Pewarnaan Gram (STIKES NASIONAL SURAKARTA)#ON_TLM2020

Introduction

This tutorial provides a step-by-step guide on the Gram staining technique, a crucial procedure in microbiology for identifying and classifying bacteria based on their cell wall characteristics. Understanding Gram staining is essential for laboratory technicians and healthcare professionals, as it influences treatment decisions for bacterial infections.

Step 1: Prepare the Slides

• Gather the necessary materials:
  • Glass slides
  • Bacterial cultures
  • Inoculating loop
  • Bunsen burner or alcohol lamp
• Sterilize the inoculating loop by heating it until red hot and allow it to cool.
• Use the loop to collect a small amount of the bacterial culture.
• Smear the culture onto the slide to create a thin film.
• Air dry the slide completely.

Step 2: Heat Fix the Bacteria

• Pass the slide through the flame of a Bunsen burner three times to fix the bacteria to the slide.
• Allow the slide to cool before proceeding to the staining process. This step ensures that the bacteria adhere to the slide and helps preserve their structure.

Step 3: Stain the Slide with Crystal Violet

• Cover the heat-fixed smear with crystal violet stain for about 1 minute.
• Rinse the slide gently with distilled water to remove excess stain.

Step 4: Apply Iodine Solution

• Add iodine solution to the slide and let it sit for 1 minute. The iodine acts as a mordant, forming a complex with the crystal violet that will help in the differentiation of the bacteria.
• Rinse the slide again with distilled water.

Step 5: Decolorize with Alcohol

• Apply 95% ethanol or acetone to the slide for 10-30 seconds. This step is critical, as it differentiates between Gram-positive and Gram-negative bacteria:
  • Gram-positive bacteria will retain the crystal violet and appear purple.
  • Gram-negative bacteria will lose the color and become colorless.
• Rinse immediately with distilled water to stop the decolorization process.

Step 6: Counterstain with Safranin

• Stain the slide with safranin for about 30 seconds. Safranin will color the decolorized Gram-negative bacteria pink.
• Rinse the slide again with distilled water and gently blot it dry with bibulous paper.

Step 7: Observe Under the Microscope

• Place the slide under a microscope and observe using the oil immersion lens.
• Record the color and shape of the bacteria:
  • Purple indicates Gram-positive.
  • Pink indicates Gram-negative.

Conclusion

Gram staining is an essential technique for identifying bacterial species in the laboratory. By following these steps—preparing slides, heat fixing, staining with crystal violet and iodine, decolorizing, counterstaining with safranin, and finally observing under a microscope—you can effectively classify bacteria based on their Gram reaction. For further studies, consider exploring the implications of identifying different bacterial types in clinical settings.