Branching Gram-Positive Bacilli: Introduction, Principle of Gram stain, Procedure and Result Interpretation

Branching gram-positive bacilli

 Introduction of Gram-Positive Bacilli

Branching gram-positive bacilli in Gram stain as shown above image. The possibility of organisms in a class of actinomycetes and the genus of this class are Actinomyces, Actinomadura, Nocardia, Streptomyces, Rothia, Arcanobacterium, etc.

Gram stain is a differential stain and therefore it uses to differentiate Gram-positive and Gram-negative bacteria. It was devised originally by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria in his laboratory.

Principle of Gram Stain

The reaction is dependent on the permeability of the bacterial cell wall and cytoplasmic membrane, to the dye–iodine complex. In Gram-positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. The alcohol /acetone mixture which acts as a decolorizing agent causes dehydration of the multi-layered peptidoglycan of the cell wall. This causes a decrease in the space between the molecules causing the cell wall to trap the crystal violet iodine complex within the cell. Hence the Gram-positive bacteria do not get decolorized and retain primary dye appearing violet.

Also, Gram-positive bacteria have more acidic protoplasm and hence bind to the basic dye more firmly. In the case of Gram-negative bacteria, the alcohol, being a lipid solvent, dissolves the outer lipopolysaccharide membrane of the cell wall and also damages the cytoplasmic membrane to which the peptidoglycan attaches. As a result, the dye-iodine complex does not retain within the cell and permeates out of it during the process of decolonization. Hence, when a counterstain uses, they take up the color of the stain and appear pink.

Requirements for Gram Stain of Branching Gram-Positive Bacilli

a) Compound light microscope

b) Reagents and glasswares

  • Bunsen flame
  • Wire loop
  • Clean grease-free slides
  • Marker pen
  • Crystal violet (Basic dye)
  • Gram’s iodine(mordant)
  • 95% ethanol (decolorizing agent)
  • 1% safranin or dilute carbol fuchsin or neutral red

c) Quality control strains

Positive Control (PC) : Staphylococcus aureus (ATCC 25923)

Negative Control (NC): Escherichia coli (ATCC 25922)

d) Specimen ( 3 days old culture of unidentified organism on blood agar was used for Gram staining.)

Preparation of Bacterial Smear for Branching Gram-Positive Bacilli

  • Take a clean, and grease-free slide for making a smear.
  • Take a loopful of 0.85% saline i. e. physiological saline and place it on the Center of the slide.
  • With a straight wire touch the surface of a well-isolated colony from the solid media and emulsify in the saline drop forming a thin film.
  • Allow the smear to air dry.
  • Heat fix the smear while holding the slide at one end, and by quickly passing the smear over the flame of the Bunsen burner two to three times.


  1. Cover the smear with crystal violet and allow it to stand for one minute.
  2. Rinse the smear gently under tap water.
  3. Cover the smear with Gram’s iodine and allow it to stand for one minute.
  4. Rinse smear again gently under tap water.
  5. Decolorize the smear with 95% alcohol for 20-30 seconds.
  6. Rinse the smear again gently under tap water.
  7. Cover the smear again gently with safranin for one minute.
  8. Rinse the smear again gently under tap water and air dry it.
  9. Observe the smear first under the low power (10X) objective, and then under the oil immersion (100X) objective.

Observation of branching gram-positive bacilli

Positive Control:   violet color, round in shape in single, pairs and cluster

Test: Purple color, coccoid  and pleomorphic forms to branched filament

Negative Control: red in color and rod in shape

Result and Interpretation of Gram staining

Gram-positive: purple or violet color

Gram-negative: Pink or red in color

Cocci: round in shape

Bacilli: rod in shape

Positive Control(PC): Gram-positive cocci in single, pairs and cluster

Test: Gram-positive coccoid  and pleomorphic forms to branched filament

Negative Control(NC): Gram-negative bacilli as shown above image.

Further Readings

  1. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  2.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
  3.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  4. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  5. Clinical Microbiology Procedure Handbook Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
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