1. Observing Microorganisms Through a Microscope
Chapter 3
Observing Microorganisms Through a Microscope

2. Units of Measurement 1 µm = 10-6 m = 10-3 mm 1 nm = 10-9 m = 10-6 mm

3. Microscopy: The Instruments
A simple microscope has only one lens.
Figure 1.2b

4. Microscopy: The Instruments
In a compound microscope the image from the objective lens is magnified again by the ocular lens.
Figure 3.1b

5. Microscopy: The Instruments
Total magnification = objective lens  ocular lens

6. Microscopy: The Instruments
Resolution is the ability of the lenses to distinguish two points.
A microscope with a resolving power of 0.4 nm can distinguish between two points ≥ 0.4 nm.
Shorter wavelengths of light provide greater resolution.

7. Microscopy: The Instruments
Refractive index is the light-bending ability of a medium.
The light may bend in air so much that it misses the small high-magnification lens.
Immersion oil is used to keep light from bending.
Figure 3.3

8. Preparation of Specimens for Light Microscopy
A thin film of a solution of microbes on a slide is a smear.
A smear is usually fixed to attach the microbes to the slide and to kill the microbes.
The smear are fixed by passing it 4-5 times through the flame.

10. The advantages of fixing a smear are:
The smear gets stuck (fixed) to the slide.
The penetration of stains into bacteria is better.
Vegetative bacteria get killed and become non-infectious.
Bacteria retain their original size and shape on staining.

11. Preparing Smears for Staining
Live or unstained cells have little contrast with the surrounding medium. However, researchers do make discoveries about cell behavior looking at live specimens.

12. Preparing Smears for Staining
Stains consist of a positive and negative ion.
In a basic dye, the chromophore is a cation.
In an acidic dye, the chromophore is an anion.
Staining the background instead of the cell is called negative staining.

13. Simple Stains Use of a single basic dye is called a simple stain.
Simple stain is an aqueous or alcoholic solution of a single basic dye.
Shape
size .
Arrangement of bacterial cells.
A mordant may be used to hold the stain or coat the specimen to enlarge it.

14. Differential staining
Gram stain
Acid fast stain

15. Gram Stain
The Gram stain classifies bacteria into gram-positive and gram-negative.
Gram-positive bacteria tend to be killed by penicillin and detergents.
Gram-negative bacteria are more resistant to antibiotics.

16. Gram Stain Color of Gram + cells Gram – cells Primary stain:
Crystal violet
Purple
Mordant:
Iodine
Decolorizing agent:
Alcohol-acetone
Colorless
Counterstain:
Safranin
Red

17. Gram Stain
Figure 3.10b

18. Gram positive and gram negative

19. Gram positive and Gram negative

20. Acid-Fast Stain
Some of the bacteria take up red color, when stained with carbol fuchsin and withstand decolorization by acid alcohol.
These bacteria are called acid fast bacilli. The bacillus responsible for tuberculosis: Mycobacterium tuberculosis belongs to this group
Figure 3.11

22. Acid-Fast Stain
Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counter stained (with a different color basic stain methylene blue) to see them.

23. Acid fast bacteria and non acid fast bacteria

25. Special staining Negative staining Endospore staining
Flagella staining

26. Negative Staining Negative staining is useful for capsules.
Capsule can be detected by India ink staining which is a negative staining in which the capsule stands out as a halo.
Capsulated bacteria such as Pneumococci, Klebsiella, Escherichia coli.
Production of capsule is better in vivo as compared to in vitro environment.
Figure 3.12a-c

27. Endospore staining
Heat is required to drive a stain into endospores.
These endospores are capable of survival under adverse conditions such as heat, drying, freezing, radiation and action of toxic chemicals.

29. Endospore staining

30. Examples Positive ( spore forming bacteria )
Clostridium perfringens (Gas gangrene )
Clostridium botulinum (Botulism poisoning )
Clostridium tetani (Tetanus)
Bacillus anthracis (Anthrax)
Negaive ( non spore forming bacteria )
Escherichia coli 

31. Endospore

32. Flagella staining
Flagella too small to be seen with a light microscope without staining.
Flagella staining requires a mordant to make the flagella wide enough to see.
Microbiologist use the number and arrangement of flagella as diagnostic aids.

33. Flagella
These are the organ of locomotion and provide motility to the bacterium.

34. Motile and non motile bacteria
Flagella
Organisms which are motile by flagella include
Vibrio cholerae 
Pseudomonas
Escherichia coli
Organisms which are non motile
Klebsiella
Shigella

35. Types of flagellar arrangement