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Observing Microorganisms through a Microscope

Observing Microorganisms through a Microscope. Chapter 3. Basic techniques needed to study Bacteria. 1. Grow Bacteria 2. Isolate Bacteria 3. Grow Bacteria in pure culture 4. Observe Bacteria 5. Identify Bacteria. Microscope. Resolving Power - ability to distinguish two distinct points

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Observing Microorganisms through a Microscope

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  1. Observing Microorganisms through a Microscope Chapter 3

  2. Basic techniques needed to study Bacteria • 1. Grow Bacteria • 2. Isolate Bacteria • 3. Grow Bacteria in pure culture • 4. Observe Bacteria • 5. Identify Bacteria

  3. Microscope • Resolving Power - ability to distinguish two distinct points • absolute limit of the Resolving Power is about 1/2 the wavelength of light that is used to illuminate the specimen

  4. Preparing smears for staining • 1. Bacteria on slide • 2. Air Dry • 3. Bacteria are HEAT FIXED to the slide • 4. Stain is applied

  5. Staining Reaction • Stains - salts composed of a positive and negative ion, one of which is colored (chromophore) • Basic Dyes - chromophore is the positive ion • dye+ Cl- • Acid Dyes - chromophore is the negative ion • Na+dye-

  6. Bacteria are slightly negative, so are attracted to the positive chromophore of the BASIC DYE • Common Basic Dyes • crystal violet • methylene blue • safranin • basic fuchsin

  7. Acid Dyes - used for Negative Staining (background is stained) Mordant - intensifies the stain or coats a structure to make it thicker and easier to see after it is stained Example: Flagella - can not normally be seen, but a mordant can be used to increase the diameter of the flagella before it is stained Salmonella typhosa

  8. Differential Stains • React differently with different types of bacteria • 2 Most Common • Gram Stain • Acid-Fast Stain

  9. Gram Stain • 1884 Hans Christian Gram • most important stain used in Bacteriology • Divides all Bacteria into 2 groups: • Gram (+) • Gram (-)

  10. Gram Stain 1. Crystal violet

  11. Gram Stain 2. Grams Iodine (mordant)

  12. Gram Stain 3. Alcohol

  13. Gram Stain 4. Safranin (Counterstain)

  14. Results • Gram (+) Purple • Gram (-) Red • Difference - due to structure of cell wall • Gram (+) Thick cell wall • Gram (-) Thin cell wall

  15. Identification of a Bacteria Unknown • 1. Gram Reaction • 2. Morphology

  16. Acid - Fast Stain • Differential Stain - divides bacteria into 2 groups • Acid - Fast • Non Acid - Fast • Used to identify organisms in the Genera Mycobacterium(high lipid and wax content in cell wall)

  17. 2 Important Pathogens Mycobacterium tuberculosis

  18. Mycobacterium leprae

  19. Acid - Fast Stain • 1. Carbolfuchsin (Red) • 2. Acid Alcohol • 3. Counterstain with Methylene Blue • Acid - Fast Cells Red • Non Acid - Fast Blue

  20. Special Stains Capsule Stain Klebsiella pneumoniae

  21. Flagella Stain Spirillum volutans

  22. Endospore Stain Bacillus cereus

  23. Clostridium botulinum

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