Gram Stain

Gram Stain

Majority of bacteria are classified on the basis of Gram staining. We can divide the bacteria into two groups basic types on the basis of Gram staining characteristics i.e. Gram positive and Gram negative. This is basically due to the difference in the cell wall of bacteria.

Principle of Gram staining

In Gram positive bacteria the cell wall is rich in peptidoglycan. In Gram negative the peptidoglycan layer is relatively thin. The bacteria with thick peptidoglycan layer in their cell wall don’t retain crystal violet when exposed to acetone iodine & thus take up counter stain becoming pink in color.

Reagents

1. Four reagents are used in this staining.
2. Crystal violet
3. Gram iodine (Mordant)
4. Acetone iodine (Decolorizer)
5. Diluted carbol fuchsin/safaranin (Counter stain)

Procedure

1.  Prepare a smear of test organism on a glass slide.
2. Air dry or fix it by passing one to two times over the flame.
3. Pour staining crystal violet dye over smear & let it remain there for 30 secs.
4. Wash it with tap water.
5. Pour Grams iodine & then keep it for 30 sec.
6. Wash it with tap water
7. Pour decolorizer, acetone iodine & let it remain there for 30 secs
8. Wash it with tap water.
9. Pour freshly diluted (1:20) carbol fuchsin or safranin over the smear & let it remain there for 30 secs
10. Wash it with tap water.
11. Put the slide on the rack for air drying.

Microscopy

Slide should be seen first in 4x objective then pour a drop of oil on smear and then focus it under 100x or oil immersion lens.

Result/ Interpretation

1. The report should include the following information
2.  Number of bacteria present as many, moderate, few or scanty.
3. Whether gram positive (purple) or gram negative (pink or red).
4. Morphology of bacteria. Whether cocci, bacilli or cocco bacilli
5. Their arrangement.
6. Presence of pus cells if there are any.

Comments

False Gram negative reaction

1. .      Cell wall damaged due to antibiotics or excessive heat fixation.
2. .      Over decolorization with acetone iodine.
3. .      Use of old Grams iodine solution which acts as a poor mordant.

Quality control

 Positive and negative controls should be applied on each test slide.

 Gram Positive control:     Staphylococcus aureus

 Gram Negative control:   Escherichia coli

This page was last modified on March 18, 2015.