In recent reports, the impact of Gram staining results on patient mortality has been documented. It is therefore highly likely that the information provided by the Gram staining will help to assess the adequacy of preliminary diagnosis and antimicrobial therapy selected after collecting culture specimens and before final identification of the microorganism. The most important and primary test to perform directly on some special samples such as cerebrospinal fluid and positive cultures is Gram staining which serves as the most rapid and simplest test to characterize microorganisms. Isolation, identification of pathogenic microorganisms in cultures and subsequent antimicrobial susceptibility testing always assists in selecting appropriate antimicrobial agent and prevention of unnecessary complications. This teaching activity uses a step-wise process to aid the student in familiarity with the use of the microscope as well as to increase the success rate with which they are able to stain and view microorganisms.Clinical microbiology laboratory plays several important roles in the management of bacterial infections. Core Microscopy Skills: Instructional Scaffolding for the Gram Stain.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. 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. Wash with a gentle stream of water for a maximum of 5 seconds. Add the secondary stain, safranin, to the slide and incubate for 1 minute.However, if the alcohol remains on the sample for too long, it may also decolorize Gram positive cells. The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet. Rinse sample/slide with acetone or alcohol for ~3 seconds and rinse with a gentle stream of water.Add Gram's iodine for 1 minute- this is a mordant, or an agent that fixes the crystal violet to the bacterial cell wall.Rinse slide with a gentle stream of water for a maximum of 5 seconds to remove unbound crystal violet. Add the primary stain (crystal violet) to the sample/slide and incubate for 1 minute.Heat fix the sample to the slide by carefully passing the slide with a drop or small piece of sample on it through a Bunsen burner three times. Make a slide of cell sample to be stained.Iodine solution/Gram's Iodine (mordant that fixes crystal violet to cell wall).However, the decolorized Gram negative cells are stained red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. A counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red.Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it.This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water. Next, a Gram's iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine. Cells are stained with crystal violet dye. ![]() Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process. Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and counterstaining, usually with safanin. Alternatively, Gram negative bacteria stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process. 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. The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |