The cell walls of gram positive bacteria have a thick layer of proteinsugar complexes called peptidoglycan and lipid content is low. 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. Gram in 1884 it remains an important and useful technique to this day. 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 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. This is a stabilizer that causes the dye to form large complexes in the peptidoglycan meshwork of the cell wall.
A common procedure, the gram stain, differentiates. 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 is a differential staining technique that provides an easy differentiation of bacteria into one of two groups. In the absence of an informative gram stain, the predictive value of sputum culture is very low. The gram stain and differential staining journal of. Oct 24, 2019 the primary stain crystal violet binds to peptidoglycan, coloring cells purple. Of all the different classification systems, the gram stain has withstood the test of time.
The gram stain is the most important staining procedure in microbiology. This method has been recommended to increase gram stain sensitivity and to decrease centrifugation and examination time for more rapid results. The 100x oil immersion lens is essential for viewing individual bacteria. As a result, bacteria are classified as either grampositive or gramnegative. Some laboratories may choose to use a cytospin slide centrifuge to concentrate body fluids for s mear preparation. Gram staining involves a fourpart process, which includes. Gram staining technique gram positive bacteria prokaryote. Apr 12, 2016 schematic showing the modified gram stain procedure. It is one of the most important and widely used differential staining techniques in microbiology. Gram smear preparation in an automated microbiology laboratory.
Gram staining is a differential staining technique that differentiates bacteria into two groups. 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. Primary care update for obgyns vol 10, issue 5, pages. Gram stain lab 2 exercise science prof online free. Flood the slide with crystal or methyl violet stain and allow to act for about 5 seconds. Differential staining is one type of staining technique used to differentiate bacteria. Cells that do not retains the colour of primary stain and takes up the colour of counter stain are gram negative. Flood slide with crystal or gentian violet 60 seconds. Gram s iodine iodine and potassium iodide is applied as a mordant or fixative. Responsibility microbiologist or above of microbiology laboratory. Primary care update for obgyns vol 10, issue 5, pages a1. Get a printable copy pdf file of the complete article 3. Gram stain, grampositive, gramnegative, heat fixation, methanol fixation. Gram staining technique free download as powerpoint presentation.
Gram staining is a differential technique that provides an easy differentiation ppt. Gram stain, gram positive, gram negative, heat fixation, methanol fixation. The gram stain remains one of the oldest, trusted, simplest and most reliable testing techniques in the microbiology laboratory. Applying a primary stain crystal violet to a heatfixed smear of a bacterial culture. For example, staphylococci, streptococci and pneumocci are grampositive and stain a deep blue, whereas coliforms and neisseria are gramnegative and stain pink. It is used to differentiate between gram positive organisms and gram negative organisms. Jul 28, 2017 visualization of microorganisms in living state is difficult.
Feb 03, 2015 gram staining gram staining is most widely used differential staining in microbiology. Visualization of microorganisms in living state is difficult. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. The gram stain is the first test performed on specimens and often leads to clinical decision making and antibiotic treatment. 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. Sop on gram staining objective to lay down the procedure for gram staining. History and explanation of the fundamental technique of determinative bacteriology article pdf available march 2004 with 22,205 reads how we measure reads. 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 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. It is recommended that, in order to determine the tendency of an organism with regard to the gram stain, more than one staining procedure be used, and that preparations of the culture be prepared at various stages of growth from 12 hours to several days in age.
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. Scope this sop is applicable for gram staining in microbiology laboratory of pharmaceutical company name. An optimized staining technique for the detection of gram. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. This will allow you to focus quickly and to look for areas of purulence. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. Gram staining uses crystal violet to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain to mark all bacteria. The links on this page provide the virtual microbiology classroom documents, web pages and videos useful in preparing for this lab. Gram positive and gram negative, which are identified in a technique called the gram stain, named after its developer, the danish bacteriologist hans christian. Misinterpretation of gram stain from the stationary growth phase of. Both gram positive and gram negative cells have peptidoglycan in their cell walls, so initially, all bacteria stain violet. Gram staining is the common, important, and most used differential staining techniques in microbiology, which was.
The gram stain is a differential stain commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall structure. Csf and other body fluids requiring centrifugation. The thicker grampositive cell walls are able to more firmly trap the large complexes than those of the gramnegative cells. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. To learn the techniques of gram staining, nigrosin staining and koh test. The name comes from the danish bacteriologist hans christian gram, who developed the technique gram staining differentiates bacteria by the chemical and physical. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. In 1884, hans christian gram, a danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. It is a primary test which quickly and cost effectively divides bacteria into one of two types. Ziehl nielsen stain is used to identify acidfast bacilli, including mycobacteria tuberculosis and leprosy, which appear as black rods.
The name comes from the danish bacteriologist hans christian gram, who developed the technique. Microbiology laboratory exercise powerpoint on how to perform gram stain technique and. From the teaching laboratory to the clinical microbiology laboratory, one of the bestknown tools to study microbes is the gram stain. Gram staining differentiates the bacteria into 2 groups. Sputum examination an overview sciencedirect topics. The addition of grams iodine, which binds to crystal violet and traps it in the cell. Principle, procedure, interpretation, examples and. The gram stain is a differential staining technique used to classify categorize. In spite of his defective method, his name lives on jay hardy, cls, sm ascp. Tip off the stain and flood the tilted slide with iodine solution and allow to act for about 5. The iodine has no effect on the gramnegative cells. This technique was introduced in 1884 by danish physician christian gram. Google scholar mittwer t, bartholomew jw, kallman bj. A comparison of heat versus methanol fixation for gram.
Objectives to learn the technique of smear preparation. Hans christian joachim gram the gram stain was devised by the danish physician, hans christian joachim gram, while working in berlin in 1883. Staining techniques in microbiology linkedin slideshare. By exploiting this phenomenon, an alternative gram staining technique has been developed. A monograph of gramstained preparations of clinical specimens. The gram stain, the most widely used staining procedure in bacteriology. This stain, originally developed to help distinguish bacteria. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. The views expressed in this editorial do not necessarily reflect the views of the journal or of asm. Rating is available when the video has been rented. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. Bisen prakash s microbes in practice, ik international, new delhi, 2014 pp 9155.
Gram staining is still the cornerstone of bacterial identification and taxonomic division. The traditional gram stain procedure is completed in either case top box. Grampositive bacteria stain deep violet to blue and gramnegative bacteria stain pink to red. Gram positives are violet, while gram negatives are red. While most bacterial cell walls contain peptidoglycan, not all bacterial cell walls have the same structure. For example, staphylococci, streptococci and pneumocci are gram positive and stain a deep blue, whereas coliforms and neisseria are gram negative and stain pink. Therefore, biological stains and staining procedures are used extensively to study their properties. Difference between gram stain and acid fast compare the.
This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Gram positive cells form a crystal violetiodine complex. Pdf gram staining procedure find, read and cite all the research you need on researchgate. Mybook is a cheap paperback edition of the original book and will be sold at uniform, low price.
It allows a large proportion of clinically important bacteria to be classified as either gram positive or negative based on their. The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. In the first step the smear is stained with basic dye crystal violet primary stain. 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. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure. Bacterial taxonomy today depends upon the extent of dna sequence homology. If your slide is all one color either pink or blue, then the slide may either have been over or under decolorized. 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. Gram stain and acid fast stain are two differential staining techniques. The first page of the pdf of this article appears above. The gram stain is an important technique in microbiology that is often a first step in characterizing and identifying bacteria.
The gram stain was first used in 1884 by hans christian gram gram,1884. Gram was searching for a method that would allow visualization of cocci in tissue sections of lungs of. 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. An important laboratory technique for the amplification and detection of specific dna sequences as, for. Unless it is a gram stain of a pure culture, there normally should be material staining both colors somewhere on the slide. Occurrence of a phosphoric ester in certain bacteria. In addition this stain also allows determination of cell morphology, size, and arrangement. A monograph of gramstained preparations of clinical specimens marler ms mtascpsm, linda m. 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 positive bacteria stain deep violet to blue and gram negative bacteria stain pink to red.
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