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. Decolorization the critical step of the gram staining procedure is the decolorization step. Gram staining is the differential staining that is used to differentiate the bacteria majorly into two groups i. Crystal violet is first applied, followed by the mordant iodine, which fixes the stain figure. To learn the techniques of gram staining, nigrosin staining and koh test. Laboratory manual in general microbiology eleventh edition. It is a primary test which quickly and cost effectively divides bacteria into one of two types. Gram was searching for a method that would allow visualization of cocci in tissue sections of lungs of. 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. Heatfix the slide with the specimen by passing it over a heat source, such as a flame, several times using a clothes pin or forceps. An optimized staining technique for the detection of gram.
The microscope is a very important tool in microbiology, but there are limitations when it comes to using one to observe cells in general and bacterial cells in particular. Gram positive organisms contain a highly crosslinked layer of peptidoglycan that retains the primary dye, crystal violet cv. The two major groups of bacteria can be divided into gram positive and gram negative. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Place slides on staining rack, drop crystal violet stain onto tissue section, stain for 1. Gram staining is a differential staining technique, which separates bacteria into two groups known as gram positive bacteria and gram negative bacteria. Introduction observation of bacteria with conventional bright field microscopy yields relatively little useful information. 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. Pdf files contains the procedure for a special stain, a procedure card, and sample container labels for the reagents. For example, gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. 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 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. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. The gram staining method was first given in 1884 by the danish scientist and physician hans christian gram the gram stain is the differential stain that stains the bacterial cells.
Gram staining is used widely technique and the most popular method in the laboratories. Dotson md, jeanine wienerkronish md, in critical care secrets fourth edition, 2007. This differential technique separates bacteria into two groups, gram. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884.
Bartholomew 1962 has pointed out that each variation in the gram staining procedure has a definite. 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. Classic gram staining techniques involve the following steps. This will ensure that all areas are stained evenly. The gram staining technique differentiates the mixed culture cells into two terms as grampositive bacteria and gramnegative bacteria. It is used to distinguish between gram positive and gram negative bacteria, which have distinct and consistent differences in their cell walls. 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 addition this stain also allows determination of cell morphology, size, and arrangement. Staining flood the entire slide when crystal violet, iodine and safranin are applied. Through a series of staining and decolorization steps, bacteria are differentiated according to cell wall composition. The gram stain procedure was originally developed by the danish physician hans christian gram to differentiate pneumococci from klebsiella pneumonia. The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. Hold the slide in a tilted downward position and allow the.
A primary stain malachite green is used to stain the endospores. It is the type of differential staining which makes the use of more than one stains to differentiate the bacteria. This staining procedure differentiates microbes into two basic groups. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. How to do a gram s stain test gram s stain is one of the most frequently used processes in identifying bacteria and is used daily in hospitals. Gram staining helps to identify bacterial pathogens in specimens and cultures by their gram reaction gram positive and gram. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. It was devised by a danish physician, hans christian gram, in 1884. It is almost always the first test performed for the identification of bacteria. The gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. 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. The contrast between a cell, which is primarily water, and the background, which is water, is poor. The gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups. Introduction to staining live specimens are difficult to see with the bright field microscope.
Gram stain developed in 1884 by hans gram, a danish microbiologist. 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. Staining and streaking protocols for simple stain, gram stain, streak plate technique and culture maintenance lab 2a. Hold the slide in a tilted downward position and allow the decolorizer to flow over the smear.
Grams stain is a laboratory staining technique that distinguishes between two groups of bacteria b y the identification of differences in the structure of their cell walls. Because endospores resist staining, the malachite green will be forced into i. This was developed in 1884 by a danish bacteriologist, hans christian gram. The gram stain was first used in 1884 by hans christian gram gram,1884. Gram staining of bacteria from environmental sources. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. Gram positive bacteria stain blue while gram negative stain red. In 1883 hans christian gram discovered an important differential staining method that is used extensively today. Gram stain is a very important differential staining techniques used in the initial characterization and classification of bacteria in microbiology. Gram positive bacteria have cell walls that contain thick layers of peptidoglycan 90% of cell wall. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. This protocol is taken from bensons microbiological applications. The gram stain is the most widely used staining procedure in bacteriology.
Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition. 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. An infected appendix, or any tissue containing both negative and positive gram rods. The gram reaction reflects fundamental differences in the biochemical and structural properties of bacteria. Difference between gram stain and acid fast compare the.
The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Some of them have improved the method, others include some minor technical variants of no value. Overheating of smears when heat fixation is used d. Apr 04, 2016 a staining technique used to classify bacteria. Here are the following main principles of gram staining techniques. Gram staining is a simple procedure if done with precision and accuracy can guide in diagnosis of several bacterial infections. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. 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. Staining of bacterial cells objective to learn the techniques of smear preparation, gram staining, nigrosin staining and correlating the results of gram staining with koh test.
Similar to a simple stain, it allows for assessment of bacterial cell morphology e. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were grampositive bacteria while only three of the. Gram positive and gram negative on the basis of cellwall difference and by the sequential application of crystal violet, iodine, alcohol and safranin. The gram stain procedure is a differential staining procedure that involves multiple steps.
Apr 12, 2016 gram stain is the most common staining technique used diagnostically within both the clinical setting and in research laboratories to differentiate between gram positive and gram negative microorganisms in various types of tissues 17, 26. This technique is used to stain a slide such as a fecal smear to observe the bacterial microflora present based on their gram stain reaction. Gram staining is still the cornerstone of bacterial identification and taxonomic division. Gram staining tips page 2 of 2 some specimens on preparation will have areas on the slide that are thicker than others.
The name comes from the danish bacteriologist hans christian gram, who developed the technique. Modification in gram staining methods since the original procedure of gram, many variations of the gram staining technique have been published. Materials and methods aseptic technique was used throughout the experiment. Gram positive organisms contain a highly crosslinked layer of peptidoglycan that retains the primary dye, crystal violet cv, following the application of the mordant, iodine i. Jun 10, 2006 gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. Some staining techniques, such as the gram and ziel neelsen stains, although of great diagnostic value because of their differential staining properties for specific bacteria, reveal little internal structure.
Gram stain is one of the most common staining technique used for examining specimens suspected to contain bacteriologic agents. The gram stain is a classical and important staining technique that remains widely used by environmental microbiologists. The original formulation comprised of aniline gentian violet, lugols iodine, absolute alcohol and bismarck brown. The acidfast stain is a differential stain used to identify acidfast organisms such as mycobacterium from nonacid fast organisms. We will discuss this and other applications of gram staining in more detail in later chapters. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. 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 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 purpose of the gram stain is to show whether the bacteria are gram positive purplecolored, gram negative pinkcolored, or both. Types, techniques, preparations and procedures microscopy refers to the practice that involves the use of a microscope for the purposes of observing small scale structures that cannot be viewed using the naked eye and often cell staining is necessary as s tructures are. Gram staining 2 free download as powerpoint presentation.
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. 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. The name comes from its inventor, hans christian gram. A differential staining technique the schaefferfulton method is used to distinguish between the vegetative cells and the endospores. May 19, 20 gram staining or gram s method is a method of differentiating bacterial species into two large groups gram positive and gram negative.
Gram negative bacteria tend to be more resistant to certain antibiotics than gram positive bacteria. Occurrence of a phosphoric ester in certain bacteria. Module common staining technique microbiology 24 notes indeterminate groups as well. Google scholar mittwer t, bartholomew jw, kallman bj. Gram staining for differentiating bacterial species youtube. Distinguishes bacteria of two large and medically important groups. Then the slide is washed with alcohol, and the gram. Gram positive bacteria gram negative bacteria provides useful information for disease treatment. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. Gram stain principle, reagents, procedure, steps, results. Objectives to learn the technique of smear preparation. Purpose of the gram staining technique the results of the gram stain are viewed using light microscopy.
This can result in areas that are under decolorized, thus organisms that are truly gram negative will appear gram positive. 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. Oct 24, 2019 if the staining procedure is performed correctly, gram positive bacteria will be purple, while gram negative bacteria will be pink. The most useful staining procedure in medical microbiology. Gram staining is the most sensitive method by which to visualize and recognize nocardiae in clinical specimens. Method of staining used to distinguish and classify bacterial species into two large groups. Flood slide with crystal or gentian violet 60 seconds. Gram staining is used to determine gram status to classify bacteria broadly. Pdf mlt 415 lab report gram stain techniques muhamad. Bacteria that stain purple with the gram staining procedure are termed gram positive.
Difference between gram positive and gram negative cell wall is thicker in a gram positive bacteria. Principle, procedure, interpretation, examples and animation last updated. The primary stain of the gram s method is crystal violet. Fixation of clinical materials to the surface of the microscope slide either by heating or by using methanol. Gram positive bacteria has only the inner plasma membrane and no outer membrane.
Sputum gram s stain and culture can be performed noninvasively and. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. The gram stain the gram stain is the most important and universally used staining technique in the bacteriology laboratory. How to do a grams stain test grams stain is one of the most frequently used processes in identifying bacteria and is used daily in hospitals. Gram staining technique is the widely used differential staining technique in bacteriology. Gram stain, a widely used microbiological staining technique that greatly aids in the identification and characterization of bacteria.
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