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What is the purpose of heat fixing?
To fix the specimen and kill the bacteria
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Describe how to perform the simple staining procedure, include equipment, materials, and the steps involved?
- Equipment: Primary stain (such as crystal violet or safranin), Rinse bottle, Slide, Stock Culture, Microscope, Rack, Loop, Bunsen Burner
- Procedure: Transfer stock culture to slide
- Air dry
- Heat fix
- Place slide on rack, let slide stain for the required time.
- Rinse slide with water, blot dry and observe under the microscope
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How is simple staining differ from gram stain in the sense of purpose for each procedure?
- Both contrast between the bacteria and background
- Both give information about cell shape, size and arrangement.
- Gram stain differs from simple stain, in that gram stain differentiates by dividing bacteria into gram positive and gram negative
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Why does a gram positive organism stain differently than a gram negative one?
- Gram Positive bacteria have more peptidoglycan so retains more crystal violet
- Gram Negative has less peptidoglycan so looses more crystal violet.
- Primary stain crystal violet interacts with the peptidoglycan in cell wall.
- The mordant iodine increase the interaction in cell wall.
- The decolorizer removes the crystal violet from gram negative bacteria cell wall but not gram postive.
- The 2nd safronin counterstains, gram negative stains with a red or pink color
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Describe the Gram stain procedure step by step, and the purpose of each reagent used in the staining procedure, including the equipment and materials?
- Equipment: Crystal violet, mordant, decolorizer, safranin, rinse bottle, slide, rack, bunsen burner, stock culture, loop, microscope
- Procedure: Transfer a loop of stock culture to a slide
- Air dry, heat fix and place on a rack
- Flood slide with crystal violet for 1 min, rinse slide with water and place back on rack
- Flood slide with iodine for 1 min, rinse with water
- Drip decolorizer onto the slide and let it run off for 2 sec, rinse with water and place back on rack
- Flood slide with safronin for 1 min, rinse with water, blot dry and observe under microscope
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What are the problems with using agar specimens vs broth specimens in making a smear?
- Agar and inoculum results in the occurence of large agrigates of bacteria pile on top of each other therefore making it hard to see morphological and gram staining results.
- Broth eventually disburses bacteria
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What does gram variable mean?
Some bacteria of a pure culture will have some cells stain gram positive and some cells gram negative
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Why must young cultures be used in Gram staining?
When gram positive bacteria become too old they stain gram negative
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What is the purpose of the 10% KOH(potassium hydroxide) procedure?
- If a gram stain of bacteria from a pure culture is inconclusive KOH prep by mixing colony on a slide for 30 sec.
- Gram negative bacteria will produce mucoid string and gram positive bacteria will not.
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