The main purpose of capsule stain is to distinguish capsular material from the bacterial cell. 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. This differential staining procedure separates most bacteria into two groups on the basis of cell wall composition. This slide set is an introduction to the use of grams stain as a rapid tool in the laboratory diagnosis of infectious disease. These organisms have waxy envelopes that make them difficult to stain and decolourise. Mycobacterium tuberculosis is the most important of this group, as it is responsible for the disease called tuberculosis tb along. Negative staining electron microscope protocol for rash illness. The main purpose of endospore staining is to differentiate bacterial spores from other vegetative cells and to differentiate spore formers from nonspore formers. Heat the slide for few seconds until it becomes hot to the touch so that bacteria are firmly mounted to the slide. The gram stain procedure distinguishes between gram positive and gram negative groups by coloring these cells red or violet. Some laboratories may choose to use a cytospin slide centrifuge to concentrate body fluids for s mear preparation. The gram stain involves staining bacteria, fixing the color with a mordant, decolorizing the cells, and applying a counterstain.
The original formulation comprised of aniline gentian violet, lugols iodine, absolute alcohol and bismarck brown. Apr 04, 2016 flood the slide with basic fuchsin counter stain and allow it to act for about 5 seconds. Full text full text is available as a scanned copy of the original print version. Gram positive and gram negative, based on the differences of the chemical and physical properties of the cell wall. In addition this stain also allows determination of cell morphology, size, and arrangement. 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. As a result of the use of 2 dyes, making this procedure a differential stain, bacteria will either become purpleblue or pink during the procedure. Place slides on staining rack, drop crystal violet stain onto tissue section, stain for 1. Spore stain schaefferfulton method staining bacteria. The gram stain is a differential stain, as opposed to the simple stain which uses 1 dye. Place a single drop of nigrosin on a clean microscope slide. Although simple stains are useful, they do not reveal details about the bacteria other than morphology and arrangement. Laboratory perspective of gram staining and its significance in investigations of infectious diseases yunusa thairu 1, idris abdullahi nasir 1, yahaya usman 2 1 department of medical microbiology, university of abuja teaching hospital, gwagwalada, abuja, nigeria 2 department of medical laboratory science, faculty of medicine, ahmadu bello university, zaria, kaduna, nigeria.
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 quick procedure used to look for the presence of bacteria in tissue samples and to characterise bacteria as grampositive or gramnegative, based on the chemical and physical properties of their cell. It is used to distinguish between grampositive and gramnegative bacteria, which have distinct and consistent differences in their cell walls. Staining is a technique used to enhance contrast in samples, generally at the microscopic level. As with most techniques, there are levels of complexity which yeild increasingly better results. The acid fast stain was discovered by ehrlich, who found that after staining with aniline dyes, tubercle bacilli resist decolourisation with acids. Gram staining helps to identify bacterial pathogens in specimens and cultures by their gram reaction gram positive and gram negative and morphology coccirod. Gram stain the previous lab introduced simple staining techniques that enable microbiologists to observe the morphological characteristics of bacteria. May 01, 2015 gram stain lab report by asem shadid 1.
The gram staining technique differentiates the mixed culture cells into two terms as gram. 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. Distinguishes bacteria of two large and medically important groups. Gram staining is an empirical method of differentiating bacterial species into two large groups grampositive and gramnegative based on the chemical and physical properties of their cell walls. Gram staining is a differential staining technique that differentiates bacteria into two groups. Mechanism of gram variability in select bacteria pdf. It is also used for staining bone marrow aspirates, urine samples and to demonstrate malarial parasites in blood smears. Differential staining is a procedure that takes advantage of differences in the physical and chemical properties of different groups of bacteria. Jan 08, 2020 the gram staining technique is the most important and widely used microbiological differential staining technique. It allows us to differentiate between different kinds of bacterial cells or different parts of a bacterial cell. The gram stain is a complex and differential staining procedure that remains a useful test performed in microbiology laboratories. Gram staining is an essential procedure used in the identification of bacteria and is frequently the only method required for studying their morphology. Learn vocabulary, terms, and more with flashcards, games, and other study tools.
Hans christian gram, is one of the most important staining techniques in microbiology. The gram stain procedure is a differential staining procedure that involves multiple steps. Get a printable copy pdf file of the complete article 2. The most useful staining procedure in medical microbiology. To differentiate between the two major categories of bacteria. Christian gram in 1884, and categorizes bacteria according to their gram character gram positive or gram negative. Our stain removal guide will help you remove even the most stubborn stains regardless of the location, fabric and type of stain. Principle, procedure, reporting and modifications by editorial team on january 11, 2020 in bacteriology, microbiology the ziehlneelsen stain zn stain, also called the hot method of afb staining, is a type of differential bacteriological stain used to identify acidfast organisms, mainly mycobacteria. This preparation method is useful for visualizing suspensions of small particles, which includes viruses, purified proteins, liposomes and small vesicle fractions. Get a printable copy pdf file of the complete article 3. Gram staining is still the cornerstone of bacterial identification and taxonomic division. Endospore staining principle, reagents, procedure and result. The name comes from the danish bacteriologist hans christian gram, who developed the technique.
The primary stain of this method is crystal violet, which can be sometimes substituted with equally effective methylene blue. Wrights stain is named for james homer wright, who devised the stain in 1902 based on a modification of romanowsky stain. In 1884, hans christian gram, a danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. Cell staining in microscopy types, techniques, preparations and procedures. 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.
A capsule is a gelatinous outer layer secreted by bacterial cell and that surrounds and adheres to the cell wall. Gram stain reagents are evaluated by staining the following recommended bacterial strains. The gram stain, the most widely used staining procedure in bacteriology. Different stains have different affinities for different organisms, or different. Most capsules are composed of polysaccharides, but some are composed of polypeptides. To learn the techniques of gram staining, nigrosin staining and koh test. Impregnation it is the deposition of salts of heavy metals on or around cells, tissue constituents etc. The primary stain, carbolfuchsin, is applied to the cells and phenol is used to allow the stain to penetrate into the waxy surface of acidfast microorganisms.
Introduction observation of bacteria with conventional bright field microscopy yields relatively little useful information. Make a slide of tissue or body fluid that is to be stained. To understand how the gram stain reaction affects gram positive and gram negative bacteria based on the biochemical and structural di. Giemsa stain is a buffered thiazineeosinate solution designed to provide coloration of blood cells similar to the original product described by giemsa. At this point, slides were immersed in alcoholic saffron american master tech scientific, inc. Some staining techniques, such as the gram and ziel neelsen stains, although of great diagnostic value because of their. After staining, an acid decolorizing solution is applied. Spore stain schaefferfulton method free download as powerpoint presentation. This was developed in 1884 by a danish bacteriologist, hans christian gram. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. It does not allow for high resolution examination of samples more technically demanding methods, using sample vitrification or rapid freezing are used for this. Fixation and staining procedure pipette all reagents down the side of the transwell insert so that the cell layer will not be disrupted.
Add the primary stain crystal violet and incubate 1 minute. It is the type of differential staining which makes the use of more than one stains to differentiate the bacteria. College of medicine alimam mohammad bin saud islamic university first year 20 2014 1 by. The gram stain the gram stain is the most important and universally used staining technique in the bacteriology laboratory. It is the differential staining techniques which was first developed by ziehl and later on modified by neelsen. Gram staining technique free download as powerpoint presentation. 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. Histologic staining options available include, but are not limited to.
Gram staining technique gram positive bacteria prokaryote. Cotton cotton can endure hot water, cold water, repeated washing and scrubbings. Stains may be used to define biological tissues highlighting. Common staining technique microbiology module microbiology notes 2. Laboratory perspective of gram staining and its significance. The procedure is named for the person who developed the technique, danish bacteriologist hans christian gram. Gram staining method, the most important procedure in microbiology, was developed by danish physician hans christian gram in 1884. Gram iodine mordant forms crystal violet iodine complexes 3. Allow smear to airdry, and heat fix in the usual manner. Negative staining is a simple technique for routine examination of structure. Gram stain the most commonly used differential stain is the gram stain, first described. The capsule differs from the slime layer that most.
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. Gram stain developed in 1884 by hans gram, a danish microbiologist. The presence of color gives the cells significant contrast so they are much more visible. Staining flood the entire slide when crystal violet, iodine and safranin are applied. For instance, a special stain technique highlights the flagella of bacteria by coating the flagella with dyes or metals to increase their width. 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. A secondary stain, methylene blue, is then applied to the cells. Wrights stain is a type of romanowsky stain, which is commonly used in hematology laboratory for the routine staining of peripheral blood smears. Tissue sections known to be pas positive andor contain glycogen should be included each time a stain sequence is performed. Csf and other body fluids requiring centrifugation. Objectives to learn the technique of smear preparation.
Crystal violet primary stain violet colored, stains all microorg 2. The century old gram stain remains the mainstay of rapid diagnosis. Links to pubmed are also available for selected references. This procedure is extensively used in the diagnosis of tuberculosis and leprosy.
Acidfast stain principle, procedure, interpretation and examples. 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. Polymorphonuclear leukocytes will show an intense red cytoplasmic stain. The gram stain dallas county community college district. The gram staining method was first given in 1884 by the danish scientist and physician hans christian gram the gram stain is the differential stain that stains the bacterial cells. 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. Oct 24, 2019 the procedure is based on the reaction between peptidoglycan in the cell walls of some bacteria.
It is a primary test which quickly and cost effectively divides bacteria into one of two types. A fluorescent stain is used in this method and the method is more. Gram staining is used widely technique and the most popular method in the laboratories. Stain removal guide printable stain removal guide pdf. Other stain techniques seek to identify various bacterial structures of importance. 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. Gram stain is a very important differential staining techniques used in the initial characterization and classification of bacteria in microbiology. Modification in gram staining methods since the original procedure of gram, many variations of the gram staining technique have been published. 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. Modified bismarck brown staining for demonstration of soft tissue mast cells pdf. This will ensure that all areas are stained evenly.
Differentiation in regressive staining differentiation is the removal of washing out of the excess stain until the colour is retained only in the tissue components to be studies. The gram staining method, named after the danish bacteriologist who. Apr 12, 2016 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. Before staining, the specimen must be mounted and fixed on the slides, as previously done in the simple. In spite of his defective method, his name lives on jay hardy, cls, sm ascp.
Unfortunately, however, when nisslstaining is combined with immunocytochemistry, the basic dyes stain primarily the cell nuclei with only faint labeling of cytoplasm of neurons. Atcc 25923, staphylococcus aureus and attc 25922, escherichia coli. Hold the slide in a tilted downward position and allow the. 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 or gram staining, also called grams method, is a method of staining used to. An optimized staining technique for the detection of gram. Improved method for combination of immunocytochemistry and. If the stain is on cotton, you have the most removal options. Gram positive bacteria that include most of the firmicutes, and gram negative bacteria that include the rest. Gram staining a important technique a staining technique used to classify bacteria. Grampositive bacteria gramnegative bacteria provides useful information for disease treatment. 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 technique is the widely used differential staining technique in bacteriology.
Spore stain principle, procedure and result interpretation. Simple and rapid, requiring only minutes as opposed to hours to even a day for sophisticated immunological techniques. Procedure of spore stain schaefferfulton method prepare smears in the usual manner using aseptic technique. Scribd is the worlds largest social reading and publishing site. Jun 10, 2006 gram stain was described by danish bacteriologist hans christian gram in 1884 to differentiate streptococcus pneumoniae from klebsiella pneumoniae in lung tissue. This method has been recommended to increase gram stain sensitivity and to decrease centrifugation and examination time for more rapid results. Flood smears with malachite green and place on top of a beaker of water sitting on a warm hot plate, allowing the preparation to steam for 2 to 3 minutes. Gram staining is a common technique used to differentiate two large groups of bacteria based on their different cell wall constituents. Stains and dyes are frequently used in histology the study of tissue under the microscope and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses disease at a microscopic level.
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. Negative staining electron microscope protocol for rash illness introduction electron microscopic em visualization of negatively stained poxvirus virions was a valuable technique for confirming poxvirus infections during the smallpox eradication campaign. Decolorization the critical step of the gram staining procedure is the decolorization step. In the cold technique known as kinyoun method, stain are not heated but the penetration is achieved by increasing concentration of basic fuchsin and phenol and incorporating a wetting agent chemical. An infected appendix, or any tissue containing both negative and positive gram rods. The data obtained from this procedure serves only as an aid to diagnosis and should. This staining technique divides bacteria into two groups namely acidfast and non acidfast. Oct 30, 2009 the nissl staining is a very useful histological method to facilitate the identification of cell populations in histological preparations. The stain binds to the mycolic acid in the mycobacterial cell wall. Acidfast stain principle, procedure, interpretation and. The excess stain is removed with treatment by 1% sulfuric acid.