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. 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. I have explained the difference between cell wall structure of gram positive and gram negative bacteria along with the function of. This was developed in 1884 by a danish bacteriologist, hans christian gram. 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. Gram staining is an empirical method of differentiating bacterial species into two large groups grampositive and gramnegative based on the chemical and.
It is categorised into two types that are given below. To learn the techniques of gram staining, nigrosin staining and koh test. Get a printable copy pdf file of the complete article 3. In most microbiological staining procedures, the bacteria are first fixed to the slide by the heat fixed smear figure 1. In addition this stain also allows determination of cell morphology, size, and arrangement. Gram was a modest man, and in his initial publication he remarked, i have therefore published the method, although i am aware that as yet it is very.
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. Prepare and fix the specimen to the microscope slide before staining. Gram positive bacteria have high content of peptidoglycan in its cell wall while the lipid content is quite low. To understand how the gram stain reaction affects gram positive and gram negative bacteria based on the biochemical and structural di. 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. Gram staining principles gram staining is used to determine gram status to classify bacteria broadly.
In gram positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. Gram stain principles, method and value flashcards quizlet. It was a few years later, that the german pathologist carl weigert 1845. Here are the following main principles of gram staining techniques. Google scholar mittwer t, bartholomew jw, kallman bj. Unless it is a gram stain of a pure culture, there normally should be material staining both colors somewhere on the slide. 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. Microbiology lecture 2 gram positive and gram negative. Cell wall structure identifies either cell is gram positive or negative in nature. The gram staining technique differentiates the mixed culture cells into two terms as grampositive bacteria and gramnegative bacteria. The name comes from the danish bacteriologist hans christian gram, who developed the technique.
Links to pubmed are also available for selected references. 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. What is gram stain test staining procedure, principle. The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. It was first developed by the danish scientist hans christian gram in an effort to make bacteria more visible in stained sections of lung tissue.
Spores can generally be discerned on grams stains endospores do not stain and appear as refractile, nonstaining bodies, but occasionally specific spore stains may be helpful. 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. For demonstrating gramnegative and grampositive in tissue. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were grampositive bacteria while only three of the. Learn vocabulary, terms, and more with flashcards, games, and other study tools. We will discuss this and other applications of gram staining in more detail in later chapters.
The cell walls of gram positive bacteria have a thick layer of proteinsugar complexes called peptidoglycan and lipid content is low. As the cell wall of bacteria is very different from the cell wall of other organisms. Principle, procedure, interpretation, examples and animation last updated. If your slide is all one color either pink or blue, then the slide may either have been over or under decolorized. Principle of gram staining when the bacteria is stained with primary stain crystal violetcv and fixed by the flme, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. 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. 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 staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as grampositive bacteria.
The composition of the cellular wall of grampositive and gramnegative bacteria tells the gram staining differences. Apr 04, 2016 a staining technique used to classify bacteria. Principle and purpose the gram stain is perhaps the most useful test conducted in the clinical microbiology laboratory. Certain bacteria are stained with aniline dyes, like gentian violet. 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. 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.
Gram stain principle, reagents, procedure, steps, results. Principle the gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. Jun 12, 2018 acidfast stain principle, procedure, interpretation and examples. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. The most common stain being gram staining, acidfast staining, endospore staining. It identifies and classifies two major groups of bacteria, i. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were.
Each of these stains aims at identifying and characterizing bacteria based on their morphologies. Flood slide with crystal or gentian violet 60 seconds. Principle, procedure, interpretation, examples and. Gramnegative bacteria tend to be more resistant to certain antibiotics than grampositive bacteria. The structure of the organisms cell wall determines whether the organism is gram psitive or negative. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. I have explained the difference between cell wall structure of gram positive. 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. The most useful staining procedure in medical microbiology. The purpose of the gram stain is to show whether the bacteria are gram positive purplecolored, gram negative pinkcolored, or both. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. Bacteria that are grampositive have a thick layer of peptidoglycan.
The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than. Hot zn stain is the usual method in which we heat the smear to enhance the dye penetration. Dr hans christian joachim gram introduced it in 1884. 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. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results.
In this procedure living, potentially pathogenic bacteria are smeared on the glass slide and allowed to air dry. Gram staining and the steps needed to complete the procedure. Jun 26, 2017 principles of different staining techniques 1. The gram staining technique differentiates the mixed culture cells into two terms as gram positive bacteria and gram negative bacteria. Gram staining is used to determine gram status to classify bacteria broadly. Today, hardy diagnostics is manufacturer in the us. Principle of gram stain the two major groups of bacteria can be divided into gram positive and gram negative. 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. Most bacteria can be differentiated by the gram reactions due to difference in their cell wall structure. Objectives to learn the technique of smear preparation. 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. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique.
To differentiate between the two major categories of bacteria. Distinguishes bacteria of two large and medically important groups. The stain is retained because the cell wall of a grampositive bacteria is not as permeable as the cell wall of a gramnegative bacteria. Its application aim at read more albert staining principle, reagents, procedure, results, interpretation. Differentiate into gram positive and gram negative bacteria. For example, gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties. To perform this staining, it requires the use of multiple or more than one stains.
The staining procedure differentiates organisms of the domain. Most bacteria have no colour, so they generate little contrast in the microscope field. These organisms are usually filamentous, gram positive rods of varying length that often form chains, but occasionally, they may stain gram negative. Gram stain principle, laboratory continuing education. Gram stain hans christian gram history and principles of gram. Whereas in cold zn staining, instead of heating, we increase the concentration of the basic dye and phenol and incorporate a wetting agent chemical. It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. Gram staining technique is the widely used differential staining technique in bacteriology.
Differential staining such as gram staining and afb staining are commonly used to identify and differentiate the bacterial isolates. Gram staining of bacterial cells is neither an accurate nor elaborate technique, but nevertheless it is practically useful to distinguish two big domains of eubacteria species. The gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups. 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. The most widely used staining procedure in microbiology is the gram stain. 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. Yet there are differences in the cell walls within the species of bacteria. The cell walls for grampositive microorganisms have a higher peptidoglycan and lower lipid content than gramnegative bacteria. The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable.
This video explains the principle behind gram staining procedure. 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 grams is prevented. Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. 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, wbcs as well as the parasites.
Method of staining used to distinguish and classify bacterial species into two large groups. Pdf mlt 415 lab report gram stain techniques muhamad. Staining is an auxiliary technique used in microscopic techniques used. Materials and methods aseptic technique was used throughout the experiment.
The filter gram stain was approximately 100fold more sensitive than the slide gram stain. 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. Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition. 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. Full text full text is available as a scanned copy of the original print version. 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, procedure, interpretation and animation. Principle, reagents, procedure, step, result interpretation of gram stain. Acidfast stain principle, procedure, interpretation and. Gram positive bacteria have high content of peptidoglycan in. The process of histological staining takes five key stages which involve. Principle the gram reaction is dependent on permeability of the bacterial cell wall and cytoplasmic membrane, to the dyeiodine complex. What is gram stain test staining procedure, principle and. Gram stain developed in 1884 by hans gram, a danish microbiologist. Gram positive bacteria stain deep violet to blue and gram negative bacteria stain pink to red. Principle, preparation of reagents and interpretation using this link s. 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 chained, clustered of gram negative and gram positive bacteria. Faintly staining gramnegative organisms, such as campylobacter and brucella, may be visualized using an alternative counterstain e. Detailed principle, reagent preparation, procedure, result interpretation, examples and animation video for gram staining technique.
Simple staining can be useful in the following circumstances. Start studying gram stain principles, method and value. Oct 05, 2018 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 chained, clustered of gram negative and gram positive bacteria. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. Gram positive bacteria that include most of the firmicutes, and gram negative bacteria that include the rest. Therefore, to see bacteria with the microscope, it is necessary to apply colour by using a staining reagent. 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. Grampositive bacteria that include most of the firmicutes, and gramnegative bacteria that include the rest. Occurrence of a phosphoric ester in certain bacteria. The lipoid capsule of the acidfast organism takes up carbolfuchsin and resists decolorization with a dilute acid rinse. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. They have lipopolysaccharide containing a compound known as lipid a or. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram.
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