1. The Tools of Microbiology
Chapter 3
The Tools of Microbiology

2. Microscopy 1. Light microscopy Structure of the light microscope
Two lenses – Ocular and Objective
Image formation
Uses glass or plastic lenses to focus the image
Resolution and determining resolving power
Resolution: the ability to distinguish between two objects that are very close together.
Working distance
The distance from the end of the objective lens to the slide
Oil-immersion microscopy
Prevents further refraction of light because the oil has the same refractive index as the slide – therefore increasing resolution

3. Fig

4. The Five I’s of Microbiology
Inoculation – Obtaining a specimen and placing it in media
Incubation – Placing specimen at optimal for growth
Isolation – separating one organisms from other
Inspection – viewing object under microscope
Identification – using differential staining, biochemical and genetic testing to identify the organisms

5. Brightfield Microscopy

8. 3. Dark-field and phase-contrast microscopy
Use of star diaphragm - refracts light around the object – slide is dark, objects that are 3 dimensional are light
Phase contrast – special objective lenses put the image slightly out of “phase” to give more resolution
4. Fluorescent and electron microscopy
The fluorescent antibody technique –
fluorescent antibody will attach to a specific structure and the pigment will fluoresce so that the specific structure can be distinguished from other structures.

9. Darkfield

10. Fluorescent
Fluorescent pigments attached to antibodies bind to particular structures (ie. Microtubules, Nucleic Acid, Cell wall Structures, etc… used to identify structures

11. Confocal – many layers in focus

12. Light vs. Electron Microscopes

13. Resolution differences

14. Electron Microscopy Transmission electron microscopy
Transmission of electrons through the object – gives a 2-D look at the inside of a cell – uses magnets to focus the electron beam. Photographic film collects the beams that go through the object – giving various areas of “light” and “dark” depending on whether the electrons exposed the film or not.
Scanning electron microscopy
Bounces electrons off of the object – gives a 3-D look at the surface of objects – works like “SONAR” but uses electron beam instead of sound waves to detect the shape of objects. – uses magnets to focus the electron beam – collects information to form a “picture” through a television monitor or computer.

15. TEM – Transmission Electron Microscope

16. SEM – Scanning Electron Microscope

17. 2. Staining techniques Simple stain technique Negative stain technique
Basic dyes – pigment is on the positive charged ions attracted to the negatively charged cell
Stain cell
Negative stain technique
Acidic dyes – pigment is on the negative charged ions and are repelled by the negatively charged cell
Stain background
Gram stain technique
Primary Dye – Crystal Violet
Mordant – Iodine
Decolorizer – Ethanol
Secondary Dye - Safranin

18. Simple Stain

20. Gram Stain

21. Acid Fast Stain

22. Capsule Stain

23. Spore Stain

24. Fluorescent Microscopy