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What is the purpose of heat fixing?
To fix the specimen and kill the bacteria
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
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
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
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
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
What does gram variable mean?
Some bacteria of a pure culture will have some cells stain gram positive and some cells gram negative
Why must young cultures be used in Gram staining?
When gram positive bacteria become too old they stain gram negative
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.