What are the 4 steps of Gram staining?
The performance of the Gram Stain on any sample requires four basic steps that include applying a primary stain (crystal violet) to a heat-fixed smear, followed by the addition of a mordant (Gram’s Iodine), rapid decolorization with alcohol, acetone, or a mixture of alcohol and acetone and lastly, counterstaining with …
What step can you omit in Gram staining?
Gram Staining
| Question | Answer |
|---|---|
| can iodine be added before the primary stain in gram stain? | No, it must complex with primary stain |
| which step can be omitted without affecting determination of the gram reaction | the counter stain safranin as differentiation can still be made |
What is the correct order of staining reagents in Gram staining?
Explanation: Gram staining is a type of differential staining. In this process the fixed bacterial smear is subjected to the following staining reagents in the order listed: crystal violet, iodine solution, alcohol (decolorizing agent), and safranin. 3.
What is the Decolorizer in the Gram stain?
The decolorizer, ethyl alcohol, is the most crtitical step. Ethyl alcohol is a nonpolar solvent, and thus penetrates the cell walls of Gram negative cells more readily and removes the crystal violet-iodine complex.
What is the most time sensitive step in Gram stain?
Flood slide with 95% alcohol for 10 seconds and wash off with tap water. (Smears that are excessively thick may require longer decolorization. This is the most sensitive and variable step of the procedure, and requires experience to know just how much to decolorize). Drain the slide.
What is the correct order of staining reagents in Gram staining quizlet?
Terms in this set (8) List the 4 reagents used for the gram stain in the proper order? Crystal violet stains gram positive and gram negative cells. Iodine is used next as mordant. Alcohol decolorizes the gram negative cells, and lastly Safranin counterstains gram negative cells.
Which bacteria Cannot be Gram stained?
Atypical bacteria are bacteria that do not color with gram-staining but rather remain colorless: they are neither Gram-positive nor Gram-negative. These include the Chlamydiaceae, Legionella and the Mycoplasmataceae (including mycoplasma and ureaplasma); the Rickettsiaceae are also often considered atypical.
What color is a gram negative bacteria?
A Gram stain is colored purple. When the stain combines with bacteria in a sample, the bacteria will either stay purple or turn pink or red. If the bacteria stays purple, they are Gram-positive. If the bacteria turns pink or red, they are Gram-negative.
What are the major differences between Gram positive and Gram negative bacteria?
Gram positive bacteria have a thick peptidoglycan layer and no outer lipid membrane whilst Gram negative bacteria have a thin peptidoglycan layer and have an outer lipid membrane.
What color is E. coli when Gram stained?
pink
Gram Staining Coli will appear pink in color. The absence of this (of purple color) is indicative of Gram-positive bacteria and the absence of Gram-negative E.
How are Gram positive and Gram negative bacteria stained?
The primary stain ( crystal violet) binds to peptidoglycan, coloring cells purple. Both gram-positive and gram-negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet. Gram’s iodine ( iodine and potassium iodide) is applied as a mordant or fixative.
When to apply Gram staining to a culture?
When to Apply Gram Staining Gram staining procedure is used to determine if bacteria are gram-positive or gram-negative. This is usually the first step in the process of identifying bacteria that are present in cultures. This procedure is important in giving a clue to the diagnosis in people with infectious diseases.
How to remove a Gram stain from a microscope slide?
Procedure of Gram Stain. Prepare and fix the specimen to the microscope slide before staining. Cover the smear with crystal violet, the primary stain, for 20 seconds. Gently rinse off the stain with water. Cover the smear with Gram’s iodine, the mordant, for 1 minute. Pour off the excess Gram’s iodine.
What’s the best way to fix a Gram stain?
Heat Fixing This step kills the bacteria on the smear, and makes them firmly adhere to the slide, allowing the sample to take up stains more readily. Air-dry your smear. While holding your slide at one end (smear-side up), pass the slide through flame from a Bunsen burner, 2-3 times.
When to Apply Gram Staining Gram staining procedure is used to determine if bacteria are gram-positive or gram-negative. This is usually the first step in the process of identifying bacteria that are present in cultures. This procedure is important in giving a clue to the diagnosis in people with infectious diseases.
1. Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal Violet in the cell wall. 2. Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called Safranin. Example: Escherichia coli
Procedure of Gram Stain. Prepare and fix the specimen to the microscope slide before staining. Cover the smear with crystal violet, the primary stain, for 20 seconds. Gently rinse off the stain with water. Cover the smear with Gram’s iodine, the mordant, for 1 minute. Pour off the excess Gram’s iodine.
Heat Fixing This step kills the bacteria on the smear, and makes them firmly adhere to the slide, allowing the sample to take up stains more readily. Air-dry your smear. While holding your slide at one end (smear-side up), pass the slide through flame from a Bunsen burner, 2-3 times.