocal microscopy and scanning probe microscopyhave extremely high resolutioncan be used to observe individual atoms*Figure 2.20


Confocal Microscopyconfocal scanning laser microscopelaser beam used to illuminate spots on specimencomputer compiles images created from each point to generate a 3-dimensional image*

*Figure 2.29

*Figure 2.30

Scanning Probe Microscopyscanning tunneling microscopesteady current (tunneling current) maintained between microscope probe and specimenup and down movement of probe as it maintains current is detected and used to create image of surface of specimen*

Scanning Probe Microscopyatomic force microscopesharp probe moves over surface of specimen at constant distanceup and down movement of probe as it maintains constant distance is detected and used to create image*

*Ebola*Fly head

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Microscope