Staining

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Observing Microorganisms through a Microscope:

Observing Microorganisms through a Microscope Chapter 3

Basic techniques needed to study Bacteria:

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:

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

Preparing smears for staining:

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

Staining Reaction:

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-

Bacteria are slightly negative, so are attracted to the positive chromophore of the BASIC DYE:

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

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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

Differential Stains:

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

Gram Stain:

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

Slide 10:

Gram Stain 1. Crystal violet

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Gram Stain 2. Grams Iodine (mordant)

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Gram Stain 3. Alcohol

Slide 13:

Gram Stain 4. Safranin (Counterstain)

Results:

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

Identification of a Bacteria Unknown:

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

Acid - Fast Stain:

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)

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2 Important Pathogens Mycobacterium tuberculosis

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Mycobacterium leprae

Acid - Fast Stain:

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

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Special Stains Capsule Stain Klebsiella pneumoniae

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Flagella Stain Spirillum volutans

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Endospore Stain Bacillus cereus

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Clostridium botulinum