1. Tools of the Laboratory:
The Methods for Studying Microorganisms
Chapter 3

2. The 5 I’s of culturing microbes
Inoculation – introduction of a sample into a container of media
Incubation – under conditions that allow growth
Isolation – separating one species from another
Inspection – looking at specimen (microscope)
Identification

3. 1. Inoculation 3

4. 2. Incubation Isolation

5. 4.Inspection 5.Identification

6. Inspection

7. Identification

8. Identification

9. Media Three categories: Physical state Chemical composition
Liquid
Semisolid
Solid (can go back to liquid)
Solid (cannot go back to liquid)
Chemical composition
Synthetic
Nonsynthetic, complex
Functional type
General
Enriched
Selective
Differential
Anaerobic
Assay

10. Most commonly used media:
nutrient broth – liquid medium containing beef extract & peptone
nutrient agar – solid media containing beef extract, peptone & agar
agar is a complex polysaccharide isolated from red algae
solid at room temp, liquefies at boiling (100oC), does not resolidify until it cools to 42oC
provides framework to hold moisture & nutrients
not digestible for most microbes

11. Enriched media

12. Selective & Differential Media

13. Differential media

14. Microscope
Magnification – ability to enlarge objects
Resolving power – ability to show detail

15. Compound Light Microscope

16. Effect of wavelength on resolution

17. Oil immersion lens

18. Types of light microscopes
Bright-field – most widely used, specimen is darker than surrounding field
Dark-field – brightly illuminated specimens surrounded by dark field
Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures

19. 3 views of the same cell a. Bright-field b. Dark-field c
3 views of the same cell a. Bright-field b. Dark-field c. Phase-contrast

20. Fluorescence Microscope
Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewer’s eye
Uses dyes that emit visible light when bombarded with shorter uv rays.
Useful in diagnosing infections

22. Electron microscopy
Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds.
Electron waves are 100,000X shorter than the waves of visible light.
Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength.
Magnification between 5,000X and 1,000,000X+

23. 2 types of electron microscopes
Transmission electron microscopes (TEM) – transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts
Scanning electron microscopes (SEM)– provides detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it. (1987)

24. Transmission Electron Microscope

25. Transmission Electron Micrograph

26. Scanning Electron Microscope

27. Scanning Electron Micrograph

30. “Carbon monoxide man” (Scanning Tunneling Microscope-Made of 28 CO molecules)

31. Specimen preparation wet mounts & hanging drop mounts – fixed mounts –
allow examination of characteristics of live cells: motility, shape, & arrangement
fixed mounts –
are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.

32. Staining
cationic dyes – basic dye, with positive charges on the chromophore
Chromophore: chemical(s) that absorb light that is responsible for color in a molecule
anionic dyes – acidic dye, with negative charges on the chromophore
surfaces of microbes are negatively charged and attract basic dyes – positive staining.
negative staining – microbe repels dye & it stains the background

33. Staining simple stains –one dye is used
differential stains – use a primary stain and a counterstain to distinguish cell types or parts. examples: Gram stain, acid-fast stain and endospore stain
special stains: capsule and flagellar stains

34. Types of stains