Gram’s staining

Bacteria differ from one another chemically and physically and may react differently to a given staining procedure. This is the principle of differential staining. Differential staining can distinguish between types of bacteria. The Gram stain is the most useful and widely employed differential stain in bacteriology. The method was developed by Hans Christian Gram in 1884. It divides bacteria into two major groups: Gram negative and Gram positive.

The first step in the procedure involves staining with the basic dye crystal violet. This is the primary stain. It is followed by treatment with an iodine solution, which functions as a mordant; that is, it increases the interaction between the bacterial cell and the dye so that the dye is more tightly bound or the cell is more strongly stained. The smear is then decolorized by washing with an agent such as 70% ethanol or isopropanol-acetone. Gram-positive bacteria retain the crystal violet-iodine complex when washed with the decolorizer, whereas gram-negative bacteria lose their crystal violet-iodine complex and become colorless. Finally, the smear is counterstained with a basic dye, different in color than crystal violet. This counterstain is usually safranin. The safranin will stain the decolorized gram-negative bacteria pink. The end result is that gram-positive bacteria are deep purple in color and gram-negative bacteria are pinkish to red in colour.


1. Primary Stain – Crystal Violet
2. Mordant – Grams Iodine
3. Decolourizer – Ethyl Alcohol
4. Secondary Stain – Safranin

1. Prepare heat-fixed smears of the given bacterial culture.
2. Place the slides on the staining rack.
3. Flood the smears with crystal violet and let stand for 1 minute.
4. Rinse with water for 5 seconds.
5. Cover with Gram‘s iodine mordant and let stand for 1 minute.
6. Rinse with water for 5 seconds.
7. Decolorize with 70% ethanol for 15 seconds. Do not decolorize too long. Add the decolorizer drop by drop until the crystal violet fails to wash from the slide.
8. Rinse with water for 5 seconds.
9. Counterstain with saffranin for about 30 seconds.
10. Rinse with water for 5 seconds.

Blot dry with tissue paper and examine under oil immersion. Gram-positive organisms stain blue to purple; gram-negative organisms stain pink to red. There is no need to place a coverslip on the stained smear.

Acid-Fast staining by hot and cold method

A few species of bacteria in the genera Mycobacterium and Nocardia, and the parasite Cryptosporidium do not readily stain with simple stains. However, these microorganisms can be stained by heating them with carbolfuchsin. The heat drives the stain into the cells. Once the microorganisms have taken up the carbolfuchsin, they are not easily decolorized by acid-alcohol, and hence are termed acid-fast. This acid-fastness is due to the high lipid content (mycolic acid) in the cell wall of these microorganisms.

Ziehl-Neelsen staining, also known as acid-fast or hot stain method was developed by Franz Ziehl, a German bacteriologist, and Friedrich Neelsen, a German pathologist, in the late 1800s. It is a differential staining technique used to identify acid-fast organisms, mainly Mycobacterium. Acid-fast microorganisms retain carbolfuchsin and appear red on staining. Microorganisms that are not acid-fast, termed non-acid-fast, will appear blue or brown due to the counterstaining with methylene blue after they have been decolorized by the acid-alcohol.


A) Hot staining Method

1. Prepare a smear consisting of a mixture of E. Coli and M. smegmatis.
2. Allow the smear to air dry and then heat-fix.
3. Place the slide on a hot plate that is within a chemical hood (with the exhaust fan on), and cover the smear with a piece of paper toweling that has been cut to the same size as the microscope slide. Saturate the paper with Ziehl‘s carbolfuchsin. Heat it for 3 to 5 minutes. Do not allow the slide to dry out, and avoid excess flooding. Also, prevent boiling by adjusting the temperature.
4. Remove the slide, let it cool, and rinse with water for 30 seconds.
5. Decolorize by adding acid-alcohol drop by drop. This requires 10 to 30. Rinse with water for 5 seconds.
7. Counter stain with alkaline methylene blue for about 2 minutes.
8. Rinse with water for 30 seconds.
9. Blot dry with bibulous paper.
10. Examine the slide under oil immersion

B) Cold staining Method

The cold staining method is a modification of Ziehl-Neelsen procedure. This method employs a wetting agent (Tergitol No. 4 or Triton X 100) rather than heat to ensure stain penetration is known as the Kinyoun staining procedure (developed by Joseph Kinyoun, German bacteriologist, in the early 1900s).

1. Heat-fix the smear.
2. Stain the smear for 5 minutes with carbolfuchsin prepared with Tergitol No. 7 (heat is not necessary).
3. Decolorize with acid-alcohol and then wash with tap water. Repeat this step until no more color runs off the slide.
4. Counterstain with alkaline methylene blue for 2 minutes. Wash and blot dry.
Examine under oil. Acid-fast organisms stain red; the background and other organisms stain blue.