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# 3. microscopy

## on Sep 27, 2012

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Simple review of the light microscope and staining for microbiology

Simple review of the light microscope and staining for microbiology

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## 3. microscopyPresentation Transcript

• Microscopy Chapter 3 Back
• Microscopy Resolution and magnification Different types of light microscopy: principle and use Electron microscope: principle StainsSep-12 2
• Units of Measurement 1 µm = 10-6 m = 10-3 mm 1 nm = 10-9 m = 10-6 mm 1000 nm = 1 µm 0.001 µm = 1 nmSep-12 3
• Principles of Light Microscopy Light passes through specimen and through a series of magnifying lenses Important factors in light microscopy include Magnification Resolution ContrastSep-12 4
• Principles of Light Microscopy: Magnification Compound Microscope : microscope has two magnifying lenses  Lenses include ocular lens and objective lens Lenses combine to enlarge objects  Magnification is equal to the product of the ocular lens x the objective lens: 10x X 100x = 1,000xSep-12 5
• Principles of Light Microscopy: Resolution Resolving power is defined as the minimum distance existing between two points where they still appear as separate Resolving power determines how much detail can be seen Naked eye ≈ 0.1mm Light microscope ≈ 0.2μm Electron microscope ≈ 2.5nmSep-12 6
• Principles of Light Microscopy: Resolution Resolution o Resolution depends on the quality of lenses and wavelength of illuminating light  How much light is released from the lens o Maximum resolving power of most brightfield microscopes is 0.2 μm (1x10-6)  This is sufficient to see most bacterial structures  Too low to see virusesSep-12 7
• Principles of Light Microscopy Contrast Reflects the number of visible shades in a specimen Higher contrast achieved for microscopy through specimen stainingSep-12 8
• Microscopy: The Instruments Two lenses in the compound microscope: ocular lens Objective lens Resolution and contrast are controlled by the condenser lens and iris diaphragm Bacteria are transparent against a brightly illuminated background and must be stained for bright- field microscopy.Sep-12 9
• Microscopy: The Instruments Refractive index is the light- bending ability of a medium. The light may bend in air so much that it misses the small high-magnification lens. The refractive indexes of oil and glass are similar. Immersion oil is used to keep light from bending.Sep-12 10 Figure 3.3
• Principles of Light Microscopy Examples of light microscopes that increase contrast o Phase-Contrast Microscope o Interference Microscope o Dark-Field Microscope o Fluorescence Microscope o Confocal Scanning Laser MicroscopeSep-12 11
• Principles of Light Microscopy Dark-Field Microscope o Reverse image  Specimen appears bright on a dark background Like a photographic negative o Achieves image through a modified condenserSep-12 12
• Electron Microscopy Resolution is a function of wavelength: the shorter the wavelength the higher the resolution. Uses electrons instead of visible light. The shorter wavelength of electron beam gives greater resolution.Sep-12 13
• Principles of Electron Microscopy Uses electromagnetic lenses, electrons and fluorescent screen to produce image Resolution increased 1,000 fold over brightfield microscope o To about 0.3 nm (1x10-9) Magnification increased to 100,000x Two types of electron microscopes o Transmission o ScanningSep-12 14
• Disadvantages of Electron Microscope No true color Artifacts Large depth of field Destroys sampleSep-12 15
• Staining Live or unstained cells have little contrast with the surrounding medium. Cells are stained with dyes to make them visible. Unstained specimens are used to observe cell behavior: motility.Sep-12 16
• Preparation of Specimens for Light Microscopy A thin film of a solution of microbes on a slide is a smear. A smear is usually fixed by heating the slide to attach the microbes to the slide.Sep-12 17
• Preparing Smears for Staining Stains consist of a positive ion and negative ion. In a basic dye, the chromophore is a cation (Chr+). In an acidic dye, the chromophore is an anion (Chr -). Staining the background instead of the cell is called negative staining.Sep-12 18
• Simple Stains Bacteria cell surfaces are slightly negatively charged and basic dyes are used as stains. Use of a single basic dye is called a simple stain. Common basic dyes include o Methylene blue o Crystal violet o Safranin o Malachite greenSep-12 19
• Differential Stains: Gram Stain Used to distinguish one bacterial group from another. The Gram stain is the most frequently used procedure to stain bacteria. There are as many variations on the Gram stain procedure as there are labs. Mostly the timing of each step and the decolorizer composition differ. The Gram stain classifies bacteria into Gram-positive and Gram-negative bacteria.Sep-12 20
• Differential Stains: Gram Stain Color of Color of Gram + cells Gram – cells Primary stain: Crystal violet Purple Purple Mordant: Iodine Purple Purple Decolorizing agent: Purple Colorless Alcohol-acetone Counterstain: Purple Pink or Red Safranin/Carboxyl fuchsinSep-12 21
• Gram Stain Procedure Animation This animation shows all the steps involved in the Gram stain. http://www.medschool.lsuhsc.edu/microbiology/Flash/g stainN.htmSep-12 22
• Differential Stain Acid-fast Stain o Used to stain organisms that resist conventional staining o Used to stain members of genus Mycobacterium: e.g. Mycobacterium tuberculosis  High lipid concentration in cell wall prevents uptake of dye  Uses heat to facilitate staining  Once stained difficult to decolorizeSep-12 23
• Special Stains Capsule stain o Example of negative stain o Allows capsule to stand out around organism Endospore stain o Staining enhances endospore o Uses heat to facilitate staining Flagella stain o Staining increases diameter of flagellaSep-12 24
• Morphology of Prokaryotic Cells Prokaryotes exhibit a variety of shapes o Most common  Coccus  Spherical  Bacillus  Rod or cylinder shaped  Cell shape not to be confused with Bacillus genusSep-12 25
• Morphology of Prokaryotic Cells Prokaryotes exhibit a variety of shapes o Other shapes  Coccobacillus  Short round rod  Vibrio  Curved rod  Spirillum  Spiral shaped  Spirochete  Helical shape  Pleomorphic  Bacteria able to vary shapeSep-12 26
• Morphology of Prokaryotic Cells Prokaryotic cells may form groupings after cell division Cells adhere together after cell division for characteristic arrangements Arrangement depends on plan of division especially in the cocciSep-12 27
• Morphology of Prokaryotic Cells Division along a single plane may result in pairs or chains of cells Pairs = diplococci o Example: Neisseria gonorrhoeae Chains = streptococci o Example: species of StreptococcusSep-12 28
• Morphology of Prokaryotic Cells Division along two or three perpendicular planes form cubical packets Example: Sarcina genus Division along several random planes form clusters Example: species of StaphylococcusSep-12 29
• Morphology of Prokaryotic Cells Some bacteria live in groups with other bacterial cells o They form multicellular associations  Example: myxobacteria These organisms form a swarm of cells » Allows for the release of enzymes which degrade organic material » In the absence of water cells form fruiting bodies  Other organisms for biofilms Formation allows for changes in cellular activitySep-12 30