Key Facts about Gram Staining Techniques that you might not know

I hope you all are well aware about the Gram staining techniques, its protocol (procedure), principles, interpretation of gram stained slides. So in this blog post I am writing some thing about Gram Stain which is less discussed or less memorized by most of the students, but which are very useful and worth knowing.

Fig: Gram Staining Technique and result of Gram Stain

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. We know how to perform gram stain but we may not know the procedure we applied is adequate or not. You may fail to recover organism in Gram Stain from clinical specimen but same clinical specimen yield organism when cultured.So lets find why this happens.

Limitation of Gram Stain: Mycobacteria stain weakly with gram stain and bacteria such as mycoplasmas, 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 104 to 105 organisms per milliliter of uncentrifuged fluid.Adequacy of Gram Staining method: After performing 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.