GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

1. Gather all equipment and supplies– Gram stain– Microscope slides– Heat source– Sterile swab– Culture or specimen– Microscope

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

2. Label the slide with the patient’s initials or the specimen number.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

3. Choose an isolated colony off of the agar plate and obtain bacteria with a sterile swab.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

4. Place the swab on the microscope slide and spread the colonies in a circular motion.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

5. Heat fix the microorganisms to the slide by placing the bottom of the slide to heat for approximately 30 seconds.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

6. Place slide on staining tray or hold with forceps above the sink.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE7. Flood the surface of the slide with

Crystal Violet stain and let sit for one minute.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

8. Rinse the slide with distilled water.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

9. Flood the slide with Gram’s Iodine and time for one minute.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

10. Rinse the slide with distilled water.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

11. Flood the slide with Gram’s decolorizer and time for 30 seconds.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

12. Rinse the slide with distilled water.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

13. Flood the slide with the counterstain, Safranin, and let sit for one minute.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

14. Rinse the slide with distilled water.

GRAM STAIN PROCEDUREGRAM STAIN PROCEDURE

15. Blot the slide and read with the oil immersion lens of the microscope. Look for Gram-negative and Gram-positive bacteria.