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