Identification of Bacteria Using Staining Techniques

Staining Techniques:

Stain: A stain is a substance that adheres to a cell which gives cell color because bacteria are slightly negative charge at pH 7.0. The presence of color gives the cells significant contrast so that they are prominently visible. Different strains have different affinities for different organisms. Hence, the staining technique enhances the contrast in the microscopic image. Generally, crystal violet, Gram’s Iodine, acetone, and safranin are used as stains. The table indicates the color changes for different bacteria.

Table 1: Different stains for different bacteria

Crystal violetPurple or bluePurple or blue
Gram’s iodinePurple or bluePurple or blue
Acetone or AlcoholPurple or blueColorless
SafraninPurple or blueRed or Pink


  • Bacteria have nearly the same refractive index as water so when they are observed under the microscope they are invisible to the naked eye. Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope.
  • Staining is the primary recognition of bacteria.
  • It is of importance in the diagnosis of infectious diseases in culture and directly from clinical samples.

Types of Staining Technique:

Classification of bacterial staining
Flowchart 1: Classification of bacterial staining

Simple Staining

Simple staining is carried out to visualize bacteria and to compare morphological shapes and arrangements of the bacterial cell. In this technique, the bacterial smear is stained with a single basic dye such as crystal violet, safranin, methylene blue, etc.

Principle: These single basic dyes will give up a hydroxyl ion or accept a hydrogen ion that leaves the stain positively charged. Since the surface of bacteria is negatively charged so these charges are strongly bound to the cell surface (cell wall). Then the cells are visible against a light background.

Steps (Fig.1):

1. Clean and dry microscope slides thoroughly.

2. Flame the surface on which the smear is to be spread.

3. Flame the inoculating loop.

4. Transfer a loop full of tap water to the flamed slide surface.

5. Reflame the loop making sure the entire length of the wire that will enter the tube has been heated to redness.

6. Remove the tube cap with the fingers of the hand holding the loop.

7. Flame the tube mouth.

8. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over an area the size of a dime. It should be a thin, even smear.

9. Reflame the inoculating loop to redness including the entire length that entered the tube. 10. Allow the smear to dry thoroughly.

11. Fix the smear cautiously bypassing the underside of the slide through the burner flame two or three times.

12. Stain the smear by flooding it with one of the staining solutions and allowing it to remain covered with the stain for the time designated below.

Methylene blue – 1 minute

Crystal violet – 30 seconds

Carbol fuchsin – 20 seconds

During the staining, the slide may be placed on the rack or held in the fingers.

13. At the end of the designated time rinse off the excess stain with gently running tap water. Rinse thoroughly.

14. Wipe the back of the slide and blot the stained surface with a paper towel.

15. Place the stained smear on the microscope stage smear side up and focus the smear using the 10X objective.

16. Choose an area of the smear in which the cells are well spread in a monolayer. Center the area to be studied, apply the oil directly to the smear, and focus the smear under oil with the 100X objective.

Simple staining
Fig.1: Simple staining

Observation: Spherical-shaped bacteria coccus is turned blue color whereas rod-shaped bacteria, bacilli turn a red color.

Use: To study morphology and arrangement of bacteria cells.

Gram’s Staining

It is a type of differential staining where two or more stains are used to differentiate between types of bacteria. This staining technique was discovered by Hans Christian Gram in the year 1884.


  • It is an important test for the rapid diagnosis of infectious agents.
  • It differentiates types of bacteria whether Gram-positive or Gram-negative.
  • It helps in the study of the morphology of bacteria.
  • It helps in finding the evidence of capsule, spores, pus cells, epithelial cells, Yeast cells, etc.
  • To understand how the Gram stain reaction affects Gram-positive and Gram-negative bacteria based on the biochemical and structural differences of their cell walls.


1. Crystal violet: It is the primary stain which is used as a simple stain because it dyes the cell wall of bacteria cell.

2. Gram’s Iodine: It acts as a mordant which helps to fix the primary dye to the cell wall.

3. Decolourizer: It is used to remove the stain of primary dye (crystal violet) from the Gram-negative bacterial cell wall. Decolourizer is composed of organic solvents like acetone, ethanol, or in combinations.

4. Safranin: Finally, a counterstain is applied to stain those cells that have lost their primary stain (especially Gram-negative bacteria) as a result of decolorization.

The procedure is based on the ability of microorganisms to retain the color of the stains used during the gram stain reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary stain, purple.

Gram-positive bacteria are not decolorized by alcohol and will remain purple. After the decolorization step, a counterstain is used to impart a pink color to the decolorized gram-negative organisms.

Gram-positive bacteria: Stain dark purple due to retaining the primary dye called Crystal violet in the cell wall.

Example: Staphylococcus aureus.

Gram-negative bacteria: Stain red or pink due to retaining the counterstaining dye called Safranin.

Example: Escherichia coli.

There are some differences between Gram-positive and Gram-negative bacteria which are listed in Table 2.

Table 2: Differences between Gram-Positive and Gram-negative bacteria

Gram-Positive bacteriaGram-Negative bacteria
1. They retain crystal violet dye and stain dark violet or purple, they remain colored blue or purple with gram stain when washed with absolute alcohol and water.1. They are decolorized to accept counterstains such as Safranin or Fuchsine. They stain red or pink and they do not retain the Gram stain when washed with absolute alcohol and acetone.
2. Peptidoglycan is multilayered thick.2. Peptidoglycan is single-layered thin.
3. Outer membrane is absent.3. Outer membrane is present.
4. Periplasmic space is absent.4. Periplasmic space is present.
5. Teicholic acids are present.5. Teicholic acids are absent.
6. Lipid and lipoprotein content is low.6. Lipid and lipoprotein content is high.
7. They have high resistance to physical disruption and drying.7. They have a low resistance to physical disruption and drying.
8. Cell wall is 100-120 Å thick; single layer. Lipid content of the cell wall is low, whereas Murein content is 70-80%.8. Cell wall is 70-120 Å thick; two-layered. Lipid content is 20-30% (high), Murein content is 10-20%.
9. More susceptible to antibiotics.9. More resistant to antibiotics.

Acid Fast Staining

The acid-fast staining method is one of the differential staining techniques. This technique was first developed by Ziehl and later on modified by Neelsen and hence this method is also known as Ziehl-Neelsen staining techniques. In the year 1883, Neelsen used Ziehl’s carbol-fuchsin and heated then decolorized it with acid alcohol followed by counterstained with methylene blue. Thus Ziehl-Neelsen staining technique was developed as acid-fast staining (Table 3).


  • To differentiate bacteria into the acid-fast group and non-acid fast groups.
  • It is used for those microorganisms which are not staining by simple or Gram staining method, particularly the member of genus Mycobacterium which is resistant to other stains.


The smear is prepared and stained with carbol fuchsin which solubilizes the lipoidal material present in the Mycobacterial cell wall and by heating, carbol fuchsin further penetrates through the lipoidal wall and enters into the cytoplasm, resulting in all cells appearing red. Then the smear is decolorized with 3% HCl in 95% alcohol (decolourizing agent) but the acid-fast cells are resistant due to the presence of a large amount of lipoidal material in their cell wall that prevents the penetration of the decolourizing solution. Then the smear is stained with a counterstain, methylene blue, and only decolorized cells are absorb and appear blue while acid-fast cells retain the red color (Table 3).

Table 3: Cell colors with various dyes

Cell colors with various dyes

Procedure (Fig.2):

1. Prepare bacterial smear on a clean and grease-free slide, using sterile technique.

2. Allow smear to air dry and then heat fix.

3. Cover the smear with carbol fuchsin stain.

4. Heat the stain until vapor just begins to rise about 60°C. Allow the heated stain to remain on the slide for 5 minutes.

5. Wash off the stain with clean water.

6. Cover the smear with 3% v/v acid alcohol for 5 minutes or until the smear is sufficiently decolorized, i.e. pale pink.

7. Wash well with clean water.

8. Cover the smear with malachite green stain for 1–2 minutes, using the longer time when the smear is thin.

9. Wash off the stain with clean water.

10. Wipe the back of the slide clean, and place it in a draining rack for the smear to air dry.

11. Examine the smear microscopically, using the 100 X oil immersion objective.

Acid-fast: Mycobacterium tuberculosis, Mycobacterium smegmatis.

Non-Mycobacterial bacteria: Nocardia; Coccidian Parasites: Cryptosporidium.

A. Non-acid-fast Blue color; B. Bright red to intensive purple, Red, straight, or slightly curved rods
Fig.2: A. Non-acid-fast: Blue color; B. Bright red to intensive purple, Red, straight, or slightly curved rods
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