Differential staining is one type of staining technique used to differentiate bacteria. The links on this page provide the virtual microbiology classroom documents, web pages and videos useful in preparing for this lab. The views expressed in this editorial do not necessarily reflect the views of the journal or of asm. It is one of the most important and widely used differential staining techniques in microbiology. While there is a wide range of staining methods available, the procedures for those are similar to that of grams stain. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price. The first page of the pdf of this article appears above.
While most bacterial cell walls contain peptidoglycan, not all bacterial cell walls have the same structure. In 1884, hans christian gram, a danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. Gram was searching for a method that would allow visualization of cocci in tissue sections of lungs of. Gram stain and acid fast stain are two differential staining techniques. Staining techniques in microbiology linkedin slideshare. The gram stain remains one of the oldest, trusted, simplest and most reliable testing techniques in the microbiology laboratory. History and explanation of the fundamental technique of determinative bacteriology article pdf available march 2004 with 22,205 reads how we measure reads. Of all the different classification systems, the gram stain has withstood the test of time. Google scholar mittwer t, bartholomew jw, kallman bj. A comparison of heat versus methanol fixation for gram. Bacterial taxonomy today depends upon the extent of dna sequence homology.
Oct 24, 2019 the primary stain crystal violet binds to peptidoglycan, coloring cells purple. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. This is a stabilizer that causes the dye to form large complexes in the peptidoglycan meshwork of the cell wall. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. From the teaching laboratory to the clinical microbiology laboratory, one of the bestknown tools to study microbes is the 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. Jul 28, 2017 visualization of microorganisms in living state is difficult. Gram positives are violet, while gram negatives are red. Gram stain, gram positive, gram negative, heat fixation, methanol fixation. A comparison of this new method with well known gram stain methods demonstrates its superiority in differentially staining grampositive and gramnegative bacteria in control experimental tissue lesions when types of fixatives, duration of tissue storage prior to paraffin embedment, types of embedding agents, and section thickness are varied. Sop on gram staining objective to lay down the procedure for gram staining. Gram staining bacteria requires the use of aseptic technique to ensure the sterility of the experiment. Visualization of microorganisms in living state is difficult. Both gram positive and gram negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet.
Gram positive bacteria stain deep violet to blue and gram negative bacteria stain pink to red. Flood slide with crystal or gentian violet 60 seconds. Gram staining involves a fourpart process, which includes. Flood the slide with crystal or methyl violet stain and allow to act for about 5 seconds. In the first step the smear is stained with basic dye crystal violet primary stain. Primary care update for obgyns vol 10, issue 5, pages.
A monograph of gramstained preparations of clinical specimens. Primary care update for obgyns vol 10, issue 5, pages a1. Gram staining is the common, important, and most used differential staining techniques in microbiology, which was. Some laboratories may choose to use a cytospin slide centrifuge to concentrate body fluids for s mear preparation. Grampositive bacteria stain deep violet to blue and gramnegative bacteria stain pink to red.
In spite of his defective method, his name lives on jay hardy, cls, sm ascp. Difference between gram stain and acid fast compare the. The gram stain was first used in 1884 by hans christian gram gram,1884. It allows a large proportion of clinically important bacteria to be classified as either gram positive or negative based on their. Occurrence of a phosphoric ester in certain bacteria. The 100x oil immersion lens is essential for viewing individual bacteria. Microbiology laboratory exercise powerpoint on how to perform gram stain technique and. The name comes from the danish bacteriologist hans christian gram, who developed the technique. 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. In addition this stain also allows determination of cell morphology, size, and arrangement. Gram staining technique free download as powerpoint presentation. Gram staining technique gram positive bacteria prokaryote. A monograph of gramstained preparations of clinical specimens marler ms mtascpsm, linda m.
It is a primary test which quickly and cost effectively divides bacteria into one of two types. Apr 12, 2016 schematic showing the modified gram stain procedure. Sputum examination an overview sciencedirect topics. The gram stain and differential staining journal of. Pdf gram staining procedure find, read and cite all the research you need on researchgate. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure. Objectives to learn the technique of smear preparation. The gram stain is a differential stain commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall structure. 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. 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.
The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. The iodine has no effect on the gramnegative cells. Therefore, biological stains and staining procedures are used extensively to study their properties. The modified gram stain technique was performed in the same fashion as described in the gram stain above, with the only addition of a counterstain after the first dehydration step of 100 % alcohol fig.
It is used to differentiate between gram positive organisms and gram negative organisms. Nov 03, 20 cells that do not retains the colour of primary stain and takes up the colour of counter stain are gram negative. Gram positive and gram negative, which are identified in a technique called the gram stain, named after its developer, the danish bacteriologist hans christian. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. Quick gram method by this method fairly good results may be obtained with very short staining times, which are convenient when only one slide has to be stained. Gram staining is a differential staining technique that provides an easy differentiation of bacteria into one of two groups. Scope this sop is applicable for gram staining in microbiology laboratory of pharmaceutical company name. A fundamental laboratory technique that is introduced in general biology and microbiology courses is staining of bacterial cells on glass slides for visualization and characterization purposes. Tip off the stain and flood the tilted slide with iodine solution and allow to act for about 5.
Get a printable copy pdf file of the complete article 3. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. This technique was introduced in 1884 by danish physician christian gram. Hans christian joachim gram the gram stain was devised by the danish physician, hans christian joachim gram, while working in berlin in 1883. The thicker grampositive cell walls are able to more firmly trap the large complexes than those of the gramnegative cells. Gram in 1884 it remains an important and useful technique to this day. Rating is available when the video has been rented.
This stain, originally developed to help distinguish bacteria. Gram smear preparation in an automated microbiology laboratory. To learn the techniques of gram staining, nigrosin staining and koh test. Gram staining is still the cornerstone of bacterial identification and taxonomic division. When the bacteria is stained with primary stain crystal violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol.
Gram stain set instructions introduction one of the most frequently cited criteria by which bacteria are classified involves the reaction of individual species to the gram staining procedure. Responsibility microbiologist or above of microbiology laboratory. An important laboratory technique for the amplification and detection of specific dna sequences as, for. Csf and other body fluids requiring centrifugation. 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. For example, staphylococci, streptococci and pneumocci are gram positive and stain a deep blue, whereas coliforms and neisseria are gram negative and stain pink. The major difference in the modified gram stain procedure occurs during the dehydration process and is the application of alcoholic saffron lower right box, highlighted in red.
This will allow you to focus quickly and to look for areas of purulence. The gram stain is a differential staining technique used to classify categorize. Gram positive cells form a crystal violetiodine complex. Gram staining differentiates the bacteria into 2 groups. An optimized staining technique for the detection of gram.
The latest idsaats guidelines 58 recommend obtaining a sputum sample for gram stain and culture in hospitalized patients with the clinical indications listed in table 336 but are optional for patients without these conditions. As a result, bacteria are classified as either grampositive or gramnegative. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. This method has been recommended to increase gram stain sensitivity and to decrease centrifugation and examination time for more rapid results. The gram stain is an important technique in microbiology that is often a first step in characterizing and identifying bacteria.
Gram s iodine iodine and potassium iodide is applied as a mordant or fixative. In the absence of an informative gram stain, the predictive value of sputum culture is very low. Principle, procedure, interpretation, examples and. Feb 03, 2015 gram staining gram staining is most widely used differential staining in microbiology. A comparison of this new method with well known gram stain methods demonstrates its superiority in differentially staining gram positive and gramnegative bacteria in control experimental tissue lesions when types of fixatives, duration of tissue storage prior to paraffin embedment, types of embedding agents, and section thickness are varied. 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. Misinterpretation of gram stain from the stationary growth phase of. The gram stain is the most important staining procedure in microbiology. The name comes from the danish bacteriologist hans christian gram, who developed the technique gram staining differentiates bacteria by the chemical and physical. Ziehl nielsen stain is used to identify acidfast bacilli, including mycobacteria tuberculosis and. Gram staining is a differential staining technique that differentiates bacteria into two groups. A common procedure, the gram stain, differentiates.
For example, staphylococci, streptococci and pneumocci are grampositive and stain a deep blue, whereas coliforms and neisseria are gramnegative and stain pink. If your slide is all one color either pink or blue, then the slide may either have been over or under decolorized. The traditional gram stain procedure is completed in either case top box. Gram staining is a differential technique that provides an easy differentiation ppt. At this point, slides were immersed in alcoholic saffron american master tech scientific, inc. The addition of grams iodine, which binds to crystal violet and traps it in the cell. Applying a primary stain crystal violet to a heatfixed smear of a bacterial culture. The cell walls of gram positive bacteria have a thick layer of proteinsugar complexes called peptidoglycan and lipid content is low. The gram stain is the first test performed on specimens and often leads to clinical decision making and antibiotic treatment. 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.
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