Overview
This transcript outlines a step-by-step Gram stain procedure for preparing a slide for microscopic examination.
Gram Staining Procedure
- Add methyl violet to the prepared slide; allow to stain for 30 seconds.
- Gently wash slide with distilled water into the metal dish provided.
- Add Gram’s iodine to the slide; leave for one minute to fix the primary stain.
- Gently wash slide again with distilled water into the metal dish.
- Rinse slide gently with alcohol; direct flow away from the labeled side.
- Note: Alcohol can remove labeling ink if it contacts the label area.
- Wash slide gently with distilled water to stop decolorization.
- Add neutral red to the slide; leave to stain for one minute as counterstain.
- Wash slide gently with distilled water; remove excess water by tapping.
- Tap off water against the staining bowl edge or onto tissue; air dry fully.
- Once fully dry, the slide is ready for microscope examination.
Gram Stain Timing and Reagents
| Step | Reagent | Purpose | Duration |
|---|
| 1 | Methyl violet | Primary stain for Gram-positive cells | 30 seconds |
| 2 | Distilled water | Rinse to remove excess stain | Until clear |
| 3 | Gram’s iodine | Mordant to fix crystal violet complex | 1 minute |
| 4 | Distilled water | Rinse after iodine | Until clear |
| 5 | Alcohol | Decolorizer; remove stain from Gram-negative cells | Brief, gentle rinse |
| 6 | Distilled water | Stop decolorization | Until clear |
| 7 | Neutral red | Counterstain to visualize Gram-negative cells | 1 minute |
| 8 | Distilled water, dry | Final rinse and drying | Until fully dry |
Safety and Handling Notes
- Direct alcohol rinse away from the labeled side to preserve slide labeling.
- Perform all rinses gently to avoid washing off the specimen.
- Collect rinse water in the metal dish to manage waste properly.
Action Items / Next Steps
- Allow slide to air dry completely before microscopy.
- Proceed to examine the stained slide under the microscope.