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Chapter 3

Chapter 3. Observing Microorganisms Through a Microscope. Units of Measurement. 1 µm = 10 -6 m = 10 -3 mm 1 nm = 10 -9 m = 10 -6 mm 1000 nm = 1 µm 0.001 µm = 1 nm. Microscopy: The Instruments. A simple microscope has only one lens. Figure 1.2b. Microscopy: The Instruments.

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Chapter 3

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  1. 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 • 1000 nm = 1 µm • 0.001 µm = 1 nm

  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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Differential staining • Gram stain • Acid fast stain

  14. 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.

  15. Gram Stain

  16. Gram Stain Figure 3.10b

  17. Gram positive and gram negative

  18. Gram positive and Gram negative

  19. 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

  20. 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.

  21. Acid fast bacteria and non acid fast bacteria

  22. Special staining • Negative staining • Endospore staining • Flagella staining

  23. 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

  24. 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.

  25. Endospore staining

  26. 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

  27. Endospore

  28. 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.

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

  30. 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

  31. Types of flagellar arrangement

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