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. 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. Jun 12, 2018 principle of acidfast stain when the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through lipoidal wall and enters into cytoplasm. Most bacteria have no colour, so they generate little contrast in the microscope field. Gram staining is a microbiologic procedure used to differentiate gram negative from gram positive bacteria. The cell walls for gram positive microorganisms have a higher peptidoglycan and lower lipid content than gram negative bacteria. 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 be gramnegative. If your slide is all one color either pink or blue, then the slide may either have been over or under decolorized. These online assessment tools will help you strengthen your understanding of the gram stain method. 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. The gram staining technique differentiates the mixed culture cells into two terms one which retains the color of the primary stain is known as grampositive bacteria and the cells which get decolorized and takes the counterstain are known as gramnegative bacteria. The gram reaction reflects fundamental differences in the biochemical and structural properties of bacteria.
It oxidizes the carbon to carbon bonds between two adjacent hydroxyl groups. Gram staining is a microbiologic procedure used to differentiate gramnegative from grampositive bacteria. Summary and explanation the gram stain was originally devised by christian gram in 1884. Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. The first step of the modern gram stain involves using crystal violet, a basic dye fig.
Iodine forms complexes with crystal violet to fix the stain. Mordant plays role in intensifying the primary stain and properly fix it onto the cells. Gram stain hans christian gram history and principles of gram. Add the primary stain crystal violet and incubate 1 minute.
The gram stain, the most widely used staining procedure in bacteriology. Principle, procedure and results february 2, 2015 acharya tankeshwar bacteriology, staining techniques in microbiology 45 gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. Natural dye hematoxylin is the nuclear stain which stains cell nuclei. Principle of gram stain the two major groups of bacteria can be divided into gram positive and gram negative. When the bacteria is stained with primary stain crystal violet and fixed by the mordant, some of the bacteria are. Cover the fixed smear with crystal violet stain for 30 seconds and then wash off with water. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction.
The name comes from the danish bacteriologist hans christian gram, who developed the technique. Giemsa stain is a differential stain that is used to variably stain the various components of the cells and it can be used to study the adherence of pathogenic bacteria to the human cells. Christian gram in 1884 introduces gram staining technique and classified bacteria on its gram characters which might be gram negative or gram positive. In the microbiology field, extensive staining techniques are used. 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. The gram stain was first used in 1884 by hans christian gram gram,1884. If you continue browsing the site, you agree to the use of cookies on this website.
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. The staining procedure differentiates organisms of the domain bacteria according to cell wall structure. On the basis of their reaction to the gram stain, bacteria can be divided into two large groups. Unless it is a gram stain of a pure culture, there normally should be material staining both colors somewhere on. The gram stain procedure was originally developed by the danish physician hans christian gram to differentiate pneumococci from klebsiella pneumonia. The filter gram stain was approximately 100fold more sensitive than the slide gram stain. Make a slide of tissue or body fluid that is to be stained. Therefore, to see bacteria with the microscope, it is necessary to apply colour by using a staining reagent. Cryptococcus neoformans is an encapsulated fungal organism and it can cause disease in apparently immunocompetent, as well as. Gramnegative cells have a thin peptidoglycan layer and stain red to pink. The gram stain is fundamental to the phenotypic characterization of bacteria. Its teleomorph is filobasidiella neoformans, a filamentous fungus belonging to the class tremellomycetes.
Principle of flagella stain flagella are too thin to be visualized using a bright field microscope with ordinary stains, such as the gram stain, or a simple stain. Azure and eosin are acidic dye which variably stains the basic components of the cells like the cytoplasm, granules etc. A microorganism is grampositive if the stain remains violet secondary to the presence of. Acidic dye stains the basic components of the cell and basic dye stain the acidic components of the cell. Principle, reagents, procedure, step, result interpretation of gram. The cells are colored red or violet so as to distinguish its group. In the gramnegative cell wall, the amount of lipids is very high, when the cell is dissolved in alcohol, it leads to the formation of large pores in the cell. The gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups. Gram staining is a differential staining technique that differentiates bacteria into two groups. Giemsa stain is commonly used when there is need to examine the blood smear for the parasites but is a good stain for routine examination of blood smear and used to differentiate nuclear and cytoplasmic morphology of the various cells of the blood like platelets, rbcs giemsa staining technique. The cell walls of gram positive bacteria have a thick layer of proteinsugar complexes called peptidoglycan and lipid content is low. Occurrence of a phosphoric ester in certain bacteria. Principle, procedure, interpretation, examples and.
This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. Gram stain principle, laboratory continuing education. Gram stain principles, method and value flashcards quizlet. 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. 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. A wet mount read more flagella stain principle, procedure and result interpretation. You will be asked about differential staining, grampositive and. Principle the gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions.
The different response of the two groups to the gram stain is based on fundamental differences in cell wall structure and composition. It is one of the differential stains that are used to characterize bacteria in one of two groups. The gram stain, page 2 of 9 plasma membrane and a somewhat thicker outer membrane. Gram stain is probably one of the most commonly used staining procedures used in the field of microbiology.
Pdf mlt 415 lab report smear preparation and gram stain. Most bacteria can be differentiated by the gram reactions due to difference in their cell wall structure. The stain is retained because the cell wall of a grampositive bacteria is not as permeable as the cell wall of a gramnegative bacteria. The polychromatic pap stain involves five dyes in three solutions. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. Cell wall of gram positive and gram negative bacteria.
In 1884, while examining lung tissue from patients who had died of pneumonia, gram had discovered that certain stains were preferentially taken up and. Google scholar mittwer t, bartholomew jw, kallman bj. The structure of the organisms cell wall determines whether the organism is gram psitive or negative. What is gram stain test staining procedure, principle. History and explanation of the fundamental technique of determinative bacteriology article pdf available march 2004 with 23,000 reads how we measure reads. Gramnegative bacteria are decolorized by the alcohol. Grampositive cells have a thick peptidoglycan layer and stain blue to purple. Gram staining principle, procedure, function of reagents. Cryptococcus neoformans is an encapsulated yeast and an obligate aerobe that can live in both plants and animals. Bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as grampositive bacteria. 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. Bacteria that retain the initial crystal violet stain purple are said to be gram positive, whereas those that are decolorized and stain red with carbol fuchsin or safranin are said to be gram negative.
Microbiology module common staining technique microbiology 22 notes 2. Gram stain protocols american society of microbiology. Acidfast stain principle, procedure, interpretation and. It is also known as gram staining or gram s method. How the cell wall contributes to these staining differences is described below. Flood slide with crystal or gentian violet 60 seconds. The cell walls for grampositive microorganisms have a higher peptidoglycan and lower lipid content than gramnegative bacteria. Pdf gram staining procedure find, read and cite all the research you need on researchgate. In accumulation, these staining techniques also help in the determination of cell size, structure. Here are the following main principles of gram staining techniques. It was devised by a danish physician, hans christian gram, in 1884. Function of crystal violet primary stain in gram staining the first stain which is used is the primary stain or we can call it as crystal violet stain. The gram stain learning the basics richard tom thomson, jr.
Principle of acidfast stain when the smear is stained with carbol fuchsin, it solubilizes the lipoidal material present in the mycobacterial cell wall but by the application of heat, carbol fuchsin further penetrates through lipoidal wall and enters into cytoplasm. The standard grams staining method can be used to differentiate intact, morphologically similar bacteria into two groups. 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. The most important and widely used differential stain for bacteria is the gram stain. 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. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than. Pas stain principle initial reaction the first reaction makes use of a periodic acid that acts as an oxidizing agent. It differentially stains the human and bacterial cells and appeared as purple and pink colored bodies respectively. Nov 03, 20 it includes introduction, history, principle, method of gram staining procedure slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. Grampositive cell wall contains a thick layer of peptidoglycan with numerous teichoic acid crosslinking which resists the decolorization. The gram stain differentiates bacteria into two fundamental varieties of cells. Jan 30, 2019 function of crystal violet primary stain in gram staining the first stain which is used is the primary stain or we can call it as crystal violet stain.
The gram stain procedure distinguishes between gram. Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. 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. Giemsa stain is a differential stain and contains a mixture of azure, methylene blue, and eosin dye. Gram positive bacteria stain deep violet to blue and gram negative bacteria stain pink to red. Pas stain periodic acidschiff stain procedure and uses. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. The filtergram stain detected 2 to 100 bacteria, whereas the slidegram stain failed to detect less than. For demonstrating gramnegative and grampositive in tissue. 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. Microbiology lecture 2 gram positive and gram negative. It is specific for the phosphate groups of dna and attaches itself to where there are high amounts of adeninethymine bonding. Primary stain gives blue or purple colour when it binds to the cell wall of bacteria.
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 filtergram stain was approximately 100fold more sensitive than the slidegram stain. Gram stain, a widely used microbiological staining technique that greatly aids in the identification and characterization of bacteria. Grampositive bacteria stain deep violet to blue and gramnegative bacteria stain pink to red. Gram stain principle, reagents, procedure, steps, results. 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. Papanicolaou stain includes both acidic and basic dyes. Gram was searching for a method that would allow visualization of cocci in tissue sections of lungs of. Unless it is a gram stain of a pure culture, there normally should be material staining both colors somewhere on the slide. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall.
This is the primary stain which is initially applied to a heatfixed smear of bacteria. What is gram stain test staining procedure, principle and. In brief, the procedure involves the application of a solution of iodine potassium iodide to cells previously stained with crystal violet or gentian violet. The principle of gram staining relies on the reaction of a bacterial cell with the gram stain which finally differentiates the bacteria into grampositive and gramnegative. The gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions.
The gramreaction rely upon the chemical nature of the bacterial cell wall, especially the lipids which comprise 1122% in gramnegative cell wall and 14% in the grampositive cell wall. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. Principle of gram stain the two major groups of bacteria can be divided into grampositive and gramnegative. 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 includes introduction, history,principle, method of gram staining procedure slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. For use in the grams staining method for the initial differentiation of gram positive and gram negative bacteria. 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. Giemsa staining technique principle, preparation, procedure.
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 gram staining method is named after hans christian gram, the. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. 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. The differences in cell wall composition of grampositive and gramnegative bacteria account for the gram staining differences.