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GRAM STAINING AND CLINICAL UTILITY Dr.T.V.Rao MD Gram staining is one of the oldest procedure in Microbiology which continues to be practised by wise and young alike yet many of us have not perfected with a definitive opinion and difference of opinion by different observers leading to many conflicts in the work place We teach many of our Undergraduate students in Medicine as the basis of diagnostic work for understanding the matters when they become interns or practising physicians. WHAT IS A GRAM STAINING - Gram staining is a bacteriological laboratory technique used to differentiate bacterial species into two large groups based on the physical properties of their cell walls. Many clinicians ask us whether gram stain of the specimen sent us is positive or negative then a question arises then -is-better-for-my-gram-stain-to-be-positive-or-negative but not correct to express without giving further details and expressing the limitations of Gram staining in clinical decision making. Many years of working on the table clarifies many matters including conflicts in observers opinion However the Clinical Microbiologists should be familiar with many scientific facts before giving valued opinion to make clinical decision making. Many test observations and the experiment conducted was based upon the known attributes of two different groups of bacteria those that are gram positive and those that are gram negative. Using a specific staining procedure it is possible to differentiate the two types under a microscope The gram stain method of differentiation is possible because of differences in the cell membrane between the two categories of bacteria The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain purple are said to be Gram-positive whereas those that are decolorized and stain red with carbol fuchsin or safranin are said to be Gram-negative. This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick relatively impermeable wall that resists decolourization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane which can be disrupted by decolourization. Some bacteria have walls of intermediate structure and although they are officially classified as Gram-positives because of their linage they stain in a variable manner. A gram stain continues to guide us on basic bacteriology and no day goes in laboratory without doing this basic procedure as A Gram stain and culture of the material from an infected site are the most commonly performed microbiology tests used to identify the cause of a bacterial infection. Often detecting the presence of bacteria and determining whether an infection is caused by an organism that is Gram-positive or Gram-negative will be sufficient to allow a health practitioner to prescribe treatment with an appropriate antibiotic while waiting for more specific tests such as a culture to be completed. Absence or presence of white blood cells in the Gram stain can help establish that an adequate sample was obtained as white blood cells are frequently present with an infection and presence of neutrophils and lymphocytes express the inflammatory reaction. Many times we forget to read the association of Neutrophils pus cells and epithelial cell as the criteria of the specimen is well flourished with infected material in situ while it is important or biological elements as mould and yeast associated with the specimens as colonizers or infectious elements. WE GET MANY REQEUSTS Gram stain is typically ordered along with a culture when a bacterial or sometimes fungal infection is suspected. It is also usually performed when the result of a culture is positive on a sample of the bacteria grown in the culture. Test results are reported promptly to help guide treatment The Gram stain continues to be more requested opinion when we get the CSF

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Body fluids otherwise sterile it makes a great sense if we observe many fields even finding very few bacteria makes a greater sense in decision making than awaited culture reports after 24 to 48hours NEGATIVE FOR MICROBES MEANS - A negative Gram stain is often reported as "no organism seen." This may mean that there is no bacterial infection present or that there were not enough bacteria present in the sample to be seen with the stain under a microscope and subject the specimens further with culturing on selective simple or enriched media POSITIVE FOR MICROBES MEANS- Positive Gram stain results usually include a description of what was seen on the slide. This typically includes: •Whether the bacteria are Gram-positive purple or Gram-negative pink •Shape — round cocci or rods bacilli •Size relative quantity and/or arrangement of the bacteria if relevant •Whether there are bacteria present within other cells intracellular •Presence of red blood cells or white blood cells Fungi in the form of yeasts or molds may be seen on a Gram stain and are reported. Yeast may appear as single cells that may have buds while molds may appear as a wide variety of plant-like branches called hyphae. Further testing may be performed to identify the specific type Cautious - when reporting many other specimens Gram stains are not helpful when the disease- causing bacteria pathogen cannot be differentiated from normal bacterial flora. For example a Gram stain of a sample from the throat cannot tell the health practitioner what bacteria are causing the infection because both the "commensal bacteria" and the "pathogenic bacteria" look the same under the microscope. The idea stands good in reporting of specimen of stools as we many times cannot differentiate many pathogens and commensal present in plenty WHAT MICROBIOLOGISTS SHOULD DECIDE ON GRAM STAINING the microbiology laboratory must use objective criteria by Gram stain screening for purulent / septic material before inoculation in to culture media. Hence the routine sputum Gram stain is essential to provide meaningful culture report. All the specimens we receive for culture should have a basic evaluation of Gram staining on 1 st day of receiving the specimen in the laboratory to avoid much confusion on the 2 nd day of culture reporting most important specimen for Gram staining continues to be observation of Sputum for Microscopy. Clinicians are interested in rapid simple inexpensive and readily available tests that will assist them in prescribing proper medications for lower respiratory tract infections. Sputum Gram stain served this function in the management of lower respiratory tract infection if the Microbiologists observe the sputum with power to discriminate many uncommon microbes in the sputum can be visualised and more scientific decisions on selecting culture Media as the matters warrant . Ref Use of the Gram stain in microbiology- Research Article Biotechnic Histochemistry Volume 76 Issue 3 2001 Dr.T.V.Rao MD Professor of Microbiology Freelance writer

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