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 is less discussed/ memorized by the naive students, but which are important in troubleshooting Gram staining technique in the diagnostic laboratories.
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, 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
- 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.
- 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
(Source: Koneman’s)
| Organism | Classic Presentation | Variant Presentation | Comments |
| Streptococccus pneumoniae | Gram-positive, lancet-shaped, diplococci | Elongated cocci, resembling short bacilli | May be misinterpreted as mixed organisms; over-decolorized cells may be mistaken for gram-negative coccobacilli |
| Acinetobacter spp. | Gram-negative coccobacilli | Gram-negative cocci; gram-variable staining is common | May 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 perfringens | Boxcar-shaped gram positive bacilli | Gram-positive cocci | May 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 perfringens | Boxcar-shaped gram positive bacilli | Gram-variable or Gram –negative bacilli | May be 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 neoformans | Gram-positive round or oval cells with budding | Gram-variable cells | May be mistaken for artifacts; size and shape distinguish them from bacteria |
I have a question. We do a manual gram stain on FFPE tissue. It is a kit from polyscientific. Every once in awhile, the negatives get over differentiated and we have to restain. What are the pros and cons of taking the specimen back to water and starting from the beginning versus taking the specimen back to the step before basic fuschin (ether-acetone) and restaining just the negatives because the positives are good. The positives never differentiate out and they are stained with crystal violet.
Marie Land