Chapter 2 Laboratory Techniques: Microscopy PDF

Chapter 2 Laboratory Techniques: Microscopy 1  Microscopy: The Instruments Simple microscope has one lens (van Leeuwenhoek’s) Compound microscope has multiple lenses Ocular lens (in eyepieces) helps magnify Objective lenses (on nosepiece) have multiple...

Chapter 2 Laboratory Techniques: Microscopy 1  Microscopy: The Instruments Simple microscope has one lens (van Leeuwenhoek’s) Compound microscope has multiple lenses Ocular lens (in eyepieces) helps magnify Objective lenses (on nosepiece) have multiple magnifications so you can choose which you want to use Condenser lens directs the light (or electrons) and does NOT magnify Total magnification = objective lens  ocular lens We use compound microscopes Types of Microscopy Light Microscopy (max TM=1000x) Brightfield Darkfield Phase-contrast Fluorescence Electron Microscopy (max TM=100,000x) Transmission (TEM) Scanning (SEM) TM=total 3 The Light Microscopes  many types brightfield microscope darkfield microscope phase-contrast microscope fluorescence microscope  are all compound microscopes image formed by action of 2 lenses Unlike simple microscopes by van Leeuwenhoek 4  Lenses and the Bending of Light light is refracted (bent) when passing from one medium to another refractive index a measure of how greatly a substance slows the velocity of light direction and magnitude of bending is determined by the refractive indices of the two media forming the interface (like from air to glass and glass to water for through a fish tank) 5 Lenses  focus light rays at a specific place called the focal point  distance between center of lens and focal point is the focal length  strength of lens related to focal length short focal length more magnification 6 The Brightfield Microscope produces a dark image against a brighter background has several objective lenses parfocal microscopes remain in focus when objectives are changed total magnification product of the magnifications of the ocular lenses and the objective lenses (see slide 8) Crystal violet stain (simple stain) of Escherichia coli (brightfield microscope 7 image)  Microscope Resolution  Resolution is the ability of a lens to separate or distinguish small objects that are close together- or distinguish two points  The 1. wavelength of light used and 2. Electron refraction of the light are both major vs. light factors in resolution microsco shorter wavelength  greater resolution py Less refraction  greater resolution Using oil immersion = 8 Microscopy: The Instruments Refractive index: The amount of bending light undergoes for a particular medium. The light may bend in air so much that it misses the small high-magnification lens. Immersion oil is used to keep light from bending when using the 100x objective (reducing refraction, thus increasing resolution). The Light Microscopes  many types brightfield microscope darkfield microscope phase-contrast microscope fluorescence microscope  are all compound microscopes image formed by action of 2 lenses Unlike simple microscopes by van Leeuwenhoek 10  Brightfield/Darkfield Illumination Brightfield: Stained objects are visible against a bright background. Specimens are usually fixed and stained so they are visible. Figure 3.4a–b Darkfield Microscopy Image is formed by light reflected or refracted by the specimen produces a bright image of the object against a dark background Often used to observe living, unstained preparations (not quite as good as phase- contrast) For eukaryotes, it has been used to observe internal structures For prokaryotes, it has been used to identify bacteria such as Treponema pallidum, the causative agent of syphilis 12  Phase-Contrast Microscope Phase-Contrast Microscopy is a contrast-enhancing optical technique that can produce high-contrast images of transparent specimens such as living cells, thin tissue slices, etc. It is the best type of microscopy for viewing cells that are alive; living cells can be examined in their natural state without being killed, fixed, and stained enhances the contrast between intracellular structures having slight differences in refractive index 13 The Fluorescence Microscope exposes specimen to ultraviolet, violet, or blue light Specimens are usually stained with fluorochromes– molecules that emit light of different colors shows a bright image of the object resulting from the fluorescent light emitted by the “stained “specimen Has applications in medical microbiology and microbial ecology studies 14  Electron Microscopy Beams of electrons are used to produce images Wavelength of electrons is much shorter than light (photons), resulting in much higher resolution The first electron microscope was constructed by Ernst Ruska in 1931. Requires complicated preparation of samples, cannot be used for living organisms 15 Electron Microscopy vs. Light Microscopy Beams of electrons are Beams of light (photons) used to produce images are used to produce images Wavelength of electrons Wavelength of photons is is much shorter than light much longer than (photons), resulting in electrons, resulting in much higher resolution lower resolution Can get up to 100,000x Can get up to 1000x TM TM can be used for living Requires complicated organisms and also for preparation of samples, dead/fixed organisms cannot be used for living organisms 16 Transmission Electron Microscopy (TEM) Ultra thin sections of specimens (usually). Electrons pass through the specimen, then an electromagnetic lens, to a screen or film. Specimens may be stained with heavy metal salts. Figure 3.9a (1 of 2) Fixation preserves internal and external structures and fixes them in position organisms usually killed and firmly attached to microscope slide heat fixation – routine use with bacteria preserves overall morphology but not internal structures Kills organisms and makes them adhere to the slide chemical fixation – used with larger, more delicate organisms protects fine cellular substructure 18  Dyes and Staining Dyes make internal and external structures of cell more visible by increasing contrast with background Acidic dyes stain the background; basic dyes stain the specimens Simple stains Use ONE dye (stain) to color either the specimen or the background Positive (direct) stains Color the specimen (background is unstained) Negative stains Color the background (specimen is unstained) 19 Staining  Simple stains a single stain is used Can be used to determine size, shape and arrangement of bacteria  Differential stains More than one stain is used Used to separate bacteria into groups based on properties (such as cell wall type) [can also be used to determine size, shape and arrangement of bacteria] Gram-stain and acid-fast stain are examples 20 The Gram Stain  most widely used differential staining procedure  divides bacteria into two groups based on differences in cell wall structure: Gram-positive and Gram-negative bacteria. 21  Differential Stains: Gram Stain Step Reagent Color of Color of Gram–positive Gram–negative cells cells 1 Primary stain Crystal Purple Purple violet 2 Mordant Iodine Purple Purple 95% 3 Decolorization Purple Colorless Ethanol 4 Counterstain Safranin Purple Pink/Red Acid-fast Stain particularly useful for staining members of the genus Mycobacterium (and Nocardia) Mycobacterium tuberculosis – causes tuberculosis Mycobacterium leprae – causes leprosy high lipid content in cell walls (mycolic acid) is responsible for their staining characteristics: Acid-fast bacteria have a waxy substance called mycolic acid in their cell walls which makes them impermeable to many staining procedures, including the Gram stain. 23 Special Stains Capsule stain Capsule staining uses negative staining and positive staining together to show bacterial capsules. endospore stain Heat is required to drive a stain into endospores. Flagella staining requires a mordant to make the flagella flagella stain wide enough to see. Figure 3.13a–c Capsule staining 1. perform negative stain, air dry; 2. brief heat-fix, 3. perform positive (direct) stain Negative staining – stain does not penetrate the bacterial cell but causes the background to appear dark or opaque Cells are not heat-fixed before negative staining so no shrinkage of the cell occurs (and no damage to capsule). After a BRIEF heat-fix, a positive simple stain is performed to stain thecapsule cell insidestains: the capsule. (negative negative and positive stains both stain only done) 25  Staining Specific Structures Endospore staining double staining technique like acid-fast stain, uses heat bacterial endospore is one color and vegetative cell (aka normal cell) is a different color Flagella staining mordant applied to increase thickness of flagella Flagellar stain of Proteus vulgaris 26