1 Chapter Microscopy

2 Light Microscopes – uses light passed through a specimen Types include:  Brightfield  Darkfield  Phase-Contrast  Differential Interference Contrast  Flourescence (UV Light)  Confocal (Laser light) Electron Microscopes – use a beam of electrons which are either passed through or bounced off of a specimen Types include:  Transmission (TEM)  Scanning (SEM)  Scanning Tunneling (STEM)  Atomic Force (SEM also)

3 Light Microscopy see images on pg Bright-field: dark image against a brighter background (what we use in lab) Dark-field: only refracted light enters objective, making specimen bright, background dark (useful for observing living cells, particularly spirochetes) Phase-contrast: converts slight changes in refraction into easily detected variations of light intensity (useful in observing living cells) Fluorescence: specimen exposed to ultraviolet light, compounds in the sample fluoresce, giving off visible light

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5 Microscope Compound Light Microscope : a microscope with a series of lenses Total magnification: calculated by multiplying the objective lens magnification by the ocular lens magnification Resolution: ability to distinguish between two points a specified distance apart. The smallest distance that can be distinguished equals the resolving power of the scope. About 0.2 microns is the limit of white light resolution

6 Light microscope Electron microscope Visible to eye

7 Ocular lens look in here! Second magnification Prism bends light Objective Lens First magnification Condenser Focuses light Illuminator ‘the light’ Focus knob Stage Move specimen Specimen

8 Oil Immersion Lenses for High Magnification

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10 BUT, if our specimen is too small for light Must use electron microscope

11 TEM SEM

12 Staining Fixation: process by which internal and external structures of cells are preserved Heat-fixation - fix an air-dried thin film (smear) by passing through flame Dyes: have chromophore groups (give color) and bind to cells by ionic, covalent, or hydrophobic bonding Hydrophobic – e.g. Sudan black, stains lipids Ionic stains can be either: (remember ions?) Basic (+ charged dye attracted to – charged bacteria) OR Acidic (- charged dye attracted to + background)

13 Staining continued… Simple Stain: uses one staining agent  Methylene blue  Crystal violet  Safranin  Carbolfuchsin Mordant – chemical added to make the dye bind more tightly (increased affinity for specimen)

14 Differential Stains divide bacteria into separate groups based on different staining properties – different groups stain differently Gram stain - developed by Dr. Christian Gram, 1884 Our most important/useful staining procedure: Crystal Violet - 1 minute (primary stain) Gram's Iodine - 1 minute (this is a mordant!) 95% ethanol sec (Rinse to decolorize) Safranin - 1 minute (secondary stain)

15 Gram Staining

16 Interpreting Gram Stains Purple cells = Gram Positive (G+) Gram positive bacteria - trap crystal violet-iodine complex, due to thick layer of peptidoglycan in cell wall Red cells = Gram Negative (G-) Gram negative bacteria - large amount of lipids in cell wall dissolved by the 95% ethanol, allowing crystal violet-iodine complex to escape leaving cells colorless. The safranin counterstain turns these cells red

17 Acid Fast Stain Acid-fast stain: Carbolfuchsin used to stain Mycobacterium, which have high content of mycolic acid (waxy substance) Must use steam heat to force carbolfuchsin stain into cells Acid-alcohol decolorizer removes stain from non-acid fast cells counterstain with methylene blue

18 Negative/Capsular Stain: reveals capsule layer around cells. Capsules often indicate a bacterium’s virulence, or ability to cause disease! Dye = Nigrosin or India Ink mix bacteria with the dye & spread on clean slide, dry Bacteria are then often stained with a simple stain The capsule shows as a clear halo around the bacterial cells useful to observe Klebsiella pneumonia & other capsule producers

19 Spore Stain used to stain endospores of Clostridium and Bacillus Endospores do not readily take up dye, must be ‘forced’ but once it penetrates the stain is not easily decolorized heat smear over steam with Malachite green stain Then rinse with water & counterstain with safranin (red stain) RESULT = Endospores - Green; Vegetative cells - Red

20 Flagellar Stain Some bacteria have flagella, whip-like structures with which they move. Some stains specifically stain these structures Flagella of bacteria are coated with tannic acid or potassium alum and stained with basic fuchsin (Gray Method)

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