Gram staining of bacteria from environmental sources. It is one of the differential stains used to characterize bacteria as either gram positive bacteria or. Gently flood the smear with gram s iodine and let stand for 1 minute. Full text full text is available as a scanned copy of the original print version. The gram stain is a differential stain commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall structure. Module common staining technique microbiology 24 notes indeterminate groups as well. The gram stain and differential staining journal of. 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. Gram stain is one of the most common staining procedures.
Cover the smear with crystal violet, the primary stain, for 20 seconds. 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. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. The gram stain is used to classify bacteria on the basis of their forms, sizes, cellular morphologies, and gram reactions. Get a printable copy pdf file of the complete article 3. A common procedure, the gram stain, differentiates. Gram variable observations in gram staining the gram staining procedure does not always give clearcut results. Overheating of smears when heat fixation is used d. Prepare slide following directions for the appropriate sample source. 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 stain purpose, procedure and preparation microscopemaster. Flood airdried, heatfixed smear of cells for 1 minute with crystal violet staining reagent. Place slide with heat fixed smear on staining tray. Preparing serial dilutions 40 basic pract book 2006 21106 11. The gram stain is fundamental to the phenotypic characterization of. An infected appendix, or any tissue containing both negative and positive gram rods. Gram positive bacteria stain deep violet to blue and gram negative bacteria stain pink to red. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. Place slides on staining rack, drop crystal violet stain onto tissue section, stain for 1. In brief, the procedure involves the application of a solution of iodine potassium iodide to cells previously stained with crystal violet or gentian violet. 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. This standard operating procedure is applicable to all technical staff in the microbiology section that have been trained in performing this procedure and are competent and authorized to perform this procedure. Flood slide with crystal or gentian violet 60 seconds. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet.
The word gram is always spelled with a capital, referring to hans christian gram, the inventor of gram staining gram staining principles gram staining is used to determine gram status to classify bacteria broadly. The procedure steps of gram stain prepare and fix the specimen to the microscope slide before staining. Gram staining technique is the widely used differential staining technique in bacteriology. Place a drop of water in the middle of each circle with a dropper. Named after hans christian gram who developed the method in 1884, the gram stain allows one to distinguish between gram. Identifying microorganisms in normally sterile body fluids screening sputum specimens for acceptability for bacterial culture guiding initial antimicrobial therapy. The staining technique, developed in the late 1700s by christian gram classifies the rigid cell walled bacteria into one of two groups based on whether they are able to resist the decolorizing action of an alcoholic.
Decolorization the critical step of the gram staining procedure is the decolorization step. The gram stain procedure was originally developed by the danish physician hans christian gram to differentiate pneumococci from klebsiella pneumonia. While there is a wide range of staining methods available, the procedures for those are similar to that of grams stain. If you continue browsing the site, you agree to the use of cookies on this website. Protocols for using the gram stains in the microbiology laboratory. Some organisms are gram variable and may appear either gram negative or gram positive according to the conditions. The name comes from the danish bacteriologist hans christian gram, who developed the technique. Asm journals are the most prominent publications in the field, delivering uptodate and authoritative coverage of both basic and clinical microbiology. Through a series of staining and decolorization steps, organisms in the domain bacteria are differentiated according to cell wall composition. The reason for staining bacteria is due to the fact that most bacteria are transparent and cannot be seen through the microscope.
Carbolfuchsin solution, microwave 80 power, 45 seconds, allow slides to stand in hot solution. 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. Principle, procedure, interpretation, examples and animation last updated. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. Gram stain, gram positive, gram negative, heat fixation, methanol fixation. Acid fast staining differentiates acid fast bacteria from non acid fast bacteria based on the mycolic acid content in the cell wall. Links to pubmed are also available for selected references. It includes introduction, history,principle, method of gram staining procedure slideshare uses cookies to improve functionality and performance, and to provide you with relevant advertising. It is important to note that the expected results of the modern gram stain are best generated from young, actively growing 1824 hourold cultures. Studying on the granules of acidfast bacilli observed by using the various staining procedures, the author found the following results.
To visualize these cells, the contemporary gram stain procedure applies the counterstain safranin to the smear causing cells to appear red or pink. Full text get a printable copy pdf file of the complete article 1. A comparison of heat versus methanol fixation for gram. Label a glass microscope slide with pertinent information. Gram stain principle, reagents, procedure, steps, results. The filter gram stain was approximately 100fold more sensitive than the slide gram stain. Gram stain report introduction the purpose of this experiment is to determine the shape and gram stain of the bacteria under a microscope. Staining flood the entire slide when crystal violet, iodine and safranin are applied.
Cover different classification schemes for grouping bacteria, especially the use of the gram stain 2. The modified procedure provided greater reagent stability and better differentiation of organisms. 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 positive bacteria have cell walls that contain thick layers of peptidoglycan 90% of cell wall. Gram positive bacteria that include most of the firmicutes, and gram negative bacteria that include the rest. The gram stain is a type of differential stain that allows a microbiologist to. History and explanation of the fundamental technique of determinative bacteriology article pdf available march 2004 with 21,573 reads how we measure reads. The procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria. This is the difference between acid fast and gram stain. The gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain. Hold the slide in a tilted downward position and allow the decolorizer to flow over the smear. The gram stain, the most widely used staining procedure in bacteriology, is a complex and differential staining procedure.
Please note that the quality of the smear too heavy or too light cell concentration will affect the gram stain results. Through a series of staining and decolorization steps, bacteria are differentiated according to cell wall composition. Perform gram stain procedure quality control per standard laboratory protocol. Use millipore filtered water in the waterbath and staining procedure. In spite of his defective method, his name lives on jay hardy, cls, sm ascp. The procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain reaction. 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. Objectives to learn the technique of smear preparation. Smears for staining may be prepared in order of likely positive results i. Gram staining differentiates gram negative bacteria and gram positive bacteria based on the thickness of their cell walls. Unless it is a gram stain of a pure culture, there normally should be material staining. If your slide is all one color either pink or blue, then the slide may either have been over or under decolorized. By exploiting this phenomenon, an alternative gram staining technique has been developed. 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.
To learn the techniques of gram staining, nigrosin staining and koh test. Alternate gram staining technique using a fluorescent lectin. Gram staining is a differential staining technique that provides an easy differentiation of bacteria into one of two groups. Hold the slide in a tilted downward position and allow the. The gram stain procedure was originally developed by the. Gram stain hans christian gram history and principles of gram. The filter gram stain detected 2 to 100 bacteria, whereas the slide gram stain failed to detect less than. Gently flood smear with crystal violet and let stand for 1 minute. The gram stain is a classical and important staining technique that remains widely used by. Sentinel laboratory guidelines for suspected agents of. Gram staining is a differential staining technique that differentiates bacteria into two groups.
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