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
microbiology.scu.edu.tw

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