Gram's DecolourizerS032. Ingredients Ethyl alcohol, 95% 50.0 ml Acetone 50.0 ml **Formula adjusted, standardized to suit performance parameters. Directions. 1)Prepare a thin smear on clear, dry glass slide. 2)Allow it to air dry and fix by gentle heat. 3)Flood with Gram's Crystal Violet (S012) for 1 minute. (If over staining results in improper decolourization of known gram- negative organisms, use less crystal violet). 4)Wash with tap water. 5)Flood the smear with Gram’s Iodine (S013).
What is the purpose of Remel Gram decolorizer?
(95% ETHYL ALCOHOL) INTENDED USE Remel Gram Decolorizer (95% Ethyl Alcohol) is a reagent recommended for use in qualitative procedures to differentiate gram-negative from gram-positive organisms. SUMMARY AND EXPLANATION The Gram stain method was developed in 1884 by the Danish
What is the method of staining using a generic Gram staining technique?
Gram staining technique requires simultaneous use of chemical reagents for a fixed period followed by washing; Primary stain (crystal violet), Mordant (iodine), Decolorizer (ethanol or acid-alcohol) and Counter stain (safranin or dilute carbol-fuchsin).
What is the best counterstain for Gram staining?
Safranin and dilute carbol-fuchsin are commonly used counterstain in Gram staining procedure, another being Neutral red (it stains gonococci and meningococci well).
What is used as a Decolorizer in Gram staining?
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 Decolorizer made of?
The decolorizing agent, (ethanol or an ethanol and acetone solution), interacts with the lipids of the membranes of both gram-positive and gram-negative Bacteria. The outer membrane of the gram-negative cell is lost from the cell, leaving the peptidoglycan layer exposed.
Can you use alcohol as Decolorizer for Gram stain?
Decolorizer-Alcohol Either acetone or ethyl alcohol can be used as the decolorizing agent. The alcohol dissolves lipids found in the outer cell membrane of Gram-negative bacteria, allowing the crystal violet-iodine complex to leak out of the thinner peptidoglycan layer.
Which of the following is commonly used in a gram Decolorizer?
Remel Gram Decolorizer (95% Ethyl Alcohol) is a reagent recommended for use in qualitative procedures to differentiate gram-negative from gram- positive organisms. The primary stain, crystal violet, is a basic dye which rapidly permeates the cell wall of all bacteria, staining the protoplast purple.
How do you make a Decolorizer?
Decolorizer. Some workers prefer to use acetone by itself, ethanol 95% v/v, or ethanol-iodine as the decolorizing solution. A mixture of acetone-alcohol is recommended because it decolorizes more rapidly than ethanol 95% v/v, and is less likely to over-decolorize smears than acetone without alochol added.
What is the best procedure for decolorization?
What is the best procedure for decolorization? Add decolorizing agent until run-off is clear. This method allows the decolorizing agent to dissolve the outer membrane of Gram-negative cells and rinse out the crystal violet from the thin layers of peptidoglycan. This causes the run-off to be purple.
What happens if you don't use alcohol in Gram stain?
The alcohol will decolorize the sample if it is Gram negative, removing the crystal violet. However, if the alcohol remains on the sample for too long, it may also decolorize Gram positive cells. Add the secondary stain, safranin, to the slide and incubate for 1 minute.
Can you use isopropyl alcohol in a Gram stain?
STAIN TECHNOLOGY there has been in the Gram stain. There is no question but that the proposed substitute (50% ethyl and 50% isopropyl alcohol) is entirely satisfactory in the Gram technic.
What happens if you don't Decolorize a Gram stain?
The decolorizer should stay on the slide for no more than 15 seconds! If the decolorizer is left on too long, even gram positive cells will lose the crystal violet and will stain red.
Why is Decolorizing the most crucial step in gram staining?
The length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing agent can remove all the stains from both types of bacteria. The final step in gram staining is to use basic fuchsin stain to give decolorized gram-negative bacteria pink color for easier identification.
How do you make a Gram stain reagent?
There are six basic steps:Apply a smear of bacteria on to a slide. ... Add about 5 drops of Hucker's Crystal Violet to the culture. ... Add about 5 drops of iodine solution to the culture. ... Tilt slide and decolorize with solvent (acetone-alcohol solution) until purple color stops running. ... Add about 5 drops of Safranine O.More items...
What is a Decolorizer?
Definition of 'decolorizer' 1. a substance that removes colour. 2. a substance that removes the greeny-brown colour in glass that is caused by impurities.
What is decolorization in microbiology?
Bacterial decolorization and degradation is emerging as a cost-effective, environmentally friendly treatment method for the breakdown of these toxic dyes and the process is highly affected by various factors like temperature dye structure, concentration, etc.
What is the use of safranin solution?
Safranin is used as a counterstain in some staining protocols, colouring cell nuclei red. This is the classic counterstain in both Gram stains and endospore staining. It can also be used for the detection of cartilage, mucin and mast cell granules.
What is an acid fast organism?
Acid-fast bacteria, also known as acid-fast bacilli or simply AFB, are a group of bacteria sharing the characteristic of acid fastness. Acid fastness is a physical property that gives a bacterium the ability to resist decolorization by acids during staining procedures.