5.2 PROCEDURE Air dry, Blot dry and Observe under Microscope. 5.2.1 Gram-staining is a four part procedure which uses certain dyes to make a bacterial cell stand out against its background. I would say yes. Safranin (The Counter stain) Painstakingly put together in a simple language. Pls wat will happen when decolouriser is left out, Decolizer is needed for differencites ….so why it left out. What happen if we don’t use iodine ? 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. The results of the Gram stain are viewed using light microscopy. Mr Len artifacts results from poor methods of rinsing of slides. If too little heat is applied, the bacteria will wash off the slide during staining. 5.3.1 Take a clean glass slide. Save my name, email, and website in this browser for the next time I comment. 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.
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.
← sop for entry & exit procedure In Microbial limit test and Biosafety, sop for Monitoring of Compressed Air/gases for microbiological analysis →. The gram-negative cells will lose color, while the gram-positive cells will remain violet or blue. For example – E. coli (Gram negative short rods) S.aureus (Gram positive cocci in cluster). Allow to stand for about 1Minute. This makes the Gram stain a valuable diagnostic tool for a medical clinic or lab. She has taught science courses at the high school, college, and graduate levels. 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. 5.1.1 Crystal Violet (The Primary Stain) 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. The procedure is named for the person who developed the technique, Danish bacteriologist Hans Christian Gram. The gram-negative cells should be stained red or pink, while the gram-positive cells will still appear purple or blue.
Use a dropper to apply the primary stain (crystal violet) to the slide and allow it to sit for 1 minute.
1.1 To lay down the Procedure for Gram Staining of Bacteria. 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) …
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. Apply the secondary stain, safranin, and allow it to sit for 1 minute. A caveat in the examination of the Gram smears is the distortion in morphology that can be caused by antimicrobial therapy.
Applying too much heat or for too long can melt the bacteria cell walls, distorting their shape and leading to an inaccurate 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. Gram stain testing is a method for classifying bacteria based on their cell wall. The primary limitation of the technique is that it yields erroneous results if mistakes are made in the technique. Great! 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%). heat fixing of smear blackened and disolve the lipoid cpnt of the cell as well as it schring of cell cpnt. 5.1 Requirement
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. A magnification of 500x to 1000x may be needed to distinguish cell shape and arrangement. View the slide using a compound microscope. Neutrophil nuclei should be pink. This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations of microorganisms. Place a small drop of bacterial sample on a slide. The specimen should be mounted and heat fixed on a slide before you proceed to stain it the reagents you will need to successfully perform this operation are: 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. The slide may be stained as above.
5.4.3 Spread on the surface of the slide properly. The procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria. If the staining procedure is performed correctly, gram-positive bacteria will be purple, while gram-negative bacteria will be pink.
For example: • The patient clinically has meningitis; the Gram film of the CSF shows Gram-positive cocci in chains. What will be the final color of Gram (+) bacteria if mordant will be omitted? 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. 5.4.1 Take a clean glass slide. However, if the decolorizer is left on too long, all cells will lose color! 5.4.4 Fix the smear by heating lightly opposite side of the smear, by passing quickly over the flame two or three times.
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. 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. Your email address will not be published. 5.3.4 Put this colony on to the previously deposited drop of purified water and mix. Gently rinse with water no longer than 5 seconds. Because in medicine what accompanies practicals are viva and these questions can be very important Cheers Gud luck, Greatly impressed by this article. It allows scientists to determine whether an organism is gram-positive or gram-negative. pink color. )Until no further violet colour comes off. 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. Decolorizer (Such as ethanol) 4.
The Gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain.
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