Gram Staining procedure and Interpretation

Gram Staining This is a technique used for the differentiation of Gram-positive and Gram-negative bacteria and is usually almost  the first test performed for the identification of bacteria.

Hans Christian Gram
Hans Christian Gram

The Gram staining method, was named after the Danish bacteriologist who originally devised it in 1882 (published 1884), Hans Christian Gram and is one of the most important staining techniques in microbiology.

Differences between Gram positive and Gram Negative bacteria cell wall


Gram-positive bacteria cell walls are totally different from those Gram-negative that is Gram-positive bacteria cell walls has a thick layer of peptidoglycan associated with teichoic acids while  in Gram-negative bacteria, their cell walls is made up of a thin layer of peptidoglycan associated with a lipopolysaccharide and lipoprotein. This structural difference between their cell walls results in a different ability to retain certain dyes and a different ability to resist alcoholic decolorization during the process of gram staining.

Reagent used in Gram Staining

  • Crystal violet solution
  • Gram’s iodine (Lugol iodine solution)
  • Alcohol-acetone solution
  • Safranin OR Basic fuchsin (Carbol fuchsin)

Steps involved in Gram staining

  • Applying a primary stain(crystal violet),
  • Addition of a mordant (Gram’s iodine)
  • Rapid decolorization with alcohol or acetone
  • Counterstaining with safranin, neutral red or basic fuchsin.

Gram staining Procedure

  • Dry and heat-fix the smear then cover it with crystal violet solution then allow it to react for 1 minute then rinse with distilled water.
  • Add Gram’s iodine (Lugol solution) and allow to react for 1 minute, pour off the lugol solution and decolorize with Alcohol-acetone solution for at least 5 seconds.
  • Wait few seconds then rinse with distilled water.
  • Counter stain the slide with safranin or carbol fuchsin and allow to react for atleast 1 minute then rinse with distilled water and dry the smear.
Also read  LACTOBACILLI MRS AGAR Principle, Composition, Preparation and Colonies Characteristics
Gram staining procedure
Gram staining procedure

It should be noted that, exceeding the decolorization time could result in the crystal violet stain removal from both Gram positive and negative bacteria.

Gram staining Microscopic Observation

Gram Negative VS Gram positive
Gram Negative VS Gram positive
  • Gram-positive bacteria pink up the crystal violet color due to their thick peptidoglycan and are not decolorized by acetone hence stained Purple.
  • Gram-negative bacteria loses its purple color after decolorization due to a thin peptidoglycan unable to retain the primary stain hence pick up the counter stain and are stained Pink.

Gram-variable bacteria

Some microorganisms with Gram-positive type of cell wall may not express this character in particular circumstances (growth conditions, staining) but additional test can be conducted to determine their classification like the KOH String Test  and others

Reference: tgw1916.net

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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