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Reagents:
Gram’s crystal violet Solution
Gram’s iodine Solution
Gram’s Decolorizer Solution
Gram’s safranin Solution
Directions:
Prepare a Slide Smear:
• Transfer a drop of the suspended culture to be examined on a slide with an inoculation loop. If the culture is to be taken from a Petri dish or a slant culture tube, first add a drop or a few loopful of water on the slide and aseptically transfer a bit of the colony. It should only be a very small amount of culture. A visual detection of the culture on an inoculation loop already indicates that too much is taken.
• Spread the culture with an inoculation loop to an even thin film over a circle of 1.5 cm in diameter. It is possible to put 3 to 4 small smears on a slide, if more than one culture is to be examined.
• Hold the slide with a clothes-pin. Allow to air dry and fix it over a gentle flame, while moving the slide in a circular fashion to avoid localized overheating. The applied heat helps the cell adhesion on the glass slide to make possible the subsequent rinsing of the smear with water without a significant loss of the culture.
Gram Staining:
1. Flood the fixed smear with Gram’s crystal violet Solution. Let stand for 60 seconds.
2. Pour off the stain and gently wash with tape water from a faucet or a plastic water bottle.
3. Flood with Gram’s iodine Solution. Allow it to remain for 60 seconds.
4. Pour off the iodine solution and gently wash with tape water. Shake off the excess water from the surface.
5. Decolorize with Gram’s Decolorizer Solution until the blue dye no longer flows from the smear. Further delay will cause excess decolorization in the gram-positive cells, and the purpose of staining will be defeated.
6. Gently wash the smear with tape water.
7. Counterstain with Gram’s safranin Solution for 60 seconds.
8. Wash off the red safranin solution with water. Blot with bibulous paper to remove the excess water. Alternatively, the slide may shaken to remove most of the water and air-dried.
9. Examine the finished slide under a microscope (oil immersion objective). Attention: Wash off any spilled stain immediately with water to avoid leaving permanent marks in the sink, lab bench, or glassware.
Result:
• Gram-positive organisms are bluish purple
• Gram-negative organisms are pinkish red
References
77730 Gram Staining Kit (Bacteria Staining Kit according to Gram)
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