The gram stain is a differential method of staining used to assign bacteria to one of two groups grampositive and gramnegative based on the properties of their cell walls. Use millipore filtered water in the waterbath and staining procedure. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. Rinse the slide with a gentle jet of water from a wash bottle. Overheating of smears when heat fixation is used d.
On the basis of their reaction to the gram stain, bacteria can be divided into two groups. Gram staining was given by christian gram in 1884 and it is also called differential staining because it differentiates bacterial in different groups. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were gram positive bacteria while only three of the isolates were gram negative bacteria. Bacteria cell walls are stained by the crystal violet. Chemical mechanism of the gram stain and synthesis of a. Gram staining tips page 2 of 2 some specimens on preparation will have areas on the slide that are thicker than others. The gram stain should almost always be done as the first step in diagnosis of a bacteria infection the gram stain is named after the danish scientist hans christian. With these types of organisms, grampositive and gramnegative cells may be present within the same preparation. What are the mechanisms of gram staining, and how do they. Learn what it involves and when your doctor might order it. The cell walls for gram positive microorganisms have a higher peptidoglycan and lower lipid content than gram negative bacteria. The primary stain of this method is crystal violet, which can be sometimes substituted with equally effective methylene blue. The name comes from the danish bacteriologist hans christian gram, who developed the technique.
This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Bacterial classification, structure and function introduction the purpose of this lecture is to introduce you to terminology used in microbiology. The gram stain complex has been found to be dissociated into dye and iodine components when exposed to a differentiating solvent, thus eliminating cell membrane permeability to large molecules as a gram staining and its molecular mechanism 21 table iii quantitative estimation of crystal violet extracted from gramstained s. Gram stain set instructions introduction one of the most frequently cited criteria by which bacteria are classified involves the reaction of individual species to the gram staining procedure. Grampositive bacteria which have a thick peptidoglycan wall retain the crystal violet dye staining them violet or purple, while all other bacteria e. Corynebacterium diptheria seen in gram staining 35.
The filter gram stain was approximately 100fold more sensitive than the slide gram stain. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Gram s iodine iodine and potassium iodide is applied as a mordant or fixative. As a result, bacteria are classified as either grampositive or gramnegative.
The method is named after its inventor, the danish scientist hans christian gram 18531938, who developed the technique in 1884 gram 1884. Gram stain hans christian gram history and principles of gram. Gram staining or gram stain, also called grams method, is a method of staining used to differentiate bacterial species into two large groups gram. Gram staining is a quick procedure used to look for the presence of bacteria in tissue samples and to characterise bacteria as grampositive or gramnegative, based on the chemical and physical properties of their cell walls. Jan, 2017 gram staining or gram stain, also called gram s method, is a method of staining used to differentiate bacterial species into two large groups gram positive and gram negative. This is the most important staining technique in bacteriology. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. 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. Explain the mechanism of gram staining yahoo answers.
Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. Gram staining is still the cornerstone of bacterial identification and taxonomic division. Full text get a printable copy pdf file of the complete article 3. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. Gram positive bacteria dont lyse in 3% koh so dna is released. Details of the chemical mechanism of the gram stain were determined in. Occurrence of a phosphoric ester in certain bacteria. Feb 27, 2009 some bacteria, after staining with the gram stain, yield a gram variable pattern. The gram staining method, named after the danish bacteriologist who originally devised it in 1882 published 1884, hans christian gram, is one of the most important staining techniques in microbiology.
Differential staining is a technique which uses more than one stain to differentiate bacteria. Alternatively they may be much smaller bananalike forms with pointed ends. The name comes from the danish bacteriologist hans christian gram, who developed the technique gram staining differentiates bacteria by the chemical and physical properties of their cell walls. Gram positive cells form a crystal violetiodine complex. Gramnegative cells will stain pink with the gram stain. Some bacteria, after staining with the gram stain yeild a pattern called gramvariable where a mix of pink and purple cells are seen. Pdf gram staining procedure find, read and cite all the research. Gram staining is generally the first step in identifying an unknown. This platinum anion therefore allows the gram staining mechanism to be followed by electron microscopy. Gram staining of bacteria from environmental sources. Gram negative gently stirred into a drop of 3% koh potassium hydroxide, the bacteria lyse break open, releasing their dna making the liquid viscous in 4560 secs. This was developed in 1884 by a danish bacteriologist, hans christian gram. In a symposium on the surface of the gram positive bacteria, stacey 1949 postulated.
Gram stain or gram staining, also called gram s method, is a method of staining used to distinguish and classify bacterial species into two large groups. This can result in areas that are under decolorized, thus organisms that are truly gram negative will appear gram positive. Oct 01, 2007 i think if you had a book, it might help you better. It is one of the classic ways to determine the gram positive and gram negative bacteria. They are often thin, wispy, eyelashlike faintly staining curved gram negative rods.
I regard the cell wall as built in layers, the layer nearest to the. Gram staining is a differential staining technique that differentiates bacteria into two groups. A gram stain is a test used to learn if bacteria are present in your body fluids or tissue. Some organisms are gramvariable and may appear either gram negative or grampositive according to the conditions.
Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. Gram positive cells is dehydrated by the alcohol in the decolorizer and loses permeability. While both classes have an underlying phospholipid plasma membrane, the structure of the cellular wall varies greatly. It is one of the classic ways to determine the grampositive and gramnegative bacteria. The gram stain procedure was originally developed by the. Oct 24, 2019 both gram positive and gram negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet. Make a slide of tissue or body fluid that is to be stained.
Gram staining is an empirical method of differentiating bacterial species into two large groups gram positive and gram negative based on the chemical and physical properties of their cell walls. In addition this stain also allows determination of cell morphology, size, and arrangement. Flood slide with crystal or gentian violet 60 seconds. We have tested a number of sh reagents to determine their effect on the gram reaction. As a result, bacteria are classified as either gram positive or gram negative. The gram stain is a type of differential stain that allows a microbiologist to identify the differences between organisms andor differences within the same organism. Gram staining is the technique developed by christian gram 1 2. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than.
In spite of his defective method, his name lives on jay hardy, cls, sm ascp. Gram stain and acidfast stain are most popularly known as differential stains. The gram staining technique differentiates the mixed culture cells into two terms as gram. Gram staining depends on the thickness of the peptidoglycan layer of bacteria. Dec 20, 2017 gram staining was given by christian gram in 1884 and it is also called differential staining because it differentiates bacterial in different groups gram positive and gram negative, based on. Finally, look for and quantify the amount of mobiluncus morphotypes present. If using a tissue sample, add 12 drops onto the glass slide.
Huckers modification method gram staining procedure. The differential response to the gram stain is based on fundamental differences in the cell wall structure and composition of cells. Studying on the granules of acidfast bacilli observed by using the various staining procedures, the author found the following results. The most widely used staining procedure in microbiology is the gram stain, discovered by the danish scientist and physician hans christian joachim gram in 1884. Google scholar mittwer t, bartholomew jw, kallman bj. Recall that typical gram positive bacteria have a multilayered, thickened peptidoglycan layer, while gram positive bacteria have a thinner peptidoglycan layer sitting in the middle of the periplasmic space. Gram staining is an empirical method of differentiating bacterial species into two large groups grampositive and gramnegative based on the chemical and physical properties of their cell walls.
It is almost always the first test performed for the identification of bacteria. Jun 12, 2018 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. Add the primary stain crystal violet and incubate 1 minute. The grampositive bacteria have a thick layer of peptidoglycan whereas gramnegative bacte. Grampositive bacteria become gramnegative when their cell wall is ruptured or removed. Gram stain reagents for in vitro use only catalogue no. Alcohol or acetone is used to decolorize the cells. The gram positive bacteria have a thick layer of peptidoglycan whereas gram negative bacte. Oct 23, 2016 gram staining is the technique developed by christian gram 1 2. The primary stain of the grams method is crystal violet.
Sg5155 our gramstain reagents are intended to be used as a differential stain for the microscopic examination of bacterial cultures and laboratory specimens. Apr 04, 2016 gram negative pseudomonas aeruginosa bacteria pinkrods. Gram staining and its molecular mechanism sciencedirect. Gram staining allows for the categorization of most bacteria into two broad structural classes. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. Chemical mechanism of the gram stain and synthesis of a new. Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. The genera actinomyces, arthobacter, corynebacterium, mycobacterium, and propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in gram negative staining of these gram positive. Huckers modification method gram staining procedure is used to microscopically identify gram positive and gram negative bacteria. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution for 5 minutes. The crystal violet stain is the primary stain, which stains everything in the smear blue.
It is theorized that during the gram stain procedure, the cell wall of. A sample under 24 hours old is recommended, as older bacteria may have damaged cell walls that respond less predictably to gram staining. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were grampositive bacteria while only three of the isolates were gramnegative bacteria. Place the slides on a staining rack, smear side up and crayon markings down. Gram stain protocols american society of microbiology. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. The gram staining technique differentiates the mixed culture cells into two terms as grampositive bacteria and gramnegative bacteria. Peptidoglycan is mainly a polysaccharide composed of two subunits called nacetyl glucosamine and nacetyl muramic acid. The gram staining method, named after the danish bacteriologist who originally devised it in 1882 published 1884.
Recall that typical grampositive bacteria have a multilayered, thickened peptidoglycan layer, while grampositive bacteria have a thinner peptidoglycan layer sitting in the middle of the periplasmic space. Cover different classification schemes for grouping bacteria, especially the use of the gram stain 2. Some bacteria, after staining with the gram stain yeild a pattern called gram variable where a mix of pink and purple cells are seen. Gram positive bacteria have a thick meshlike cell wall which is made up of peptidoglycan 5090% of cell wall, which stains purple. Smears that are too thick will be difficult to decolorize and imposable to read. This test can be used to confirm and determine gram stain reaction when if gram stain reagents arent available. Slide preparation purulent material should be selected whenever possible. Add a few drops of crystal violet stain on the smears. Apr 23, 2020 in order for the gram stain to be useful, add a thin layer of the sample on the stain. In a symposium on the surface of the grampositive bacteria, stacey 1949 postulated. Gram stain or gram staining, also called grams method, is a method of staining used to distinguish and classify bacterial species into two large groups. Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram. Gram staining technique is the widely used differential staining technique in bacteriology. The chemical theories of the gram stain mechanism usually as sign to iodine an essential role in the formation of a grampositive dyeiodinecellsubstance.