Lab Gram stain Procedure and Reagents

Question 1

1st step in Gram staining procedure.

Answer 1

Place the smears on the stain tray (with or without slide holders attached).

Question 2

2nd step in Gram staining procedure.

Answer 2

Apply a generous amount of crystal violet stain; let stand for 30 seconds.

Question 3

3rd step in Gram staining procedure.

Answer 3

Hold the slide at a 45° angle and wash with distilled water for several seconds to remove the dye.

Question 4

4th step in Gram staining procedure.

Answer 4

Apply Gram’s iodine; let stand for one (1) minute.

Question 5

5th step in Gram staining procedure.

Answer 5

Hold the slide at a 45° angle and wash with distilled water for several seconds.

Question 6

6th step in Gram staining procedure

Answer 6

Hold the slide at a 45° angle; apply Gram’s alcohol for 5 - 10 seconds

Question 7

7th step in Gram staining procedure

Answer 7

Hold the slide at a 45° angle and wash with distilled water from the wash bottle for several seconds. This stops the reaction with the decolorizer.

Question 8

8th step in Gram staining procedure

Answer 8

Apply a generous amount of safranin stain for 30 seconds to 1 minute.

Question 9

9th step in Gram staining procedure

Answer 9

Hold the slide at a 45° angle and wash with distilled water for two (2) seconds.

Question 10

10th step in Gram staining procedure

Answer 10

Gently blot the slide between sheets of bibulous paper to remove excess water

Question 11

11th step in Gram staining procedure

Answer 11

Using the microscope find the smear with 10x objective, add immersion oil, and observe under
100x oil immersion objective. Remember to use only the fine adjustment knob to focus under the
oil immersion objective.