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. The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent. The lipoid capsule of the mycobacteria is of such high molecular weight that it is waxy at room temperature and successful penetration by the aqueousbased staining solutions such as gram s is prevented. Three introductory chapters cover gramstain procedures, quality control, evaluation, and more. Gram staining and the steps needed to complete the procedure. Both bacterias, positive and negative, cell wall is composed of peptidoglycan,the gram positve has a thicker wall and both will take up the crystal violet.
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. Gram stain of ocular exudate typically shows predominance of neutrophils with intracellular gram negative diplococci. Atcc 25923, staphylococcus aureus and attc 25922, escherichia coli. Gram staining was firstly introduced by cristian gram in 1883 which used to differentiate the gram positive microorganisms and gram negative microorganisms. Currently, the only control method for determining the validity of this. Principle of gram staining 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. Principle of gram stain the two major groups of bacteria can be divided into gram positive and gram negative. Romanowsky stains may also be used for wet fixed slides, but are primarily applied to airdried smears. Oct 24, 2019 the gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. The differences in cell wall composition of gram positive and gramnegative bacteria account for the gram staining.
Moraxella catarrhalis and neisseria meningitidis can be confused with n. Gram stain principle, reagents, procedure, steps, results. To a gram positive cell the counter stain stains the cell, but the cells are already so heavily stained by crystal violet that the addition of pink does not change the dark purple color of the cells. Weiss, in principles and practice of pediatric infectious disease third edition, 2008 diagnosis.
Gramnegative cells have a thin peptidoglycan layer and stain red to pink. The proceduresteps of gram stain prepare and fix the specimen to the microscope slide before staining. The composition of the cellular wall of gram positive and gram negative bacteria tells the gram staining differences. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. Some bacteria, after staining with the gram stain, yield a gram variable pattern. Gram stain reagents are evaluated by staining the following recommended bacterial strains.
Flood slide with crystal or gentian violet 60 seconds. The gram staining technique differentiates the mixed culture cells into two terms as gram positive bacteria and gram negative bacteria. The primary stain crystal violet binds to peptidoglycan, coloring cells purple. Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition. Gram staining or grams method is an emprical method of differentiating bacterial species into two large groups grampositive and gramnegative based on the chemical and physical properties of.
The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Corynebacterium diptheria seen in gram staining 35. Microbiology lecture 2 gram positive and gram negative bacteria gram staining principle this second microbiology lecture will explain the properties of g. The cell walls for gram positive microorganisms have a higher peptidoglycan and lower lipid content than gram negative bacteria. The gram stain procedure distinguishes between gram. In clinical settings, light microscopes are the most. Modified ziehl neelson for acid fast bacilli, gram staining bacteria, mucicarmine mucins, pas for. Gram stain hans christian gram history and principles of gram.
Gram positive bacteria have thicker cell walls with less fatty substances lipids than gram negative. Gram stain under microscope purpose, procedure and preparation. It allows a large proportion of clinically important bacteria to be classified as either gram positive or negative based on their. Stain uptake is often due to dyetissue or reagenttissue affinities.
Gram staining procedure animation microbiology principle. Both grampositive and gramnegative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet. Gram stain principle bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as gram positive bacteria. Staining is a biochemical process usually conducted to increase the contrast of a microscopic image. Pdf mlt 415 lab report gram stain techniques muhamad. The principle of gram staining relies on the reaction of a bacterial cell with the gram stain, which finally differentiates the bacteria into gram positive and gram negative. Wash slide in a gentle and indirect stream of tap water for 2 seconds. The staining procedure differentiates organisms of the domain. Differentiation in regressive staining differentiation is the removal of washing out of the excess stain until the colour is retained only in the tissue components to be studies. Gram staining or grams method is an emprical method of differentiating bacterial species into two large groups grampositive and gramnegative based on the chemical and physical properties of their cell wall.
The different response of the two groups to the gram stain is based on fundamental differences in cell wall structure and composition. Principle of gram staining the structure of the organisms cell wall determines whether the organism is gram psitive or negative. 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. 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. It is a primary test which quickly and cost effectively divides bacteria into one of two types.
Gram positive microorganisms stain bluepurple and gram negative. Gram stains are of little value for direct smears of stool, throat samples, and sputum from cystic fibrosis. This is applied and stains the gram negatives pink. Apr 04, 2016 gram negative pseudomonas aeruginosa bacteria pinkrods. Gram stain principle, laboratory continuing education.
Principle, procedure, interpretation, examples and. Gram staining or grams method 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 wall. On the basis of their reaction to the gram stain, bacteria can be divided into two large groups. It was discovered by danish scientist and physician hans christain joachin gram in 1884. In differential staining, specimen is subjected to a series of stains dyes in which different organisms or different parts of the cell are stained differently so that they can be distinguished from each other. Cover the smear with crystal violet, the primary stain, for. This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. The procedure is based on the ability of mo to retain colour of the stain. Gram in 1884 it remains an important and useful technique to this day. Of all the different classification systems, the gram stain has withstood the test of time. When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary stain by resisting declorization while others get decolorized by a decolorizer.
The gram stain is a type of differential stain that allows a microbiologist to. Your explanation of the principle and procedure for acid fast staining were basically correct. This technique differentiates bacteria in 2 groups i. Gram s stain is one of the most frequently used processes in identifying bacteria and is used daily in hospitals. Gram staining is a differential staining technique that differentiates bacteria into two groups. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violetiodine complex to leach out of. Principle the gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions.
Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram positive and gram negative bacteria on the basis of differential staining with a crystal violetiodine complex and a safranin counterstain. Gram stain protocols american society for microbiology. Staining is an auxiliary technique used in microscopic techniques used to enhance the clarity of the microscopic image. Certain bacteria are stained with aniline dyes, like gentian violet. Excessive rinsing during the staining procedure, especially if smear is not properly fixed. What is gram stain test staining procedure, principle. The cell walls of gram positive bacteria have a thick layer of proteinsugar complexes called peptidoglycan and lipid content is low. The differences in cell wall composition of grampositive and gramnegative bacteria account for the gram staining. The procedure for differentiating between two principal groups of bacteria. The procedure is based on the ability of microorganisms to retain colour of the stains used during the gram stain reaction. Staining samples with different dyes is an important part of microscopy where the color contrast of a magnified is increased to differentiate the stained portion of the image from the rest. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Jun 12, 2018 principle of gram staining 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.
Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope. If the control slides do not prove satisfactory, the staining procedure is not accepted. The stain is retained because the cell wall of a gram positive bacteria is not as permeable as the cell wall of a gram negative bacteria. It gets its name from the danish bacteriologist hans christian. Gram staining technique is the widely used differential staining technique in bacteriology. Staining methods are also influenced by selective uptake of reagents into tissues, and selective losses of products and or reagents from the tissues. Acidfast stain principle, procedure, interpretation and. Gram staining showing gram positive cocci in clusters, and culture of fluid from a vesicle or pustule or from beneath the lifted edges of a crusted plaque of impetigo, are usually sufficient to establish a diagnosis.
The name comes from the danish bacteriologist hans christian gram, who developed the technique. The gram staining is one of the most crucial staining techniques in microbiology. The lipoid capsule of the acidfast organism takes up carbolfuchsin and resists decolorization with a dilute acid rinse. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. Impregnation it is the deposition of salts of heavy metals on or around cells, tissue constituents etc. This was developed in 1884 by a danish bacteriologist, hans christian gram. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. The gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups. What is gram stain test staining procedure, principle and. Positive and negative control slides should be used every time the staining procedure is performed except for gram staining where positive controls may be enough unless a new batch of stain is made. The gram staining procedure is one of the most basic and important tests performed in the clinical microbiology laboratory. For demonstrating gram negative and gram positive in tissue.
Oct 05, 2020 gram staining is a differential staining technique that is used for microscopic examination of bacteria. In your procedure however, the preparation of the smear needs not be sterile but aseptically done to prevent unwanted materialsorganisms on your slide and assuring that only the specimen you intend to examine is on your slide. Doc principles of staining mohammad ehtisham academia. The procedure steps of gram stain prepare and fix the specimen to the microscope slide before staining. Gram staining was firstly introduced by cristian gram in 1883 which used to differentiate the gram positive microorganisms and. The gram stain, the most widely used staining procedure in. Microbiology lecture 2 gram positive and gram negative. Stains and dyes are widely used in the scientific field to highlight the structure of the biological. The most important and widely used differential stain for bacteria is the gram stain.
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