Gram staining

Perform Gram staining of the given bacterial smear

 

Objective: To perform Gram staining on a bacterial sample and differentiate between Gram-positive and Gram-negative bacteria based on color.

Theory: Gram staining is a widely used differential staining technique in microbiology that helps to classify bacteria into two major groups: Gram-positive and Gram-negative. In 1884, a Danish pathologist, Hans Christian Gram, discovered a method of staining bacteria using two dyes in sequence, each of a different color. he found that bacteria fall into two groups. The first group retains the color of the primary dye: crystal violet (these are called gram-positive). The second group loses the first dye when washed in a decolorizing solution but then takes on the color of the second dye, a counterstain, such as safranin  (these are called gram-negative). An iodine solution is used as a mordant (a chemical that fixes a dye in or on a substance by combining with the dye to form an insoluble compound) for the first stain.

The Gram stain is based on the ability of bacterial cell walls to retain the crystal violet dye during solvent treatment (decolorization). Gram-positive bacteria have a thick peptidoglycan layer that traps the crystal violet-iodine complex, resisting decolorization. In contrast, Gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane, allowing the dye to wash out easily during decolorization and take up the counterstain

 Requirements:

• Bacterial sample
• Crystal violet (primary stain)
• Gram’s iodine (mordant)
• 95% alcohol or acetone-alcohol (decolorizer)
• Safranin (counterstain)
• Slides
• Distilled water
• Bunsen burner
• Inoculating loop
• Microscope
• Blotting paper

Procedure:

1.     Prepare a heat-fixed bacterial smear on a clean glass slide.

2.     Flood the smear with crystal violet and leave it for 1 minute.

3.     Rinse gently with distilled water.

4.     Flood the smear with Gram’s iodine and leave for 1 minute.

5.     Rinse gently with distilled water.

6.     Decolorize with 95% alcohol by adding drops until no more purple flows off (about 10-20 seconds).

7.     Immediately rinse with distilled water.

8.     Counterstain with safranin for 1 minute.

9.     Rinse with distilled water and blot dry with blotting paper.

10.  Examine under a microscope first using 10x objective, 40x and then 100x oil immersion lens.

 Observation:

S.N	Reagents used	Shape of bacteria	Color of bacteria	Inference

 

 

Result:

Gram-positive bacteria: Appear purple due to the retention of crystal violet stain. Gram-negative bacteria: Appear pink or red due to taking up the safranin counterstain.

 

Discussion:

The Gram staining procedure differentiates bacteria based on structural differences in their cell walls.

• Gram-positive bacteria have a thick peptidoglycan wall that traps the crystal violet-iodine complex, thus retaining the purple color even after alcohol decolorization.
• Gram-negative bacteria have a thin peptidoglycan layer and a high lipid content in their outer membrane; the alcohol dissolves the lipids and removes the primary stain, allowing the cells to take up the pink counterstain.
  This method is a preliminary test widely used in clinical laboratories to help identify bacterial pathogens and to guide initial antibiotic therapy.

Conclusion:

In this experiment, Gram staining was successfully performed on a bacterial sample. The staining technique allowed the differentiation of bacteria into Gram-positive and Gram-negative groups based on their cell wall properties. Gram-positive bacteria appeared purple, indicating their ability to retain the crystal violet stain, while Gram-negative bacteria appeared pink or red due to taking up the safranin counterstain.

Precautions:

• Prepare thin and even smears to avoid false results.
• Do not overheat during heat-fixing, as it can distort bacterial shapes.
• Proper timing for each staining and decolorizing step is crucial.
• Use fresh reagents for reliable results.
• Wash gently with water to prevent washing away the smear.
• Handle the microscope and glass slides carefully to avoid damage or accidents.

References:

1. Cappuccino, J. G., & Sherman, N. (2014). Microbiology: A Laboratory Manual (10th ed.). Pearson.
2. Pelczar, M.J., Chan, E.C.S., & Krieg, N.R. (1993). Microbiology: Concepts and Applications. McGraw-Hill.