How to Perform a Gram Stain - MCCC Microbiology

Summary

This video demonstrates the steps to perform a Gram stain, a crucial laboratory technique for identifying bacteria. The procedure involves four key reagents: crystal violet, Gram's iodine, ethanol, and saffranin. The steps include adding and rinsing each reagent in a specific order, with particular attention to the decolorization process using ethanol. The video emphasizes the importance of proper technique to avoid over or under-decolorization. After staining, the slide is examined under a microscope using different lens powers, including oil immersion for high-resolution viewing. The video concludes with cleaning instructions for the microscope.

Takeaways

• 😀 Make sure to start with a clean slide that has your specimen added and heat-fixed before beginning the Gram stain process.
• 😀 You will need four reagents for the Gram stain: Crystal Violet, Gram's Iodine, Ethanol, and Safranin.
• 😀 Start by adding Crystal Violet to the smear, ensuring the entire area is covered. Let it sit for 30 seconds.
• 😀 After 30 seconds, rinse the slide with deionized water by tilting it over the tray.
• 😀 Next, add Gram's Iodine and let it sit for 60 seconds, then rinse again with deionized water.
• 😀 The ethanol step is crucial; add drops of ethanol slowly, watching for a light purple color to appear. Do not over-decolorize.
• 😀 Rinse the slide with water after the ethanol step to stop the decolorization process.
• 😀 The final reagent is Safranin, which should be applied for 30 seconds before rinsing with deionized water.
• 😀 After rinsing, blot dry the slide gently with bibulous paper.
• 😀 When preparing the slide for viewing, start by focusing under the scanning power lens, then move to low and high power lenses.
• 😀 Before using the oil immersion lens, add a drop of microscope oil and clean the oil immersion lens with acetone when done.

Q & A

What is the first reagent used in the Gram stain process?

The first reagent used is crystal violet, which is applied to the smear to stain the specimen.

How long should the crystal violet be left on the slide?

The crystal violet should remain on the slide for 30 seconds.

What is the purpose of grams iodine in the Gram stain process?

Grams iodine acts as a mordant, which helps fix the crystal violet stain inside the cells, enhancing the staining effect.

How long should grams iodine stay on the slide?

Grams iodine should stay on the slide for 60 seconds.

What is the most critical reagent in the Gram stain procedure?

The most critical reagent is ethanol, as it must be carefully applied to avoid over-decolorizing or under-decolorizing the smear.

How can you tell if too much ethanol has been added during the decolorization step?

If too much ethanol is added, the slide will be over-decolorized, and the cells may lose the crystal violet stain, leading to inaccurate results.

What happens if not enough ethanol is used during decolorization?

If not enough ethanol is used, the smear may be under-decolorized, leading to false results, as some cells may retain the crystal violet stain that should have been removed.

What is the fourth reagent used in the Gram stain process?

The fourth reagent is saffranin, a red dye that is applied to the smear after decolorization to stain any cells that have lost the crystal violet stain.

How long should the saffranin stay on the slide?

The saffranin should stay on the slide for 30 seconds.

What is the purpose of using microscope oil during the Gram stain observation?

Microscope oil is used during the oil immersion lens phase to help improve the clarity and resolution of the specimen at high magnification.

How should the slide be cleaned after using the oil immersion lens?

After using the oil immersion lens, the lens should be cleaned with acetone to remove any residual oil.

What is the proper way to focus the slide under the microscope after staining?

First, focus the slide under the scanning power lens, then move to low and high power. For the high power lens, use oil immersion, adjusting the lens to touch the oil and get a clear focus.