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Chapter 3: Observing Microorganisms Through a Microscope

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1 Chapter 3: Observing Microorganisms Through a Microscope

2 Units of Measurement Appendix D, “Exponential Notation” section
Microorganisms Appendix D, “Exponential Notation” section

3 Microscopy: The Instruments
Magnifies specimen image Light microscopy: use of any microscope that uses visible light to view a specimen Compound microscope: series of lenses Magnifies specimen image Concentrates light onto specimen MagTotal = Magobjective x Magocular Figure 3.1b

4 Microscopy: The Instruments
Resolution: the ability of the lenses to distinguish two points as separate (i.e. see fine detail) Expressed as limit of resolution (or resolving power) Limit of resolution (D): inversely proportional to resolution Smaller D = better resolution i.e. A microscope with a limit of resolution of 50 nm can distinguish between two points at least 50 nm apart Shorter wavelengths of light provide greater resolution

5 Light Microscopy Refractive index: the light-bending ability
Staining changes the refractive index of a specimen to increase its contrast with the background Immersion oil acts as an extension of the lensmore light is trapped Figure 3.3

6 Light Microscopy: Brightfield Illumination
Dark objects are visible against a bright background Staining is necessary to increase a specimen’s refractive index relative to the background’s Organisms are killed Figure 3.4a, b

7 Electron Microscopy Uses electron beam instead of lightbeam
factor increase in resolution compared to visible light microscopy

8 Transmission Electron Microscopy (TEM)
Electron beam: nm wavelength Vis light: nm Electrons pass through the specimen, then an electromagnetic lens, to a screen or film Specimens may be stained with heavy metal salts (enhance electron absorption by specimen) Figure 3.8a

9 Transmission Electron Microscopy (TEM)
10, ,000X magnification Limit of resolution 2.5 nm Micrographs are black and white, but false color can be added electronically Figure 3.8a

10 Scanning Electron Microscopy (SEM)
Beam of electrons scan the surface of a whole specimen Secondary electrons emitted from the specimen produce the image Figure 3.8b

11 Scanning Electron Microscopy (SEM)
1,000-10,000X magnification Limit of resolution 20 nm Figure 3.8b

12 Brightfield illumination
(light microscopy) Electron microscopy

13 Preparation of Specimens for Light Microscopy
Smear: a thin film of a solution of microbes on a slide A smear is usually heat-fixed to Attach the microbes to the slide Kill the microbes

14 Preparing Smears for Staining
Stains are salts that consist of a positive and negative ion Acidic dye: the chromophore is an anion Basic dye: the chromophore is a cation A mordant may be used to intensify staining in one of several ways (help retain the dye, coat a structure to enlarge it, etc.) Crystal violet:

15 Simple Staining Techniques
Use of a single basic dye Bacteria are negatively charged at neutral pH Negative stain: Use of acidic dye to stain the background instead of the cells

16 Differential Stains: Gram Stain
Differential stains react differently with different types of bacteria All bacteria are not stained the same color in the end The Gram stain classifies bacteria into gram-positive and gram-negative groups Gram-positive bacteria tend to be killed by penicillin and detergents Gram-negative bacteria tend to be more resistant to some antibiotics, but more susceptible to physical stress

17 Differential Stains: Gram Stain
Figure 3.10b

18 Differential Stains: Acid-Fast Stain
Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast Figure 3.11

19 Special Stains Special stains are used to stain specific cell structures Heat is required to drive a stain into endospores Flagella staining requires a mordant to make the flagella wide enough to see Figure 3.12a-c


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