Gram’s Staining << Back

  • Objective
  • Procedure
  • Self Evolution (MCQ)
  • Edutainment
  • Video
  • Simulation
  • Assignment
  • Reference
  • Links library
  • Feedback

 

1.       Understanding the gram staining

2.       Understanding the biochemistry of underlying the gram staining

3.       Understand the difference between grams positive and negative bacteria.

 

Requirements:

1.24 hours old culture of Staphyloccus aureus

2.24 hours old culture of Escherichia coli

3.       solutions of crystal violet

4.       Gram’s iodine*

5.       95% ethanol and/or isopropanol-acetone mixture (3:1 v/v)

6.       Safranin

7.       Clean glass slides

8.       Inoculating loop

9.       blotting paper

10.   Spirit burner or gas burner

11.   Glass cover slip

* 2 g potassium iodide in 300 ml distilled water plus 1 g iodine crystals

Procedure:

1.       Take a loop full of culture on slide

2.       Prepare a smear and air dries it

3.       Heat fixes the smear

4.       Place the slide on staining rack

5.       Flood the smears with crystal violet and let stand for 30 seconds

6.       Rinse the slide with water for 5 seconds

7.       Cover with Gram’s iodine mordant and let stand for 1 minute

8.       Rinse the slide with water for 5 seconds

9.       Decolorize with 95% ethanol for 15 to 30 seconds.

10.   Rinse the slide with water for 5 seconds

11.   Counterstain with safranin for about 60 to 80 seconds

12.   Rinse the slide with water for 5 seconds

13.   Blot dry with blotting paper