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. Grampositive bacteria produces a bluepurple color with this staining procedure while gramnegative ones are red in color 5. Ensure you use a pencil as many inks are removed by the reagents used in the staining procedure. Objectives to learn the technique of smear preparation. 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. In brief, the procedure involves the application of a solution of iodine potassium iodide to cells previously stained with crystal violet or gentian violet. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. Grampositive bacteria gramnegative bacteria provides useful information for disease treatment. 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. Gram positive vs gram negative stain gram stain procedure preparing a sample. Nonetheless, it is generally considered good practice to use young cultures of known gram positive and gram negative bacteria as controls when staining an unknown bacterium. We developed a rapid and sensitive method for the differentiation of gram positive and gram negative. Gram staining is a microbiologic procedure used to differentiate gramnegative from grampositive bacteria.
Gram positive bacteria have cell walls that contain thick layers of peptidoglycan 90% of cell wall. Gram positive bacteria will be purple and gram negative bacteria will be colorless. Distinguishes bacteria of two large and medically important groups. Cells from old cultures may stain gram negative even if the bacteria are gram positive. To use and relate the gram stain to the study of bacterial cell morphology, and as an important step in the identification of a bacterial species. The gram stain is fundamental to the phenotypic characterization of bacteria.
Gram negative cells have a thin peptidoglycan layer and stain red to pink. This protocol is taken from bensons microbiological applications. Microbiology lecture 2 gram positive and gram negative bacteria gram staining principle this second microbiology lecture will explain the properties of gram positive and gram negative bacteria. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. This staining procedure differentiates microbes into two basic groups. The cells are colored red or violet so as to distinguish its group. Oct 24, 2019 the gram stain is a differential method of staining used to assign bacteria to one of two groups gram positive and gram negative based on the properties of their cell walls. Place slides on staining rack, drop crystal violet stain onto tissue section, stain for 1 minute. The gram stain is the most important staining procedure in microbiology. 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. The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable. It is called a differential stain since it differentiates between grampositive and gramnegative bacteria. Gram variable observations in gram staining the gram staining procedure does not always give clearcut results.
To understand the benefit obtained from visualizing unstained microorganisms. Bacteria that stain purple with the gram staining procedure are termed grampositive. It is used to differentiate between gram positive organisms and gram negative organisms. Bacteria that stain purple with the gram staining procedure are termed gram positive. Grampositive bacteria have cell walls that contain thick layers of peptidoglycan 90% of cell wall. This was developed in 1884 by a danish bacteriologist, hans christian gram. The staining procedure differentiates organisms of the domain bacteria according to cell wall structure. It is one of the differential stains that are used to characterize bacteria in one of two groups. The name comes from the danish bacteriologist hans christian gram, who developed the technique gram staining differentiates bacteria by the chemical and physical. In this gram stained smear of a positive blood culture bottle one can see gram positive cocci in pairs, chains arrows, and clusters arrowhead. The counter stain used in the gram stain is safranin pink.
An infected appendix, or any tissue containing both negative and positive gram rods. The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. Another note of interest is that variations of the gram stain procedure have been developed over the years. The gram stain differentiates bacteria into two fundamental varieties of cells. Grampositive vs gramnegative bacteria difference and. Some organisms are gramvariable and may appear either gram negative or grampositive according to the conditions.
Aug 21, 2019 gram positive vs gram negative stain gram stain procedure preparing a sample. When they are seen together, however, it is best to remain noncommittal, reporting only grampositive. 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. Mode of action of gram stain at this time the mechanism of the gram stain reaction is not completely known. Cover the smear with crystal violet, the primary stain, for 20 seconds. Apr 23, 2020 gram staining is a quick procedure used to look for the presence of bacteria in tissue samples and to characterise bacteria as gram positive or 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. The procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria. Cover the smear with gram s iodine, the mordant, for 1 minute. Pdf mlt 415 lab report gram stain techniques muhamad. The gram staining procedure is one of the most basic and important tests performed in the clinical microbiology laboratory. Laboratory manual in general microbiology eleventh edition.
Label a clean glass microscope slide with your sample identification. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. An optimized staining technique for the detection of gram. To learn the techniques of gram staining, nigrosin staining and koh test. Bacteria that retain the initial crystal violet stain purple are said to be grampositive, whereas those that are decolorized and stain red with carbol fuchsin or safranin are said to. When they are seen together, however, it is best to remain noncommittal, reporting only gram positive. In this gramstained smear of a positive blood culture bottle one can see grampositive cocci in pairs, chains arrows, and clusters arrowhead. For demonstrating gramnegative and grampositive in tissue. On adding a counterstain such as safranin or fuchsine after washing, gram negative bacteria are stained red or pink while gram positive bacteria retain their crystal violet dye.
Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram. Schematic showing the modified gram stain procedure. What is gram stain test staining procedure, principle and. The gram staining technique differentiates the mixed culture cells into two terms as grampositive bacteria and gramnegative bacteria. Both bacterias, positive and negative, cell wall is composed of peptidoglycan,the grampositve has a thicker wall and both will take up the crystal violet. A microorganism is grampositive if the stain remains violet secondary to the presence of. Cell wall structure identifies either cell is grampositive or negative in nature during the procedure when we stained by primary stain and secure it by a mordant. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms.
Overheating of smears when heat fixation is used d. The isolated bacteria are stained by various methods depending upon the bacteria in focus. The gram stain is the most widely used staining procedure in bacteriology. This test differentiates the bacteria into grampositive and gramnegative bacteria, which helps in the classification and differentiation of microorganisms. Jul 01, 2019 microbiology lecture 2 gram positive and gram negative bacteria gram staining principle this second microbiology lecture will explain the properties of gram positive and gram negative bacteria. The purpose of the gram stain is to show whether the bacteria are gram positive purplecolored, gram negative pinkcolored, or both. It is also known as gram staining or gram s method. Materials and methods aseptic technique was used throughout the experiment. Gram positive vs gram negative technology networks. Negative staining principle, procedure and result interpretation objectives of negative staining to perform a negative staining procedure.
The gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. Cell wall structure identifies either cell is gram positive or negative in nature. The name comes from the danish bacteriologist hans christian gram, who developed the technique. The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Grampositive cells have a thick peptidoglycan layer and stain blue to purple. In 1883 hans christian gram discovered an important differential staining method that is used extensively today. 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. What is gram stain test staining procedure, principle.
Flood the gram s iodine for 1 minute and wash with water. Nonetheless, it is generally considered good practice to use young cultures of known grampositive and gramnegative bacteria as controls when staining an unknown bacterium. On adding a counterstain such as safranin or fuchsine after washing, gramnegative bacteria are stained red or pink while grampositive bacteria retain their crystal violet dye this is due to the difference in the structure of their bacterial cell wall. Currently, the only control method for determining the validity of this. 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. With these types of organisms, grampositive and gramnegative cells may be present within the same preparation 40. It is often possible to differentiate streptococci chains from staphylococci clusters. Staining of the bacteria forms the foremost and the most important step in the identification of bacteria. Flood slide with crystal or gentian violet 60 seconds. Occurrence of a phosphoric ester in certain bacteria. Prepare the smear of suspension on the clean slide with a loopful of sample. Principle of negative staining negative staining requires the use of an acidic stain such as india ink or nigrosin. Crystal violet was poured and kept for about 30 seconds to 1 minutes and rinse with water. Gram stain principle, reagents, procedure, steps, results.
If the slide is heat fixed, allow it to cool to the touch before applying the stain. It is called a differential stain since it differentiates between gram positive and gram negative bacteria. Gram staining is a differential staining technique that differentiates bacteria into two groups. Learn vocabulary, terms, and more with flashcards, games, and other study tools. Relatively large boxcarshaped grampositive bacilli are seen, spores are usually present but the demonstration of spores is frequently difficult. Gram staining involves a fourpart process, which includes. Bacteria that retain the initial crystal violet stain purple are said to be gram positive, whereas those that a. Thin grampositive bacilli with numerous ovoid or citronshaped, subterminal spores are present. This test differentiates the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiation of microorganisms. Gram stain is probably one of the most commonly used staining procedures used in the field of microbiology.
Gramnegative cells have a thin peptidoglycan layer and stain red to pink. During the procedure when we stained by primary stain and secure it by a mordant. There are many theories, but none are completely satisfactory. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. It was developed by danish microbiologist hans christian gram in 1884 as an effective method to distinguish between bacteria with different types of cell walls, and even today it remains one of the most frequently used staining techniques. The major difference in the modified gram stain procedure occurs during the dehydration process and is the application of alcoholic saffron lower right box, highlighted in red. They are based on the physical and chemical reactions of the cell to the staining reagents. The gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. A comparison of heat versus methanol fixation for gram. 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 gram stain should almost always be done as the first step in diagnosis of a bacteria infection.
The gram stain procedure was originally developed by the danish physician hans christian gram to differentiate pneumococci from klebsiella pneumonia. The traditional gram stain procedure is completed in either case top box. Gram stain developed in 1884 by hans gram, a danish microbiologist. 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. This is applied and stains the gram negatives pink. 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. Through a series of staining and decolorization steps, bacteria are differentiated according to cell wall composition. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. Gram stain, grampositive, gramnegative, heat fixation, methanol fixation. The gram stain procedure is a differential staining procedure that involves multiple steps.
Gram staining is a microbiologic procedure used to differentiate gram negative from gram positive bacteria. The most useful staining procedure in medical microbiology. The gram stain was developed by christian gram in 1884 and modified by hucker in 1921. Gram staining is still the cornerstone of bacterial identification and taxonomic division. If preparing your slide from a liquid bacterial culture. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Here are the following main principles of gram staining techniques.
A microorganism is gram positive if the stain remains violet secondary to the presence of. Microbiology lecture 2 gram positive and gram negative. Method of staining used to distinguish and classify bacterial species into two large groups. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. The yellow contrast in the connective tissue collagen within skin.
680 631 121 1075 819 1236 1042 1132 890 1564 1616 232 1079 1485 649 208 1569 1176 449 334 671 1407 348 1447 801 1061 498 799 653 1302 702 1155 169