Variations in Gram Staining Results

Last updated on June 21st, 2021

I hope you all are well aware of the Gram staining, its protocol (procedure), principles, and interpretation. In this blog post,  I am writing other important aspects of Gram stain which are less discussed/ memorized by the naive students, but which are important in troubleshooting Gram staining technique in the diagnostic laboratories.

Gram stain procedure

We all know that Gram stain is the most important staining technique for identifying bacteria using light microscopy but Gram staining techniques also have some limitations.

Sometimes, you may fail to see the organism in Gram Stain smear but the same clinical specimen may yield organisms when cultured. So, let’s find why this happens;

  • Limitation of Gram Stain: Mycobacteria stain weakly with gram stain and bacteria such as Mycoplasma, Rickettsiae, Chlamydiae do not take up the dyes used in Gram stain or are too small to be seen with light microscopy.
  • Sensitivity of Gram Staining Technique: To be visible on a slide, organisms that stain by the Gram method must be present in concentrations of about 10^4 to 10^5 organisms per milliliter of uncentrifuged fluid.
  • Adequacy of Gram Staining method: After performing a gram stain, the Microbiologist/Technician should first determine whether the Gram stain is adequate. In an appropriately stained specimen, the nuclei of neutrophils are red. If the nuclei are blue, the decolorization is insufficient.

Variations in Gram Reaction

  1. Gram-positive bacteria may lose their ability to retain crystal violet and stain Gram negatively for the following reasons:
    • Cell wall damage of bacteria due to antibiotic therapy or excessive heat fixation of the smear.
    • Over- decolorization of the smear
    • Use of an Iodine solution which is too old, i.e. yellow instead of brown in color (always store in a brown glass or other light opaque containers).
    • Smear has been prepared from an old culture.
  2. When smear is too thick, Gram-negative bacteria may not be fully decolorized during decolorization steps and appear as Gram-positive bacteria.

Pitfalls in the Interpretation of Gram’s Stains

OrganismClassic PresentationVariant PresentationComments
Streptococccus pneumoniaeGram-positive, lancet-shaped, diplococciElongated cocci, resembling short bacilliMay be misinterpreted as mixed organisms; over-decolorized cells may be mistaken for gram-negative coccobacilli
Acinetobacter spp.Gram-negative coccobacilliGram-negative cocci; gram-variable staining is commonMay be mistaken for Neisseria spp. and reported as gram-negative cocci; search the smear to find some organisms that demonstrate elongated forms, which are not seen in Neisseria.
Clostridium perfringensBoxcar-shaped gram positive bacilliGram-positive cocciMay be mistaken for Streptococcus pneumoniae and reported as gram-positive cocci; in addition to a coccal form, cells retain crystal violet tenaciously during decolorization
Clostridium perfringensBoxcar-shaped gram positive bacilliGram-variable or Gram –negative bacilliMaybe mistaken for gram-negative bacilli; the boxcar shape is a clue that the organism is gram-positive; other Clostridia and Bacillus spp. May also appear similar.
Yeast, especially Cryptococcus neoformansGram-positive round or oval cells with buddingGram-variable cellsMay be mistaken for artifacts; size and shape distinguish them from bacteria

References and further reading

About Acharya Tankeshwar 476 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.