Gram positive bacteria have high content of peptidoglycan in. The name comes from the danish bacteriologist hans christian gram, who developed the technique. The filter gram stain was approximately 100fold more sensitive than the slide gram stain. Grampositive bacteria that include most of the firmicutes, and gramnegative bacteria that include the rest. Acidfast stain principle, procedure, interpretation and. Principle, procedure, interpretation, examples and animation last updated. Cell wall structure identifies either cell is gram positive or negative in nature. 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 procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. Gram staining is an empirical method of differentiating bacterial species into two large groups grampositive and gramnegative based on the chemical and. This differential staining procedure separates most bacteria into two groups on the basis of 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. 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. Simple staining can be useful in the following circumstances. Gram positive bacteria gram negative bacteria provides useful information for disease treatment. Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition. It was a few years later, that the german pathologist carl weigert 1845. We will discuss this and other applications of gram staining in more detail in later chapters. Its application aim at read more albert staining principle, reagents, procedure, results, interpretation. Gram staining principle, procedure, function of reagents. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. Gram staining is used to determine gram status to classify bacteria broadly. Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. For example, gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties.
Principle, preparation of reagents and interpretation using this link s. Most bacteria have no colour, so they generate little contrast in the microscope field. Principle, procedure, interpretation and animation. 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. They have less amount of peptidoglycan in their cell wall. Method of staining used to distinguish and classify bacterial species into two large groups. Gram stain developed in 1884 by hans gram, a danish microbiologist. Gram variable observations in gram staining the gram. I have explained the difference between cell wall structure of gram positive and gram negative bacteria along with the function 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. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. 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. 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. It identifies and classifies two major groups of bacteria, i. 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 stain hans christian gram history and principles of gram. May 9, 2016 acharya tankeshwar bacteriology, laboratory diagnosis of bacterial disease.
Objectives to learn the technique of smear preparation. Gram staining and the steps needed to complete the procedure. Gram staining is a differential staining technique that differentiates bacteria into two groups. 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. 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. Gram positive bacteria have high content of peptidoglycan in its cell wall while the lipid content is quite low. Bacteria that retain the primary crystal violet stain that is first applied as part of the staining process are classified as grampositive bacteria. Here are the following main principles of gram staining techniques. Occurrence of a phosphoric ester in certain bacteria. Most bacteria can be differentiated by the gram reactions due to difference in their cell wall structure. Yet there are differences in the cell walls within the species of bacteria. The stain is retained because the cell wall of a grampositive bacteria is not as permeable as the cell wall of a gramnegative bacteria. What is gram stain test staining procedure, principle and. The gram staining technique differentiates the mixed culture cells into two terms as gram positive bacteria and gram negative bacteria.
Materials and methods aseptic technique was used throughout the experiment. 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. This video explains the principle behind gram staining procedure. The cell walls for grampositive microorganisms have a higher peptidoglycan and lower lipid content than gramnegative bacteria. It is categorised into two types that are given below.
Detailed principle, reagent preparation, procedure, result interpretation, examples and animation video for gram staining technique. Distinguishes bacteria of two large and medically important groups. They have lipopolysaccharide containing a compound known as lipid a or. Gram staining technique is the widely used differential staining technique in bacteriology. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than. What is gram stain test staining procedure, principle. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were.
Gram positive bacteria that include most of the firmicutes, and gram negative bacteria that include the rest. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. The lipoid capsule of the acidfast organism takes up carbolfuchsin and resists decolorization with a dilute acid rinse. In gram positive bacteria, the crystal violet dye iodine complex combines to form a larger molecule which precipitates within the cell. For demonstrating gramnegative and grampositive in tissue. I have explained the difference between cell wall structure of gram positive. If your slide is all one color either pink or blue, then the slide may either have been over or under decolorized.
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. Faintly staining gramnegative organisms, such as campylobacter and brucella, may be visualized using an alternative counterstain e. 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. 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. Links to pubmed are also available for selected references. Principle and purpose the gram stain is perhaps the most useful test conducted in the clinical microbiology laboratory.
Gram positive bacteria stain deep violet to blue and gram negative bacteria stain pink to red. 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. To learn the techniques of gram staining, nigrosin staining and koh test. 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. 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. Jun 12, 2018 acidfast stain principle, procedure, interpretation and examples. 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. Grampositive bacteria stain deep violet to blue and gramnegative bacteria stain pink to red. 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. Gram staining principles gram staining is used to determine gram status to classify bacteria broadly. 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. Principle, procedure, interpretation, examples and. 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. The gram staining technique differentiates the mixed culture cells into two terms as grampositive bacteria and gramnegative bacteria.
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. Pdf mlt 415 lab report gram stain techniques muhamad. Prepare and fix the specimen to the microscope slide before staining. Gram stain principle, laboratory continuing education. The cell walls of gram positive bacteria have a thick layer of proteinsugar complexes called peptidoglycan and lipid content is low. Dr hans christian joachim gram introduced it in 1884.
The process of histological staining takes five key stages which involve. Start studying gram stain principles, method and value. 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. Certain bacteria are stained with aniline dyes, like gentian violet. Gram stain principle, reagents, procedure, steps, results. Jun 26, 2017 principles of different staining techniques 1. The most useful staining procedure in medical microbiology. Therefore, to see bacteria with the microscope, it is necessary to apply colour by using a staining reagent. The most widely used staining procedure in microbiology is the gram stain. Google scholar mittwer t, bartholomew jw, kallman bj. The most common stain being gram staining, acidfast staining, endospore staining.
Full text full text is available as a scanned copy of the original print version. 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. Gram stain principles, method and value flashcards quizlet. The composition of the cellular wall of gram positive and gram negative bacteria tells the gram staining differences. The composition of the cellular wall of grampositive and gramnegative bacteria tells the gram staining differences. These organisms are usually filamentous, gram positive rods of varying length that often form chains, but occasionally, they may stain gram negative. Principle of gram stain the two major groups of bacteria can be divided into gram positive and gram negative. The purpose of the gram stain is to show whether the bacteria are gram positive purplecolored, gram negative pinkcolored, or both.
Whereas in cold zn staining, instead of heating, we increase the concentration of the basic dye and phenol and incorporate a wetting agent chemical. Principle, reagents, procedure, step, result interpretation of gram stain. 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 test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. Each of these stains aims at identifying and characterizing bacteria based on their morphologies. The gram stain technique is based on the differential structure of the cellular membranes and cell walls of the two groups.
Get a printable copy pdf file of the complete article 3. To differentiate between the two major categories of bacteria. Gramnegative bacteria tend to be more resistant to certain antibiotics than grampositive bacteria. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were grampositive bacteria while only three of the. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent.
Staining is an auxiliary technique used in microscopic techniques used. The staining procedure differentiates organisms of the domain. This was developed in 1884 by a danish bacteriologist, hans christian gram. Unless it is a gram stain of a pure culture, there normally should be material staining both colors somewhere on the slide. Microbiology lecture 2 gram positive and gram negative.
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. Differential staining such as gram staining and afb staining are commonly used to identify and differentiate the bacterial isolates. 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.
In this procedure living, potentially pathogenic bacteria are smeared on the glass slide and allowed to air dry. As the cell wall of bacteria is very different from the cell wall of other organisms. Principle the gram reaction is dependent on permeability of the bacterial cell wall and cytoplasmic membrane, to the dyeiodine complex. 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. The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure.
Bacteria that are grampositive have a thick layer of peptidoglycan. Principle the gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. Today, hardy diagnostics is manufacturer in the us. 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. In most microbiological staining procedures, the bacteria are first fixed to the slide by the heat fixed smear figure 1. Apr 04, 2016 a staining technique used to classify bacteria.
Flood slide with crystal or gentian violet 60 seconds. Hot zn stain is the usual method in which we heat the smear to enhance the dye penetration. The structure of the organisms cell wall determines whether the organism is gram psitive or negative. To perform this staining, it requires the use of multiple or more than one stains. The gram stain is almost always the first step in the identification of a bacterial organism, while gram staining is a valuable. 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. Differentiate into gram positive and gram negative bacteria. Learn vocabulary, terms, and more with flashcards, games, and other study tools.1439 1066 1597 476 337 1462 1459 1217 827 1502 1233 731 1405 930 1157 1026 1561 1027 605 511 330 424 455 68 710 1572 1229 104 902 1215 417 629 791 382 325 424