Good for for CSF’s, pus and epithelial cells. pink color. Because in medicine what accompanies practicals are viva and these questions can be very important Cheers Gud luck, Greatly impressed by this article. We can use Gram staining kit in ready to use form, from some commercial supplier such as HIMEDIA. Rinsing too long can remove too much color, while not rinsing long enough may allow too much stain to remain on gram-negative cells. 5.4.2 Flame the loop to red hot and cool in air. If too little heat is applied, the bacteria will wash off the slide during staining. Also, use acetone alcohol – 50% acetone and 50% of 95% ETOH to decolorize. Because the bacteria are colored, not only is their Gram stain group identified, but their shape, size, and clumping pattern may be observed. Flood the gram’s iodine for 1 minute and wash with water. Spread the culture with an inoculation loop to an even thin film or smear. Crystal Violet (The Primary Stain) View the slide using a compound microscope. Your email address will not be published. What will be the final color of Gram (+) bacteria if mordant will be omitted? Required fields are marked *. The Gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. Crystal Violet was poured and kept for about 30 seconds to 1 minutes and rinse with water. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in colour. 5.4 PREPARATION OF THE SMEAR FROM LIQUID GROWTH the reason why mordant is use is to grow the microorganism. Your email address will not be published. 5.4.3 Spread on the surface of the slide properly. The results of the Gram stain are viewed using light microscopy. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. If decolorizer is left out, it won’t dissolve the lipids in the cell walls, and therefore, the staining process would be incomplete and it won’t allow the cells to be stained. 5.3.2 Deposit a drop of purified water on the glass slide. Gram's iodine (mordant, to fix crystal violet in the cell wall), Safranin (secondary stain or counterstain), Water in a squirt bottle or dropper bottle. The procedure is named for the person who developed the technique, Danish bacteriologist Hans Christian Gram. Prepare the smear of suspension on the clean slide with a loopful of sample. It allows scientists to determine whether an organism is gram-positive or gram-negative. 5.5.8 See the slide under Microscope with the use of immersion oil on the smear and observe in the Microscope using oil immersion lens (100X). By using both the Basic Bacteria Identification diagrams and the Table of Bacterial Causes of Infection (following diagrams) you can identify the likely bacteria from the Gram film appearance on the microscopy result. Save my name and email in this browser for the next time I comment. A mordant fixes or binds cells intensifying stains in cell or tissue to allow for visual clarity under a microscope. This material will consistently display a few neutrophils and a mixture of Gram (+) and (-) organisms. After the decolorizing step, a counterstain is applied (usually safranin, but sometimes fuchsine) to color the bacteria pink. Neutrophil nuclei should be pink. While the stain may not definitely identify bacteria, often knowing whether they are gram-positive or gram-negative is sufficient for prescribing an effective antibiotic. But if i may ask, which step in Gram staining tech can be omited without affecting the final result? What happen if we don’t use iodine ? Allow to stand for about 1Minute. Examine the finished slide under a microscope. Gram Staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884. 2. 2.0 SCOPE 2.1 This procedure applicable for Gram Staining of Bacteria in Microbiology Laboratory 3.0 RESPONSIBILITY 3.1 Microbiologist QC 4.0 ACCOUNTABILITY 4.1 QC Manager 5.0 PROCEDURE 5.1 Requirement 5.1.1 Crystal Violet (The Primary Stain) 5.1.2 Iodine Solution (The Mordant) 5.1.3 Decolorizer (Such as ethanol) … The role of iodine is that it act as grams mordant and increases the interaction between bacterial cell wall and the dye (crystal violet) so that the dye is more tightly bound on the cell and is more stained. A caveat in the examination of the Gram smears is the distortion in morphology that can be caused by antimicrobial therapy. This makes the Gram stain a valuable diagnostic tool for a medical clinic or lab. I would say yes. For example: • The patient clinically has meningitis; the Gram film of the CSF shows Gram-positive cocci in chains. Air dry, Blot dry and Observe under Microscope. 5.3 PREPARATION OF THE SMEAR FROM SOLID GROWTH SUCH AS SLANT OR PLATE. 5.3.5 The smear should be thin enough to dry completely within a few seconds. 5.5.4 Flood the smear with iodine solution and kept for 1 minute. 5.2.1 Gram-staining is a four part procedure which uses certain dyes to make a bacterial cell stand out against its background. Add safranin  for about 1 minute and wash with water. 5.5.9 Absorb gauze in the cleaning solution and apply to the Oil immersion lens till all oil is remove and lens is clean. While it can be used on broth cultures, it's best to centrifuge them first. This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations of microorganisms. Gram Negative Bacteria: Escherichia coli (E. coli), Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas,Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella etc, Download Animation from Below: Gram Staining Animation. Practice and skill are needed to produce a reliable result. Gently rinse the slide with water no longer than 5 seconds to remove excess stain. Safranin (The Counter stain) Heat fix the bacteria to the slide by passing it through the flame of a Bunsen burner three times. Place a small drop of bacterial sample on a slide. Pls wat will happen when decolouriser is left out, Decolizer is needed for differencites ….so why it left out. ← sop for entry & exit procedure In Microbial limit test and Biosafety, sop for Monitoring of Compressed Air/gases for microbiological analysis →. The primary limitation of the technique is that it yields erroneous results if mistakes are made in the technique. 5.1.1 Crystal Violet (The Primary Stain) It can be considered a differential stain because its used to better differentiate between microorganisms and/or structures while viewed under a microscope. Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. 5.2 PROCEDURE The procedure is named for the person who developed the technique, Danish bacteriologist Hans Christian Gram. the function of the mordant is to fix (attach) the crystal violet to the cell wall (peptidoglycan cell wall)so as not to be washed away easily thereby forming a complex as stated earlier. A magnification of 500x to 1000x may be needed to distinguish cell shape and arrangement. 5.5.1 The staining is carried out with the help of Gram Staining reagent. However, even this information may be useful in narrowing down bacterial identity. For example – E. coli (Gram negative short rods) S.aureus (Gram positive cocci in cluster). Can safranin be called a differential stain. Note that only a very small amount of culture is needed. When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. 3. The gram-negative cells should be stained red or pink, while the gram-positive cells will still appear purple or blue. Painstakingly put together in a simple language. 5.4.4 Fix the smear by heating lightly opposite side of the smear, by passing quickly over the flame two or three times. 5.1.3 Decolorizer (Such as ethanol) Try fixing with 100% Methanol for one minute and letting the slide dry, rather than heat fixing..preserves cellular integrity and reduces artifact caused by heat fixation. 5.3.6 Fix the smear by heating lightly on the opposite side of the smear, by passing quickly over the flame two or three times. 2.1 This procedure applicable for Gram Staining of Bacteria in Microbiology Laboratory. 5.1.2 Iodine Solution (The Mordant) Widal Test- Introduction, Principle, Procedure,…, Benedict’s Test- Principle, Composition,…, Different Size, Shape and Arrangement of Bacterial Cells, Nutrient Agar: Composition, Preparation and Uses, MacConkey Agar- Composition, Principle, Uses,…, Differences between Gram Positive and Gram Negative Bacteria, A decolorizer made of acetone and alcohol (95%). 1. 1.0 OBJECTIVE 1.1 To lay down the Procedure for Gram Staining of Bacteria. It is also known as Gram staining or Gram's method. 5.4.1 Take a clean glass slide. The differential nature of the Gram stain is based on the ability of some bacterial cells to retain a primary stain (crystal violet) by resisting a decolorization process. 5.5.5 Wash the slide with water and decolorizes with Gram’s Decolorizer (Ethyl alcohol 95%. Moreover, the test procedure is performed to differentiate between the two groups of bacteria. Both gram-positive and gram-negative bacteria pick up the pink stain, but it is not visible over the darker purple of the gram-positive bacteria. Gram stain testing is a method for classifying bacteria based on their cell wall. In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off. The gram-negative cells will lose color, while the gram-positive cells will remain violet or blue. ThoughtCo uses cookies to provide you with a great user experience. If Gram positive organisms have such complex cell wall that could defy decolorisation,why then is a mordant used? She has taught science courses at the high school, college, and graduate levels. The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low. 5.5.3 Wash with water and drain the excess water. I studied microbiology at Polytechnic(HND) and presently a 300 level student of Lead City University,Ibadan, Nigeria studying Biology Education.This piece has really helped in solving my Assignment on Biological Techniques(BIO 315), All enterobcteriaceae are gram negative bacteria but not all gram negative are enterobcteriaceae to distinguish the enterobcteriaceae you have to do oxidase test because enterobcteriaceae are oxidase negative bacteria. Iodine Solution (The Mordant) )Until no further violet colour comes off. Apply the secondary stain, safranin, and allow it to sit for 1 minute. However, most eukaryotic cells except fungi (including yeast) fail to stick to the slide during the process. The procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria. Save my name, email, and website in this browser for the next time I comment. Applying too much heat or for too long can melt the bacteria cell walls, distorting their shape and leading to an inaccurate result. Decolorizer (Such as ethanol) Gently rinse with water no longer than 5 seconds. 5.1 Requirement Mr Len artifacts results from poor methods of rinsing of slides. Gram Staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884. Don’t you think there should be another staining similar to gram staining, Even dilute cabolfuchsin is another counter stain that (1:9)dilution, Hi Very much appreciated Would be great if u also add some points about the modifications and variations and also gram variable bacteria and some general info about why gram staining is needed and where it is not indicated !! Today we use Gram’s staining techniques to aid in the identification of bacteria, beginning with a preliminary classification into one of two groups: Gram positive or Gram negative.