Bacterial Gram Staining (

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Information about Bacterial Gram Staining (

Published on March 20, 2014

Author: PakRose1

Source: Bacterial Gram Staining Hello Guys, we are going to discuss about the Types of bacteria on the basis of their cell wall structure. Ok, Let’s 1st start with… What is Bacteria? The word bacteria is the plural of bacterium. Grammatically the headline should just verbalize "What are bacteria?" The erroneous utilization has been included in the headline to remind readers that it is erroneous - and hopefully avail rectify an increasingly mundane mistake in the English language. Bacteria are diminutive living beings (microorganisms) - they are neither plants nor animals - they belong to a group all by themselves. Bacteria are minuscule single-cell microorganisms, customarily a few micrometres in length that mundanely subsist together in millions. Now, There are 3 different types of Bacteria on the basis of their cell wall structure: Acid Fast Bacteria These bacteria contain Mycolic Acid in their cell wall, which prevents the easy penetration of Stain. So, it is usually heated after applying the stain. Very few bacteria are acid fast; this makes staining for acid-fastness particularly useful in diagnosis. Some common examples are Mycobacterium and Nocardia Species. Mycobacterium includes pathogens known to cause serious diseases in mammals, including tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). Gram Positive & Gram Negative Bacteria The other two types of Bacteria are Gram Positive & Gram Negative. For Better understanding, it’s easy to make a graph or table, comparing the characteristics of these two. Sr. Characteristic Gram Positive Gram Negative 1 Peptidoglycan layer [ Repeated layers of structure ( NAG---Peptide Chain---NAM) NAG: N-acetylglucosamine NAM: N-acetlymuramic acid ] Thick (2, 3 layers) Thin (single-layered)

2 Teichoic acids [ Act as Antigen, Two types are 1. Lipoteichoic Acid 2. Wall Teichoric Acid ] Present in many Absent 3 Periplasm Absent Present 4 Lipopolysaccharides [ Contains Antigens 1. Lipid A 2. Carbohydrates 3. Pilli ] Absent Present 5 Outer membrane Absent Present 6 Toxins produced Primarily exotoxins Primarily endotoxins 7 Gram reaction Dark Violet or Purple Pink or Red How to perform Gram Staining? 1. Make a slide of cell sample to be stained. 2. Primary Staining: Add the primary stain (crystal violet) to the sample/slide and incubate for 1 minute and then wash it. 3. Mordant: Add Gram's iodine for 1 minute- this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall. 4. Decolourization: Add alcohol for ~3 seconds and rinse with a gentle stream of water. The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet. (Note: Do not allow the decolourizer to stay on the sample for too long, as it may also decolorize Gram positive cells) 5. Counter Staining: Add the secondary stain (counter stain), safranin, to the slide and incubate for 1 minute. Wash with a gentle stream of water for a maximum of 5 seconds. How to Check Results? If the bacteria is Gram positive, it will retain the primary stain (crystal violet) and not take the secondary stain (safranin), causing it to look violet/purple under a microscope. If the bacteria is Gram negative, it will lose the primary stain and take the secondary stain, causing it to appear red when viewed under a microscope.

How Does Gram Staining Work? Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decolouring process. If you Like this Post, Please do Share it. Also must give your comments please.

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