Mechanisms to assess gram stain interpretation proficiency of. In gram staining and afb staining we use alcohol or acidalcohol or acid as a decolorizer but in spore staining, water is sufficient to be used as decolorizer because. The first step, before staining, is to heat fix the bacteria. The abovementioned research applications of the safranin stain show that the scope of safranin staining is not only limited to plant histology, but also microbiology, oncology, stem cell differentiation, and arthrology. Oct 23, 2016 gram staining is the technique developed by christian gram 1 2. The primary stain of the gram s method is crystal violet. Cover different classification schemes for grouping bacteria, especially the use of the gram stain 2. Gram stain reagents are evaluated by staining the following recommended bacterial strains. Flood slide with crystal or gentian violet 60 seconds. Cells from old cultures may stain gram negative even if the bacteria are gram positive.
Protocols for using the gram stains in the microbiology laboratory. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Gram stain is one of the most common staining technique used for examining specimens suspected to contain bacteriologic agents. Most bacteria can be differentiated by the gram reactions due to difference in their cell wall structure. It is one of the most useful staining procedures because it classifies bacteria into two large groups. The gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. Gram negative bacteria tend to be more resistant to certain antibiotics than gram positive bacteria. After staining with methyl violet and treatment with iodine, a dyeiodine. Gram positive and gram negative on the basis of cellwall difference and by the sequential application of crystal violet, iodine, alcohol and safranin.
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. It is theorized that during the gram stain procedure, the cell wall of. Solvents such as aniline, dimethylaniline, and nitrobenzene, which form chargetransfer complexes with iodine, can extract crystal violet from gram stained staphylococcus aureus s. The gram stain procedure is a differential staining procedure that involves multiple steps.
The gram stain procedure was originally developed by the danish physician hans christian gram to differentiate pneumococci from klebsiella pneumonia. These bacteria have a cell wall containing a thick layer of. 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. To address gram stain interpretation proficiency in a satellitecentralized microbiology laboratory paradigm, two programs were devised. Gram positive bacteria retain the color of the primary stain crystal violet in the gram staining procedure and appear as purpleviolet under a light microscope. The gram stain reaction and koh test results were compared with the antibiotic disk susceptibilities vancomycin and colistin the preliminary. This stain, originally developed to help distinguish bacteria from host cells in tissue, has evolved as a key assay to help clinicians decide which antibiotics should be used to treat infections, given that gram positive and gram negative organisms display differential responses to some classes of antimicrobial agents. Contrary to the recent staceys views, burke and barnes have concluded from their extensive investigations that if any combination is formed between crystal violet and the cellular proteins, this combination is destroyed upon the addition of iodine in the gram staining procedure. Gram staining is a differential staining technique that differentiates bacteria into two groups. In quality assurance program 1, nonmicrobiology technologists at satellite laboratories were required to interpret standardized gram stained specimens of clinical material prepared by an experienced microbiologist at a central laboratory. 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.
Get a printable copy pdf file of the complete article 3. It is the type of differential staining that is used to differentiate the bacteria majorly into two groups i. To learn the techniques of gram staining, nigrosin staining and koh test. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. It is one of the classic ways to determine the grampositive and gramnegative bacteria. Take the inoculating loop and disinfect this inoculating loop by the heating it on the bunsen burner flame.
Google scholar mittwer t, bartholomew jw, kallman bj. Jan, 2017 gram staining or gram stain, also called gram s method, is a method of staining used to differentiate bacterial species into two large groups gram positive and gram negative. In this procedure living, potentially pathogenic bacteria are smeared on the glass slide and allowed to air. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. Pdf crystal violet hexamethylpararosaniline chloride interacts with aqueous kii2 during the gram stain via a simple metathetical anion. A discussion of the mechanism of gram staining and how it differentiates bacteria on the basis of their cell wall structure should also be included 1 point. 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. 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. Mechanisms to assess gram stain interpretation proficiency. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents.
Recall that typical gram positive bacteria have a multilayered, thickened peptidoglycan layer, while gram positive bacteria have a thinner peptidoglycan layer sitting in the middle of the periplasmic space. The microscopicanalysis using gram staining technique showed that majority of the bacterial isolates were grampositive bacteria while only three of the. The chapter discusses the physicochemical studies on gram staining in relation to permeability theory. Pdf chemical mechanism of the gram stain and synthesis. Furthermore, since a platinum salt replaced the iodine mordant of the gram stain, energydispersive xray spectroscopy could evaluate the stain intensity and location by monitoring the platinum signal. Explain the mechanism of the gram stain why gram negative cells lose the primary dye during decolorization because gram negative cell walls dissolve during decolorization due to their high lipid content allowing the primary dye to leak out of the cell wall. The gram stain permits the separation of all bacterial species into two large groups, those which retain the primary dye gram positive, and those which lose the primary dye and take the color of the secondary dye gram negative. It is used to distinguish between gram positive and gram negative bacteria, which have distinct and consistent differences in their cell walls. 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. J a davies, g k anderson, t j beveridge, and h c clark.
It is a technique used to differentiate types of bacteria using certain physical and chemical characteristics of their cell walls. Dec 20, 2017 gram staining was given by christian gram in 1884 and it is also called differential staining because it differentiates bacterial in different groups gram positive and gram negative, based on. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Gram staining is the technique developed by christian gram 1 2. 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. The gram positive bacteria have a thick layer of peptidoglycan whereas gram negative bacte. Chemical mechanism of the gram stain and synthesis of a. The name comes from the danish bacteriologist hans christian gram, who developed the technique. Gram staining lab introduction gram staining is a very important technique used in biology labs all over the world.
The probable mechanism of the gram stain is attributed to differences in the permeability of the two essential cell wall types. Gram stain reagents are checked weekly and also when a new lot of stain is to be put into use, as per the clia quality control regulatory notice published july 18, 1997. Therefore, the mechanism of the gram stain appears to be related to the presence of an intact cell wall able to act as a barrier to decolorization of the primary stain. The mechanism of gram differentiation attempts have been made from time to time to find a satisfactory solution for the mechanism of gram staining, and many theories have been put forward, gram staining and its molecular mechanism ii both chemical and physical. This test differentiate the bacteria into gram positive and gram negative bacteria, which helps in the classification and differentiations of microorganisms. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. In this report, we describe a mechanism for objectively assessing the gram stain interpretation competency of nonmicrobiology laboratorians and present a quality assurance program for monitoring and increasing the proficiency of gram staining. Occurrence of a phosphoric ester in certain bacteria. Pdf chemical mechanism of the gram stain and synthesis of a. Gram staining and its molecular mechanism sciencedirect. Pdf gram staining procedure find, read and cite all the research you need on researchgate.
The gram staining method, named after the danish bacteriologist who originally devised it in 1882 published 1884, hans christian gram, is one of the most. Here are the following main principles of gram staining techniques. This paper deals with an investigation of the effect of these reactions on bacteria. The gram staining method is named after hans christian gram, the danish. Principle, procedure, interpretation, examples and animation last updated. Gram staining procedure, mechanism, explanation introduction the gram stain was developed in 1884 by the danish bacteriologist hans christian gram. The gram staining technique differentiates the mixed culture cells into two terms as gram positive bacteria and gram. Oct 01, 2007 i think if you had a book, it might help you better. To start the staining process, pour crystal violet dye on the slide. For example, gram staining can help clinicians classify bacterial pathogens in a sample into categories associated with specific properties.
The gram stain has also been explained entirely on the basis of cell membrane permeability, in which case various roles have been assigned to iodine. The gram stain, the most widely used staining procedure in bacteriology. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. Gram staining or gram stain, also called gram s method, is a method of staining used to differentiate bacterial species into two large groups gram positive and gram. The grampositive bacteria have a thick layer of peptidoglycan whereas gramnegative bacte. 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 gram positive and gram negative. What are the mechanisms of gram staining, and how do they. Cell staining in microscopy types, techniques, preparations. Remember that a hypothesis need not be correct, but it must be tested by the. 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 difficult to discern due to insufficient contrast. In brief, the procedure involves the application of a solution of iodine potassium iodide to cells previously stained with crystal violet or gentian violet.
It is one of the classic ways to determine the gram positive and gram negative bacteria. It is almost always the first test performed for the identification of bacteria. Apr 04, 2016 a staining technique used to classify bacteria. 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.
Fischer, 1951 between intact wool and gram negative bacteria, medium degraded wool and gram variable bacteria, as well as between highly degraded wool and gram positive bacteria suggested that reversal of gram. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. This platinum anion therefore allows the gram staining mechanism to be followed by electron microscopy. 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.
Atcc 25923, staphylococcus aureus and attc 25922, escherichia coli. These gram variable bacteria could be split into two groups on the basis of their staining responses. A third experiment was performed with two cellulose diffusion cells, one. Gram staining is a bacteriological laboratory technique used to differentiate bacterial species into two large groups grampositive and gramnegative based on the physical properties of their cell walls. Feb 27, 2009 some bacteria, after staining with the gram stain, yield a gram variable pattern. Overheating of smears when heat fixation is used d. Solubilities of dyeiodine precipitates and further studies of primary dye substitutes. 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. 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. The gram stain the gram stain is the most important and universally used staining technique in the bacteriology laboratory. Chemical mechanism of the gram stain and synthesis of a new. Chemical mechanism of the gram stain and synthesis of a new electronopaque marker for electron microscopy which replaces the iodine mordant of the stain. Gram staining depends on the thickness of the peptidoglycan layer of bacteria. Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. Not only the common staining behavior but also certain other similarities fischer et al. Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope. It is one of the differential stains that are used to characterize bacteria in one of two groups. How would you explain the mechanism of gram staining. Bacterial cell morphology, staining reactions, classification of bacteria.
Mode of action of gram stain at this time the mechanism of the gram stain reaction is not completely known. Gram stain is probably one of the most commonly used staining procedures used in the field of microbiology. In most microbiological staining procedures, the bacteria are first fixed to the slide by the heat fixed smear figure 1. They are based on the physical and chemical reactions of the cell to the staining reagents. 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. There are many theories, but none are completely satisfactory. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. With safranin staining, the gram negative bacteria can be easily distinguished from gram positive bacteria.
These results would indicate that the formation of a specific proteindye complex is not an essential factor in the mechanism of the gram stain. The genera actinomyces, arthobacter, corynebacterium, mycobacterium, and propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in gram negative staining of these gram positive. Explain the mechanism of gram staining yahoo answers. The gram staining method, named after the danish bacteriologist who originally devised it in 1882 published 1884, hans christian gram, is one of the most important staining techniques in microbiology. Gram staining technique is the widely used differential staining technique in bacteriology. It was devised by a danish physician, hans christian gram, in 1884. In addition this stain also allows determination of cell morphology, size, and arrangement. Bacterial classification, structure and function introduction the purpose of this lecture is to introduce you to terminology used in microbiology.
The gram staining technique differentiates the mixed culture cells into two terms as grampositive bacteria and gramnegative bacteria. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope. We will discuss this and other applications of gram staining in more detail in later chapters. Cell staining in microscopy types, techniques, preparations and procedures. Full text get a printable copy pdf file of the complete article 3. This means putting the slide with the sample on a slide warmer, and let it sit for 5 minutes. These gram variable bacteria could be split into two groups on the basis of their staining. This was developed in 1884 by a danish bacteriologist, hans christian gram. Generally, the cell wall is nonselectively permeable. Apr 26, 2019 gram staining protocol smear preparation first of all, take a lubricant permitted dry slide. Gram staining protocol for bacterial differentiation. The gram reaction reflects fundamental differences in the biochemical and structural properties of bacteria. Cell structure in relation to the gram reaction as shown during lysis of bacillus subtilis. Gram staining is still the cornerstone of bacterial identification and taxonomic division.
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