1. Observing Microorganisms Through a Microscope
Chapter 3
Observing Microorganisms Through a Microscope
Lectures prepared by Christine L. Case
© 2013 Pearson Education, Inc. 1

2. Tick Actual size Red blood cells
Figure 3.2 Microscopes and Magnification.
Unaided eye
≥ 200 m
Light microscope
200 nm – 10 mm
Tick
Actual size
Scanning
electron
microscope
10 nm – 1 mm
Red blood cells
Transmission
electron
microscope
10 pm – 100  m
E. coli bacteria
T-even bacteriophages
(viruses)
Atomic force
microscope
0.1 nm – 10nm
DNA double helix

3. Preparing Smears for Staining
Live or unstained cells have little contrast with the surrounding medium. Researchers do make discoveries about cell behavior by observing live specimens.
ANIMATION Microscopy and Staining: Overview

4. Preparing Smears for Staining
Staining: coloring the microbe with a dye that emphasizes certain structures
Smear: a thin film of a solution of microbes on a slide
A smear is usually fixed to attach the microbes to the slide and to kill the microbes

5. Simple Stains Simple stain: use of a single basic dye
A mordant may be used to hold the stain or coat the specimen to enlarge it
ANIMATION Staining

6. Differential Stains Used to distinguish between bacteria Gram stain
Acid-fast stain

7. Gram Stain Classifies bacteria into gram-positive or gram-negative
Gram-positive bacteria tend to be killed by penicillin and detergents
Gram-negative bacteria are more resistant to antibiotics

8. Gram Stain Color of Gram-Positive Cells Gram-Negative Cells
Primary Stain:
Crystal Violet
Purple
Mordant:
Iodine
Decolorizing Agent:
Alcohol-Acetone
Colorless
Counterstain:
Safranin
Red

9. Application of crystal violet (purple dye)
Figure 3.12 Gram staining.
Gram-positive
Gram-negative
Application of crystal violet (purple dye)
Application of iodine (mordant)
Alcohol wash (decolorization)
Application of
safranin (counterstain)
Rod
(gram-negative)
Cocci
(gram-positive)

10. Acid-Fast Stain
Stained waxy cell wall is not decolorized by acid-alcohol
Mycobacterium
Nocardia

11. Acid-Fast Stain Color of Acid-Fast Non–Acid-Fast Primary Stain:
Carbolfuchsin
Red
Decolorizing Agent:
Acid-alcohol
Colorless
Counterstain:
Methylene Blue
Blue

12. Figure 3.13 Acid-fast bacteria.
M. bovis

13. Special Stains Used to distinguish parts of cells Capsule stain
Endospore stain
Flagella stain

14. Negative Staining for Capsules
Cells stained
Capsule is not stained--negative stain

15. Figure 3.14a Special staining.
Capsules
Negative staining

16. Figure 3.14b Special staining.
Endospore
Endospore staining

17. Figure 3.14c Special staining.
Flagellum
Flagella staining

18. Table 3.3 A Summary of Various Stains and Their Uses