1. Observing Microorganisms Through Microscopes

2. Units of measurement Or Nanometer = 0.000000001 m = 10-9
Metric system
The standard unit of length in metric system is the meter (m)
--the advantage of m.s is that the units are related to each other by factors of 10
1m=100 cm or 1000mm
Micrometer = m = 10-6 Or
Nanometer = m = 10-9

3. Microscopes
There are two basic types of microscopes (according to source of illumination) that are commonly used in Microbiology:
light microscopes
and electron microscopes. 

4. illuminator = light source Condenser = has lenses that direct the light rays through the specimen Objective lenses = the lenses closest to the specimen Ocular lens = eyepiece = the image is magnified again by ocular lens

5. Magnification = enlargement
Resolution = is the ability of the lenses to distinguish fine detail & structure
Refraction index = is a measure of the light bending ability of a medium

6. Calculate the magnification. = multiplying the obj. L. power by the ocu.l. power – 10X
Low power – 10X == 100X
High power – 40X == 400X
Oil immersion – 100X == 1000X

7. Greater resolution can be achieved by
using oil immersion,
by filtering out with-blue light,
and by replacing light with electrons.

8. Blue Light Increases Resolution
Blue light has shorter wavelength than other visible regions of the electromagnetic spectrum.
Shorter wavelength results in higher resolution.
Blue Light Increases Resolution
Blue filter is inserted between light source and condenser.

9. Oil Immersion Increases Resolution

10. Oil Immersion Increases Resolution
Air has a different Index of Refraction from water (so light bends).
Air has a different Index of Refraction from glass (so light bends).
Oil Immersion Increases Resolution
The Mineral Oil has the same Index of Refraction as glass (so light does not bend).

11. B- Electron Microscope abeam of electrons is used –free electrons travel in waves (100.000)

12. EM = Increased Resolution
Transmission Electron Microscopy (TEM): electrons are transmitted through substance.
EM = Increased Resolution
Scanning Electron Microscopy (SEM): electrons bounce off the surface of specimen resulting in a more 3-D image.

13. Types of Microscopes

14. Dark field Microscope a- Light Microscopy:
Types of Microscopes

15. Fluorescent Microscope
The ability of substance to absorb short wave length (ultraviolet) and give off light at a longer wave length (visible) 15

16. In fluorescence microscopy specimens are first stained with fluorochromes and then viewed through a compound microscope by using an uv light source
The m.o. appear bright objects against a dark background
Fluorescence microscopy is used primarily in a diagnostic procedure called f.ab. tequnique

17. Observation of microorganisms

18. Observation of microorganisms
Colorless
A smear must be
prepared
stained
Staining = coloring -Increase contrast of microorganisms
Fixed = attached
fixing – kills , fix & preserves various parts of m.o. in their natural state

19. Preparation of smear for light field Microscope

20. Dyes
Organic salts compose of a positive and a negative ion, one of which is colored and is known as the chromophore
In Basic dye: positive ion is colored
Cystal violet, methylen blue, malachite green, safranin.
In Acidic dye: negative ion is colored
eosin, acid fuchsine, and nigrosin.
As bacteria are slightly negatively charge at pH7, it colored with basic dye

21. Staining Types of stains Simple stain: Differential stain:
Structural or special stains

22. Differential Stains React differently with different kinds of bac.
More than one dye
Example : as Gram stain, acid fast
Primary dye
Mordant
Decolorizing step
Counter stain

23. Differential Stain: The Gram Stain

24. Gram Stain 24

25. Acid fast stain Mycobacterium Red dye=carbol-fuchsin
Gentile heating =enhance penetration & retention of the dye
Acid alcohol=decolorizing – removes the red color from not AF bac.
Methylene blue

26. Acid-Fast Staining
Note that the acid-fast bacteria are found as red clumps of filamentous cells.
Mycobacterium avium complex (MAC) with acid fast stain often has the characteristic appearance shown here with numerous mycobacteria filling macrophages. Such macrophages may be distributed diffusely or in clusters.

27. India ink Capsule stain
Steps
India ink Capsule stain 27

28. Special Stain: Capsule Staining
Note that the background is stained as well as the bacteria, plus there is a “halo” around the bacteria. The halo represents the capsule.
Special Stain: Capsule Staining

29. Spore stain
Malachite green

30. Flagella stain
Mordant----- iodine