Detailed principle, reagent preparation, procedure, result interpretation, examples and animation video for gram staining technique. The purpose of the gram stain is to show whether the bacteria are gram positive purplecolored, gram negative pinkcolored, or both. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. 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. The gram stain differentiates bacteria into two fundamental varieties of cells. It is the type of differential staining which makes the use of more than one stains to differentiate the bacteria. It is also known as gram staining or gram s method. 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.
Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. If everything shows negative red you can shorten the decolorization time. Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution. This was developed in 1884 by a danish bacteriologist, hans christian gram. Bacterial cell wall may have higher lipid content or the protein content. Microbiology lecture 2 gram positive and gram negative. The difference between these shcauffer fulton and klein staining techniques is the application of dyes i. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were grampositive bacteria while only three of the. First of all, the gram staining was named after dr. Jul 28, 2008 the staining technique is based on the difference between the cell wall composition of different bactweria. Stains are used to highlight structures in clinical specimens, often.
Endospore staining types, principle, procedure and. Certain bacteria are stained with aniline dyes, like gentian violet. The gram stain, the most widely used staining procedure in bacteriology. Use millipore filtered water in the waterbath and staining procedure.
In 1883 hans christian gram discovered an important differential staining method that is used extensively today. The gram staining method was first given in 1884 by the danish scientist and physician hans christian gram. In 1884, hans christian gram, a danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. The simple stain can be used as a quick and easy way to determine cell shape, size and arrangements of bacteria. If everything shows positive purple, try a slightly longer decolorization period. Gram stain hans christian gram history and principles of gram.
At the end of the procedure, spores stain green and other cells stain red. To differentiate between the two major categories of bacteria. Principle, procedure, reporting and modifications by editorial team on january 11, 2020 in bacteriology, microbiology the ziehlneelsen stain zn stain, also called the hot method of afb staining, is a type of differential bacteriological stain used to identify acidfast organisms, mainly mycobacteria. 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.
Principle, reagents, procedure, step, result interpretation of gram. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. The gram stain procedure used for differentiating bacteria into two groups. In negative staining technique, an acidic stain such as nigrosin, india ink, eosin or congo red is used in which the bacterial culture or the specimen is mixed well and then spread over the microscopic glass slide to form a thin smear. These organisms are usually filamentous, gram positive rods of varying length that often form chains, but occasionally, they may stain gram. Get a printable copy pdf file of the complete article 2. Flood slide with crystal or gentian violet 60 seconds. Gram stain principle, reagents, procedure, steps, results. Make a slide of tissue or body fluid that is to be stained. Jun 26, 2017 principles of different staining techniques 1. 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. For demonstrating gram negative and gram positive in tissue. Occurrence of a phosphoric ester in certain bacteria. Another differential stain technique is the acidfast technique.
Distinguishes bacteria of two large and medically important groups. Acidfast stain principle, procedure, interpretation and examples. 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 direct microscopic examination of specimens and cultures can provide a rapid presumptive diagnosis meanwhile gram staining is a differential stain that provide morphological information regarding the shape of cell,the type of cell arrangement that is single. Gram staining is used for the identification and differentiation of different types of bacteria. What is the principle and procedure of gram staining. To learn the techniques of gram staining, nigrosin staining and koh test. Negative staining technique principle, procedure, results. 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. Giemsa staining technique principle, preparation, procedure. Bacterial capsules are nonionic, so neither acidic nor basic stains will adhere to their surfaces. A counterstain, safranin, is then used to give color to the nonsporeforming bacteria. Jan 30, 2019 gram staining is basically a staining technique which is broadly used in microbial and molecular biology. The word gram is always spelled with a capital, referring to hans christian gram, the inventor of gram staining gram staining principles gram staining is used to determine gram status to classify bacteria broadly.
Gram positive bacteria gram negative bacteria provides useful information for disease treatment. This technique differentiates species of mycobacterium from other bacteria. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. 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 differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. If only one result shows up, either your staining method was flawed or there was a problem with the reagent used. Google scholar mittwer t, bartholomew jw, kallman bj. The direct microscopic examination of specimens and cultures can provide a rapid presumptive diagnosis meanwhile gram staining is a differential stain that provide morphological information regarding the shape of cell,the type of cell arrangement that is. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. To understand how the gram stain reaction affects gram positive and gram negative bacteria based on the biochemical and structural di.
Add the primary stain crystal violet and incubate 1 minute. June 12, 2018 by sagar aryal 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. Overheating of smears when heat fixation is used d. Staining is an auxiliary technique used in microscopic techniques used to enhance the clarity of the microscopic image.
The two major groups of bacteria can be divided into grampositive and gramnegative. The gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups. Used as a mordant in gram s staining, iodine enhances the entrance of the dye through the pores present in the cell wallmembrane. Another endospore staining technique that is not commonly used is known as the klein method of endospore staining.
The cell walls of gram positive bacteria have a thick layer of proteinsugar complexes called peptidoglycan and lipid content is low. Full text is available as a scanned copy of the original print version. Each of these stains aims at identifying and characterizing bacteria based on their morphologies. This technique was developed by a danish bacteriologist hans christian gram in 1884. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were gram positive bacteria while only three of the. Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. In addition this stain also allows determination of cell morphology, size, and arrangement. Gram staining is a differential staining technique that differentiates bacteria into two groups. The gram staining technique is the most important and widely used microbiological differential staining technique. Christian grams to differentiate between gram positive and negative bacteria.
There are a variety of staining methods like simple, differential and special staining that are used in labs to examine the bacteria. Starch is a substance common to most plant cells and so a weak iodine solution will stain starch present in the cells. The solution should be standardized by staining with a control slides. It is a technique that is widely used for the examination of cells, tissues and cellular components. Negative staining principle, reagents, procedure and result. What is the principle of negative staining technique. Gram stain is one of the most common staining technique used for examining specimens suspected to contain bacteriologic agents. Gram stain developed in 1884 by hans gram, a danish microbiologist. In capsule staining procedure we do not heat fix and rinse the smear with water as heat and water may dislodge capsules from bacteria. The gram staining technique differentiates the mixed culture cells into two terms as gram positive bacteria and gram. Why social distancing is the best way to combat covid 19.
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. Histological staining is a series of technique processes undertaken in the preparation of sample tissues by staining using histological stains to aid in the microscope study anderson, 2011. Also the stains used in gram staining have different affinity for these components and they bind with them reversibly or irreversibly. In the microbiology field, extensive staining techniques are used. The gram stain the gram stain is the most important and universally used staining technique in the bacteriology laboratory. The gram staining technique differentiates the mixed culture cells into two terms as grampositive bacteria and gramnegative bacteria. Its application aim at read more albert staining principle, reagents, procedure, results, interpretation. Gram staining procedure animation microbiology principle. The gram staining method, named after the danish bacteriologist who.
The process of histological staining takes five key stages which involve. Iodine is one component in the staining technique known as gram staining, used in microbiology. The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. It is used to distinguish between grampositive and gramnegative bacteria, which have distinct and consistent differences in their cell walls. Stains and dyes are frequently used in histology the study of tissue under the microscope and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses disease at a microscopic level. Module common staining technique microbiology 24 notes indeterminate groups as well. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. 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. Objectives to learn the technique of smear preparation. It is used to distinguish between gram positive and gram negative bacteria, which have distinct and consistent differences in their cell walls. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. This is the primary phase in the process of identification of an organism.
Here are the following main principles of gram staining techniques. Staining is an auxiliary technique used in microscopic techniques used to enhance the clarity of the. 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. Flood the slide with basic fuchsin counter stain and allow it to act for about 5 seconds. I would like to give an elaborate explanation for the reagents being used in this gram staining and why it is used. Jun 11, 2018 negative staining principle, reagents, procedure and result.
Histologic staining options available include, but are not limited to. Apr 04, 2016 a staining technique used to classify bacteria. Pdf mlt 415 lab report gram stain techniques muhamad. Gram staining technique is the widely used differential staining technique in bacteriology. 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. This staining procedure differentiates microbes into two basic groups. Cell wall structure identifies either cell is gram positive or negative in nature during the procedure when we stained by primary stain and secure it by a mordant. Materials and methods aseptic technique was used throughout the experiment. Gram staining procedure animation principle, procedure, interpretation principle. Step by step procedure with explanation gram staining principle. Oct 05, 2018 gram stain is one of the most common staining technique used for examining specimens suspected to contain bacteriologic agents. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. This protocol is taken from bensons microbiological applications.
The main purpose of negative staining is to study the morphological shape, size and arrangement of the bacteria cells that is difficult to stain. I have explained the difference between cell wall structure of gram positive and gram negative bacteria along with the function of. Links to pubmed are also available for selected references. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. Staining is a valuable technique used in microscopy to enhance contrast in the microscopic image. Acidfast stain principle, procedure, interpretation and. Staining is a technique used to enhance contrast in samples, generally at the microscopic level.
Gram staining of bacteria from environmental sources. Laboratory manual in general microbiology eleventh edition. 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. Principle, procedure, interpretation, examples and animation last updated.
Principle, procedure, interpretation and animation. True to its name, the simple stain is a very simple staining procedure involving single solution of stain. Gram staining is basically a staining technique which is broadly used in microbial and molecular biology. Any basic dye such as methylene blue, safranin, or.
Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. Gram staining principle, procedure, function of reagents. Gram staining is used widely technique and the most popular method in the laboratories. 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. The most common stain being gram staining, acidfast staining, endospore staining.
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