Staining techniques - Pharmaceutical Microbiology Third Semester PDF Notes

Identification of bacteria – Staining techniques

Intended learning objectives

At the end of this lecture, the student will be able to:

       Classify the staining techniques

       Explain the principle and procedure involved morphological and gram staining

       Outline the significance of staining in identification of bacteria

       Explain the principle and procedure of acid fast staining

       Explain the significance and principle of structural staining

Need for staining

       Most microorganisms appear almost colourless when viewed through a standard light microscope

       Hence must be fixed and stained to

      Increase visibility

      Accentuate specific morphological features

      Preserve them for future study

       Staining simply means colouring the microorganisms with a dye that emphasizes certain structures

Basic staining procedure

Step 1: Smear preparation

       A thin film of material containing the microorganisms is spread over the surface of the slide. This film, called a smear.

       It is allowed to air dry.

Step 2: Fixation

       By passing it through the flame of a bunsen burner several times, smear side up, or by covering the slide with methyl alcohol for one minute.

       Fixing simultaneously kills the microorganisms and fixes them to the slide.

       It also preserves various parts of microbes in their natural state with only minimal distortion

Step 3: Staining

       Stain is applied and then washed off with water

       Slide is blotted with absorbent paper.

       The stained microorganisms are now ready for microscopic examination.


       Stains are salts composed of a positive and a negative ion,

       The colored ion is known as the chromophore.


1.       Positive ion à Colored chromophore àBasic stain

2.       Negative ion à Colored chromophore àAcidic stain

  • Bacteria are slightly negatively charged at pH 7.
  • Thus, the colored positive ion in a basic dye is attracted to the negatively charged bacterial cell.
  • Basic dyes include crystal violet, methylene blue, malachite green and safranin
  • Acidic dyes are not attracted to most types of bacteria 
  • The dye's negative ions are repelled by the negatively charged bacterial surface
  • The dye  colors the background instead
  • Preparing colorless bacteria against a colored background is called negative staining.
  • Examples of acidic dyes are eosin, acid fuchsin, and nigrosin

Negative staining

       Valuable for observing overall cell shapes, sizes, and capsules

       The cells are made highly visible against a contrasting dark background

       Distortions of cell size and shape are minimized because fixing is not necessary and the cells do not pick up the stain

Simple staining

       A simple stain is an aqueous or alcohol solution of a single basic dye

       The primary purpose of a simple stain is to highlight the entire microorganism so that cellular shapes and basic structures are visible

       The stain is applied to the fixed smear for a certain length of lime and then washed off

       The slide is dried and examined

       Simple stains commonly used in the laboratory - methylene blue, carbolfuchsin, crystal violet, and safranin.


       Mordant - A chemical that intensifies the stain

Functions of mordant

       To  increase the affinity of a stain for a biological specimen

       To coat a structure (such as a flagellum) to make it thicker and easier to see after it is stained with a dye.

Differential stains

       Differential stains react differently with different kinds of bacteria 

       Can be used to distinguish them

       The differential stains most frequently used for bacteria are the Gram stain and the acid-fast stain

Gram stain

       Gram stain was developed in 1884 by the Danish bacteriologist Hans Christian Gram

       Most useful staining procedures - classifies bacteria into two large groups: gram-positive and gram-negative.

Ø  Step 1: Primary stain

Ø  Step 2: Mordant

Ø  Step 3: Decolorization

Ø  Step 4: Counter stain

       The purple dye and the iodine combine in the cytoplasm of each bacterium and color it dark violet or purple.

       Bacteria that retain this color after the alcohol has attempted to decolorize them are classified as gram-positive

       Because gram-positive bacteria retain the original purple stain, they are not affected by the safranin counterstain

       Bacteria that lose the dark violet or purple color after decolorization are classified as gram negative

       Because gram-negative bacteria are colorless after the alcohol wash, they are no longer visible.

       This is why the basic dye safranin is applied; it turns the gram-negative bacteria pink.

       Stains such as safranin that have a contrasting color to the primary stain are called counterstains

Principle of Gram stain

       Different kinds of bacteria react differently to the Gram stain

       Structural differences in their cell walls affect the retention or escape of a combination of crystal violet and iodine, called the crystal violet- iodine (CV-I)complex

       Gram-positive bacteria have a thicker peptidoglycan cell wall than gram-negative bacteria

       Gram- negative bacteria contain a layer of lipopolysaccharide (lipids and polysaccharides) as part of their cell wall

Crystal violet + Iodine

       Enters Gram positive cell wall à Peptidoglycan layer retains   CV-I during alcohol decolorization à Gram-positive cells retain the color of the crystal violet dye

       Enters Gram negative cell wall à Alcohol wash disrupts the outer lipopolysaccharide layer à CV- I complex is washed ou tthrough the thin layer of peptidoglycan à Gram negative Cells are colorless à Turn pink upon safranin staining

Clinical significance of Gram staining

       Gram reaction of a bacterium can provide valuable information for the treatment of disease.

       Gram-positive bacteria tend to be killed easily by penicillins and cephalosporins.

       Gram-negative bacteria are generally more resistant because the antibiotics cannot penetrate the lipopolysaccharide layer.


Acid-Fast Stain

       Used to identify all bacteria in the genus Mycobacterium and Nocardia

      Mycobacterium tuberculosis, the causative agent of tuberculosis

      Mycobacterium leprae the causative agent of leprosy

       Acid-fast stain binds strongly only to bacteria that have a waxy material in their cell walls

Acid fast staining Procedure

  1. The red dye carbol fuchsin is applied to a fixed smear
  2. The slide is gently heated for several minutes ((Heating enhances penetration and retention of the dye).

       The slide is cooled and washed with water.

       Smear is next treated with acid-alcohol, a decolorizer, which removes the red stain from bacteria that are not    acid -fast.

       The acid-fast microorganisms retain the red color because the carbol fuchsin is more soluble in the cell wall lipids than in the acid-alcohol

Acid fast staining Principle

       The smear is then stained with a methylene blue counterstain.

       Non acid-fast cells appear blue after application of the counterstain

Special stains

       Special stains are used to color and isolate specific parts of microorganisms, such as endospores and flagella, and to reveal the presence of capsules.

  1. Negative Staining for Capsules
  2. Endospore (Spore) Staining
  3. Flagella Staining

Negative Staining for Capsules

       Many microorganisms contain a gelatinous covering called a capsule

       Demonstrating the presence of a capsule is a means of determining the organism's virulence, the degree to which a pathogen can cause disease.

       Step 1: Mix the bacteria in a solution containing a fine colloidal suspension of colored particles (usually India ink or nigrosin)

       Step 2: Provide a contrasting background and then stain the bacteria with a simple stain, such as safranin

       Due to their chemical composition, capsules do not accept most biological dyes, such as safranin, and thus appear as halos surrounding each stained bacterial cell.

Endospore (Spore) Staining

       Endospore is a special resistant, dormant structure formed within a cell

       Protects a bacterium from adverse environmental conditions

       Endospores cannot be stained by ordinary methods

       The dyes do not penetrate the wall of the endospore

Schaeffer- Fulton endospore stain

       Malachite green, the primary stain, is applied to a heat-fixed smear

       Heated to steaming for about 5 minutes (i.e, malachite green permeate the spore wall)

       Washed for about 30 seconds with water to remove the malachite green from all of the cells parts except the endospores.

       Safranin, a counterstain, is applied to the smear to stain portions of the cell other than endospores.

       The endospores appear green within red or pink cells

Acid-Fast Staining

       Note: In Gram Staining and AFB Staining we use Alcohol or Acid Alcohol or Acid as a decolorizer but in spore staining  water is sufficient ( to be used as decolorizer) because:

Ø  malachite green dye is water-soluble and does not adhere well to the cell wall

Ø  vegetative cells have been disrupted by heat,

Ø  because of these reasons, the malachite green rinses easily from the vegetative cells

Flagella staining

       Bacterial flagella (singular: flagellum) are structures of locomotion too small to be seen with a light microscope without staining.

       A tedious and delicate staining procedure uses a mordant and the stain carbol fuchsin to build up the diameters of the flagella until they become visible under the light microscope


       Morphological stains help in identifying the cell size, shape and structure

       Simple stains color the cells

       Negative staining color the background

       Gram staining differentiates between gram positive and gram negative cells

       The difference in gram staining is due to difference in cell wall composition

       Stages of  gram staining –

       Primary stain




       Acid fast staining is used to identify bacteria containing mycolic acid in cell wall – belonging to genus Mycobacterium and Nocardia

       It differentiates between acid fast and non-acid fast organisms

       Special structural stains like capsule spore and flagella staining, help in visualization of these bacterial structures

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