GRAM STAINING

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Edited by Dr.T.V.Rao MD Importance and Limitations of Gram Staining in Diagnostic Microbiology
Majority of the Microbiology Laboratories do a culture work for Bacterial identification from several specimens arise from the Hospitals. Gram staining is a common traditional procedure and an age old procedure since Christian Grams contribution in 18th Century, for Bacterial studies. The differentiation of bacteria into either the gram-positive or the gram-negative group is fundamental to most bacterial identification systems. This task is usually accomplished through the use of Grams Staining Method. Unfortunately, the gram stain methodology is complex and prone to error. The technique is used as a tool for the differentiation of Gram-positive and Gram-negative bacteria, as a first step to determine the identity of a particular bacterial sample. The Gram stain is not an infallible procedure for diagnosis, identification, or phylogeny. However it is of extremely limited use when saprophytes and commensals are associated with careless specimen collection AND PRECESSING IN OUR Laboratories however it is a procedure which helps when Gram stains are performed on body fluids, CSF or biopsy when infection is suspected. It yields results much more quickly than culture, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis. Many Grams stains from specimens with Polymicrobial flora are fallacious as it is in Diabetic foot, Sputum, faecal, and urinary specimens. Many juniors and even the Seniors Microbiologists believe the first impressions and come to hasty conclusion in advising the therapeutic options. My experience proves that when we process THE BACTERIA from the Culture plates, by Macroscopic observation many of our conclusions may not be true, and have little determination and patience to think all you see and define by naked eye ( Macroscopic Observations ) may not prove correct when we really do a Grams staining, many Micrococci, Diptheroids, and Candida spp and other normal flora mimic as pathogenic isolates and processed with confusing uncharacterised Biochemical reaction, and tested for Antibiograms, this is an area where the Grams staining plays wonders to enlighten us. Problems with Grams Method have led to a search for other tests that correlate with the cell wall structure of the gram-positive and the gram-negative cells. Several improvements/alternatives to the classical gram stain have appeared in the literature. Many conflicts in our Laboratories between members of the staff and technicians are due to ignorance as when we have a little interest to go in a Methodical and Scientific way. However Microbes prosper with advantage by our ignorance in Diagnosis and Treatment. D.S. Murthy from Osmania Medical College, Hyderabad India true sir. But, at the same time, Gram stain may provide the clues, which we have to confirm by further processing. It is only a preliminary test or the first step but not a conclusive evidence of the infecting agent. While the technique is error prone, we can reduce the errors by standardizing the protocol in the laboratory with different specimens and periodic quality assessment. Most errors are because of overconfidence (and resulting negligence) than because of the technique per se.

Sridhar Rao JJM Medical College, Davangere India Yes sir, Gram stain is the most fundamental
step in the laboratory diagnosis of bacterial infectious disease. The smear from the clinical specimen not only provides useful information on the likely aetiology but also reveals the quality of the specimen. Gram stained smear examination (in my opinion) is a highly useful preliminary examination that MUST be followed on all exudate specimens (irrespective of requisition). This will help the identification and interpretation of culture results. Unfortunately, it is overlooked in many laboratories. Sir, as you have rightly pointed out, it helps in getting rid of misidentification of organisms by their colony morphology alone. Many times organisms identified as Staphylococcus turn out to be something else on Gram stained smear examination. This age-old staining technique certainly has its limitations, but under an experienced microbiologist, it is still an indispensable tool. T.V.Rao Travancore Medical College, Kollam India I request all the Postgraduate students to take greater interest in doing and reading Gram's stains by themselves, Many Case reports published by people who are keenly interested in Microscopic observations. As we are all aware we have no modern facilities in Medical colleges, Good observation can definitely give many clues in diagnosing Bacterial infections Vijendra Kwale yes Post Graduate in MD Microbiology Osmania Medial College Hyderabad Sir. We face this very frequently - colony morphologies very similar to Staphylococcus But it turns out to be something else in the smear! , many times Diptheroids/micro & sometimes Candida too when we cross check with the direct gram stain of sample, it does also help rule out contamination done by us during plating. But as a PG, i must admit sir, that its very difficult at this stage for me to make out organisms properly in directs especially when they are scanty in the sample still, we keep trying to search more carefully and differentiate from things other than the organisms .. T.V.Rao Travancore Medical College, Kollam Kerala India Very Nice Kawle about your writing on Gram staining, At least in 10 - 20 % of predictions by Macroscopic observation may not be true when we see under Microscope, I have seen at many departments these matters are left to Technicians and Juniors Microbiologists Many organisms which are just contaminants and normal flora are processed as assumed pathogens giving a conflicting Biochemical reaction then inexperienced name them as rare isolates for Academic presentation. Please remember human body contains 10 times more normal floral bacteria than our own cells. I request the message known to many this is the reason where Bacteriology reporting went wrong in many Microbiology Departments. I wish that you contribute more good ideas for the benefit of many with good wishes Email; doctortvrao@gmail.com